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10352011 | Activated heptahelical receptors are phosphorylated by a family of G protein–coupled receptor kinases (GRKs). 1 Following phosphorylation, the receptors bind to another family of proteins called arrestins . The regions of the receptors that arrestins bind to, generally the third intracellular loop and the portion of the carboxyl-terminal tail closest to the membrane, are also primary determinants for G protein interaction. Arrestin binding to receptors thus results in desensitization of G protein–mediated signaling by preventing interaction of receptors with G proteins. An emerging view, however, is that the binding of arrestins to heptahelical receptors also initiates a new set of signaling pathways in addition to blocking those mediated by G protein activation. It was proposed recently, for example, that β-arrestin can act as an adaptor protein to recruit the tyrosine kinase Src into a signaling complex organized around the β 2 -adrenergic receptor . It is well known that stimulation of many heptahelical receptors can lead to the activation of MAP kinases, but the mechanisms involved have been difficult to define. While G protein activation is clearly necessary, activation of tyrosine kinases of the Src family is required in many cases as well . The most recent findings reveal that Src associates in cells with agonist-activated β 2 -adrenergic receptors, as assessed by immunofluorescence and coimmunoprecipitation. The recruitment of cellular Src to β 2 -adrenergic receptors is potentiated by overexpression of β-arrestin, and in vitro pull-down studies reveal a direct high-affinity association between Src and β-arrestin. β-Arrestin–mediated association of Src with β 2 -adrenergic receptors is a key step in mitogenic signaling by these receptors, since inhibition of the binding of β-arrestin to either the β 2 -adrenergic receptor or Src attenuates β 2 -adrenergic activation of MAP kinase. These results indicate that the association of arrestins with heptahelical receptors does not simply uncouple receptors from G protein pathways, but rather induces a switch in receptor signaling from classical second messenger–generating G protein–mediated pathways to other pathways such as those involving Src and leading to the activation of MAP kinase. Moreover, arrestins have also been found to interact with a number of cellular proteins involved in endocytosis such as clathrin heavy chain , the clathrin adaptor AP-2 , and NSF ( N -ethylmaleimide sensitive fusion protein) . These interactions represent potential mechanisms by which heptahelical receptors might directly regulate the cellular endocytic machinery. Thus, arrestins may well represent multifunctional adaptor proteins that mediate a number of aspects of heptahelical receptor signaling. GRKs may also be signaling intermediates for heptahelical receptors rather than just proteins involved in receptor desensitization. Recently, it was found that GRK2 can associate with and phosphorylate tubulin . GRKs have also been shown to associate with actin and a novel ARF GTPase-activating protein (ARF GAP) called GIT1 . These findings illustrate at least two ways in which the recruitment of GRKs to activated heptahelical receptors may lead directly to cytoskeletal regulation or to modulation of other intracellular processes: (a) allosteric activation of GRKs by ligand-occupied receptors may catalyze the phosphorylation of key nonreceptor substrates such as tubulin; and (b) GRKs may act as noncatalytic adaptors to recruit key signaling intermediates (e.g., an ARF GAP) into complex with the receptors at the plasma membrane. Several subtypes of heptahelical receptors have been proposed to organize SH2 domain–based signaling complexes in a manner analogous to that seen for receptor tyrosine kinases. The heptahelical angiotensin AT 1 receptor, for example, activates the Jak2 tyrosine kinase following stimulation with angiotensin II . The mechanism underlying this effect involves Src-mediated tyrosine phosphorylation of the AT 1 receptor itself . It is interesting to speculate that this phosphorylation might result from β-arrestin–mediated recruitment of Src to the receptor, but at present this idea has not been tested. When Tyr319 on the AT 1 receptor carboxyl-terminal tail is phosphorylated, Jak2 coimmunoprecipitates with the AT 1 receptor in an agonist-dependent fashion; mutation of Tyr319 to Phe blocks coimmunoprecipitation of Jak2 with AT 1 receptors and also attenuates Jak2 activation mediated by angiotensin II stimulation . Originally, it was thought that Jak2 interaction with the AT 1 receptor was direct. Jak2 does not have an SH2 domain, however, so it was not clear how it could bind to the AT 1 receptor tail in a phosphotyrosine-dependent manner. Subsequent studies revealed that the Jak2/AT 1 receptor tail interaction can be blocked by antibodies to the SHP family of SH2 domain– containing tyrosine phosphatases , indicating that SHP proteins probably act as adaptors to facilitate the association of Jak2 with the AT 1 receptor. It has also been shown that another SH2 domain–containing protein, phospholipase Cγ1, can be coimmunoprecipitated with the tyrosine-phosphorylated AT 1 receptor , although the significance of this interaction for downstream signaling by the receptor has not yet been clarified. The β 2 -adrenergic receptor is phosphorylated on tyrosine by the insulin receptor tyrosine kinase . Several tyrosines in the β 2 -adrenergic receptor have been shown to be phosphorylated, and it has also been reported that the SH2 domain–containing adaptor protein Grb2 can associate with β 2 -adrenergic receptors following phosphorylation of Tyr350/354 on the receptor . It is not yet known, however, if this association mediates any downstream signaling by the β 2 -adrenergic receptor. Nonetheless, given these provocative findings with the AT 1 and β 2 -adrenergic receptors, a significant point of future interest will be to see if other heptahelical receptors may be tyrosine-phosphorylated and thus capable of hosting SH2 or PTB domain–based signaling complexes. Heptahelical receptor–mediated regulation of small GTP-binding proteins, such as Ras, Rab, Rho, and ARF, has been studied for years but has typically been viewed as a downstream consequence of heterotrimeric G protein activation . Recently, however, it has been shown that activation of phospholipase D by certain heptahelical receptors, including M 3 muscarinic acetylcholine receptors and H 1 histamine receptors, is not blocked by inhibitors of heterotrimeric G protein pathways, such as pertussis toxin or phospholipase C inhibitors, but is sensitive to the ARF inhibitor brefeldin A and the Rho inhibitor C3 botulinum toxin . ARF and Rho can also be immunoprecipitated in an agonist-dependent fashion in association with M 3 muscarinic receptors and AT 1 angiotensin receptors. The receptors capable of binding ARF and Rho exhibit a conserved motif (N-P-x-x-Y) in their seventh transmembrane span. Mutation of this motif prevents association of the receptors with ARF and Rho and also alters receptor signaling to phospholipase D. While it is not clear at present if the association of ARF and Rho with the heptahelical receptors is direct, it is clear that these small GTP-binding proteins can form a complex with some heptahelical receptors and that formation of this complex can mediate signaling of these receptors to phospholipase D. The heptahelical receptor-binding proteins discussed so far (heterotrimeric G proteins, arrestins, GRKs, SH2 proteins, and small GTP-binding proteins) all bind to either the receptor third intracellular loop or the portion of the receptor tail nearest the plasma membrane. Many heptahelical receptors, however, have quite long intracellular carboxyl-terminal tails, suggesting that the distal portions of some receptor tails may also be capable of mediating association with various intracellular signaling proteins. Moreover, the carboxyl-terminal tails of some heptahelical receptors terminate in variants of the T/S-x-V motif required for binding to PDZ domain–containing proteins such as PSD-95 . One example of a heptahelical receptor with a long intracellular tail is the β 2 -adrenergic receptor. Overlay studies demonstrated that the tail of this receptor binds with very high affinity to a single protein in tissue extracts; subsequent purification and sequencing revealed this binding partner to be a PDZ domain–containing protein, the Na + / H + exchanger regulatory factor (NHERF) . NHERF binds not only to the β 2 -adrenergic receptor tail in vitro, but also to the full-length β 2 -adrenergic receptor in cells in an agonist-dependent fashion as assessed by immunofluorescence studies. β 2 -Adrenergic regulation of renal Na + /H + exchange has long been known to be opposite of what would be expected from a G s -coupled receptor. Activation of G s -coupled receptors such as parathyroid hormone receptors increases cellular cyclic AMP, which in a PKA-dependent fashion facilitates the association of NHERF with renal Na + /H + exchangers and thus leads to inhibition of Na + /H + exchange . Activation of β 2 -adrenergic receptors also increases cellular cyclic AMP, yet paradoxically leads to stimulation of renal Na + /H + exchange . A point mutant of the β 2 -adrenergic receptor with the final residue of the receptor changed from leucine to alanine, which cannot bind NHERF but which exhibits normal G protein coupling, inhibits the activity of the renal Na + /H + exchanger in cells rather than stimulating it like the wild-type receptor . These findings suggest that the ability of the β 2 -adrenergic receptor to bind NHERF is critical for β 2 -adrenergic regulation of renal Na + /H + exchange in vivo. Rhodopsin is another heptahelical receptor that has been found to associate with a PDZ domain–containing protein in a functionally relevant manner. Rhodopsin binds to InaD , a multi-PDZ domain scaffolding protein that also associates with a number of signaling intermediates involved in rhodopsin-initiated pathways, such as phospholipase Cβ, protein kinase C, and the TRP ion channel . Mutations in InaD profoundly distort photon-induced rhodopsin signaling . The physical association of rhodopsin and InaD has been demonstrated by coimmunoprecipitation and by in vitro fusion protein pull-down experiments , but it is not known at present if the association of InaD and rhodopsin in cells occurs constitutively or if instead it is promoted by photoactivation of rhodopsin. In any case, it seems that rhodopsin can facilitate the assembly of intracellular protein complexes involved in phototransduction via its interaction with InaD. The interactions of PDZ domains with the carboxyl termini of their target proteins are quite specific . As demonstrated by the β 2 -adrenergic receptor point mutant, a change of a single amino acid can be enough to completely disrupt an otherwise high-affinity association. Only a small number of heptahelical receptors terminate in the carboxyl-terminal motif (S/T-x-L) required for high-affinity NHERF binding . However, since the >50 known PDZ domain–containing proteins recognize diverse target motifs, it is probable that some of these proteins associate with specific heptahelical receptors in a functionally relevant manner. Signaling through PDZ domain–mediated associations may therefore be a feature common to many heptahelical receptors. Several heptahelical receptors exhibit polyproline regions on either their third intracellular loops or carboxyl-terminal tails. Polyproline regions are known to mediate binding to a variety of conserved protein domains such as SH3 domains, WW domains, and EVH domains . Recently, several subtypes of heptahelical metabotropic glutamate receptor (mGluR) were shown to bind members of the Homer family of EVH domain–containing proteins through a polyproline region found in the mGluR tail region . This binding has been shown in yeast two-hybrid studies, fusion protein pull-downs, and coimmunoprecipitation studies. Some members of the Homer family can dimerize, and are thus capable of linking mGluRs to other proteins with appropriate polyproline motifs. For example, Homer proteins can facilitate a functional interaction between mGluRs and endoplasmic reticulum–based inositol trisphosphate (IP3) receptors, which control intracellular calcium release. When the mGluR/ Homer association is blocked, the ability of mGluRs to mobilize intracellular calcium is attenuated . These findings suggest that Homer is a key intermediate in mGluR regulation of intracellular calcium levels, and thus shed light on the puzzling observation made shortly after the cloning of the mGluRs that alternative splicing of the mGluR1 carboxyl-terminal tail results in profound differences in the ability of this receptor to mobilize intracellular calcium . Another heptahelical receptor that can bind signaling proteins through a polyproline region is the dopamine D4 receptor, which contains a stretch of prolines in its third intracellular loop. This polyproline region in the D4 receptor can mediate in vitro binding to a number of SH3 domain–containing proteins, including Grb2 and Nck, as assessed by yeast two-hybrid and protein pull-down assays . It is not clear at present, however, which polyproline-binding proteins are the relevant cellular partners for D4 receptors or for other polyproline-containing heptahelical receptors such as β 1 -adrenergic receptors and M4 muscarinic receptors. Further work in this area should reveal which polyproline-binding proteins couple to which receptors in cells, as well as what the consequences of these interactions are for receptor signaling. Several heptahelical receptor binding partners have been identified for which no clear roles in downstream signaling have yet been demonstrated. Examples include the interaction of Grb2 with the β 2 -adrenergic receptor and dopamine D4 receptor, as described above, as well as the interaction of the β 2 -adrenergic receptor and some α-adrenergic receptor subtypes with the α subunit of the eukaryotic initiation factor 2B , and the interaction of the bradykinin B2 receptor with endothelial nitric oxide synthase . The recent proliferation of techniques for detecting protein–protein interactions is likely to lead to an increase in the number of known binding partners for various heptahelical receptors. Each of these interactions will represent a new potential mechanism of heptahelical receptor signaling, although the true physiological significance of each interaction may not be immediately obvious. While such lines of research are describing novel mechanisms by which heptahelical receptors may generate intracellular signals, other lines of research are describing physiological effects mediated by heptahelical receptors for which the molecular mechanisms are unknown. Genetic studies in invertebrates, in particular, have yielded a number of examples of heptahelical receptors mediating physiological actions through pathways that are apparently independent of G proteins. For instance, the cyclic AMP receptors of the slime mold Dictyostelium discoideum are heptahelical receptors that induce chemotaxis of undifferentiated Dictyostelium cells into an aggregated fruiting body. These chemotactic effects of Dictyostelium cyclic AMP receptor stimulation are known to be mediated through G protein activation . However, aggregated Dictyostelium cells undergo a number of cyclic AMP receptor–mediated transcriptional changes that are independent of G protein activation, since cells with G protein subunits deleted still exhibit these changes following stimulation by cyclic AMP . The mechanisms by which this class of heptahelical receptors might mediate G protein–independent effects, however, are completely unknown. More genetic evidence for signaling by heptahelical receptors through means other than traditional G protein pathways comes from the study of a family of receptors known as frizzled. In many species, ranging from C. elegans to Drosophila to mammals, tissue polarity during development is regulated by the Wnt family of secreted proteins, which exert their effects on developing cells by binding to members of the frizzled family . Activation of some frizzled family heptahelical receptors results in increases in cellular calcium that can be inhibited by modulators of G protein function such as pertussis toxin and GDP-β-S . Thus, it seems that frizzled receptors can couple to G proteins. However, genetic studies have identified a number of signaling intermediates downstream of frizzled , such as dishevelled , glycogen synthase kinase-3, β-catenin, and the product of the adenomatous polyopsis coli (APC) gene , and none of these proteins resemble known components of classical G protein signaling pathways. Dishevelled is the most proximal frizzled signaling intermediate identified. It is not known if the interaction between frizzled and dishevelled is direct, but it is interesting to note that dishevelled contains a PDZ domain and many frizzled family members possess carboxyl-terminal motifs appropriate for PDZ domain association. Therefore, it is possible that members of the frizzled family may signal through direct coupling to PDZ domain–containing proteins like dishevelled in a manner analogous to the PDZ domain–mediated interaction of the β 2 -adrenergic receptor with NHERF. Some components of frizzled signaling pathways have been identified as oncogenes in mammalian tissues , emphasizing the importance of understanding frizzled signaling. Another genetically identified heptahelical receptor that signals via unknown mechanisms is smoothened. This receptor is a relative of the frizzled family of receptors, and is a key mediator of hedgehog signaling . Hedgehog , a soluble protein first identified as a regulator of patterning during Drosophila development, binds to a cell surface receptor known as patched , which leads to regulation of the activity of smoothened to exert control over cell proliferation and differentiation. Since smoothened is a heptahelical receptor, much attention has been focused on the possibility that it might couple to heterotrimeric G proteins, but at present there is no conclusive evidence for such coupling. Indeed, genetic studies have identified several key proteins, such as the serine/threonine kinase fused and the putative transcriptional factor cubitus interruptus , as intermediates in the smoothened signaling pathway; none of these proteins resemble known components of G protein signaling pathways . Activating mutations in the mammalian homologue of smoothened have been identified recently as underlying causes of sporadic basal-cell carcinoma , revealing that smoothened , like frizzled , may be involved in carcinogenesis. The intracellular signaling mechanisms used by both frizzled and smoothened are thus of interest not just as novel examples of heptahelical receptor signaling, but also as potential points of clinical intervention in the treatment of some cancers. Over the past several years, evidence has emerged that heptahelical receptors can signal through associations with intracellular partners other than G proteins. In some cases, these partners are known receptor-interacting proteins, such as arrestins and GRKs, which were thought previously to be involved only in receptor desensitization. In other cases, they are novel partners such as NHERF or Homer, which were not known previously to interact with heptahelical receptors. For heptahelical receptors that seem to mediate physiological effects via unknown G protein–independent pathways, such as frizzled and smoothened , it might be useful to consider analogies with other heptahelical receptors for which the early steps of various G protein–independent signaling mechanisms have been elucidated. Some of these mechanisms are likely to be quite general: for example, arrestins and GRKs can bind to many heptahelical receptors, and arrestin- and GRK-mediated formation of signaling complexes may therefore be a feature common to many heptahelical receptors. Other mechanisms, such as the activation of small GTP-binding proteins or the formation of SH2-based signaling complexes organized around tyrosine-phosphorylated residues, may be relevant to a small number of heptahelical receptors but not to the majority. Still other mechanisms are likely to be highly receptor-specific: the binding of NHERF to the β 2 -adrenergic receptor and the binding of Homer to metabotropic glutamate receptors, for example, depend on the presence of precise motifs that are likely to be found in few other heptahelical receptors, although other receptors are likely to contain slightly modified motifs that mediate binding to other specific PDZ or polyproline-binding domains. There are >1,000 heptahelical receptors but only ∼20 different heterotrimeric G proteins. Such an arrangement would seem to place limitations on the specificity of heptahelical receptor signal transduction, if G proteins were the only mediators of heptahelical receptor–initiated signaling. However, it now seems likely that each heptahelical receptor may activate its own relatively specific set of intracellular signaling pathways, including both G protein– dependent and G protein–independent mechanisms . The net physiological effect of stimulation of a particular heptahelical receptor will thus reflect the sum of the various intracellular pathways it can activate, with some of the pathways being quite general, others being fairly specific, and some being unique to the individual receptor. The near future is likely to yield a number of new examples of heptahelical receptor signaling through means other than classical G protein pathways. Some of these new receptor-initiated signaling pathways may be variations on a theme already seen in other heptahelical receptors, while others are likely to be completely novel. In any case, the old view of heptahelical receptors as simple G protein activators is currently being replaced by a new view of these receptors as complicated signal-transducing machines capable of directly coupling to a host of intracellular signaling pathways. | Study | biomedical | en | 0.999996 |
10352012 | The strain YK113 ( MAT a , ura3-52 , lys2-801 , ade2-101 , his3Δ200 , trp1Δ1 , leu2Δ1 , Δctf13::HIS3 , pCTF13 ( URA3 ), C.F. TRP , Rad2 distal; Doheny et al., 1993 ) was used to assay the function of ctf13 F-box mutations. Mutant ctf13 alleles were integrated at the leu2 locus and resulting LEU + transformants were tested for growth in the presence of 5-fluororotic acid (5-FOA). Protein half-lives were determined by promoter shut-off experiments in the protease deficient strain JB811 . Extracts were prepared as described . F-box point mutations in p58 and Cdc4p were created by site-directed mutagenesis of plasmids pIR112 and pIR329 (pRS316::PY 2 -HA 3 -CDC4), respectively, essentially as described . Mutant sequences were confirmed by sequencing through the region homologous to the mutagenic oligonucleotide. For targeted integration at leu2 , pIR112 was linearized using HpaI. Yeast manipulations were performed as described . CBF3 genes were subcloned into the pFastBac plasmid ( GIBCO BRL ) and baculovirus DNA was prepared as recommended. p23 Skp1 , p64, and p110 constructs were created containing the sequence MRGSH 6 fused to the initiator methionine. p110 has methionines at residues 1 and 12 of the published sequence, and we have found that p110 fused at Met-12 is more active in DNA binding than p110 fused at Met-1. Thus, p110 fused at Met-12 was used for the work described here. As in yeast, p58 produced in insect cells has a short half-life and is expressed at lower levels than the other CBF3 subunits. The relatively low levels of p58 in insect cell lysates made it difficult to purify and, in this paper, we primarily use recombinant p58 in unfractionated extracts. Nevertheless, in several cases we show that the properties of unpurified and purified CBF3 proteins are indistinguishable. Proteins were expressed in High Five insect cells (Invitrogen Corp.) by infecting cells with one or more baculoviruses and harvesting after 48–72 h. To minimize protein degradation, all steps of the extraction were performed on ice. Cytoplasmic extracts were prepared by swelling cells in hypotonic lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM KCl, 1.5 mM MgCl 2 , 10 mM β-mercaptoethanol, 10 μg/ml each of leupeptin, pepstatin, and chymostatin, 50 mM TPCK, 1 mM PMSF), breaking the cells with a Dounce tissue grinder (Wheaton), and centrifuging the lysate to pellet the nuclei. Supernatants were adjusted to 10% glycerol, 150 mM KCl, and 50 mM β-glycerophosphate before freezing for storage. The nuclear pellet was extracted for 30 min in nuclear extraction buffer (10 mM Hepes, pH 8.0, 50 mM β-glycerophosphate, 0.1 mM EDTA, 0.5 M KCl, 5 mM MgCl 2 , 10% glycerol, 50 mM NaF, 10 mM β-mercaptoethanol, 10 μg/ml each of leupeptin, pepstatin, and chymostatin, 50 mM TPCK, 1 mM PMSF) and centrifuged to remove DNA and other insoluble material. Under these extraction conditions, p64, p58, and p110 were found primarily in nuclear fractions while p23 Skp1 partitioned mostly to the cytoplasmic fraction. Nuclear extracts used for purification were prepared in the absence of EDTA so that material could be incubated directly with metal chelate resin (see below). 1% Triton X-100 was included in nuclear extraction buffer when p110 was prepared for purification to improve solubility. His 6 -tagged proteins were purified using Ni-NTA Superflow resin (Qiagen, Inc.). Extracts were adjusted to 3 mM imidazole, added to resin, and bound in batch overnight at 4°C. For p64 and p23 Skp1 , resin was washed in Ni buffer (20 mM Hepes, pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM β-mercaptoethanol, 50 mM imidazole) before elution in the same buffer but containing 500 mM imidazole. For p110, resin was washed in Ni buffer (3 mM imidazole), eluted in batch with Ni buffer (250 mM imidazole), and diluted to 150 mM NaCl before loading onto a column containing a small volume of Poros 20 HQ resin (PerSeptive Biosystems) to concentrate the protein. p110 was then eluted in Ni buffer containing 800 mM NaCl and no imidazole. Gel filtration chromatography was performed on a SMART System ( Pharmacia Biotech ) using Superose 6 and Superose 12 columns. To determine diffusion coefficients, standard curves were generated by plotting elution volume versus 1/D, where D is the diffusion constant, using the following protein standards (Bio-Rad): thyroglobulin (molecular mass = 670 kD, D = 2.63 × 10 −7 cm 2 s −1 ), bovine gamma globulin (molecular mass = 158 kD, D = 4.1), chicken ovalbumin (molecular mass = 44 kD, D = 7.8), and equine myoglobulin (molecular mass = 17 kD, D = 11.3). Diffusion coefficients for protein standards were obtained from Sober and represent values determined in water at 20°C (D 20,w ). Elution from a gel filtration column correlates with Stokes radius . All columns were run at 4°C with column buffer (10 mM Hepes, pH 8.0, 6 mM MgCl 2 , 10% glycerol, 150 or 300 mM NaCl, 10 mM β-mercaptoethanol). Analyses were repeated at least two times and values for 1/D varied by no more than 4% between experiments. Purified p23 Skp1 and p110 were diluted to 150 mM NaCl with column buffer lacking NaCl. Because p64 was only partially soluble in 150 mM NaCl, it was diluted to 300 mM NaCl and separated using column buffer containing 300 mM NaCl. Diluted proteins were centrifuged to remove insoluble material before loading onto the column. Elution was monitored by absorbance at 280 nm and fractions were collected and analyzed by immunoblotting and by bandshift assays. Because it was difficult to express and purify sufficient quantities of p58, all analyses of p58-containing mixtures were performed using insect cell extracts. Samples were prepared by dialyzing nuclear extracts into column buffer using a microdialysis system ( GIBCO BRL ) and then centrifuging to remove insoluble material. To compare directly the elution profiles of different sets of coexpressed proteins, the fraction size, the number of fractions, and the starting point for fraction collection were kept constant. Gradients were prepared in 2-ml volumes by layering successive 0.4-ml aliquots of column buffer containing decreasing concentrations of glycerol and incubating the gradient at 4°C for 1 h to equilibrate. Purified p64 was analyzed in 300 mM NaCl and all other proteins were analyzed in 150 mM NaCl. p110 and p64 were sedimented in 15–35% gradients while p23 Skp1 and p23 Skp1 /p58 were sedimented in 5–25% gradients. Purified p23 Skp1 , p64, and p110 were prepared by diluting to the same salt concentration as the gradient using column buffer that lacked glycerol and NaCl. Insect cell extract containing p58 and p23 Skp1 was dialyzed into column buffer lacking glycerol but containing 150 mM NaCl. For p110 and p64, gradient standards were added directly to the protein samples; for p23 Skp1 and p58, the standards were run separately. Gradients were centrifuged at 50,000 rpm 10–16 h at 4°C in a TL-S55 swinging bucket rotor ( Beckman Instruments ) and fractionated by removing 100-μl aliquots from the top of the gradient. Fractions were then analyzed either by bandshifts or by TCA precipitation followed by immunoblotting. Protein standards ( Boehringer Mannheim ) cytochrome c (molecular mass = 12.5 kD, s = 1.9 S), chymotrypsinogen A (molecular mass = 25 kD, s = 2.58), hen egg albumin (molecular mass = 45 kD, s = 3.55), bovine serum albumin (molecular mass = 68 kD, s = 4.22), aldolase (molecular mass = 158 kD, s = 7.4), and catalase (molecular mass = 240 kD, s = 11.3) were separated on SDS-polyacrylamide gels, stained with Coomassie blue and quantified using IPLab Gel image processing software (Signal Analytics). All samples were analyzed at least two times, and sedimentation coefficients varied by no more than 15% between experiments. Native molecular mass was calculated using the expression molecular mass = RTs / D (1 − νρ), where R is the ideal gas constant; T, absolute temperature; s, sedimentation coefficient; D, diffusion coefficient; ν, partial specific volume; and ρ, density of water at 20°C. Partial specific volumes (0.715 cm 3 g −1 for p110, 0.724 for p64, 0.697 for p23 Skp1 , and 0.719 for a p23 Skp1 /p58 heterodimer) were estimated from amino acid content. Stokes radii were calculated using the equation a = kT /6πη D , where a is Stokes radius and η is viscosity of water at 20°C. f/f 0 was calculated using the relationship f / f 0 = a /(3ν M /4π N ) 1/3 , where M is molecular mass and N is Avogadro's number. Bandshift assays were performed as described . Bacterial GST-fusion proteins were expressed in the strain BL21ΔE3 using the plasmid pGEX-4T-2 as recommended ( Pharmacia Biotech ). Cells were lysed by sonication in breakage buffer (20 mM Hepes, pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM DTT, and 1 mM PMSF), the cell extracts were cleared by centrifugation and the concentration of GST-fusion was estimated by comparing Coomassie-stained bands on a 10% SDS-PAGE gel to a protein standard. Extracts containing GST-fusion proteins were adjusted to 1% Triton X-100 and bound to glutathione-Sepharose beads for 1 h at 20°C as recommended ( Pharmacia Biotech ). Beads were spun down and washed three times with IP buffer (1% Triton X-100, 50 mM Hepes, pH 8.0, 150 mM NaCl, 50 mM NaF, 50 mM β-glycerophosphate, and 1% glycerol). 40 μl of a 50% bead slurry containing 2.5 μg of immobilized GST-fusion protein was used for each binding reaction. p58 mutants and fragments were amplified from plasmids by PCR using 5′ primers designed to include a T7 promoter and Kozak consensus start ATG codon (5′-tgtaatacgactcactatagggccaccatg-3′). To generate 35 S-labeled protein in vitro, mRNA was synthesized from the PCR products using T7 RNA polymerase as recommended ( Promega ). Approximately 500 ng of mRNA was incubated in a 100-μl rabbit reticulocyte lysate in vitro translation reaction for 2 h at 30°C. 200-μl binding reactions containing 20 μl of 35 S-labeled protein, 40 μl of 50% bead slurry, 2 μl of PMSF, and 138 μl of IP buffer were incubated for 4 h at 4°C with end-over-end tube rotation. The beads were then recovered by centrifugation, washed three times with 1 ml IP buffer, and bead associated 35 S-labeled protein analyzed by SDS-PAGE and autoradiography. Yeast strain JB811 containing plasmid borne GAL1 driven GST- CTF13 or GST- ctf13 truncations were grown to late-log phase in media containing 2% galactose and 2% raffinose at 30°C for ∼14 h. A sample was chilled at time 0 and the remaining cells were washed into media containing 2% glucose, grown at 30°C, and aliquots placed on ice at the indicated times after addition of glucose. Extracts were prepared and duplicate samples of GST-fused proteins were purified from 2 mg of total yeast protein by binding to glutathione-Sepharose ( Pharmacia Biotech ). Beads were washed three times in IP buffer (see above) and bead-bound protein was loaded on 12.5% SDS-PAGE gels. Protein was transferred to nitrocellulose membranes and the GST moiety was detected by immunoblotting with an anti-GST antibody. Antibody binding was quantified using 125 I-labeled protein A ( DuPont /NEN) and a PhosphorImager (Molecular Dynamics). p64 was treated with dilute proteolytic enzymes in 50 mM Hepes, pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM EDTA at 30°C. Aliquots were removed at different time points and proteolysis stopped by boiling the sample in SDS-PAGE loading buffer containing 5 mM PMSF. Microsequencing of proteolytic fragments was performed at the MIT Biopolymers Lab from PVDF blots of SDS-PAGE gels. To understand the regulation and function of CBF3, we need to determine its mode of assembly and its structure when bound to DNA. In this paper, we use a combination of biochemical and molecular techniques to study CBF3 complexes in solution and in association with CDEIII. Hydrodynamic measurements have proven particularly valuable for determining the shape and composition of various CBF3 complexes. Protein hydrodynamics generates values for the native mass and size (the radius of gyration or Stokes radius) of a complex. By systematically determining the native masses of complexes produced by mixtures of two or three CBF3 subunits, we have deduced which subunits touch each other. To extend this interaction map and determine the consequences of disrupting various subunit-subunit interactions, we have used molecular techniques to map binding domains. The method is quite laborious but the net result is simple: an experimentally derived model for the organization of CBF3. To obtain CBF3 in reasonable yield and free of contaminating yeast proteins, recombinant CBF3 proteins were produced in insect cells. We have shown previously that recombinant CBF3 is indistinguishable in its DNA-binding properties from CBF3 isolated from yeast . The native molecular masses and radii of gyration of proteins were calculated using hydrodynamic data obtained from gel filtration columns and glycerol velocity gradients. Fractions from columns or gradients were collected and the levels of a particular CBF3 subunit determined either by Western blotting, absorbance measurements, or “bandshift complementation” assays. In the complementation assay, the amount of an active CBF3 subunit was measured by adding an excess of the other CBF3 proteins and radiolabeled CEN DNA, and then quantifying CBF3-DNA complexes on nondenaturing bandshift gels. We have determined previously that CBF3 subunits can exchange in and out of various complexes before binding to centromeric DNA. Thus, the complementation approach permits a selective analysis of a single active protein in a multicomponent mixture. The p110 subunit of CBF3 eluted as a single symmetrical peak from gel filtration columns and glycerol velocity gradients, indicating that it was present as a relatively homogenous species . Preparations of recombinant p110 often contained a major p110 breakdown product, but its presence did not interfere significantly with molecular mass determinations: on gel filtration columns and gradients it fractionated away from full-length p110. On sizing columns, the apparent mass of p110 was nearly 670 kD but on velocity gradients it sedimented near the 158-kD aldolase standard. Such differences in apparent mass are expected for proteins that are not globular, but a simple formula can be used to calculate a native molecular mass independent of protein shape (see Materials and Methods). For these calculations, a diffusion constant was obtained from the elution volume of p110 on Superose sizing columns relative to the elution volumes of protein standards , a sedimentation coefficient was obtained from the migration of p110 on glycerol velocity gradients relative to standards , and a partial specific volume was estimated by extrapolation from the amino acid composition. Based on these data, we obtained a native molecular mass for p110 of 197 kD ± 10%, leading us to conclude that p110, whose calculated monomer molecular mass is 112 kD, forms a homodimer in solution (Table I ). We have shown previously that p110 is in close contact with bases in the major groove of CDEIII DNA and the hydrodynamic data reveal that the protein is indeed elongated and has a long dimension about the same as 4.5 helical turns of B-form DNA. To obtain further evidence that p110 can self-associate, we expressed native and GST-tagged p110 either individually or in combination, and then isolated GST p110 using glutathione-Sepharose. We observed that native p110 bound GST-tagged p110 but not, as a negative control, GST alone (lane 3). The ability of GST p110 to pull down native p110 is good evidence that p110 indeed forms multimers. From these findings, we conclude that p110 exists in solution as a dimer, and possibly also as higher-order oligomers. Next, we analyzed p64 using similar methods . The calculated native molecular mass of p64 was 132 kD, suggesting that p64 is a homodimer in solution (the p64 homodimer seems to be roughly spherical; Table I ). In addition to the dimer peak, complementation assays revealed a second, later eluting peak of p64 on sizing columns . We have not been able to determine a sedimentation coefficient for this material, but its migration on gel filtration columns suggests that it represents monomeric p64. To confirm that p64 can self-associate, we asked whether GST p64 and His 6 -tagged p64 would interact when coexpressed. Pull-downs using glutathione-Sepharose beads showed that p64 binds to GST p64 but not (as a control) to GST alone . We conclude that p64 is primarily a dimer in solution but that it is in equilibrium with a small pool of monomeric protein. p64 is the only subunit of CBF3 that is detectably homologous to proteins known to bind to DNA. The amino terminus of p64 contains a Zn 2 Cys 6 “zinc cluster” DNA-binding module similar in sequence to the zinc clusters found in other fungal DNA binding proteins . The Gal4p zinc cluster, whose structure has been determined by x-ray crystallography, forms a compact domain that is attached to the rest of the protein by a flexible linker . To determine whether p64 has a similar domain organization, we treated purified protein with limiting amounts of protease for varying times and looked for the accumulation of discrete protein fragments . In multidomain proteins, proteolytic mapping can be used to identify domain boundaries because exposed loops between domains are often significantly more susceptible to digestion than the domains themselves. After cleaving p64 with limiting amounts of trypsin for 10 min, essentially all of the intact protein was converted into a 58-kD form ; more extensive proteolysis led to the accumulation of 51- and 48.5-kD species . The amino terminus of the 58-kD fragment was residue 48 (as determined by NH 2 -terminal sequencing), indicating that cleavage had occurred just downstream of the zinc cluster domain . Similarly sized cleavage products were generated, albeit less efficiently, by digestion with chymotrypsin or V8 protease . From these data, we conclude that the zinc cluster of p64 is attached to the remainder of the protein via a proteolytically accessible and potentially flexible arm. Well characterized zinc cluster proteins such as Gal4p and Hap1p bind, in the absence of other proteins, to direct or inverted repeats of the sequence CCG . CDEIII contains only one CCG half-site, but DNA-protein cross-linking shows that p64 contacts this CCG and a second sequence, TGT (in CEN3 ), 12 bp to the left . We wondered whether this TGT might represent a degenerate CCG half-site and thus whether p64 might be able to bind on its own to a CDEIII sequence in which a second complete CCG half-site had been introduced. Two CDEIII variants were synthesized so as to create a second CCG half-site in one of three different positions while retaining the highly conserved 8G residue that contacts p64 . Competition experiments showed that neither variant CDEIII was able to bind efficiently to CBF3 and in no case could we obtain evidence from bandshift gels that p64 could bind to the variant CDEIIIs . Moreover, it appeared that mutations in the bases surrounding 8G were nearly as deleterious for CBF3 binding as mutations in the absolutely conserved CCG centered on 20C (compare mutants 2 and 3). We therefore conclude that the inability of p64 to bind to DNA on its own is unlikely to be a simple consequence of the absence of a second CCG half-site. We have shown previously that p58 is active for DNA binding only if phosphorylated in a p23 Skp1 -dependent manner. p58 has proven quite difficult to purify in large quantities, and we therefore analyzed nuclear extracts from insect cells coexpressing p23 Skp1 and p58. Only some of the p58 expressed in insect cells is active , and we therefore used a complementation assay to detect specifically the active pool of p58. The calculated native molecular mass of p58 derived from insect cells coexpressing p23 Skp1 and p58 was 76 kD . p58 has been shown previously to associate with p23 Skp1 in yeast and in insect cells , but following p23 Skp1 -mediated phosphorylation, p58 can dissociate from p23 Skp1 and remain active . Thus, we needed to determine whether our active p58 was actually associated with p23 Skp1 . We reasoned that by generating a fusion between p23 Skp1 and GST we would alter the mobility of p23 Skp1 sufficiently that free and p23 Skp1 -bound p58 could be distinguished. As shown in Fig. 3 b, the coexpression of p58 with GST p23 Skp1 shifted all of the p58 to a higher molecular mass, demonstrating that p58 forms a stable complex with GST p23 Skp1 and, thus, probably also with untagged p23 Skp1 . We conclude that the 76-kD native molecular mass of p58 reflects the formation of a stable heterodimer between one p58 polypeptide and one p23 Skp1 . We are somewhat uncertain about the oligomeric state and mass of p23 Skp1 . Data for bacterially expressed p23 Skp1 are clear: at a concentration of ∼100 μM, p23 Skp1 is monomeric and has the mass predicted from its amino acid sequence. However, the data for insect cell–expressed p23 Skp1 are more ambiguous. The majority of p23 Skp1 is monomeric, but ∼30% of the protein migrates as a larger species that may represent an oligomer. Unfortunately, in the absence of a reliable assay for functional p23 Skp1 , it is not possible to determine whether the activity of the potentially oligomeric forms is different from that of monomer. A great advantage of hydrodynamic measurements is that they not only allow protein-protein interactions among CBF3 subunits to be probed, but the stoichiometry of the hetero-oligomeric complexes to be determined. We used mobility shifts on gel filtration columns as an assay for the interaction of p58 with p64 and p110. First, we looked for an association between p58 and p64 by coexpressed p58 either with p23 Skp1 alone or with both p23 Skp1 and p64, and then determined the elution volume of p58 on gel filtration columns using bandshift complementation assays . Active p58 eluted earlier from columns in the presence of p64 than in its absence , indicating that interaction with p64 had shifted p58 into a larger complex. To confirm this interpretation, we performed a similar experiment but assayed for p64 by bandshift complementation. On a gel filtration column, we observed a clean shift in the elution of p64 from fraction 12 to fraction 10 . Thus, the p58-shifted p64 elutes in the same fraction as the p64-shifted p58, confirming the reliability of the methodology. The calculated native mass of the putative p58/p64/ p23 Skp1 complex was ∼200 kD (data not shown), consistent with the binding of one p58-p23 Skp1 heterodimer (77 kD) to one p64 homodimer (132 kD). From these data we conclude that p58, p23 Skp1 , and p64 can form a stable heterotetramer in solution in the absence of p110 and centromeric DNA. Moreover, when other CBF3 proteins are present in excess, essentially all of the p58 is found in a p58/p23 Skp1 /p64 hetero-oligomeric complex. Applying the same logic, we looked for the association of p58/p23 Skp1 with p110 by coexpressing the three proteins and assaying for p58 activity in gel filtration eluates. We observed that the coexpression of p58 with p110 shifted a fraction of the p58 activity to a larger sized complex . Multiple earlier-eluting peaks were present but a significant amount of the p58 was not altered in mobility. We conclude that p58 and p23 Skp1 can bind to p110 in solution. The existence of several peaks on the gel filtration column may reflect the presence of multiple p58-p23 Skp1 -p110 complexes or it may arise from the formation of a single relatively unstable complex that partially dissociates during chromatography. Finally, we mixed extracts from cells expressing p64 and p110, and, by determining the position of active p64 on a gel filtration column, examined whether p64 and p110 would bind to each other in solution. We could detect no difference in the elution behavior of p64 in the presence or absence of p110 . In addition, the elution of p23 Skp1 from columns was not altered by coexpression with either p64 or p110 (data not shown). While these are negative results, they suggest that p23 Skp1 , p64, and p110 are unable to form subcomplexes in the absence of p58. Having determined that several different p58-containing CBF3 subcomplexes can form, we asked whether all four CBF3 proteins would associate in the absence of centromeric DNA (as discussed below, we cannot always be certain that p23 Skp1 is present in p58-containing complexes, but our data suggest that the majority of p58 is p23 Skp1 -associated). It has been shown previously that p58, p64, and p110 coelute from a gel filtration column , but these earlier experiments did not demonstrate that the proteins were associated in a single complex. To test for the formation of a p58/p64/p110 complex, we looked for a shift in p64 mobility on columns that could arise only from the formation of a three-way p58-p64-p110 complex (or a four-way complex also containing p23 Skp1 ). When extracts containing p23 Skp1 , p58, p110, and limiting amounts of p64 were analyzed on a Superose 12 column, one population of p64 peaked at fraction 12, the position of p64 alone, and a second population peaked at fraction 5 . Because p58/p23 Skp1 /p64 complexes peak in fraction 10 , the peak of p64 activity in fraction 5 must represent a complex containing p58, p64, p110, and probably also p23 Skp1 (although we cannot rigorously prove, even with GST/p23 Skp1 , that p23 Skp1 is present in the complex). From these data we conclude that a CBF3 complex containing three, and probably four, subunits can form in the absence of CEN DNA. What is the relationship between the CBF3 subcomplexes detected in solution and DNA-bound CBF3? To investigate this, extracts from insect cells expressing all four CBF3 proteins were incubated with a 56-bp probe spanning CDEIII and the migration of the resulting DNA-protein complexes on sizing columns and glycerol gradients was determined by analyzing fractions directly on nondenaturing bandshift gels. We were unable to recover activity from a gel filtration column, perhaps because the complex dissociated during chromatography. However, following fractionation on velocity gradients, we observed a single peak of CBF3-CDEIII with a sedimentation coefficient of 15.3 S . These data show that CBF3 forms a single compact structure on a 56-bp fragment of CDEIII DNA. As we will discuss in detail in the discussion, these data can be combined with CBF3-DNA protein cross-linking that we have reported previously to generate a preliminary but compelling model for the architecture of CBF3. To determine the significance of the protein-protein interactions we had detected among CBF3 proteins, we wanted to disrupt them one-by-one first in vitro and then in yeast. We focused on mapping binding domains in p58 because the hydrodynamic data suggest that p58 forms a core subunit responsible for mediating interactions among the other three CBF3 proteins. Mapping binding domains involves a simpler assay than hydrodynamic measurements. We fused GST to the amino termini of p23 Skp1 , p64, and p110 and expressed the resulting fusion proteins in either bacteria or yeast. Purified fusion proteins were incubated with p58 translated in reticulocyte lysate, and the amount of p58 associated with the GST fusion was quantitated following GST capture on glutathione-Sepharose. Using this assay, we observed p58 binding to GSTp23 Skp1 , GSTp64, and to GSTp110 but not to GST alone or to a series of p110 truncations . In a typical reaction, 15–20% of the input p58 bound to GSTp23 Skp1 or to GSTp64 following a 4-h incubation at 4°C; binding to GSTp110 was considerably less efficient but still well above background levels. The selectivity in the binding of p58 to the CBF3-GST fusions relative to GST alone and the relatively low concentrations of the soluble and immobilized proteins (∼0.25 pM soluble p58 and 0.2 μM immobilized GST fusion in a reaction with a total protein concentration of 10 mg/ml) confirm that the binding of p58 to p23 Skp1 , p64, and p110 is specific and of high affinity. To map regions of the p58 protein responsible for binding to other CBF3 proteins, a series of NH 2 - and COOH-terminally deleted p58 proteins was translated in vitro and incubated with bead-bound GSTp23 Skp1 , GSTp64, or GSTp110 . There was considerable variability in the efficiency with which different p58 fragments were translated, so the extent of binding to p58 was expressed in a manner that corrects for translation efficiency. Both the NH 2 -terminal p58 truncation (139–479) and the COOH-terminal truncation (1–336) bound as efficiently to GSTp64 as to full-length p58 . This finding suggested that a region sufficient for p64 binding might lie in the sequence common to the two deletions. This was confirmed by using an internal fragment spanning residues 139–336. The 197–amino acid polypeptide containing p58 residues 139–336 is the smallest linear fragment of p58 to which we can detect efficient p64 binding: nine smaller derivatives of p58 were inactive in p64 binding. GSTp110 bound efficiently to p58(1–336) but not to p58(1–271) or p58(139–336) . We conclude from these data that p64 binds to p58 in a region between amino acids 139 and 336. The p110 binding site appears to span a region that lies in the primary structure of p58 just COOH-terminal to the p64 binding site, but partially overlaps it. p58 contains an apparent match to the F-box consensus, a sequence thought to constitute a binding site for p23 Skp1 . The failure of an F-box–deleted p58 protein, p58(61–479), to bind to p23 Skp1 but not to GSTp64 or GSTp110 shows that the F-box is required for p58-p23 Skp1 association . To probe sequence requirements in the F-box, 7 single and double point mutations were introduced into residues that are highly conserved among F-box proteins, and 10 mutations were introduced into nonconserved but charged residues. Mutations that changed conserved F-box amino acids (L12A, L12D, P13A, L12A/ P13A, V20A, Y21R, and L24A) abolished the binding of p58 to p23 Skp1 . These mutations did not appear to disrupt the overall structure of p58, because binding to p64 was unaffected . None of the mutations made at nonconserved but charged residues had any effect on p58 binding to either p23 Skp1 or to p64 . We conclude that the F-box in p58 is required for p23 Skp1 binding and that this binding primarily involves amino acids conserved among F-box family members. The majority of these conserved F-box residues are nonpolar, suggesting that interaction of p23 Skp1 with p58 is mediated largely by hydrophobic contacts. To compare our in vitro observations to data obtained in cells, we examined the ability of p58 proteins carrying F-box mutations to function in vivo in place of wild-type p58. Mutant p58 genes were integrated at the leu2 locus in yeast strains carrying a deletion of the gene encoding p58 (using plasmid shuffling, see Materials and Methods). Of the mutations that impaired p58-p23 Skp1 binding in vitro, we found that three mutations in consensus F-box residues (L12D, L12A-P13A, and Y21A) either prevented or severely impaired cell growth, and that two others caused a 10–100-fold increase in the rate of chromosome loss . In contrast, mutations in nonconsensus F-box residues had no effect on cell growth . We conclude from these observations that the F-box in p58 is required for function in vivo, presumably because p23 Skp1 -p58 binding is required for p58 activation in cells just as it is in vitro. Is the p58 F-box sufficient for p23 Skp1 binding? We observed that an NH 2 -terminal fragment of p58 encompassing the F-box alone (residues 1–58) did not bind to p23 Skp1 either in reticulocyte lysates or in yeast extracts . Binding of p23 Skp1 was observed to p58 proteins lacking the extreme COOH terminus (1–412) but not to more extensively truncated p58 derivatives. We conclude that the F-box in p58 is not sufficient for binding to p23 Skp1 and that at least one additional region of p58, probably located between residues 336 and 412, is also required. Having mapped regions of p58 involved in binding to p23 Skp1 , p64, and p110 in vitro, we wanted to determine the consequences of disrupting various interactions for p58 activation and destruction in cells. First, it was necessary to determine that the contacts we had mapped using recombinant proteins were also important in yeast. We therefore examined the state of association of p58 in wild-type yeast by fractionation on a gel filtration column. We observed that the bulk of activated p58 was present in a peak centered on fraction 10, the same position as recombinant p58-p23 Skp1 -p64 . Some activated p58 in yeast extracts eluted earlier from the column, indicating that a portion of the protein is probably bound to p110 or to other unidentified proteins. Thus, p58 in wild-type yeast is predominantly complexed with p64 and very little is present as a simple p23 Skp1 -p58 heterodimer. Consistent with this, Western blotting shows that GSTp58 is associated with p23 Skp1 and with p64 when isolated from yeast extracts . Next, we examined various mutated p58 proteins in yeast. As expected from our in vitro data, a double point mutation in the p58 F-box abolishes binding to p23 Skp1 but not to p64. p58-p64 association was eliminated, however, in the GSTp58(Δ139–336) protein by the deletion of the presumed p64 binding site between residues 139 and 336 . These data show that the p58 sequences determined to be important for protein-protein interactions among recombinant CBF3 subunits are also required in vivo. In particular, we find (a) that the p58 F-box is required for the binding of p23 Skp1 to p58 in vivo but that it is not sufficient and (b) that an internal region of p58 between residues 139 and 336 is required for p64 binding. To estimate the half-life of p58 when it is a part of various binary complexes containing other CBF3 subunits, we raised the levels of the complexes in cells by overexpressing p58 and either p23 Skp1 , p64, or p110 under control of the GAL1 promoter in otherwise wild-type cells. In all cases, Western blotting confirmed that CBF3 proteins were being expressed at elevated levels (data not shown). The task of comparing the half-lives of full-length and truncated p58 proteins was made easier by fusing p58 to GST. GST is normally very stable in yeast but the half-life of GSTp58, ∼15 min, was indistinguishable from that of wild-type p58 , showing that the instability of p58 can be transferred to a fused GST moiety. The stability of p58 was measured by placing the protein under the control of the GAL1 promoter, repressing transcription by the addition of glucose, preparing cell extracts at various times following transfer to glucose media, and then determining GSTp58 levels by Western blotting. The relative stability of p23 Skp1 , p64, and p110 ensured that they were present at similar levels at the start and end of the p58 transcriptional pulse-chase. We observed that overexpression of p64, but not p23 Skp1 or p110, increased the half-life of GSTp58 dramatically . To rule out the possibility that p64, which is a DNA-binding protein, was altering p58 transcription, we measured p58 message levels by Northern blotting in wild-type cells and in cells overexpressing p64 and determined that they were similar (data not shown). We conclude from these findings that the overexpression of p64 has a direct effect on the half-life of p58 protein. Next, we asked whether overexpressed p64 was in fact forming a complex with GSTp58 and whether complex formation was required for p58 stabilization. We isolated GSTp58 on glutathione beads and measured the level of bound p64 by Western blotting (see below). This showed that the amount of GSTp58/p64 complex was three- to fourfold higher in cells overexpressing p64 than in wild-type cells. To demonstrate that the formation of the GSTp58/p64 complex was required for p58 stabilization, we analyzed the GSTp58(Δ139–336) deletion, which does not bind to p64 . At steady state, GSTp58(Δ139– 336) was two- to threefold less abundant than wild-type p58 and was slightly less stable. Importantly, the half-life of GSTp58(Δ139–336) was unaltered by p64 overexpression demonstrating that p64 exerts its effect on p58 by directly binding to the p58 protein. Why are the kinetics of p58 degradation biphasic? In cells overexpressing p64, p58 levels fell as rapidly as in wild-type cells ( t 1/2 15 min) between 0 and 30 min but then remained essentially constant for >2 h (a similar effect was observed with unfused p58 in the presence of overexpressed p64, data not shown). The simplest explanation is that there are two pools of p58 in the cell, free p58 and p64-associated p58, and that the half-life of the p64-associated p58 is substantially longer than that of free p58. If this hypothesis is correct, the levels of the p58/p64 complex should remain nearly constant following a transcriptional shut-off, even as the levels of total p58 protein fall rapidly (with a half-life of 15 min). If the hypothesis is wrong, both free p58 and p64-bound p58 should be degraded with similar kinetics. As a test, we expressed GSTp58 under the control of the GAL1 promoter and then isolated GSTp58 and tightly bound p64 on glutathione-Sepharose at various times after transcriptional shut-off. The levels of bead-bound GSTp58 and p64 were determined by Western blotting. We observed that the amount of GSTp58 fell sixfold in 90 min, consistent with the previously determined 15 min half-life, but the amount of p58/p64 complex fell by <20% . Furthermore, in cells overexpressing p64, a similar phenomenon was observed, except that the absolute levels of p58/p64 were four- to fivefold higher and p58 was much longer lived . We interpret this to mean that there is indeed a special pool of p64-bound p58 that is more stable than the free p58 . Thus, we have shown that the overexpression of p64 stabilizes p58. This effect is specific to p64, and is not observed with overexpressed p23 or p110 . Moreover, the stabilization of p58 requires the formation of a p58/p64 complex . From these data we conclude that the stabilization of the p58 polypeptide is linked to its assembly into a p58/p64 CBF3 subcomplex that, while not competent to bind DNA on its own , is probably an intermediate in kinetochore formation. Instability is conferred on proteins such as mitotic cyclins by a discrete destruction box that is necessary and sufficient for promoting ubiquitin-mediated degradation. However, in many other proteins, instability determinants are found throughout a protein. To explore instability determinants in p58, we determined the half-lives of truncated p58 variants following fusion to GST. Because the p23 Skp1 -containing SCF complex is required for the destruction of a number of cell cycle regulators in yeast, including G 1 cyclins and the p34 Cdc28 kinase inhibitor Sic1p , we wondered whether p23 Skp1 binding was required for p58 degradation. We found that a p58 variant lacking the F-box, p58(139–479) was unstable , as was p58 with a double point mutant in the F-box p58 . In addition, an apparent PEST sequence, identified by the PEST-FIND algorithm of Rechsteiner and Rogers as lying between residues 211 and 231, was not necessary for p58 instability . Intriguingly, however, the p58 F-box (residues 1–58) was sufficient to confer instability on GST . In all, we examined seven different fusions between GST and p58 and observed that all had a half-life of 15–20 min . Considered together, these data show that sequences conferring rapid degradation on p58 are present at multiple locations in the p58 polypeptide and that neither p23 Skp1 binding nor PEST sequences are required. CBF3 is an essential component of the yeast kinetochore that binds to the critical CDEIII region of centromeric DNA. An important goal of this work was to probe the structure of the CBF3 proteins and to investigate how they associate to form a functional CBF3 complex. To this end, we have used hydrodynamic methods to determine the stoichiometries and approximate physical dimensions of individual CBF3 proteins and of complexes containing two or more different proteins. The primary advantages of using calibrated gradients and columns to examine complex formation are that one can follow active subsets of proteins using functional assays and that the stoichiometries of various complexes can be determined directly. In addition, however, we have confirmed the existence of the critical protein-protein interactions using pull-down assays from reticulocyte lysates and from yeast extracts. We have found that all of the CBF3 proteins are multimeric. p110 exists as an elongated homodimer, p64 forms a homodimer, and p23 Skp1 and p58 form a stable heterodimer. Protein hydrodynamics yield a fairly crude picture of a protein's structure, but our calculations for the stoichiometries of individual subunits are close to integral, as one would hope, and estimates for the stoichiometries of various binary and ternary complexes yield numbers that are the sums of the masses of the individual subunits. We therefore believe that the information is sufficiently precise to build a low resolution model for the CBF3 complex. To represent this information pictorially, we have modeled the CBF3 proteins as ellipsoids . In solution, p58 forms a complex with p23 Skp1 , p64, and p110, but these proteins do not appear to associate stably with each other in the absence of p58. This suggests that p58 forms the core of CBF3 and mediates stable interactions among the other CBF3 proteins . The primary complex formed by native p58 in yeast extracts and recombinant p58 appears to be a stable heterotrimeric assembly containing one copy of p23 Skp1 , one copy of p58, and two copies of p64. Reflecting its role as a bridging subunit, p58 can be divided into distinct functional regions. The amino terminus of p58 contains a fairly good match to the F-box consensus that is necessary but not sufficient for binding to p23 Skp1 ; a COOH-terminal region of p58 is also required. The binding of p64 and p110 to p58 appears to be mediated by sequences that lie approximately in the center of the p58 polypeptide (residues 139–336). The observation that the p23 Skp1 binding sites do not overlap p64 and p110 binding sites is consistent with our finding that p58 binds independently to p23 Skp1 , p64, and p110, and, at the resolution of our current assay, the presence of p23 Skp1 neither reduces nor enhances p64 and p110 binding (data not shown). However, preliminary data obtained from the analysis of recombinant CBF3 suggest that the efficient formation of a four-protein CBF3 complex requires p23 Skp1 -mediated activation of p58, perhaps because unactivated p58 assumes a structure in which p64 and p110 exclude each other from a shared binding site (Kaplan, K., and P.K. Sorger, unpublished observations). At least 10 proteins from S. cerevisiae contain potential F-boxes . These proteins function in physiologically diverse pathways including methionine biosynthesis , glucose repression , protein ubiquitination , and chromosome segregation . In three yeast proteins, Cdc4p , Grr1p , and p58 (this work), and in one human protein, Skp2 , point mutations or deletions in the F-box have been shown to impair p23 Skp1 binding. In p58, we have examined 15 point mutations in a 45-residue sequence that spans the F-box and shown that 6 mutations abolish p23 Skp1 binding in vitro. Three of these mutations also severely impair cell growth and several others increase the rate of chromosome loss. All of the F-box residues shown by mutagenesis to be important for p23 Skp1 binding are nonpolar, suggesting that the interaction of p23 Skp1 with the F-box is mediated primarily by hydrophobic contacts. It is not clear whether the COOH-terminal region of p58 that is also required for p23 Skp1 binding has a counterpart in other F-box proteins. p58 is not detectably similar in sequence to Cdc4p and Grr1 outside the F-box. The amino terminus of p64 contains a Zn 2 Cys 6 zinc cluster that in Gal4p contains six cysteines coordinating two zinc ions and fits into the major groove of DNA . Well-characterized members of the fungal zinc cluster family bind to DNA as dimers, and recognize sequences that contain either direct or inverted repeats of the DNA sequence CCG . In Gal4p, dimerization is mediated in part by coiled coils found in an arm that lies COOH-terminal to the zinc cluster. We have found that p64 is also dimeric, and that its zinc cluster is linked to the remainder of the p64 protein by a proteolytically accessible sequence that may represent a linker arm. Despite these similarities with Gal4p, p64 is unique among studied zinc cluster proteins in that it does not bind to DNA except as part of a multiprotein complex. Does this reflect an inherent property of p64 or is it a feature (such as low affinity) of the CDEIII binding site? DNA-protein cross-linking shows that p64 contacts a CCG sequence at the center of CDEIII that is essential for centromere function and a TGT (or A C A on the bottom strand) 12 bp to the left that is essential for CBF3 binding . Among Zn 2 Cys 6 zinc cluster proteins, the requirement for two intact CCGs is not absolute: in vitro selection yielded Hap1p binding sites that contain a single intact CCG and a second degenerate CCG in which only the central C is conserved . When CDEIII is mutated to generate sequences with two “perfect” CCG sites in various orientations, not only is p64 unable to bind to the mutant CDEIII on its own, but CBF3 binding is largely abolished . We therefore postulate that p64 binds to CDEIII only in the context of a CBF3 complex not because CDEIII contains a low-affinity binding site, but because free p64 is inactive. To build a preliminary structural model for the CBF3-DNA complex we have combined the protein data presented in this paper with earlier results from DNA-protein cross-linking . Based on the stoichiometries of the CBF3 proteins in solution, the simplest composition for CBF3 bound to a 56-bp CDEIII fragment is one copy of p23, one p58, two p64s, and two p110s. The stoichiometries and apparent shapes of the CBF3 proteins are consistent with the pattern of DNA-protein cross-linking. For example, p64 cross-links to two putative half-sites about 40 Å apart, p58 cross-links at bases midway between the p64 cross-link sites, and p110 is apparently in contact with an extended region of CDEIII, consistent with its elongated shape . Thus, the close association observed in solution between p58 and p64 also exists on DNA. The predicted mass of a CBF3 complex with the composition shown in Fig. 10 c is 450 kD (including DNA). Although problems with the analysis of CBF3-DNA complexes on gel filtration columns have prevented us from confirming this mass by hydrodynamic methods, we can use the measured 15 S sedimentation coefficient and the 450-kD predicted mass to arrive at a proposed Stokes radius for DNA-bound CBF3 of 70 Å. This radius implies that the CDEIII DNA is bent and we have therefore bent CDEIII at an arbitrary position in our model. The CBF3-DNA core complex we have modeled is the smallest complex that can associate stably with DNA. We have detected additional binding sites for p110 in the centromeric DNA that lies outside the 56-bp fragment used here and believe that the CBF3-DNA structure that assembles in cells is considerably more complicated than the structure shown in Fig. 10 . In addition, yeast kinetochores contain other DNA-binding proteins including CBF1 and specialized histones and these proteins probably interact with CBF3. Nevertheless, much of the sequence specificity in CBF3-centromere interaction lies in the core complex. Thus, the structural model we have developed is a critical first step in determining the architecture of the larger yeast kinetochore. In the future, by probing longer fragments of centromeric DNA, including additional kinetochore proteins and using methods such as atomic force microscopy that are suitable for the analysis of large DNA-protein complexes, we intend to extend our structural model beyond the core CBF3 complex. p58 is subject to both positive and negative regulation: it is activated by phosphorylation and degraded by the proteosome . We have proposed previously that coupled activation and destruction of p58 may control the amount of active CBF3 and thereby increase the fidelity of kinetochore assembly. We imagine an editing process in which p58, correctly incorporated into functional CBF3 and assembled on centromeric DNA, is protected from degradation, whereas soluble, unassembled p58 is degraded. Implicit in this proposal are two key postulates: that p58 plays a critical role in CBF3 assembly and that assembly regulates p58 stability. Evidence for the first postulate is the finding that p58 constitutes the structural core of the CBF3 complex. Because p23 Skp1 , p64, and p110 form stable complexes in solution only with p58 and not with each other, the regulation of p58 abundance and activity directly controls that state of association among all of the CBF3 subunits. Evidence for the second postulate is the observation that raising the intracellular concentration of the p58/p64 complex (but not other p58-containing complexes) greatly stabilizes p58. A similar type of regulation has been observed with the mating type specific transcription factor a1/α2 . When not associated with a1 (in a cells for example), α2 is ubiquitinated and rapidly degraded. Similarly, a1 is rapidly degraded when not associated with α2. However, when assembled into an a1/α2 complex, both a1 and α2 are protected from proteolysis. One of the instability determinants in α2 lies in the α helix that mediates interaction with a1; thus, the stabilization of α2 by a1 involves a direct masking of an instability determinant. A similar mechanism may be operating in p58 but we do not yet have enough data to localize the key regulatory sequences. We propose the following pathway for CBF3 assembly . Newly synthesized p58 is activated by phosphorylation in a p23 Skp1 -dependent manner. Both active and inactive p58 are subject to ubiquitination by SCF cdc4 , and thus to degradation by the 26 S proteosome . Those p58 molecules that bind to p64 are protected from degradation, perhaps because they are no longer substrates for SCF cdc4 . The p58/p64 complex is not particularly stable and can dissociate into free p58 and p64, again exposing p58 to the degradation pathway. However, p58/p64 complexes that bind to p110 and to centromeric DNA assemble into a CBF3 complex that dissociates only very slowly from DNA . Thus, the half-life of p58 increases as CBF3 assembles, but it is only when a high-affinity CBF3 complex forms on centromeric DNA that p58 is fully protected from degradation. To our knowledge, there are few structures in a cell for which the copy number of active complexes must be as closely controlled as the number of active kinetochores. We believe that the high selectivity of kinetochore assembly is ensured not only by the intrinsic sequence specificity of CBF3-CDEIII interactions, but also by the regulation of CBF3 abundance and activity. This regulation of abundance and activity appears to comprise an assembly-dependent editing mechanism that balances two competing demands. Following the replication of chromosomes in S-phase, sufficient active CBF3 must be present to occupy the centromeres on all chromosomes. However, CBF3 must be maintained at low concentrations to reduce the possibility of ectopic kinetochore formation and the generation of highly unstable dicentric chromosomes. Linking p58 stability to CBF3 assembly and to DNA binding would couple the number of active CBF3 complexes to the number of centromeres. | Study | biomedical | en | 0.999996 |
10352013 | The following materials were purchased as indicated: Mouse Multiple Tissue Northern Blot ( Clontech ), Ready-to-Go DNA labeling kit ( Pharmacia ), MitoTracker (Molecular Probes), Triton X-100 and guinea pig IgG ( Sigma Chemical Co. ), SSC buffer (5′→ 3′ Inc.), enzymes used for DNA manipulations (Life Technologies, Inc.), the DNA sequencing kit (U.S. Biochemical Corp.), monoclonal 12-CA-5 anti-HA tag antibody (BAbCo), biotinylated donkey anti–mouse IgG antibody, FITC donkey anti–mouse IgG antibody, donkey anti–rabbit IgG antibody, and Cy-5 conjugated anti–guinea pig IgG antibody (Jackson ImmunoResearch), Texas red streptavidin ( Amersham Corp. ), and erythrocyte lysate transcription-and-translation kit ( Promega ). Antibodies against D-AKAP1 were generated in female rabbits at Cocalico Corp. All oligonucleotides were synthesized at the Peptide and Oligonucleotide Facility at the University of California, San Diego. Blots containing 2 μg of immobilized mRNAs from selected adult mouse tissues were probed with 32 P-radiolabeled cDNA for the various splice variants. Nucleotides 1–90 encoding the first 30 residues of D-AKAP1b were amplified by PCR and used to make the cDNA probe specific for the N1 splice, nucleotides 2071–2366 of D-AKAP1a were used for the C1 splice, and nucleotides 2953–3317 of D-AKAP1c were used for the C2 splice. Fig. 2 a was taken from Huang et al. , in which nucleotides 854–1226 encoding the kinase binding domain in the core region of D-AKAP1 were used for a probe common to all the splice variants. Nitrocellulose filters were prehybridized in 6× SSC (750 mM sodium chloride and 75 mM sodium citrate, pH 7.0), 1% milk, and 0.5 mg/ml salmon sperm DNA for 4 h at 65°C, then hybridized to 1.5 × 10 6 cpm/ml of denatured radiolabeled cDNA probe in the same buffer. Hybridization was performed at 65°C for 16 h and nonhybridized probe was removed with 0.1× SSC and 0.1%SDS at 65°C. Hybridizing mRNA signals were detected by autoradiography. To determine the subcellular localization of the two NH 2 -terminal splices of D-AKAP1, an epitope tag derived from the influenza hemagglutinin protein (HA) was engineered at either the NH 2 terminus or the COOH terminus for immunofluorescent detection using monoclonal antibodies to the HA tag . To make NH 2 -terminal HA fusion proteins of N0 and N1, a linker containing the initiation codon of the luciferase gene and the nine–amino acid HA-tag sequence YPYDVPDYA was subcloned into the pcDNA3 vector using the BamHI and EcoRI sites. Another linker was then subcloned into the NotI and XbaI sites down-stream from the HA tag to make a one nucleotide frameshift at the NotI site. This new vector was named pcDNA3ML. Finally, cDNAs encoding D-AKAP1a and D-AKAP1b were subcloned into pcDNA3ML using the newly engineered NotI site and XhoI. D-AKAP1a and D-AKAP1b both use the C1 splice but have either N0 or N1, respectively. These two constructs, designated as [HA]-N0 and [HA]-N1 for their different NH 2 -terminal splices, correspond to [HA]-D-AKAP1a and [HA]-D-AKAP1b, respectively. To make COOH-terminal HA fusion proteins of D-AKAP1, the cDNA encoding the first 144 residues of D-AKAP1a was first excised from pBS-D-AKAP1a and subcloned into pcDNA3 using EcoRI. A linker containing the HA tag epitope flanked by a blunt end and a XhoI restriction site was then ligated in using EcoRV and XhoI. The final construct was designated N0(1-144)-[HA]. To make [HA]-N0(Δ1−30), which lacks the first 30 amino acids, a NotI site was engineered at residue 30 of [HA]-N0 by site directed mutagenesis , and the cDNA encoding residues 1–30 was excised using NotI. To make deletion mutants of [HA]-N1, a NotI site was engineered at residues Gly13 or Cys24 and cDNAs encoding sequences 1–13 or 1–24 were then excised using NotI. These two constructs lacking the first 13 or 24 residues of [HA]-N1 were designated N1(Δ1-13) and N1(Δ1-24), respectively. To make (1-30)N0-[GFP], the first 30 residues of N0 were amplified by PCR with EcoRI and BglII engineered at the 5′ and 3′ termini and subcloned into vector pEGFP-N1 ( Clontech ) between EcoRI and BamHI sites. To make (1-63)N1-[GFP] and (1-33)N1-[GFP], the first 63 or 33 residues of D-AKAP1b were amplified by PCR with EcoRI and BglII engineered at their 5′ and 3′ termini, respectively. These two fragments were then subcloned into vector pEGFP-N1 ( Clontech ) between EcoRI and BamHI sites. Expression vectors for [HA]-N0 and [HA]-N1 were tested using an in vitro transcription-translation kit (TNT T7 Quick coupled system; Promega ). In brief, purified DNA was mixed with rabbit reticulocyte extract and 10 μCi [ 35 S]methionine, and incubated at 30°C for 2 h. The assay mixtures were separated by SDS-PAGE and visualized by autoradiography for 48 h. These products were also subjected to immunoblot analysis using antibodies specific for N0 (anti-RPP7) or N1 (anti-N1), and visualized by ECL, which takes <1 min for exposure. Mouse 10T1/2 fibroblasts were maintained in DME containing 10% FBS, and plated at a density of 1 × 10 5 cells per 10-cm plate 24 h before transfection. Cells were transiently transfected, using the calcium phosphate method with 10 μg of DNA per plate . 20 h after transfection, cells were visualized by fluorescent microscopy either directly or after fixing with 4% paraformaldehyde. For microinjection, cells were plated on glass coverslips and grown to 70% confluence in DME + 10% FBS. The coverslips were then transferred to DME containing 0.05% calf serum. After 24 h, the cells were injected into their nuclei with solutions of injection buffer (20 mM Tris, pH 7.2, 2 mM MgCl 2 , 0.1 mM EDTA, and 20 mM NaCl) containing 100 μg/ml expression plasmid DNA. In some experiments, 6 mg/ml guinea pig IgG ( Sigma Chemical Co. ) was coinjected as a marker. Experiments were performed using an automatic micromanipulator (Eppendorf) with glass needles pulled on a vertical pipette puller (Kopf). For detection of expressed proteins, cells were fixed in 4% paraformaldehyde 5 h after injection for 10 min, and then washed with 0.1% Tween in PBS. The cells were then incubated successively with appropriate antibodies. In general, cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS before staining with antibodies. HA-tagged proteins were stained with primary monoclonal anti-HA tag antibodies (dilution 1:100) for 1 h at 37°C then incubated with rhodamine-conjugated donkey anti–mouse IgG antibodies (dilution 1:200). In some experiments, the secondary antibody is biotinylated donkey anti–mouse IgG, which is in turn stained by Texas red streptavidin. To stain mitochondria, two methods were used. 200 nM of a mitochondrion-specific dye (Mitotracker; Molecular Probes Inc.) was incubated with the microinjected cells for 30 min before fixation. Alternatively, a vector encoding a mitochondrial-specific green fluorescent protein was comicroinjected with the HA-tagged proteins, and mitochondria were visualized directly by GFP. In addition, cells were stained with Cy-5 conjugated anti–guinea pig IgG (dilution 1:100) to discriminate injected from noninjected cells. To stain ER, cells were incubated with rat anti-Bip antibodies (dilution 1:100), washed three times with 0.1% Tween in PBS, and probed with fluorescein-conjugated donkey anti–rat IgG (dilution 1:100). Between incubations with different antibodies, cells were washed three times with 0.1% Tween in PBS for 5 min each time at room temperature to eliminate nonspecific staining. Samples were visualized by confocal microscopy using an MRC-1024 system (Bio Rad Laboratories) attached to an Axiovert 35M ( Zeiss AG ) and a 40× NA objective. Excitation illumination was with 488-, 568-, and 647-nm light from a krypton/argon laser. Individual images were converted to PICT format and merged as pseudo-color RGB images using Adobe Photoshop (Adobe Systems). Digital prints were from a Fujix Pictrography 3000 printer (Fuji). To investigate tissue-specific expression of the different splice variants of D-AKAP1, Northern blots were probed with 32 P-labeled splice-specific cDNAs. As described previously, probing the blot with cDNA to the core region of D-AKAP1 showed that the overall expression of the 3.8-kb D-AKAP1 message is highest in heart, liver, skeletal muscle, and kidney. In addition, a strong signal at 3.2-kb was detected only in testis . Probing the same blot with cDNA probes derived from unique regions of the various splice variants allowed detection of splice-specific expression . Proteins containing the N1 splice were expressed exclusively in liver, while the C1 splice was expressed predominantly in testis. The C2 splice had a more general expression pattern. Therefore, in addition to the distinct features revealed by amino acid sequences, these splice variants also were expressed in a tissue-specific manner. Because the N0 splice contained sequences shared by the core region, the expression pattern specific for this NH 2 -terminal splice could not be obtained. However, since the N1 splice was only expressed in liver, the D-AKAP1s that are expressed in all the other tissues most likely use the N0 splice. To determine the location of D-AKAP1 in cells, [HA]-N0 and [HA]-N1 that had an HA-tag at the NH 2 terminus of D-AKAP1a and D-AKAP1b were constructed as described in the Materials and Methods. D-AKAP1a is comprised of the N0 splice and the C1 splice; D-AKAP1b is comprised of the N1 splice and the C1 splice. To determine whether the vectors can express the appropriate proteins, expression was tested first in a cell-free translation system. As seen in Fig. 3 , in vitro transcription-translation of [HA]-N0 or [HA]-N1 gave protein products with the apparent molecular mass of 107 and 114 kD, respectively. These are approximately what would be predicted from the sequence. To further verify these expressed products, they were also subjected to immunoblot analysis using antibodies raised against a core region of D-AKAP1 (RPP7) or generated specifically to N1. Only the protein product expressed from [HA]-N1 reacted with antibodies specific for N1 . The NH 2 -terminal 30 amino acids of N0 has a putative mitochondrial-targeting sequence in S-AKAP84 . To further characterize the localization of D-AKAP1 that used this N0 splice, the vector expressing D-AKAP1a with the HA tag at the NH 2 terminus was microinjected into 10T1/2 mouse fibroblasts. Expressed proteins were visualized by immunofluorescence coupled with confocal microscopy. As seen in Fig. 4 A, cells expressing [HA]-N0 had a punctate immunofluorescent staining concentrated around the nucleus, This labeling was mimicked by a fluorescent mitochondrial marker (Mitotracker), indicating its localization to the mitochondria. To test if the HA-tag affected localization, N0-[HA] was constructed. In this vector, the first 144 amino acids of D-AKAP1a were fused to a COOH-terminal HA-tag. The staining pattern was identical for both [HA]-N0 and N0-[HA], and was thus independent of epitope tags attached at either end . To confirm the mitochondrial localization in living cells as well as in fixed cells, GFP was fused to the targeting sequence of cytochrome oxidase . This [GFP]-mitochondrial marker, [GFP]-mito, was used to preclude the possibility of staining artifacts and confirm localization to the mitochondria. Cells microinjected with the expression vector for GFP alone had a diffuse distribution, filling the entire cell as previously reported . [GFP]-Mito, in contrast, displayed typical mitochondria localization . To determine whether the first 30 residues of N0 are necessary and/or sufficient to target mitochondria, two additional constructs were engineered. [HA]-N0(Δ1−30) contained HA-tagged full-length D-AKAP1a lacking the first 30 residues, and (1-30)N0-[GFP] contained only the first 30 residues of D-AKAP1a fused to GFP. As shown in Fig. 4 C, when cDNA of [HA]-N0(Δ1−30) was microinjected into the nucleus of 10T1/2 cells, a diffused cytoplasmic staining pattern was observed indicating the loss of mitochondrial targeting. In contrast, (1-30)N0-[GFP] showed a characteristic mitochondrial pattern . Thus, as summarized in Fig. 4 E, the first 30 residues of N0 were not only necessary, but also sufficient for mitochondrial localization. In addition to the N0 splice, D-AKAP1 has at least one alternative NH 2 -terminal splice variant, N1, that includes 33 extra amino acids NH 2 -terminal to the N0 splice. To investigate if these 33 residues affect the localization of D-AKAP1, [HA]-N1 was constructed. [HA]-N1 encodes HA-tagged D-AKAP1b, which consists of the N1 splice in addition to D-AKAP1a. Cells microinjected with an expression construct for [HA]-N1 had a more “lacy” staining pattern. Colocalization of [HA]-N1 with Bip, an hsp70 homologue that resides in the lumen of the ER , indicated [HA]-N1 is targeted to the ER . Therefore, the extra 33 residues of the N1 splice not only abolished mitochondrial targeting of D-AKAP1, but also exhibited an ER-targeting function. Cells microinjected with the [HA]-N1 expression vector were also cross-stained with [GFP]-mito and showed no overlapping staining (data not shown). To further characterize this ER-targeting function of N1, two deletion mutants, N1(Δ1-13) and N1(Δ1-24) were engineered. N1(Δ1-13) contained the full-length D-AKAP1b with the first 13 residues deleted, while N1(Δ1-24) contained the full-length D-AKAP1b with the first 24 residues deleted. Both had the HA tag fused to the NH 2 -terminus. N1(Δ1-13), like full-length [HA]-D-AKAP1b, colocalized with Bip, indicating its localization to the ER , whereas N1(Δ1-24) had a mixed localization pattern between ER and mitochondria. Fig. 5 C shows fluorescence imaging of two cells microinjected with the N1(Δ1-24) expression construct that exhibited a more prominent mitochondrial pattern. Since N1(Δ1-24) had decreased ability to target D-AKAP1 to ER, residues 14–33 must participate in dictating the ER localization of N1. Residues 14–33 of N1, as shown in Fig. 5 d, contain a potential PKA phosphorylation site (RRCSY). To test if phosphorylation of this site participated in the ER-targeting function of N1, three mutants with the potentially phosphorylatable Ser changed to Ala, Glu, and Asp were engineered in the construct [HA]-N1. All three mutants exhibited ER localization (original data not shown). To further localize the ER-targeting signal, two GFP fusion constructs were engineered. (1-63)N1-[GFP] contains the 33 extra amino acids of N1 plus the 30 residues of N0, sufficient for mitochondria targeting, fused to GFP. (1-33)N1-[GFP] contains only the 33 residues of N1 fused to GFP. As shown in Fig. 6 A, cells microinjected with the expression vector encoding (1-63)N1-[GFP] had a lacy staining pattern, characteristic of ER localization, whereas cells microinjected with the (1-33)N1-[GFP] expression vector showed a diffuse staining pattern, similar to GFP alone . Cells expressing (1-63)N1-[GFP] were also stained with the anti-N1 antibody and ER localization was detected (data not shown). These results indicated that the 33 residues of N1, when fused to the mitochondrial-targeting sequence of N0, were sufficient to switch its localization to the ER. However, these 33 amino acids alone were not able to dictate ER localization. Therefore, the ER-targeting signal is at least partially located within the NH 2 -terminal 30 residues of N0. The 33 additional amino acids at the NH 2 terminus of N1 thus were able to (a) suppress the mitochondrial-targeting function of N0 and (b) generate a new ER-targeting function. Since HA-tagging at the NH 2 terminus of N0 did not alter its mitochondrial localization, it does not appear as though mitochondrial targeting requires the insertion of the NH 2 terminal N0 motif into the membrane. Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. These targeting proteins recruit active enzymes into signaling “modules” or place specific enzymes close to their substrates. For example, proteins such as src are brought to the plasma membrane by a myristylation motif at its NH 2 terminus. In addition, src homology 2 (SH2) domains bind to specific phosphotyrosines, thus permitting the assembly of signaling complexes in response to activation of cell surface receptors . Similarly, subcellular compartmentalization of serine/threonine kinases and phosphatases occurs through interactions with targeting subunits or anchoring proteins that localize these enzymes to specific sites . AKAPs play major roles in regulating the compartmentalization of PKA through specific interactions with the NH 2 -terminal dimerization domain of the R subunits . D-AKAP1 represents a family of AKAPs with splice variants at both the NH 2 terminus and COOH terminus. We showed here that, based on Northern blot analysis, three of these splice variants are expressed in a tissue-specific manner of the tissues tested. N1 was expressed exclusively in liver, while C1 was expressed predominantly in testis. C2 was expressed in all tissues tested but spleen, consistent with the overall expression pattern of D-AKAP1 core. Furthermore, we demonstrated here that the two NH 2 -terminal splice variants, N0 and N1, act as localization switches to dictate subcellular localization of D-AKAP1 to either mitochondria or endoplasmic reticulum. Fig. 7 summarizes the functional properties of the motifs at the NH 2 terminus of N0 and N1. The first 30 residues of D-AKAP1 were shown to be necessary for mitochondrial targeting of mouse S-AKAP84 by cellular fractionation . Using microinjection of the various expression constructs coupled with immunocytochemistry, we directly investigated the localization of D-AKAP1. [HA]-N0 and N0-[HA] both exhibited mitochondrial localization, whereas a construct lacking the first 30 residues of N0 lost the targeting ability. These results confirmed that for D-AKAP1, the first 30 residues of N0 are necessary for proper targeting to mitochondria. Moreover, we took advantage of GFP-fusion proteins that can be visualized without artificial staining procedures in living cells. (1-30)N0-[GFP], which contains only the first 30 residues of N0 fused to GFP, also exhibited mitochondrial localization. Therefore, the first 30 residues of N0 are not only necessary but also sufficient to target D-AKAP1 to mitochondria. When the first 30 amino acids of D-AKAP1 were compared with another protein that localizes to the outer mitochondrial membrane, hexokinase I, substantial similarities were identified . As shown in Fig. 4 E, in addition to the lack of negatively charged residues, hydrophobic residues and consecutive aromatic amino acids were homologous between the two proteins. This conserved stretch of residues (residues 1–15 in hexokinase I) was also identified to be necessary and sufficient to confer mitochondrial binding . Recently, the crystal structure of the mammalian hexokinase I was determined, revealing that this stretch of mitochondrial-targeting residues forms an amphipathic helix . Whether D-AKAP1 uses the same mechanism for mitochondrial targeting awaits further investigation. However, since the attachment of an epitope tag at the beginning of N0 did not abolish targeting to mitochondria, it is unlikely that the NH 2 -terminal motif simply inserts as an amphipathic helix into the membrane bilayer as has been proposed for hexokinase. Surprisingly, despite the fact that N1 also contains the stretch of hydrophobic residues necessary for mitochondrial targeting, it exhibited a dramatically different localization. [HA]-N1 colocalized with Bip and showed a prominent ER staining, indicating the localization to the ER. Therefore, the extra 33 amino acids in N1, including a potential myristylation site followed by a potential PKA phosphorylation site, not only abolished mitochondrial targeting of N0 but also generated an ER-targeting function. The two distinct localizations for the two NH 2 -terminal splice variants of D-AKAP1 defines the first example of an AKAP that shows a localization switch. Since N1 is only expressed in the liver, this switch of mitochondrial to ER localization may have physiological implications. D-AKAP1 does not contain a conventional Lys-Asp-Glu-Leu (KDEL) sequence that was shown to be essential for ER retention of most soluble proteins . Therefore, this ER localization of N1 might define a novel mechanism for ER retention. Furthermore, with the NH 2 -terminal HA-tagged N1 still localized to ER, myristylation at the NH 2 terminus of N1 is thus not likely to be necessary for its ER targeting. To further dissect N1, two deletion mutations were constructed and examined. N1(Δ1-13) shows localization to the ER, similar to the full-length [HA]-N1, whereas N1(Δ1-24) has a mixed staining pattern at mitochondrial and ER. Because N1(Δ1-33), i.e., [HA]-N0, localizes exclusively to the mitochondria, residues 14-33 of N1 are especially important for the ER-targeting function. A potential PKA phosphorylation site was identified within this region of N1. Nevertheless, three mutations with the potentially phosphorylatable Ser changed to Ala, Asp, and Glu all localized to ER. The Ser to Ala mutant localized to ER, indicating that the unphosphorylated D-AKAP1 is likely to reside in ER. Since Asp and Glu sometimes do not fully mimic the phosphate moiety, whether phosphorylation affects targeting to ER is still unclear. However, the ER localization is not altered in the presence of exogenous cAMP analogue (data not shown), suggesting that phosphorylation may not be important for targeting. By investigating the localization of (1-33)N1-[GFP], (1-63)N1-[GFP], and (1-30)N0-[GFP], the localization switch was further characterized. (1-33)N1-[GFP] showed a diffuse pattern in the cytoplasm, similar to GFP, whereas (1-63)N1-[GFP] localizes to ER. (1-30)N0-[GFP], i.e., (34-63)N1-[GFP], exhibited mitochondrial localization. These results not only indicate that the extra 33 residues do not contain an ER-targeting sequence, they also suggest that at least part of the ER-targeting function is embedded within the first 30 residues of N0 that also contain the mitochondrial-targeting signal. Adding N1 to the NH 2 terminus of N0 somehow is able to suppress its mitochondrial-targeting ability and also to generate a new ER-targeting signal. Whether this ER-targeting signal resides exclusively in N0 and is only exposed upon addition of N1, or whether this ER-targeting signal is instead generated by a composite of N0 and a portion of N1 is still under investigation. The alternative splicing that alters the subcellular localization of D-AKAP1 may represent a novel and important regulatory mechanism to increase regulatory versatility and allow for cell type–specific gene regulation for these anchoring proteins and their signal transduction pathways. Further effort to dissect the molecular mechanism for this ER/mitochondria localization switch is underway. | Other | biomedical | en | 0.999996 |
10352014 | For expression of Tim44 in vivo, a 2.7-kb HindIII fragment containing the TIM44 gene was cloned into YEplac33 and YEplac181 . tim44 Δ18 was constructed using the Promega Altered Sites System. The 2.7-kb HindIII TIM44 fragment was cloned into pSELECT-1 (= pALTER-1) and mutagenized with the mutagenic oligonucleotide 5′CAAAGGAGACTTAAACGTGCAGGAACAGCAGTGG3′. The 54-bp deletion was verified by DNA sequence analysis. The mutagenized HindIII fragment was subsequently cloned into YEplac181 (as a multi-copy vector) or YCplac111 (as a single-copy vector), respectively. The following Saccharomyces cerevisiae strains were used: PK82 , PK81 (MATα ade2-101 lys2 ura3-52 leu2-3,112 Δtrp1 ssc1-2(LEU2) ; Gambill et al., 1993 ), MB3 , and MB20 (MATa ade2- trp1- ura3- leu2- TIM44::LYS2 + YEplac33::TIM44(URA3) ; this study). For plasmid shuffling, double transformed cells were grown in rich liquid broth (YPD) and then plated on solid medium containing 5-fluoro-orotic acid according to Boeke et al. . The cDNA constructs encoding mitochondrial preproteins were cloned into pGEM4 ( Promega ), the transcription was performed using SP6-RNA-polymerase (Stratagene), and the precursor proteins were subsequently synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine and [ 35 S]cysteine ( Amersham ). For import experiments, mitochondria were isolated from strains of S . cerevisiae following a standard protocol . The wild-type strain PK82 or the strain MB3 containing different expression plasmids was used, as indicated in the figure legends. The import assays were carried out following standard procedures as published previously . In brief, the mitochondria (20 μg protein/100-μl assay) were incubated with reticulocyte lysate containing the preproteins in import buffer (3% [wt/vol] BSA, 250 mM sucrose, 80 mM KCl, 5 mM MgCl 2 , 10 mM MOPS-KOH, pH 7.2) at 25°C for up to 30 min. Treatment with proteinase K (50 μg/ml) was performed at 0°C for 10 min. The protease was stopped by addition of 1 mM PMSF. The mitochondria were reisolated by centrifugation at 16,000 g at 2°C for 10 min. For cross-linking experiments, the hybrid protein Su9-DHFR was accumulated in import sites by import into ATP-depleted mitochondria. Mitochondria (in the presence of 20 μM oligomycin to inhibit the F 0 F 1 -ATPase) and reticulocyte lysate were depleted of ATP by incubation with apyrase (10 U/ml; Sigma ) for 10 min at 0°C. The import was performed in the presence of 20 μM oligomycin. The mitochondria were washed by centrifugation through a sucrose cushion (500 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2) and resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2 (SEM buffer). The mitochondria (100 μg protein) were incubated at 0°C for 20 min in 1 ml SEM containing 0.1 mg/ml ethylene glycolbis succinimidylsuccinate (EGS). The reaction was stopped by addition of 100 mM Tris-HCl, pH 7.2, and a second incubation of 20 min at 0°C. Proteins were precipitated by addition of 10% trichloroacetic acid in the presence of 0.0125% deoxycholate. For immunoprecipitation, the precipitates were lysed in 20 μl 1% SDS, 60 mM Tris-HCl, pH 6.8. The samples were diluted 40-fold by addition of 1% (wt/vol) Triton X-100, 0.3 M NaCl, 10 mM Tris-HCl, pH 7.5, and the antibodies bound to protein A–Sepharose ( Pharmacia ) were added. Blue native electrophoresis was essentially performed following the protocols of Schägger and von Jagow . The samples were prepared as described by Dekker et al. . The procedures of protease treatment, swelling of mitochondria, and carbonate extraction were published previously . The tendency of mitochondrial proteins to form aggregates was tested by lysis of the organelles and subsequent centrifugation. Mitochondria from the wild-type and from the strain expressing Tim44 Δ18 in addition to the authentic Tim44 (40 μg protein) were lysed in 0.1% Triton X-100, 250 mM sucrose, 5 mM EDTA, 80 mM KCl, 10 mM MOPS, pH 7.5. The samples were divided into three parts, one part was incubated for 1 h at 0°C, the second part at 25°C, the third part was immediately mixed with trichloroacetic acid. After 1 h, the samples which had been left without trichloroacetic acid were subjected to a centrifugation of 10 min at 16,000 g and 2°C. Proteins were precipitated from the supernatants by addition of 10% trichloroacetic acid. For sonication the mitochondria (40 μg protein) were suspended in 500 μl 1 mM EDTA, 1 mM EGTA, 30 mM Tris, pH 7.5, 1 mM PMSF, 0.5 mM o -phenantroline and sonicated by a Branson Sonifier 10 times for 10 s (30% duty cycle, output 3) with intervals of 6 min for cooling of the samples. The membranes were pelleted by centrifugation at 100,000 g for 30 min. Proteins were precipitated from the supernatants by addition of 10% trichloroacetic acid. For coimmunoprecipitations, mitochondria (25 μg/sample) were lysed in 200 μl 250 mM sucrose, 80 mM KCl, 20 mM MOPS-KOH, pH 7.2, 0.1% (vol/vol) Triton X-100, 5 mM EDTA, 0.5 mM PMSF. In the experiment shown in Fig. 6 F, the mitochondria were lysed in 150 mM NaCl, 0.1% Triton X-100, 0.5 mM PMSF, 10 mM Tris-HCl, pH 7.4. After a spin of 16,000 g for 10 min, the lysates were incubated with antibodies directed against mtHsp70. The antibodies were covalently coupled to protein A–Sepharose as described previously . The membrane potential (Δψ) of isolated yeast mitochondria was determined by recording the fluorescence decrease of the voltage-sensitive dye 3,3′-dipropylthiacarbocyanine iodide [DiSC 3 (5); Molecular Probes; Sims et al., 1974 ]. The assays were performed using a Perkin-Elmer 640-40 fluorescence spectrometer at 25°C (excitation at 622 nm, slit width 5 nm). The mitochondria (100 μg protein) were incubated in 1 ml of 0.6 M sorbitol, 0.1% (wt/vol) BSA, 80 mM KCl, 10 mM MgCl 2 , 0.5 mM EDTA, pH 7.4. The final concentration of the fluorescent dye DiSC 3 (5) was 3.6 μM. The membrane potential was dissipated by the addition of 3 μM (final concentration) KCN. The difference between the fluorescences before and after the addition of KCN represents a rough assessment to the membrane potential. The region of sequence similarity between Tim44 and characteristic J-domains is shown in Fig. 1 A. The similarity extends from residue 185 to residue 202 and is depicted for the two yeast proteins Sec63p and Sis1p , and for the Escherichia coli protein DnaJ . The structure of the J-domain of DnaJ has been determined by NMR spectroscopy . The J-similarity segment of Tim44 corresponds to α helix II of DnaJ (residues 18–30) and the first half of the following turn region. In DnaJ, the turn is followed by helix III comprising residues 41–55. The secondary structures which are predicted for the corresponding part of Tim44 similarly show two hydrophilic α helices which are connected by a putative turn element. However, it should be emphasized that the similarity of Tim44 to J-domains is still very limited. Tim44 obviously does not belong to the family of homologous J-proteins. To determine the relevance of the J-similar segment for the function of Tim44 in vivo, we constructed a plasmid encoding a Tim44 with a deletion of residues 185–202 (Tim44 Δ18 ). We then tested whether Tim44 Δ18 can substitute for authentic Tim44 in a genetic assay . In a strain of S . cerevisiae expressing both forms of Tim44 from different plasmids, we used the URA3 /FOA technique to deplete the authentic Tim44. It turned out that the cells lacking the authentic Tim44 were not viable. Tim44 Δ18 could not substitute for wild-type Tim44, and neither on glycerol nor on glucose was any growth observed. Thus, this result demonstrates that the 18-residue segment of Tim44 is essential for viability of yeast. The lethality of the 18-residue deletion could be caused by two different reasons. Either the 18-residue segment is crucial for the biogenesis of Tim44, or this segment is required for the function of Tim44 within the mitochondria. To address the first possibility, we expressed Tim44 Δ18 in reticulocyte lysate and tested whether the protein could be imported into isolated mitochondria. In parallel we imported the authentic Tim44 . Both preproteins were processed to a mature form and translocated into a location protected against externally added proteinase K (lanes 3 and 7). The import was dependent on the mitochondrial membrane potential, confirming the specificity of the reaction. The result demonstrates that the 18-residue segment of Tim44 is not required for efficient transport into mitochondria in vitro. To test whether Tim44 Δ18 is similarly imported in vivo, Tim44 Δ18 was expressed in a S . cerevisiae wild-type strain from a multi-copy vector, and mitochondria were isolated to determine the amount of imported protein . We found that the mitochondria had imported both the authentic Tim44 and Tim44 Δ18 . The amount of authentic Tim44 was not significantly reduced as compared with the original wild-type strain; Tim44 Δ18 was overexpressed about threefold. The location of the imported Tim44 proteins was confirmed by a fractionation experiment. Both proteins were resistant against externally added trypsin in intact mitochondria and after opening of the intermembrane space by swelling . Only after disruption of the inner membrane by sonication did the proteins become accessible for the protease , in agreement with results which were obtained previously for the wild-type Tim44 . After lysis of the mitochondria by detergent, both proteins were similarly soluble . Even after prolonged incubation at 0 or 25°C, no formation of aggregates was observed . We then tested whether the deletion of the 18-residue segment may affect the association of Tim44 with the inner membrane. The extraction of Tim44 and Tim44 Δ18 was monitored by sodium carbonate and again both proteins showed the same behavior . In contrast to the ADP/ATP carrier (AAC) which was resistant against this treatment, both Tim44 proteins were extracted. The association of Tim44 with the inner membrane was also determined in the presence of different salt concentrations . Mitochondria were sonicated in the presence of up to 500 mM KCl and the membranes were subsequently pelleted by centrifugation. Tim44 and Tim44 Δ18 were found stably associated with the membranes; only minor amounts of both proteins were released at higher ionic strength. As a control, we followed the distribution of Mge1p which was soluble in all samples. The AAC was completely resistant against extraction. Eventually, we investigated the involvement of both forms of Tim44 in the formation of high molecular weight complexes using the method of blue native electrophoresis . As shown in Fig. 2 F, Tim44 and Tim44 Δ18 both showed the same distribution, suggesting that both proteins participate in the same interactions with the components of the Tim machinery. In a previous study we found that Tim44 is mainly associated with Tim23 . Therefore, we also determined the complex formation of Tim23 and found that it showed the same running behavior in the BN-PAGE, irrespective if only the authentic Tim44 was present or if Tim44 Δ18 was overexpressed in addition. We conclude from these experiments that Tim44 Δ18 is efficiently imported into mitochondria and acquires the correct topology at the inner side of the inner membrane. The results of the BN-PAGE indicate that Tim44 Δ18 adopts the native folding state and engages in the same interactions with the Tim machinery as the authentic Tim44. These conclusions are corroborated by the interactions of Tim44 Δ18 with different forms of mtHsp70 . If Tim44 Δ18 is recruited by the Tim machinery, is it also present at import sites during translocation of preproteins? We addressed this question by chemical cross-linking . As a substrate we synthesized the hybrid protein Su9-DHFR, containing the presequence of subunit 9 of the mitochondrial ATP synthase fused to the complete DHFR. The import of this protein is dependent on the membrane potential Δψ and ATP . After depletion of ATP, Su9-DHFR is accumulated in import sites as a membrane-spanning translocation intermediate. Following this protocol, Su9-DHFR was accumulated in mitochondria of both the Tim44 Δ18 overproducing strain and the wild-type . The translocation intermediates were cross-linked to the proteins in the vicinity by addition of the reagent EGS. In wild-type mitochondria, two products were formed, corresponding to the precursor form and the processing intermediate of Su9-DHFR . In mitochondria, which in addition contained Tim44 Δ18 , a third cross-linking product was formed which could be precipitated by antibodies against Tim44 . No reaction product was precipitated by the preimmune serum. According to its size in the SDS-PAGE, the additional product corresponds to cross-linking of Tim44 Δ18 to the processing intermediate of Su9-DHFR. The cross-linking of a protein in transit across the mitochondrial membranes confirms that Tim44 Δ18 is present at protein import sites. To determine the possible role of the 18-residue segment in Tim44, additional preproteins were imported and tested for defects in distinct steps of translocation across the mitochondrial membranes. In a first series of experiments, the β subunit of the mitochondrial ATP synthase (F 1 β) was synthesized in reticulocyte lysate in the presence of [ 35 S]methionine/[ 35 S]cysteine and imported into mitochondria which were isolated from the Tim44 Δ18 -overexpressing strain and the corresponding wild-type . It is known from previous studies that the import of F 1 β is very sensitive against defects in the import machinery and requires the mtHsp70-dependent unfolding machinery of the mitochondria . We now observed only a slight reduction in processing of F 1 β, suggesting that the deletion of the 18-residue segment of Tim44 does not cause major changes in the insertion of the presequence into the Tim machinery of the inner membrane . However, a protease-protection assay revealed a delay in the translocation of the mature part of the preprotein , indicating that the deletion affected the completion of translocation. After an import time of 20 min the efficiency of translocation was reduced by 75–80% . Some reduction was also observed after overexpression of the authentic Tim44, but the effect was much less pronounced . Modifications of mitochondrial inner membrane proteins can easily lead to a reduction of the mitochondrial membrane potential and thereby indirectly cause reduced efficiencies of protein transport. To address this possibility, we compared the membrane potential of the mitochondria which had been used in the previous experiments. As a sensitive assay we determined the membrane potential-dependent uptake of the dye DiSC 3 (5) . The uptake is reversible and can be quantified by following the change in the fluorescence of the dye. With mitochondria from the Tim44 Δ18 mutant strain no reduction in the membrane potential was observed . An indirect effect of the mutation on mitochondrial protein import mediated by a weakened membrane potential can thus be excluded. We then asked if the effect of the deletion of the 18-residue segment on protein import is dependent on the folding state of the preprotein. Previous studies have shown that the heme-binding domain of cytochrome b 2 is tightly folded and requires an intact mtHsp70 system to drive the unfolding of this domain . Following this principle, we now imported two different preproteins, b 2 (167)Δ-DHFR and b 2 (220)Δ-DHFR . Both constructs contain amino-terminal parts of cytochrome b 2 of different length fused to DHFR. The intermembrane space sorting signal is deleted to allow passage into the mitochondrial matrix. The hybrid protein b 2 (167)Δ-DHFR only contains an incomplete and therefore loosely folded heme-binding domain. A step of active unfolding of this preprotein is not required to allow the import reaction. The situation is different with the longer construct b 2 (220)Δ-DHFR. This protein contains the complete heme-binding domain (residues 81–179) and requires an unfolding reaction to allow the import. Thereby, the heme-binding domain causes restrictions if imported by a weakened translocation machinery . We compared the import kinetics of b 2 (167)Δ-DHFR and b 2 (220)Δ-DHFR in wild-type mitochondria and in mitochondria containing Tim44 Δ18 in addition to the intact Tim44 . With both preproteins, the import was clearly reduced in the Tim44 Δ18 mitochondria . To assess the effect of the heme-binding domain on the import efficiency we calculated the relative amounts of the imported proteins . We found that after 10 min of import, both constructs were imported into wild-type mitochondria with the same efficiency . With mitochondria from a Tim44-overproducing strain, the relative import efficiency of b 2 (220)Δ-DHFR was slightly improved . However, with mitochondria from the strain overproducing Tim44 Δ18 , the relative import efficiency of b 2 (220)Δ-DHFR was reduced to ∼35%, as compared with the import efficiency of b 2 (167)Δ-DHFR . Similar ratios were found at other time points of the import reaction . The 18-residue segment of Tim44 appears to be required to permit the efficient import of partially folded preproteins which require the full activity of the import machinery. The effect on loosely folded preproteins is less pronounced but also in this case the translocation is clearly facilitated if the activity of mtHsp70 is exclusively mediated by intact Tim44. The experiments shown in Figs. 2 and 3 have indicated that the interactions of Tim44 Δ18 with the Tim machinery are not disturbed by the deletion of the 18-residue segment. We now ask if Tim44 Δ18 shows an altered interaction with mtHsp70. We lysed mitochondria from the wild-type in the presence of detergent and performed coimmunoprecipitations using specific antibodies raised against mtHsp70. The precipitates were analyzed by immunoblotting and demonstrated the association of mtHsp70 with Tim44 . We then lysed mitochondria from the strain expressing both forms of Tim44. The mitochondria contained about three- to fourfold more Tim44 Δ18 than Tim44 . By coimmunoprecipitates from these lysates we compared the association of mtHsp70 with Tim44 Δ18 and the authentic Tim44. The ratio of Tim44 Δ18 to Tim44 in the precipitates was close to 1:1, demonstrating that complex formation of Tim44 Δ18 to mtHsp70 was reduced about three- to fourfold by the deletion of the 18-residue segment . Both forms of Tim44 were stable upon prolonged incubation after lysis, confirming that the reduced amount of Tim44 Δ18 in the immunoprecipitates was due to a reduced complex formation with mtHsp70 . The reduced affinity of mtHsp70 to Tim44 Δ18 as compared with the authentic Tim44 was confirmed by systematic quantifications . Complex formation of mtHsp70 with Tim44 Δ18 was reduced by ∼70%. In the presence of ATP, Tim44 Δ18 and Tim44 were both efficiently released from mtHsp70, confirming the specificity of the precipitations . ATP at a concentration of 2.5 nM was sufficient to cause the dissociation of 50% of the complexes with Tim44 Δ18 as well as with Tim44 (not shown). We conclude that the 18-residue segment of Tim44 is not the only structure which is involved in complex formation with mtHsp70. But the segment appears to be required to allow binding of sufficient efficiency and, as suggested by the results of the import experiments, for optimal cooperation of both proteins in mitochondrial protein import. The allele ssc1-2 encodes a mutant form of mtHsp70 which shows a reduced affinity for Tim44 but an enhanced affinity for substrate proteins . We expressed Tim44 Δ18 in a ssc1-2 strain and determined the interactions of the mutant mtHsp70 with both Tim44 proteins and a substrate protein by coimmunoprecipitations . While the efficiency of binding of the mutant mtHsp70 to the substrate protein Su9-DHFR was more than fourfold higher than that of wild-type mtHsp70 , the association of the mutant mtHsp70 with both Tim44 and Tim44 Δ18 was blocked . This result implies that not only the authentic Tim44 but also the truncated Tim44 Δ18 is recognized by mtHsp70 as a partner protein of special properties, and not as a substrate protein. In this study we have characterized the role of complex formation of mtHsp70 with Tim44 in mitochondrial protein import. A system of reduced binding between both proteins was created by the deletion of an 18-residue segment in Tim44 (residues 185–202) which shows a limited similarity to one of the two α helices of J-domains. The intracellular localization of Tim44 Δ18 is not altered by the deletion. In all fractionation experiments Tim44 Δ18 showed the same behavior as the authentic Tim44, and by chemical cross-linking we found that Tim44 Δ18 is localized at the inner membrane protein import sites. The correct topology of Tim44 Δ18 within the mitochondria allowed us to test whether the 18-residue segment of Tim44 is required to recruit mtHsp70 to the Tim machinery of the inner membrane. We found that binding of mtHsp70 to Tim44 Δ18 was reduced by ∼70% as compared with the authentic Tim44. The 18-residue segment of Tim44 is obviously not the only site for binding to mtHsp70. In contrast to the integral membrane protein Sec63p which is exposed to the ER lumen only by short segments of its sequence , Tim44 is a peripheral protein and in larger parts exposed to the matrix . This topology may allow the formation of multiple binding sites. While this manuscript was in preparation, a publication appeared by Greene et al. showing that the binding site of DnaJ for DnaK is the helix II of the J-domain, which corresponds exactly to the segment of similarity to Tim44. Since Tim44 seems not to belong to the family of J-proteins we assume that the J-related segment of Tim44 does not represent a J-homology in the strict sense but rather a J-analogous development to facilitate the interaction with mtHsp70. According to Greene et al. , the helix II of DnaJ interacts with the ATPase domain of DnaK. Following the analogy between Tim44 and DnaJ, Tim44 should similarly bind to the ATPase domain of mtHsp70. However, other J-proteins were found to interact with the carboxy-terminal domain of Hsp70s or to require both domains for binding . Therefore, it may be speculated that the interaction between mtHsp70 and Tim44 is mediated by multiple attachment sites, as was shown recently by the x-ray structure for the complex of DnaK with GrpE . We cannot completely rule out allosteric effects of the deletion of the 18-residue segment. However, the only difference to the wild-type protein we observed was restricted to the interaction with mtHsp70. The very sensitive assays of chemical cross-linking and blue native electrophoresis demonstrate that the oligomeric state of Tim44, and the direct interactions with preproteins and other components of the Tim machinery were retained. The comparison to the DnaJ-DnaK complex as analyzed by Greene et al. suggests that the 185–202 segment of Tim44 provides the major binding site for mtHsp70. Several data indicate that Tim44 binds to Tim23 and provides a dynamic link between the Tim proteins which form the protein import channel and the soluble mtHsp70 system of the matrix . With Tim44 Δ18 the function of this link is specifically impaired in the interactions of Tim44 Δ18 with mtHsp70. Our import experiments demonstrate that the presence of Tim44 Δ18 causes a significant reduction in the import efficiencies of different preproteins, including proteins which are regarded as loosely folded. The import of all of these preproteins is strictly dependent on mtHsp70 as demonstrated by previous studies using temperature-sensitive strains of SSC1 (encoding mtHsp70) . The strongest inhibition of import was observed with preproteins which contain a tightly folded domain. Such domains cause restrictions in the translocation across the mitochondrial membranes which are due to the requirement of unfolding within the import channel . To overcome these restrictions, the mtHsp70 system of the matrix has to exert a force on the translocating protein which is sufficient to pull the protein across the membranes. Studies to elucidate the mechanism by which this force is generated made use of the ssc1-2 mutant of mtHsp70 . The mtHsp70 of this mutant binds efficiently to translocating preproteins but is impaired in binding to Tim44. This defect correlates with an inhibition in the import of tightly folded protein domains. However, conclusions could only be drawn with reservation. The Tim machinery seems to contain at least two binding sites for mtHsp70, one at Tim44 and a second site at the Tim23/ Tim17 import channel, and both interactions are inhibited by the ssc1-2 mutation . The principle which governs the mechanism of mtHsp70-dependent protein import is still unknown. A Brownian ratchet mechanism and a mechanism of mtHsp70/Tim44-mediated pulling have been suggested. In this context it is remarkable that the effect of Tim44 Δ18 on the import of different preproteins and on the viability of yeast resembles the effects of ssc1-2 . This similarity in the phenotype thus corroborates and specifies the concept that the cooperation of Tim44 with mtHsp70 is of particular importance in the import of tightly folded protein domains. In a previous study on a complete inactivation of functional Tim44 in isolated mitochondria we showed that Tim44 acts at the inner side of the inner membrane . The results obtained with the Tim44 Δ18 construct suggest that in this location the functions of Tim44 in protein import may be confined to specific interactions with mtHsp70. In summary, the results of this study demonstrate that the J-related segment of Tim44 (residues 185–202) is required for the essential functions of Tim44 in mitochondria. This segment is not the only element involved in the interaction of Tim44 with mtHsp70, but it is required for productive cooperation of both proteins and the optimal efficiency of mitochondrial protein import. mtHsp70 is an essential motor protein in the translocation of all proteins which are imported into the mitochondrial matrix, irrespective of whether or not they contain tightly folded domains . In contrast, the requirement for an interaction of mtHsp70 with Tim44 seems to be less strict and appears to play an important role primarily in situations which require the full activity of the import motor, for example in overcoming stronger restrictions in the translocation of preproteins. The import of loosely folded preproteins is facilitated by Tim44, but the effect is much more pronounced in the case of tightly folded domains. | Study | biomedical | en | 0.999997 |
10352015 | Previous articles describe the routine procedures used in this study. These include isolation of rat heart mitochondria, transcription-translation of plasmids encoding VDAC and preornithine carbamyl transferase (pOCT), and import of 35 S-labeled translation products into mitochondria in vitro. Additional details are provided in the figure legends. Recombinant glutathione S-transferase (GST)–human Tom20Δ1-29 (formerly named GST-Δ30hTom20), lacking the NH 2 -terminal transmembrane segment of hTom20, was expressed in TOPP2 cells and purified . Protein immobilized on glutathione-Sepharose 4B was washed and suspended in 20 mM NaPO 4 , pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM CaCl 2 , and incubated with 5 μg/ml thrombin for 18 h at 4°C, generating hTom20Δ1-29 with an extra gly residue at the NH 2 terminus derived from the thrombin cleavage site. The mixture was passed through a Mono S HR 5/5 column and protein was eluted with a linear gradient (buffer A, 10 mM MES, pH 5.0; buffer B, 10 mM 3-cyclohexylamino-1-propane sulfonic acid, pH 10, 500 mM NaCl). Peak fractions containing hTom20Δ1-29 were resolved on a Superdex 200 column in 10 mM Hepes, pH 7.0, and 100 mM NaCl. Fractions containing the purified protein were dialyzed against the same buffer containing 5 mM DTT and the protein concentrated to 1.5 mg/ml. Plasmid encoding GST-hTom20Δ1-29 was manipulated by standard recombinant DNA procedures to substitute cys at hTom20 codon position 100 with ser and to introduce ser-cys after gly at the thrombin cleavage site. The thrombin cleavage product, hTom20Δ1-29/N-GSC/C100S, was generated and purified as above. Approximately 100 nm large unilamellar vesicles (LUVs) (lipid composition: PC, phosphotidylcholine; PE, phosphotidylethanolamine; PI, phosphotidylinositol; PS, phosphotidylserine; PC/PE/PI/PS at molar ratios of 55:28:13:3 or PC/PE/PI/PS/PE-bmps at molar ratios of 54.5:27.5:13:3:1) were prepared in 170 mM sucrose, 20 mM Tris acetate, pH 7.0, and 2 mM CaCl 2 . LUVs containing β-maleimidopropionic acid N -hydroxysuccinimide ester of PE (PE-bmps) were incubated for 12 h at 4°C with hTom20Δ1-29/N-GSC/C100S (10-fold molar excess relative to PE-bmps; coupling efficiency, 85–95%) followed by two 30 min incubations with excess LUVs generated in medium lacking sucrose (100 mM NaCl, 20 mM Tris acetate, pH 7.0, and 2 mM CaCl 2 ) to competitively remove unincorporated hTom20. Sucrose-loaded LUVs containing the covalently attached cytosolic domain of hTom20 were recovered by centrifugation at 50,000 × g for 60 min in a Beckman 75Ti rotor. They were used in protein import reactions in place of mitochondria. Standard protein import was conducted by combining rat heart mitochondria with human VDAC synthesized in reticulocyte lysate. In this system, the preprotein translocation pore can be jammed by partially importing a chimeric protein containing a matrix-targeting signal fused to dihydrofolate reductase (DHFR). Unfolding of the DHFR moiety on the cytosolic side of the outer membrane is prevented by the high-affinity DHFR active site inhibitor, methotrexate . In the presence of MTX, excess pODHFR , a chimeric protein consisting of the matrix targeting signal of pOCT fused to DHFR and purified from expressing bacteria, blocked import and processing of pOCT , whose processed form otherwise acquires resistance to external trypsin . In addition to inhibition of pOCT import, pODHFR/MTX inhibited outer membrane insertion of yTom70(1-29)DHFR , also designated pOMD29, a chimeric protein comprised of the transmembrane signal-anchor domain of yeast Tom70 fused to DHFR, whose insertion into the outer membrane of heart mitochondria in vitro has been documented previously . yTom70(1-29)DHFR is inserted into the outer membrane in the N in -C cyto orientation and therefore, its import in the absence of competing pODHFR was unaffected by MTX (not shown). In contrast to pOCT and yTom70(1-29)DHFR, import and membrane insertion of VDAC in this system, assessed by the temperature-sensitive acquisition of resistance to alkaline extraction , was relatively unaffected by pODHFR/MTX . The slight reduction in VDAC import at 5 min effected by pODHFR/MTX may relate to the fact that preproteins like pODHFR can stimulate recruitment of Tom20 into the translocation complex, thereby competitively reducing the amount of free Tom20 in the membrane that is available for interaction with VDAC . The failure of partially translocated pODHFR/MTX to block membrane insertion of VDAC suggested that VDAC may bypass the requirement for the Tom40 translocation pore during import. To address this question further, import of VDAC was examined using isolated yeast mitochondria containing a temperature-sensitive mutant of Tom40 . In contrast to control mitochondria, temperature-sensitive Tom40 mitochondria were incapable of importing the matrix preprotein form of cytochrome oxidase subunit Va (COX Va) at the nonpermissive temperature (37°C), as judged by the failure of pCOX Va to be processed by the mitochondria at 37°C , while processing of pCOX Va was observed at the permissive temperature of 23°C. In contrast to pCOX Va, membrane insertion of VDAC occurred at both 23 and 37°C . The slight decrease in membrane insertion of VDAC at 37°C compared with 23°C was similar for both wild-type and temperature-sensitive Tom40 mitochondria , suggesting that this difference was related to events other than the temperature-sensitive phenotype of Tom40. The cytosolic domain of hTom20, hTom20Δ1-29, when included as a soluble entity in excess in the import reaction, inhibited import of pOCT , presumably because it sequestered the preprotein through direct protein interaction and prevented transfer of the preprotein to the translocation machinery. hTom20Δ1-29 can also physically interact with VDAC . In distinct contrast to pOCT, however, hTom20Δ1-29 did not interfere with insertion of VDAC into the outer membrane. In fact, it had a slight stimulatory effect , suggesting that potential interactions between VDAC and hTom20Δ1-29 in the import reaction must be readily reversible. Moreover, the difference in response of pOCT and VDAC to hTom20Δ1-29 in the import reaction suggested a potential fundamental difference in the import pathways of the two proteins. However, the results also imply that a complex of VDAC and soluble hTom20Δ1-29 can make contact with the mitochondrial surface in vitro, a suggestion that is compatible with the observed binding of the cytosolic domain of hTom20 to lipid surfaces in vitro . To assess the possibility that hTom20 can mediate direct insertion of VDAC into a membrane lipid bilayer, synthetic LUVs were created in which the cytosolic domain of hTom20 was linked to the bilayer surface via covalent attachment to PE-pmbs . To that end, the protein was modified to contain a unique cys at the NH 2 terminus of hTom20Δ1-29; the other cys located at codon position 100 was converted to ser. These changes did not influence the ability of the receptor to interact with either VDAC or pOCT in vitro (data not shown). The liposomes had a phospholipid composition similar to that of total mitochondrial outer membrane in rat liver . Under the conditions used for mitochondrial protein import reactions with VDAC synthesized in reticulocyte lysate such liposomes were found to efficiently insert the protein, as determined by acquired resistance to extraction at alkaline pH or with urea . Moreover, the resulting import product was resistant to treatment with trypsin (lane 4), reflective of VDAC in native membranes . LUVs bearing the hTom20 cytosolic domain did not translocate pOCT, as judged by the failure of pOCT to acquire resistance to trypsin , nor did they insert yTom70(1-29)DHFR, as judged by its extractability from liposomes with 7 M urea (not shown). Conversely, LUVs lacking the hTom20 cytosolic domain did not insert VDAC . Furthermore, VDAC that associated with LUVs lacking the hTom20 cytosolic domain exhibited facile interliposomal transfer , indicating that this binding by VDAC was merely peripheral. As expected, VDAC that was inserted into the LUV lipid bilayer by the hTom20 cytosolic domain did not transfer to subsequently added protein-free LUVs. In contrast, pOCT that bound to LUVs containing the hTom20 cytosolic domain could subsequently transfer to protein-free LUVs, but slower than pOCT that bound to LUVs lacking hTom20 . This finding is consistent with the ability of hTom20 to physically interact with pOCT . Moreover, the relatively slow dissociation of pOCT from the hTom20 cytosolic domain explains the ability of excess hTom20Δ1-29 to inhibit import of pOCT into mitochondria in vitro . Finally, VDAC that had been inserted into LUVs by the hTom20 cytosolic domain was examined for its ability to transport a physiological substrate of this channel protein, ATP . LUVs were loaded with [ 32 P]ATP before the import reaction, and the subsequent release of ATP was measured at various times after the initiation of the reaction. Insertion of a single functional VDAC channel into the vesicle would be predicted to release encapsulated ATP. Release of radioactive ATP commenced immediately upon initiation of VDAC insertion into liposomes . Egress of ATP was dependent on functional VDAC, inhibited by a known antagonist, NADH , and required the presence of the hTom20 cytosolic domain on the liposome surface to integrate VDAC into the lipid bilayer. In contrast, the control protein pOCT did not stimulate ATP export from LUVs alone or LUVs with hTom20 . The first indication that a β-barrel protein like VDAC might not require a complex preprotein translocation machinery for insertion into a membrane lipid bilayer came with the observation that purified detergent-solubilized VDAC can spontaneously integrate into planar lipid bilayers as a functional entity. Moreover, in this system insertion is cooperative as a result of self-mediated stimulation . Although the lag period for this insertion was relatively long, the results suggest an inherent ability of the molecule to partition into a lipid bilayer, but presumably this is because the detergent-extracted entity is in a favorable state for lipid bilayer integration. However, in normal physiology, other considerations apply: the question of membrane specificity; the problem of competing interactions of VDAC with other proteins, specific or otherwise; the necessity to maintain a conformation of soluble VDAC after release from the ribosome that is compatible with subsequent membrane insertion; and the requirement for a mechanism to catalyze VDAC insertion upon contact with the mitochondrial outer membrane. In the import reaction documented here, the appropriate translocation-competent conformation for VDAC may be supplied by chaperones/factors present in reticulocyte lysate . The only other minimum requirement was provided by hTom20 on the surface of the membrane. While it is impossible to rule out any involvement of the Tom40 translocation pore, its contribution to VDAC membrane insertion was not detected in the assays described here, whereas its influence on import of matrix pOCT and pCOX Va, and on membrane insertion of outer membrane Tom70(1-29)DHFR was pronounced. In addition to its role as a receptor in directing newly synthesized VDAC exclusively to the mitochondrial outer membrane within the context of a whole cell, our results suggest that hTom20 is capable of catalyzing direct insertion of the protein into the lipid bilayer. The receptor may accomplish this simply by bringing VDAC into close proximity to the bilayer and/or by triggering release of presumptive chaperones or other factors from VDAC that otherwise maintain the protein water-soluble. In either scenario, VDAC may spontaneously insert into the proximate bilayer. Alternatively, or in addition, hTom20 may play a direct role in guiding the conformational change that allows the transbilayer β-barrel channel to form. | Study | biomedical | en | 0.999998 |
10352016 | All yeast strains were derivatives of or were backcrossed at least three times to W303 ( ade2-1 , trp1-1 , leu2-3 , 112 , his3-11,15 , ura3 , and ssd1 ). Strains used for this work are listed in Table I . Cells were grown in YEP medium (1% yeast extract, 2% bactopeptone, and 50 mg/liter adenine) supplemented with 2% glucose (YEPD). α factor was used at 2 μg/ml, unless otherwise stated, nocodazole at 15 μg/ml, benomyl at 12.5 μg/ml and hydroxyurea at 150 mM. All the experiments were performed at 25°C, with the exception of those involving the ts mutants mps1-1 and ndc10-1 , which were performed at 37°C. Standard genetic techniques were used to manipulate yeast strains and standard protocols were used for genetic manipulations . For tagging BUB2 at the COOH terminus, a NotI cassette containing nine tandem repeats of the myc epitope was inserted before the termination codon of the HindIII-EcoRI COOH-terminal fragment of BUB2 subcloned in Yiplac204. The resulting plasmid (pSP49) was cut with BclI for integration at the BUB2 locus of W303, thus generating a full-length tagged version of BUB2 flanked by a truncated untagged version of the gene (strain ySP710). For tagging NDC10 at the COOH terminus, a NotI cassette containing six copies of the myc epitope was inserted before the termination codon of the NdeI/XbaI COOH-terminal fragment of NDC10 subcloned in pRS304. The resulting plasmid (pSP18) was cut with BclI for integration at the NDC10 locus of W303, thus generating a full-length tagged version of NDC10 flanked by a truncated untagged version of the gene (strain ySP460). For Western blot analysis, protein extracts were prepared by TCA precipitation as previously described or as described in Surana et al., 1993 . Proteins were transferred to Protran membranes (Schleicher and Schuell). myc-tagged Bub2 and Pds1 were detected with 9E10 mAb, whereas polyclonal antibodies were used to detect Clb2. Anti-actin antibodies were purchased from Sigma Chemical Co. Secondary antibodies were purchased from Amersham and proteins were detected by an enhanced chemiluminescence system according to the manufacturer. Flow cytometric DNA quantitation was determined according to Epstein and Cross on a Becton Dickinson FACScan ® . In situ immunofluorescence was performed according to Nasmyth et al. . Visualization of Tet operators using GFP and chromosome spreading were performed as described in Michaelis et al., 1997 . Immunostaining of Bub2myc9 and Ndc10myc6 were detected by incubation with the 9E10 mAb followed by indirect immunofluorescence using CY3-conjugated goat anti–mouse Ab (1:500; Amersham ); signals were amplified using a CY3-conjugated donkey anti–goat Ab (1:500; Amersham ). Spindle pole bodies were visualized with polyclonal anti-Spc72 followed by incubation with FITC-conjugated anti–rabbit Ab (1:50, Jackson ImmunoResearch Labs.). Since the mitotic checkpoint is thought to play an important role while chromosomes try to reach a bipolar attachment to the spindle, and inactivation of proteins involved in the mitotic checkpoint increases the frequency of chromosome loss , we asked whether the checkpoint proteins Mad2 and Bub2 had any role in the timing of sister chromatid separation during a normal cell cycle. For this purpose, we constructed wild-type, bub2Δ , and mad2Δ strains carrying the tetR-GFP/tetO constructs which allow to monitor sisters separation at the centromeric regions of chromosome V . Cell cultures of the above strains were synchronized in G1 by α factor treatment and released into fresh medium either lacking or containing the microtubule depolymerizing drug nocodazole, which activates the mitotic checkpoint. As previously reported , in the presence of nocodazole both bub2Δ and mad2Δ cells lost the cohesion between sister chromatids, rebudded, and rereplicated, accumulating with DNA contents higher than 2C, whereas wild-type cells arrested in G2 as dumbbells with duplicated but unseparated chromosomes . Interestingly, we reproducibly found that in these conditions bub2Δ mutants separated sister chromatids and started rereplicating later than mad2Δ , suggesting that, in the absence of Bub2, entry into anaphase can be still somewhat delayed by other mechanisms. Without nocodazole, all three strains underwent anaphase and proceeded through the cell cycle with similar kinetics , thus indicating that either Mad2 and Bub2 have no role in the timing of sister chromatid separation during a normal cell cycle or bipolar chromosome attachment to spindle fibers in budding yeast might be a process too fast and efficient in order to allow detection of subtle differences in the timing of anaphase entry in the absence of surveillance mechanisms. Since Mad1, Mad2, Bub1, and Bub3 have been found associated to unattached kinetochores during prophase and prometaphase in higher eukaryotic cells , while the intracellular localization of Bub2 had not been established and little was known about its relationships with other mitotic checkpoint proteins, we analyzed the subcellular localization of Bub2. We tagged Bub2 at the COOH terminus with nine copies of the myc epitope, thus generating the Bub2myc9 variant, which was fully functional, as judged by the checkpoint proficiency of BUB2myc9 cells in nocodazole . Since Bub2myc9 staining in whole cells was fairly homogeneous and it was difficult to distinguish specific staining from the background observed with the same antibody on cells lacking tagged Bub2, we studied the protein localization by indirect immunofluorescence on chromosome spreads . By this technique, squashed nuclei stick to the slide and nucleoplasmic proteins are washed away, allowing detection of proteins bound to subnuclear insoluble structures, such as chromatin and nuclear cytoskeleton. We found that in all nuclei, Bub2myc9 staining was concentrated in one or two dots, very reminiscent of spindle pole bodies (SPBs). We, therefore, repeated the experiment by double staining the spreads with both anti-myc and anti-Spc72 antibodies, the latter of which recognize a constitutive SPB component . As shown in Fig. 2 A, the Bub2myc9 and Spc72 stainings completely overlapped, indicating that Bub2 resides at SPBs. As a control, we performed a double staining of the kinetochore protein Ndc10 and Spc72. To this purpose, we used a strain where the only functional copy of NDC10 was expressing a protein tagged with six copies of the myc epitope at the COOH terminus (Ndc10myc6). As it was previously shown for yeast centromeres , we found that Ndc10myc6 formed clusters near the SPBs, both in cycling and in nocodazole-arrested cells . However, not only Ndc10 clusters had a different shape compared with that of SPBs and Bub2, but not all Ndc10myc6 clusters colocalized with Spc72, while Bub2myc9 always did. Although we cannot fully prove that the Ndc10myc6 clusters correspond to kinetochores, these data strongly suggest that Bub2 does not localize at kinetochores. We then investigated further whether Bub2 protein levels and localization varied during the cell cycle and after nocodazole treatment. To this purpose, the BUB2myc9 strains were released from an α factor G1 arrest, and the Bub2myc9 protein levels and localization were analyzed at different time points. An aliquot of the synchronized culture was also incubated for 3 h in nocodazole. Bub2myc9 protein levels remained constant throughout the cell cycle and when the mitotic checkpoint was activated . Furthermore, the protein is constitutively localized at SPBs: G1 cells showed a single dot of Bub2myc9 staining, whereas two bright dots started appearing at the time of bud emergence and entry into S phase, which coincides with the time of SPB duplication (data not shown). About 30–40% of G1 nuclei displayed a fainter staining of Bub2 at SPBs than during the other cell cycle phases, regardless of growth conditions. About 55% of nuclei from nocodazole arrested cells showed two bright dots of Bub2myc9 staining side by side . This is, in these conditions, the typical spatial arrangement observed by electron microscopy for SPBs, which are duplicated but do not migrate apart in the absence of microtubules (Goetsch, L., and B. Byers, personal communication). Therefore, the nuclei showing single SPB signals by immunofluorescence (∼40%) under the same conditions likely had duplicated SPBs, which could not be resolved by this technique. Finally, we found that the localization of Bub2myc9 at SPBs was unaffected in the mitotic checkpoint mutants mad1Δ , mad2Δ , mad3 , bub1-1 , bub3Δ , mps1-1 , and ndc10-1 (data not shown). Therefore, Bub2 is constitutively present at SPBs during the cell cycle and its localization does not require any of the analyzed mitotic checkpoint proteins. The finding that Bub2 is localized at SPBs, and SPBs are mostly unseparated in nocodazole, prompted us to investigate whether Bub2 could be required to delay cell cycle progression only before SPB separation. For this purpose, bub2Δ , mad2Δ , and wild-type cells were synchronized by hydroxyurea (HU) treatment, which blocks DNA synthesis but allows duplication and separation of SPBs, thus arresting cells in S phase with short bipolar spindles. Cell cultures were then released from the HU block into nocodazole containing medium, in order to disassemble the previously formed spindles, and cell cycle progression of the three strains was analyzed. Formation and disassembly of the spindle in HU and in nocodazole, respectively, was confirmed by immunostaining of tubulin (data not shown). As shown in Fig. 4 , under these conditions wild-type cells arrested in G2, with 2C DNA contents and unseparated sister chromatids, while mad2Δ and bub2Δ cells attempted to undergo anaphase and entered a new cell cycle, as indicated by their ability to separate sister chromatids and rereplicate DNA. Therefore, both Mad2 and Bub2 checkpoint functions are required also after SPBs separation and bipolar spindle formation. Since we found Bub2 concentrated at SPBs, whereas Mad1 and Mad2 are localized, at least in higher eukaryotes, at unattached kinetochores, we asked whether Bub2 and Mad1, 2 might activate the mitotic checkpoint via different pathways. To address this question, we first constructed mad1Δ bub2Δ and mad2Δ bub2Δ double mutants, and compared their sensitivity to the microtubule depolymerizing drug benomyl to that of the single mutants. In fact, hypersensitivity to benomyl of mad and bub mutants correlates with their mitotic checkpoint defect , and inactivation of two proteins that lead to checkpoint arrest by different routes should result in a more severe checkpoint defect, and therefore in an increased benomyl sensitivity, compared with inactivation of either one. As shown in Fig. 5 , mad1Δ bub2Δ and mad2Δ bub2Δ cells were much more sensitive to benomyl than mad1Δ , mad2Δ , and bub2Δ cells, suggesting that BUB 2 belongs to an epistasis group different from that of MAD1 and MAD2 . We then verified whether the increased benomyl sensitivity of mad2Δ bub2Δ and mad1Δ bub2Δ double mutants correlated with an enhanced checkpoint defect. Cultures of mad1Δ , mad2Δ , bub2Δ , mad1Δ bub2Δ , mad2Δ bub2Δ , mad1Δ mad2Δ and wild-type strains were arrested with α factor and released in either the presence or the absence of nocodazole. Progression through the cell cycle in the absence of nocodazole was very similar in all strains (data not shown); however, in the presence of the drug mad1Δ bub2Δ and mad2Δ bub2Δ cells started rereplicating much faster and more efficiently than the single mutants and the mad1Δ mad2Δ double mutant . Since the timing and efficiency of rereplication likely reflect the kinetics by which cyclin B–dependent kinases are inactivated, these data suggest that mad1Δ bub2Δ and mad2Δ bub2Δ double mutants advance inactivation of Clb-kinases with respect to the other mutants. Conversely, deletion of BUB2 in mad1Δ or mad2Δ cells did not accelerate their timing of sister chromatids separation , indicating that Bub2 might play only a minor role in controlling degradation of the anaphase inhibitor Pds1. We then measured directly by Western blot analysis the levels of Pds1 and Clb2 during a similar experiment, where mad2Δ , bub2Δ , mad2Δ bub2Δ and wild-type strains expressed a myc-tagged Pds1 protein . As shown in Fig. 6 B, Clb2 was degraded in both mad2Δ and bub2Δ cells in nocodazole, suggesting that rereplication in these mutants is likely to result from their inability to prevent cyclin proteolysis. In addition, Clb2 disappeared more rapidly in the mad2Δ bub2Δ double mutant than in either single mutant, thus confirming that acceleration in the kinetics of rereplication in the double mutant correlates with an advanced inactivation of Clb/Cdk1 kinases. Although bub2Δ cells did also degrade Pds1, they did it very slowly, whereas Pds1 disappeared very rapidly and with nearly identical kinetics in both mad2Δ and mad2Δ bub2Δ cells. Thus, Pds1 degradation and the onset of anaphase appear to be mainly controlled by a Mad2-dependent pathway that does not seem to require Bub2. Altogether these data, while confirming that Mad1 and Mad2 are involved in the same pathway, indicate that Bub2 and Mad2 prevent B-type cyclin proteolysis by different routes, since the concomitant lack of Mad2 (or Mad1) and Bub2 has an additive effect on Clb2 degradation, exit from mitosis and entry into a new cell cycle. Conversely, Bub2 seems to play only a minor role, if any, in controlling the onset of anaphase. Since mitotic checkpoint arrest involves APC inactivation , it was possible that different APC subunits might be alternatively involved in the Mad2- and the Bub2-dependent checkpoint response. To address this question, we asked whether various APC subunits were required for cell cycle progression of mad2Δ or bub2Δ mutants in the presence of microtubule depolymerizing drugs. Unfortunately, most apc mutations are temperature sensitive and cause a cell cycle arrest in metaphase already during an otherwise unperturbed cell cycle . Furthermore, bub2Δ mutants show a poor checkpoint defect at 37°C (data not shown). However, we were able to test whether CDC26 , which is totally dispensable in a normal cell cycle at 25°C , might become indispensable for cell cycle progression of either mad2Δ or bub2Δ cells in the presence of nocodazole. The role of Cdc26 in the mitotic checkpoint was also of particular interest as we found that cdc26Δ mutants are hypersensitive to benomyl at the permissive temperature (Amato, S., and S. Piatti, unpublished results). Cell cycle progression of mad2Δ cdc26Δ and bub2Δ cdc26Δ double mutants was compared with that of each single mutant after release from α factor in the presence of nocodazole at 25°C. As shown in Fig. 7 , bub2Δ cdc26Δ cells failed to proceed in the cell cycle and arrested mostly in G2 with duplicated and unseparated chromosomes. Conversely, deletion of CDC26 did neither affect the time of anaphase onset nor cell cycle progression of mad2Δ cells. These results indicate that under these conditions Cdc26 becomes strictly required for both the onset of anaphase and exit from mitosis in the absence of Bub2 but not of Mad2, again consistently with the involvement of these two proteins in different mitotic checkpoint pathways. The mitotic checkpoint is likely to play a crucial role also during an unperturbed cell cycle, delaying the onset of anaphase until all chromosomes are properly aligned on the bipolar spindle and thereby preventing the occurrence of aneuploidies. Mistakes made during mitosis may be responsible for the abnormal karyotype of many human tumour cells and have an important role in oncogenesis. Loss of function of the p53 tumour suppressor gene impairs the mitotic checkpoint and the normal regulation of centrosome duplication . In addition, a number of human tumour cell lines have been recently found to be defective in the activation of the mitotic checkpoint in response to microtubule depolymerizing drugs . In addition, the chromosomal instability observed in two colorectal cancers is associated to mutational inactivation of BUB1 . Finally, expression of dominant negative mutant versions of either murine Bub1 or human Mad1 accelerates exit from mitosis and causes accumulation of multinucleate cells, respectively, even in the absence of microtubule depolymerizing drugs, whereas microinjection of anti-Mad2 antibodies into mammalian cells induces the onset of anaphase before chromosomes have congressed to the metaphase plate , suggesting that these proteins play an important role in regulating the normal timing of mitosis . We tested directly whether loss of function of MAD2 , BUB2 , or both, might accelerate progression through mitosis of yeast cells. We found that the kinetics of anaphase and exit from mitosis were unaffected in mad2Δ , bub2Δ and mad2Δ bub2Δ cells (data not shown) compared with wild-type. Since mad mutants were shown to increase the frequency of chromosome loss , our data suggest that either Bub and Mad proteins are not required for the correct timing of mitosis during an unperturbed cell cycle, or bipolar attachment of yeast chromosomes to spindle fibers is so fast and efficient that does not enable to detect subtle differences in the absence of surveillance mechanisms. In agreement with the latter hypothesis is the finding that in the absence of fission yeast Bub1 cells do not prematurely enter mitosis despite showing massive chromosome missegregation . The mechanism by which Mad1, 2, 3 block cell cycle progression in response to spindle depolymerization or monopolar attachment has been recently uncovered. Mad2 has been shown by several groups to bind and inactivate Cdc20/APC, thus causing stabilization of Pds1 and cyclins B with subsequent block of anaphase and mitotic exit . This physical interaction might occur directly at the kinetochore; consistently, the mammalian homologue of budding yeast APC1 , Tsg24, was constitutively localized at centromeres and p55CDC, homologous to Cdc20, was also found at kinetochores during mitosis . However, APC components, p55CDC and HsMad1 have also been localized at centrosomes . Our finding that BUB2 and MAD1, 2 belong to different epistasis groups, as indicated by the accelerated degradation of Clb2, exit from mitosis and rereplication of mad1 bub2 and mad2 bub2 compared with that of the corresponding single mutants, suggests that Bub2 might block cell cycle progression in case of monopolar attachment of chromosomes or spindle damage by a pathway distinct from the one dependent on Mad1, 2. This conclusion is further strengthened by the observation that cell cycle progression of bub2Δ , but not mad2Δ , cells in nocodazole depends on the unessential APC subunit Cdc26. Although Cdc26 seems to be constitutively part of the APC, it is required for anaphase and ubiquitination of B-type cyclins only at 37°C; furthermore, it is induced after heat shock . In S . pombe , overexpression of the Cdc26 homologue Hcn1 suppresses a cut9 mutation which causes defects in APC assembly . Thus, Cdc26/Hcn1 is a dispensable subunit that might be only required to modulate APC activity under stress conditions or when other APC subunits are defective . The fact that cell cycle progression of mad2 , but not bub2 , mutants tolerates loss of Cdc26 function suggests that either Bub2 acts by finally inhibiting a special form of APC which requires Cdc26, or that the function of a Cdc26-less APC is somewhat compromised already at 25°C and the lack of Bub2 is not sufficient to circumvent the defect. The conclusion that Mad1, 2 and Bub2 both contribute to the activation of the mitotic checkpoint via distinct routes can only be drawn for what concerns mitotic exit, which is a consequence of the inactivation of cyclin B–dependent kinases. Conversely, loss of cohesion between sister chromatids, which instead depends on Pds1 degradation, is mostly driven by the inactivation of the Mad1, 2 pathway, as the kinetics of sister chromatids separation of mad1Δ bub2Δ and mad2Δ bub2Δ double mutants is very similar to those displayed by mad1Δ or mad2Δ cells. Obviously, bub2Δ cells are able to degrade Pds1 and attempt to undergo anaphase to a certain extent in the absence of a functional spindle, but this might be a consequence, rather than a primary effect, of the inactivation of mitotic CDKs. Indeed, we found that degradation of Pds1 is very slow and inefficient in bub2Δ cells in the presence of nocodazole, suggesting that Bub2 plays only a minor role in controlling the onset of anaphase. The loss of sister chromatids cohesion we observe in bub2Δ cells treated with nocodazole might just reflect a pathological situation rather than shedding light on the actual role of Bub2. We therefore propose that Bub2's major, if not sole, role in the activation of the checkpoint is to prevent, by inhibiting B-type cyclins proteolysis, inactivation of mitotic CDKs and, therefore, cytokinesis and entry into a new cell cycle. The finding that the fission yeast homologue of Bub2, Cdc16, in addition to having a role in the mitotic checkpoint, is implicated in regulating septation and cytokinesis during a normal cell cycle supports this hypothesis. We currently do not know the molecular mechanism by which the Bub2-dependent pathway contributes to maintaining high levels of these CDKs in nocodazole. In fact, Bub2 might inhibit directly the APC-dependent degradation of cyclins B, by inhibiting Hct1 or Cdc5 or even by interfering with the integrity of the APC subunit composition; alternatively, it could prevent Sic1 accumulation or Cdc28 inhibitory phosphorylations on Thr18 and Tyr19, which have been previously shown to play a role in the mitotic checkpoint . Whatever the mechanism is, the absence of Bub2 in cells treated with nocodazole might cause a drop in Clb1-4/Cdc28 kinase activity, which would in turn lead to dephosphorylation and unscheduled activation of Hct1; Hct1/APC, possibly specifically involving the Cdc26 subunit, would then promote degradation of both mitotic cyclins and Pds1, despite Cdc20 is kept inactive by the Mad1, 2 pathway. Several lines of evidence strongly support this model: overexpression of Hct1 in nocodazole arrested cells is able to promote degradation of Pds1, albeit inefficiently , whereas deletion of BUB2 is sufficient to drive a slow degradation of Pds1 and continuous cell cycle progression in cdc20 mutants . Many mitotic checkpoint proteins, like Mad1-3, Bub1, and Bub3, have been localized at kinetochores not attached to spindle fibers, such as in prophase and prometaphase of a normal mitosis or in the presence of microtubule disrupting agents . Their localization is fully consistent with previous lines of evidence implicating the kinetochore in transmitting an inhibitory signal to the cell cycle machinery in case of monopolar attachment . Other observations suggested that the mitotic checkpoint also monitors the lack of tension at kinetochores . Phosphorylation of the kinetochore-associated 3F3/2 epitope correlates with the activation of the mitotic checkpoint and is activated by tension . Conversely, localization of Mad2 to kinetochores depends only on microtubule attachment but not on tension , suggesting that different proteins might be involved in sensing and transducing different signals. Localization of Bub2 at SPBs does not correlate with the activation of the mitotic checkpoint, but is fairly constitutive throughout the cell cycle, raising the questions of how Bub2 function is regulated, whether it plays any role during a normal cell cycle, and which signals it monitors. Whatever the signals are, we have shown that Bub2, as well as Mad2, is required to arrest cell cycle progression also after SPB separation. One possibility is that signals coming from single unattached kinetochores are integrated at SPBs, thus activating the Bub2 dependent checkpoint control. It is worth noting that, unlike in other organisms , human Mad1 is localized at kinetochores during interphase but at centrosomes during mitosis , suggesting that microtubule organizing centers are likely to play a central role in the mitotic checkpoint. Another alternative is that the signal detected by Bub2 might be different from that sensed by the other Mad and Bub proteins; for instance, it has been proposed that Bub2 might be involved in detecting alterations in the spindle structure, since it is not required to arrest the cell cycle in response to impaired kinetochore function . Unfortunately, this hypothesis cannot be tested directly because spindle defects would necessarily also compromise the kinetochore-microtubule attachment. Finally, since we propose that Bub2 only plays a minor role, if any, in regulating the degradation of Pds1 and separation of sister chromatids, we speculate that Bub2 might be involved in the activation of the mitotic checkpoint only later than the other Mad and Bub proteins, by preventing cytokinesis and exit from mitosis until anaphase has taken place. An intriguing hypothesis is that SPBs can somehow monitor when anaphase has been completed, and the inhibitory signal generated by Bub2 is rapidly extinguished when sister kinetochores have reached the poles. Which proteins does Bub2 interact with at SPBs in order to prevent cytokinesis? Recent work in fission yeast has started to elucidate the mechanism by which the Bub2 homologue Cdc16 might function. In fact, Cdc16 has been found to physically interact with Byr4, which was previously implicated in the control of septation . The Cdc16/Byr4 complex displays GAP activity towards Spg1 , a GTPase homologous to S . cerevisiae Tem1 . Remarkably, Spg1 is constitutively bound to SPBs and in the GTP-bound form seems to recruit at SPBs the Cdc7 protein kinase, which is involved in promoting cytokinesis . Mutations affecting budding yeast TEM1 cause cells to arrest in telophase and have been recently found to impair the APC-dependent ubiquitination of mitotic cyclins , thus providing a link between SPBs, APC function, and cytokinesis. The phenotype of tem1 mutants is very similar to that of several mutants defective in late mitotic events, like cdc15 , dbf2 , and cdc14 . Their characterization has uncovered a complex network of genetic interactions which led to the hypothesis that all the corresponding genes might be involved in the same process. In addition, Cdc14, which directly dephosphorylates both Sic1 and Hct1, has been postulated to be the downstream effector of the whole cascade . In conclusion, our current model of how the mitotic checkpoint could work is depicted in Fig. 8 . During prophase, while chromosomes start to attach to spindle fibers, Mad1, 2, 3, Bub1, and Bub3 present at unattached kinetochores prevent activation of Cdc20 and, therefore, degradation of Pds1 and inactivation of mitotic CDKs. At the same time Bub2, localized at SPBs, also prevents inhibition of Clb1-4/Cdc28 kinases and cytokinesis by an independent mechanism, which might involve inactivation of late mitotic proteins, like Tem1, Cdc15, Dbf2, and Cdc14. Exit from mitosis and cytokinesis are therefore blocked until anaphase has taken place, a process which might switch Bub2 function off. In the absence of Bub2 the ectopic activation of Hct1 triggered by Cdc14 would bring about Pds1 degradation and sister chromatid separation even if Cdc20 is kept inactive. | Study | biomedical | en | 0.999996 |
10352017 | Genotypes of the yeast strains used in this study, all isogenic or congenic with W3031a, are listed in Table I . Media, genetic techniques, cell cycle synchronization with α-factor, and lithium acetate transformation were as described ( 40 ). Integrating plasmids were integrated at the marker locus. A precise deletion of the BIM1 coding sequence ( bim1Δ ) was created by one-step gene replacement with the selectable marker Kan r ( 52 ). The DNA fragment used to generate the deletion was made by PCR of the kanMX2 module using the forward primer 5′- AAAAGCAAGGAT AATATTCCACCAAATCAGGGACGAAGCA CAGCTGAAGCTTC- GTACGC-3′ and the reverse primer 5′- AATACATATTCGAAAA CAATACTGCTTTTTAGTTCTCAAC GCATAGGCCACTAGTGGA- TCTG-3′ (homologies to the immediate upstream and downstream flanking regions of the BIM1 open reading frame are underlined). Gene replacement was verified by Southern blotting. The BIM1 promoter and coding sequence were recovered separately by PCR from a plasmid-borne genomic BIM1 allele (a gift from Bee-Na Lee and Elaine Elion, Harvard Medical School, Boston, MA). Primers for the open reading frame were 5′-CCAGGACTT GAAT TC AATGGAGTGCGGGTATCGGAG-3′ and 5′-GCAG CTCGAG TTAAAAAGTTTCCTCGTCGATGATC-3′ (EcoRI and XhoI restriction sites are underlined), and primers for 1.2 kb of noncoding sequence upstream of the open reading frame, BIM1 promoter, were 5′-GCAC GAGCTC TATGTTGGAACCACAGTGTAGATAC-3′ and 5′-GGTC GGATCC CTGATTTGGTGGAATATTATCC-3′ (SacI and BamHI restriction sites underlined). The coding sequence for the green fluorescent protein (GFP) incorporating the codon changes for optimal expression in yeast as well as the fluorescence increasing chromophore mutations S65G and S72A (yEGFP1) ( 9 ) was PCR amplified using the primers 5′-GTAC GGATCC ATGTCTAAAGGTGAAGAATTATTCACTGG-3′ and 5′- CATG GAATTC GCTTTGTACAATTCATCCATACC-3′ (BamHI and EcoRI restriction sites are underlined). The XhoI-KpnI fragment containing the CYC1 terminator was obtained from the pRS416 ( 45 )-based vector FB1545 ( 37 ). These fragments were sequentially ligated into the centromeric plasmid pRS316 ( 45 ) to create a plasmid for GFP-Bim1p expression . Functionality of this construct was confirmed by complementation of the mating defect of the bim1Δ strain. Northern blotting was performed on total yeast RNA (5 μg) from cells arrested with 5 μm α-factor and harvested at 10-min intervals after release, using a 500-bp probe generated with Prime-It II (Stratagene). Cell synchronization was verified by cell morphology. For Western blotting, denaturing whole yeast cell extracts were prepared by the NaOH/β-mercaptoethanol method of Yaffe and Schatz ( 58 ). Primary antibodies were rabbit anti–α-tubulin (345-4) and anti–β-tubulin (206-3) (gifts of Frank Solomon, Massachusetts Institute of Technology, Cambridge, MA ), rabbit anti-GFP (gift of Aaron Straight, Harvard Medical School), and rabbit antiactin (gift of Rong Li, Harvard Medical School). The fluorescence plasmids encoding GFP-Tub1p (pAFS125), a gift from Aaron Straight and Andrew Murray (University of California, San Francisco, San Francisco, CA), and Nuf2p-GFP (pKG67), a gift from Jason Kahana (Dana-Farber Cancer Institute, Boston, MA) and Pam Silver (Dana-Farber Cancer Institute), were transformed into BIM1 and bim1Δ strains; 24 transformants were screened for maximum fluorescence. GFP-Tub1p is controlled by the TUB1 promoter. Similar to GFP-Tub3p under the control of the galactose promoter ( 5 ), GFP-Tub1p cannot fully complement a tub1Δ mutation, but it does not adversely affect the growth rate of the cells. The Nuf2p-GFP fusion protein, which marks the spindle poles, complements a nuf2Δ mutation ( 14 , 24 ). Nuclear morphology was assessed by fluorescence microscopy in cells stained with the nuclear dye DAPI ( Boehringer Mannheim ). 700 cells from each strain were counted for calculation of the percentage of binucleate budded cells. Spindle orientation was measured in live cells expressing Nuf2p-GFP, or in fixed cells stained by indirect antitubulin immunofluorescence, in which the spindle pole bodies were parfocal, using a single combined DIC/GFP image in which fluorescence and visible light were viewed simultaneously through a DIC filter. 200 cells were counted for each strain. For microtubule length and number measurements, static images of fields of live cells expressing GFP-Tub1p were acquired on two or three separate days and combined, and length measurements were made from 150–300 cells. Time-lapse analysis of live cells was performed on log phase cells in G1, preanaphase (as assessed by the presence of a short bipolar spindle), and anaphase, grown on SC medium supplemented with adenine. 1.5 μl of a suspension of these cells was pipetted onto a thin layer of medium and 1% agarose, sandwiched between a glass slide and a coverslip, and sealed with a mixture of equal parts Vaseline, paraffin, and lanolin. The cells were viewed with a Nikon EC LIPSE E600 fluorescence microscope ( Nikon Inc. ), equipped with a 100-W mercury arc illuminator, a 100× Planapochromat 1.4 NA strain-free oil immersion objective, and the Endow GFP filter set (excitation 450–490 nm, dichroic 495, emission 500 nm LP) (Chroma Scientific). Images were acquired with a Hamamatsu 4742-95 Orca digital cooled charge coupled device (CCD) camera (Hamamatsu Photonics). The Ludl Biopoint z-axis focus motor, filter wheel, and shutter controller (Ludl Electronics) as well as the CCD camera were controlled by Openlab Software (Improvision). Time-lapse series were obtained using an automated protocol which acquired a series of 2 × 2 binned images every 8 s. Each time point consisted of a sequence of 8–10 fluorescence images in Z-focal planes 0.3 μm apart. To reduce photodamage, 7/8 of the light was attenuated by a neutral density filter and exposure time was limited to 250 ms (conditions that appeared to cause less photobleaching than shorter, brighter exposures). Only microtubules whose entire three-dimensional length was encompassed within the stack of Z-focal plane images were included in subsequent analyses. A single DIC image was obtained at the end of each time-lapse series for confirmation of cell outline and the presence or absence of a bud. Typical movies lasted for 2–10 min. For technical reasons, our study was limited to microtubules in the bim1Δ mutant that were long enough to measure, and hence it was biased to this longer population. Where appropriate, several cells from the same movie or multiple microtubules from the same cell were analyzed. During all experiments the ambient temperature of the room was maintained at 23°C. All image manipulations and measurements were performed using Openlab Software. For spindle orientation measurements, the angle formed between the spindle and the mother-bud axis, as well as statistical comparisons, was calculated using Microsoft Excel software (Microsoft Corp.). For time-lapse movies, each set of Z-focal plane images was combined to form a single two-dimensional projection. At each time point, the length of the microtubule in this projection was measured in quadruplicate and averaged; its height in the z-axis was determined by counting the number of sections containing an in-focus portion of the microtubule. These values were used to calculate the true three-dimensional microtubule length using Microsoft Excel. Statview software (Abacus Concepts) was used for the construction of life tables and calculation of dynamic rates. Use of these three-dimensional lengths instead of the two-dimensional projections produced up to a 30–40% increase in rates, depending on the orientation of the microtubule relative to the z-axis. Microtubule dynamics rates were calculated using the bivariate plots constructed with Statview software. Growth and shrinkage were defined by a line with an R 2 value of ≥0.85 and a net change in length of ≥0.7 μm. (This criterion resulted in inclusion of brief growth or shrinkage events with large slopes, and longer duration events with smaller slopes.) Pauses were defined as events lasting ≥24 s (four or more data points) in which no statistically significant growth or shrinkage occurred. Catastrophes were defined as transitions to shrinkage after a growth or a pause; rescues was defined as transitions to growth after a shrinkage or a pause. The frequency of catastrophe and rescue was calculated by dividing the total number of events by the total evaluable time in all movies of the category being analyzed. In general, based on fluorescence intensity, we believe that we were observing single microtubules and that we could differentiate true rescues from apparent rescues caused by superimposition of microtubules. However, we could not rule out the possibility that in a rare case two or more bundled microtubules were behaving as a group, leading to a falsely elevated rescue frequency. Time allocation measurements were calculated by adding together all time spent in a given state and dividing by the total evaluable time. Because our definitions of growth, shrinkage, and pausing generally required a sustained behavior ≥24 s, brief periods of time during some experiments remained unclassified. The dynamicity parameter ( 50 ) was expressed as total dimers gained and lost per second for each population of microtubules. Comparisons of statistical significance were by t test. BIM1 and bim1Δ strains were prepared for electron microscopy using the method described previously ( 57 ). Briefly, aliquots from log-phase cultures were collected by vacuum filtration, the cell paste was transferred to sample holders, and the samples were frozen in a Balzer's HPM010 high pressure freezer. The frozen cells were then freeze-substituted in 2% OsO 4 and 0.1% uranyl acetate in acetone at −90°C for 3 d, then warmed to −20°C. The samples were kept at −20°C overnight, then warmed to room temperature and embedded in Epon-Araldite. Serial 50-nm sections were cut using a Reichert Ultracut-E microtome and collected onto formvar-coated copper slot grids. The sections were post-stained with 2% uranyl acetate in 70% methanol and aqueous lead citrate. Sections were viewed in a CM10 electron microscope (Philips Electronic Instruments Co.) operating at 80 kV. Serial micrographs through spindle pole bodies from 10 bim1Δ and 5 BIM1 cells were collected. The serial negatives were digitized using a Dage 81 MTI video camera into a Silicon Graphics INDY computer. The digitized images were then aligned, and the cytoplasmic microtubules tracked and modeled using the IMOD software package ( 26 ). We examined the localization of Bim1p in living cells using a functional GFP-Bim1p fusion protein expressed from the native BIM1 promoter. Bim1p fluorescence was visible in nearly every cell at every stage of the cell cycle . In cells containing a short bipolar spindle, GFP-Bim1p fluoresced in a bright band along the length of the spindle . Following anaphase, fluorescence was maintained along the length of the spindle but was brightest at the poles and in the region of microtubule overlap at the spindle midzone . In the cytoplasm, fluorescence usually (80% of cells) appeared as an intense spot or spots in the expected position of cytoplasmic microtubule tips, equivalent to the plus ends ( 28 ) (arrows). This discontinuous localization contrasts the more uniform microtubule localization seen when the protein was overexpressed from the ACT1 promoter ( 43 ), but the tip fluorescence is remarkably similar to the recently reported staining of human EB1 and RP1 ( 23 , 34 ). The potential enrichment of Bim1p at microtubule ends has implications for models to explain the role of Bim1p in regulating microtubule dynamics in vivo. Observation of the microtubule cytoskeleton in bim1Δ mutant cells by either indirect immunofluorescence or GFP tubulin fluorescence revealed a cell cycle–dependent change in the length of cytoplasmic microtubules . During G1, the cytoplasmic microtubules in exponentially growing cells were shorter in bim1Δ than in BIM1 cells . Synchronization in G1 with the mating pheromone α-factor accentuated this effect on microtubule length . By contrast, during anaphase the mean length was slightly greater in bim1Δ compared to BIM1 cells (1.56 μm and 1.32 μm, P = 0.03). Cell size did not differ between the strains at any cell cycle stage (data not shown). By fluorescence microscopy, microtubules can be reliably measured once their length reaches 0.5 μm. When we performed electron microscopy of bim1Δ mutant cells, we saw additional, even shorter microtubules in the 100–200-nm range that were not present in wild-type controls. Fig. 1 B shows an example of the cytoplasmic outer plaque of the yeast spindle pole body with cytoplasmic microtubules emanating from it. The mean length of these short microtubules was 79 nm. We were also able to visualize the spindle pole bodies and the proximal (minus) microtubule ends by electron tomography, and these both appeared morphologically normal (data not shown). Based on these images, we hypothesized that Bim1p would affect microtubule assembly in a cell cycle–specific manner. Cytoplasmic microtubule dynamics were measured in living cells expressing GFP-Tub1p under the control of the TUB1 promoter. The level of expression of the GFP-Tub1p fusion protein was approximately one-fourth that of untagged α-tubulin . The GFP-Tub1p fusion protein produced uniform fluorescence along nuclear and cytoplasmic microtubules similar to that seen by indirect immunofluorescence in fixed wild-type cells, and the growth rates of strains containing GFP-Tub1p were indistinguishable from the corresponding nonfluorescent strains (data not shown). Initial observations of the microtubule movements in living cells revealed dynamic changes in the length of microtubules coupled with vigorous pivoting and swiveling of the microtubules about the spindle pole body. We used a measure of microtubule length determined from the two-dimensional projection of the microtubule in a composite of Z-focal plane sections and the path through serial Z sections at every time point (see Materials and Methods). This technique eliminated underestimates of rates based on the length of the two-dimensional projection alone and expanded the analysis to include microtubules that made excursions out of the focal plane. In BIM1 cells, cytoplasmic microtubules exhibited dynamic instability in vivo as has been described ( 5 , 44 ), but the rates were considerably faster in our system than previously reported. There was greater microtubule dynamic instability during G1 than in preanaphase or anaphase, and this was due to increases in the shrinkage rate as well as both transition frequencies. These dynamic changes, discussed in more detail below, are illustrated in the time-lapse sequences of a BIM1 cell in the G1 phase of the cell cycle, shown in Fig. 3 A, and in the life history tables in Fig. 4 A, and summarized in Tables II and III . In BIM1 cells, microtubules were most dynamic during the G1 phase of the cell cycle. The shrinkage rate was significantly faster than the growth rate during G1 (shrinkage 3.2 μm/min vs. growth 2.2 μm/min, P = 0.006, Table II ). These rates were faster than the shrinkage and growth rates in anaphase, which were 2.2 μm/min and 1.6 μm/min, respectively (the difference in shrinkage rates between G1 and anaphase was statistically significant, P = 0.007). Rates were similar in preanaphase compared to anaphase (preanaphase shrinkage rate 2.7 μm/min and growth rate 1.7 μm/min). In addition to growth and shrinkage rates, the frequency of dynamic transitions was greater in G1 compared to mitosis. In G1, catastrophe and rescue frequencies were 0.008/s and 0.007/s, respectively, compared to the preanaphase catastrophe frequency of 0.004/s and rescue frequency of 0.002/s, and the anaphase catastrophe frequency of 0.007/s and rescue frequency of 0.004/s. A comparison of time distribution between growing, shrinking, and pausing was also informative (Table III ). During G1, when the shrinkage rate was faster, the amount of time spent shrinking was similar to the rest of the cell cycle, but the amount of time spent growing was greater. BIM1 cells spent 51% of their time growing during G1, vs. 38% of their time growing during anaphase. For comparison, we also used the dynamicity parameter ( 50 ), which takes into account the total activity during the microtubule lifetime. Dynamicity was increased 1.6-fold during G1, 44 dimers/s vs. 27 dimers/s during mitosis. Thus, although microtubule length was similar in G1 and mitotic cells, microtubules in G1 cells were significantly more dynamic, with faster shrinkage rates, more frequent catastrophes and rescues, and less time spent pausing. The microtubule lengths predicted from multiplying the mean growth rate with the mean time spent growing correlated well with the lengths measured in static images of BIM1 cells (predicted length 1.7 μm vs. measured length 1.6 μm in G1; predicted length 1.6 μm vs. measured length 1.3 μm in mitosis). One explanation for the reduction in microtubule length and number in the bim1Δ mutant could be that Bim1p functions as a classical MAP and stabilizes microtubules ( 6 ). However, as alluded to above, dynamic behavior cannot be predicted from the microtubule lengths in static images. In fact, the overall effect of BIM1 deletion was to make microtubules significantly less dynamic than in BIM1 cells, demonstrating that Bim1p promotes dynamic instability. The most dramatic differences between microtubule dynamics in BIM1 and bim1Δ cells were observed during the G1 phase of the cell cycle. During G1, the shrinkage rate of microtubules in the bim1Δ mutant was 1.8 μm/min, compared to the rate of 3.2 μm/min in BIM1 cells ( P = 0.002, Table II ). By contrast with BIM1 cells, where shrinkage was significantly faster than growth, the growth rate was equivalent to the shrinkage rate in bim1Δ cells during G1. In preanaphase and anaphase, the growth and shrinkage rates showed a trend, not statistically significant, to slower rates in the bim1Δ mutant compared to BIM1 cells. Transition frequencies were also lower in the bim1Δ mutant. During G1, the catastrophe frequency in bim1Δ cells was twofold less than in BIM1 (0.004/s, vs. 0.008/s) and the rescue frequency was threefold less than in BIM1 (0.002/s vs. 0.007/s). In preanaphase, the catastrophe and rescue frequencies were more similar between bim1Δ and BIM1 (catastrophe frequencies 0.002/s in bim1Δ vs. 0.004/s in BIM1 ; rescue frequencies 0.001/s in bim1Δ vs. 0.002/s in BIM1 ). During anaphase, transition frequencies rose again slightly (catastrophe frequencies 0.005/s for bim1Δ vs. 0.007/s for BIM1 ; rescue frequencies 0.002/s for bim1Δ vs. 0.004/s for BIM1 ). During G1, the dynamicity of microtubules in bim1Δ cells, 12 dimers/s, was 3.7-fold lower than the wild-type level of 44 dimers/s. Consequently, microtubules in bim1Δ cells showed greater dynamicity during mitosis than G1. Because our analysis was limited to microtubules in the bim1Δ mutant that were long enough to measure, it might not be representative of the population as a whole; however, based on the electron micrographs which showed very short microtubules in G1 bim1Δ cells, we believe the measured dynamicity is likely to be conservative, rather than to exaggerate the effect of BIM1 deletion. The decreases in dynamic instability are illustrated in Figs. 3 and 4 and summarized in Table II . Microtubules in bim1 Δ cells exhibited a marked increase in the amount of time spent in the paused state during G1 . This increase in pausing was entirely accounted for by a decrease in the time spent growing (8% of G1 spent growing by microtubules in bim1Δ cells vs. 51% in BIM1 cells). During preanaphase, microtubules in bim1Δ cells spent 22% of their time pausing and spent 32% of their time growing (compared to 51% of time spent growing during preanaphase in BIM1 ). Microtubules in bim1Δ cells spent 21% of their time shrinking in G1, compared to 47% in preanaphase. During anaphase, the time distribution in bim1Δ was nearly identical to BIM1 (27% spent pausing, 33% spent growing, and 28% spent shrinking). Thus, microtubules in bim1Δ cells spent less time growing and more time pausing during G1. The rate of shrinkage was statistically different from BIM1 , and both catastrophe frequencies and the time distribution were markedly altered. As with BIM1 cells, the microtubule lengths predicted from the dynamics parameters correlated well with the measured lengths (predicted length 1.0 μm vs. measured length 1.1 μm in G1; predicted length 1.5 μm vs. measured length 1.6 μm in mitosis). Taken together, these data support the conclusion that, rather than acting as a microtubule stabilizing factor, Bim1p promotes microtubule dynamics. To assess whether the Bim1p-specific effect on microtubule dynamics was related to changes in the concentration of α- and β-tubulin, Western blotting for tubulin was performed in cells growing asynchronously or arrested in G1. Fig. 5 shows that α- and β-tubulin levels were equivalent in BIM1 and bim1Δ strains, both during growth and during α-factor–induced G1 arrest, as well as in the BIM1 and bim1 Δ strains expressing GFP-Tub1p used for microtubule dynamics measurements. The compactness of the yeast mitotic spindle prevented us from visualizing nuclear microtubule dynamics in the bim1Δ mutant. However, two lines of evidence suggest that Bim1p is important for nuclear microtubule function. First, cells with simultaneous deletion of BIM1 and the gene encoding the spindle assembly checkpoint protein Mad1p were inviable, suggesting that BIM1 deletion activates the spindle assembly checkpoint (data not shown). Second, electron micrographs of bim1Δ cells showed aberrant spindle structures not observed in wild-type controls. Fig. 6 shows an example of a budded bim1Δ cell whose spindle pole bodies have duplicated but a bipolar spindle is not present. Instead, cytoplasmic microtubules intersect at right angles between the two poles. Such a structure could represent an intermediate in spindle formation or a collapsed bipolar spindle. This spindle morphology has not been seen in the BIM1 control or in other wild-type cells examined. We performed Northern and Western blotting to test whether BIM1 was cell cycle regulated. bim1Δ cells expressing a functional GFP-Bim1p fusion protein from the BIM1 promoter were released from an α-factor arrest and cells harvested every 10 min through 2 cell cycles. GFP-BIM1 (and untagged BIM1 ) mRNA was cell cycle regulated, peaking during G1-S and dropping during mitosis , consistent with its observed effect on microtubule dynamics which predominates during G1. This cell cycle regulation of mRNA was similar to the fluctuations in BIM1 mRNA seen by whole genome microarray analysis ( 8 ). Western blotting for the GFP epitope showed a significantly blunted fluctuation in the amount, and no change in the electrophoretic mobility, of Bim1p during the cell cycle . The same result was obtained using Bim1p fused to an HA epitope tag (data not shown). Thus, although BIM1 was transcriptionally regulated, under these conditions the levels of epitope-tagged Bim1p were less affected. The cell cycle effect of Bim1p on microtubule dynamics may therefore be regulated by posttranslational modifications, or by a functional interaction with another protein. The principal function of cytoplasmic microtubules in yeast is to position the nucleus properly during growth and mating ( 21 ). To investigate the consequences of shorter, less dynamic cytoplasmic microtubules caused by BIM1 deletion, we examined nuclear positioning in bim1Δ cells during vegetative growth. Nuclear position, consisting of both nuclear movement close to the bud neck and alignment of the spindle angle relative to the mother-bud axis, was measured in live cells containing the spindle pole body protein Nuf2p fused to GFP ( 24 ). In the bim1Δ mutant, the localization of Nuf2p-GFP at the poles was more diffuse than in BIM1 cells (data not shown), but the growth of the bim1Δ strain containing Nuf2p-GFP was only minimally reduced (doubling time 2 h vs. 2.2 h at 24°C). As shown in Fig. 8 , cells lacking BIM1 displayed a random orientation of the preanaphase spindle relative to the mother-bud axis (the mean spindle orientation angle was 43° in bim1Δ cells vs. 32° in BIM1 cells) and an increased distance between the nucleus and the bud neck (the mean distance from the proximal spindle pole to the bud neck was 2.1 ± 1.2 μm in bim1Δ cells vs. 1.0 ± 0.4 μm in BIM1 cells). This nuclear position defect in bim1Δ cells was also observed by indirect antitubulin immunofluorescence . While spindle position in the bim1Δ mutant was abnormal, relatively few bim1Δ cells went on to execute an abnormal anaphase and produce binucleate mother cells. By DAPI staining, 4% of budded binucleate BIM1 cells retained both nuclei in the mother cell, whereas in the bim1Δ cells 5% of budded binucleate cells retained both nuclei in the mother ( n = 700, data not shown). Why are the consequences of abnormal spindle position so mild in the bim1Δ background? In bim1Δ cells with misoriented anaphase spindles, we observed a consistent pattern of spindle correction ( n = 5). Fig. 9 shows a time-lapse series of one such cell, in which a cytoplasmic microtubule enters the bud neck, contacts the lateral cortex of the bud, and appears to pull the elongated spindle through the neck. During the initial part of this correction process, sliding appears to occur without depolymerization. Cytoplasmic dynein and Kip3p are candidate motors for producing this sliding force ( 5 , 14 ), and, consistent with this idea, deletions of both of these proteins produce synthetic lethality with bim1Δ (data not shown). Once properly positioned, the yeast spindle elongates through the bud neck. Elongation of the spindle reflects concerted changes in kinetochore, pole to pole, and interdigitating microtubules that undergo simultaneous polymerization, depolymerization, and sliding within the nucleus, as well as pulling forces on the nucleus generated by cytoplasmic microtubules. It follows a biphasic pattern consisting of an initial rapid elongation phase followed by a period of slower continued elongation ( 24 , 48 , 59 ). The initial phase of spindle elongation was mildly reduced in bim1Δ cells, at 0.41 μm/min, compared to 0.59 μm/min in BIM1 cells ( P = 0.07, n = 7), while the slow phases were equivalent at 0.18 μm/min in bim1Δ cells and 0.22 μm/min in BIM1 cells. This small decrease in the initial, rapid phase of spindle elongation in bim1Δ cells suggests a subtle effect of Bim1p on some microtubule populations later in the cell cycle, and it could be due to an effect on either nuclear (pushing) or cytoplasmic (pulling) microtubules. The effect of Bim1p loss on G1 microtubule dynamics— slowing of the shrinkage rate, reduction in the frequencies of catastrophe and rescue transitions, and increase in the pause time—produced shorter microtubules. We hypothesized that BIM1 deletion might produce a net opposing effect to mutations that increase microtubule length, such as deletion of KAR3. Kar3p protein levels were unchanged by BIM1 deletion (data not shown). We investigated the synthetic phenotype of a bim1Δkar3Δ double mutant and found that deletion of BIM1 suppressed the temperature-sensitive growth defect of the kar3Δ mutant . This suppression correlated with an intermediate microtubule length during G1, the time when these mutations produce the most dramatic effects. Fig. 10 B shows the microtubule morphology in representative fields of single and double mutant cells arrested in G1 with α-factor, by indirect antitubulin immunofluorescence. kar3Δ mutant cells expressing GFP-Tub1p grew poorly relative to kar3Δ cells, so this analysis was not performed in living cells. As discussed above, bim1Δ mutant cells are defective in positioning the mitotic spindle. Examination of spindle position in single and double mutant cells by indirect antitubulin immunofluorescence revealed correction of the bim1Δ spindle position defect by simultaneous deletion of KAR3 . While the Bim1p and Kar3p mechanisms and sites of action (discussed below) may differ, these results demonstrate that the loss of opposing activities can produce normal appearing microtubule structures which correlate with suppression of defects in cell growth and spindle position. This cross-suppression between bim1Δ and kar3Δ did not extend to the microtubule-based process of karyogamy (data not shown). It was relatively specific to bim1Δ and kar3Δ , as other mutations which shortened microtubules, such as deletion of BIK1 ( 3 ), could not rescue KAR3 deletion, and other mutations which lengthened microtubules, such as deletion of DYN1 or KIP3 , could not suppress the bim1Δ phenotypes. To better understand the mechanism of the bim1Δkar3Δ interaction, we measured microtubule dynamics in the bim1Δkar3Δ double mutant expressing GFP-Tub1p, as done above for the bim1Δ single mutant. Strikingly, microtubule dynamics in the bim1Δkar3Δ double mutant during G1 were like those in the bim1Δ single mutant. During G1, the microtubule shrinkage rate was 2.1 μm/min, the rescue and catastrophe frequencies were 0.004/s and 0.003/s, dynamicity was 17 dimers/s, and the percentage time pausing was 17% (Tables II and III ). During preanaphase and anaphase, the parameters were similar to those of the wild-type strain, also as observed with the bim1Δ single mutant. These measurements suggest that simultaneous deletion of KAR3 is able to functionally suppress the effects of bim1Δ on microtubule length and spindle orientation without suppressing the effects on microtubule dynamics directly. Previous descriptions of microtubules in living wild-type yeast cells were discrepant in their conclusions regarding cell cycle changes in microtubule dynamics. Carminati and Stearns noted cell cycle-specific changes in microtubule dynamics, whereas Shaw et al. found rates to be similar in G1 and mitosis ( 5 , 44 ). Our findings that microtubules were at their most dynamic during G1, and that shrinkage rates were uniformly faster than growth rates, support the attractive hypothesis of Carminati and Stearns that increases in cytoplasmic microtubule dynamics before mitosis facilitate spindle positioning ( 5 ). Microtubules underwent frequent, rapid pivoting and swiveling motions about the spindle pole bodies, although its quantitation was beyond the scope of this study. These pivoting motions are likely to contribute substantially to the mechanism of microtubule-based searching for cortical attachment sites. Unlike the previously reported values for microtubule dynamics rates in vivo ( 5 , 44 ), rates of growth and shrinkage in our study were on the order of three- to fourfold faster. There are both technical and biological explanations which may account for the differences in rates between studies. The technical advance in our system was the determination of microtubule lengths which took into account their projection as well as their path through serial Z-focal plane sections. As noted by Shaw et al., measuring the two-dimensional projection alone results in an underestimate of rates ( 44 ). Other explanations for the differences in dynamics rates include strain background, cell ploidy (haploid or diploid), GFP construct (Dyn1p-GFP, GFP-Tub3p, or GFP-Tub1p), and sugar source (glucose or galactose). Elucidation of the relative importance of these biological differences may explain important aspects of microtubule dynamics regulation. As an example, in haploid cells which undergo the axial budding pattern, the spindle is transported to the opposite side of the cell following cell division ( 7 ), and this movement may be achieved, in part, by greater microtubule dynamic instability in haploid cells than in diploid cells. The bim1Δ mutation caused cytoplasmic microtubules to be shorter and less dynamic during G1, without affecting the total concentrations of α- and β-tubulin in the cell. BIM1 deletion dramatically decreased microtubule dynamicity, through a decrease in the shrinkage rate, decreases in both the catastrophe and rescue frequencies, and a redistribution of time from growing to pausing, and these effects were primarily observed during G1. From the bim1Δ phenotype, we infer that Bim1p belongs to the emerging class of microtubule-binding proteins which promote dynamic instability. The bim1Δ mutation dramatically increased the time microtubules spent in the paused state. The appearance of pausing could result either from treadmilling, from the interruption of dynamic instability, or from microexcursions too rapid or too small for detection. Pausing was an integral part of the lifetimes of microtubules in BIM1 cells, and it was greatly accentuated in the bim1Δ mutant, in the same cell cycle pattern as other changes, suggesting that Bim1p increases dynamic instability by interrupting these periods of pausing. Microtubule pausing has been observed under a variety of conditions. In vitro, yeast microtubules spent 51% of the time in the paused or attenuated state ( 11 ). In quiescent Swiss 3T3 cells, microtubules at the leading edges spent significantly more time pausing than in cells stimulated with serum ( 10 ). Chemotherapeutic drugs such as Taxol, Estramustine, and Vinblastine have been shown to suppress microtubule dynamics in vitro ( 12 , 38 , 56 ); treatment of living cells with Vinblastine significantly increased the duration of pauses and the total pause time ( 15 ). These results suggest that the effects of some antimitotic agents might be due to the disruption of interactions between microtubules and associated regulatory proteins. Based on the severe reduction of dynamicity in the bim1Δ mutant, members of the EB1 family are possible candidates for proteins displaced by antimitotic drugs. The finding that Taxol inhibits the in vivo association of EB1 and microtubules ( 34 ) is consistent with such a mechanism. Mechanistically, there are several ways in which Bim1p could increase the dynamic behavior of microtubules. Growing and shrinking microtubules have been found to have distinctly different structures at their ends. Because we observed an apparent concentration of Bim1p at microtubule ends, it is appealing to speculate that Bim1p may lower the energetic barrier between growing and shrinking structures at the microtubule growing end. One mechanism by which binding of Bim1p could increase the rate of growth would be through alteration of tubulin GTPase activity. Bim1p has also been identified at the spindle poles by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry ( 55 ), consistent with the GFP-Bim1p fluorescence pattern. Bim1p may also affect microtubule minus end dynamics, or it may regulate a protein such as Kar3p, which exerts its effects at the minus ends. By electron microscopy, however, the minus ends of microtubules in the bim1Δ mutant appeared capped, similar to wild-type controls, and simultaneous deletion of KAR3 did not restore wild-type dynamicity to microtubules. Thus, we favor the interpretation that Bim1p acts at the plus ends of microtubules to promote dynamic instability. A second finding that remains to be fully explained is the cell cycle specificity of Bim1p in regulating cytoplasmic microtubule dynamics. Because the localization and the concentration of epitope-tagged Bim1p varied less than twofold during the cell cycle, Bim1p activity may be regulated by posttranslational mechanisms. Alternatively, constant Bim1p activity may be counteracted by cell cycle–dependent changes in other proteins that affect microtubule dynamics. A likely consequence of the reduced dynamicity of microtubules in the bim1Δ mutant is a decrease in the opportunity of cytoplasmic microtubules to contact and search the cell cortex. These interactions may be important for nuclear positioning relative both to the bud neck in mitosis and to the projection tip and partner nucleus during mating and karyogamy (nuclear congression). Consistent with this idea, we observed a severe spindle position defect in the bim1 Δ mutant, and, as has been described ( 43 ), a dramatic bilateral karyogamy defect in bim1Δ × bim1Δ crosses . In multicellular organisms, members of the BIM1 /EB1 family may perform similar roles, positioning the nucleus or MTOC in polarized cells. A recent study proposed a novel checkpoint in yeast which delays the completion of cytokinesis until the mitotic spindle is correctly positioned ( 35 ). Bim1p was postulated to play a major role in this checkpoint, because deletion of BIM1 completely abolished the cell cycle delay which occurred in response to a defect in spindle position (generated by inactivation of the dynactin component Act5p). These findings raised the question of whether Bim1p has a role in the checkpoint mechanism independent of its function in regulating microtubule dynamics. We favor the interpretation that, by increasing microtubule dynamics, Bim1p promotes microtubule-cortex interactions that help to sense, or are sensed by, the cytokinesis checkpoint machinery. Our findings highlight an interesting paradox in the yeast spindle positioning process. We note that cells lacking Bim1p have a spindle orientation defect, similar to the type of defect that Bim1p is proposed to detect. However, spindle mispositioning in bim1Δ cells is efficiently corrected and does not lead to the accumulation of multinucleate or binucleate unbudded cells. In other words, the spindle position defect of bim1Δ cells does not require the Bim1p-dependent delay for its correction. Although the mechanism for this error correction is unknown, a likely candidate for pulling the spindle rapidly through the bud neck is the dynein/dynactin complex. The bim1Δ spindle position defect may be rapidly corrected by dynein motor activity, while the spindle position defect in dynactin mutants may elicit an alternate, less efficient pathway of spindle repositioning. Kar3p is a kinesin motor protein that promotes minus end microtubule depolymerization in vitro and localizes to the poles in vivo during vegetative growth ( 16 , 32 , 41 ). We found that the bim1Δ mutation suppressed the inability of kar3Δ mutant cells to grow at 37°C, and that the double mutant cells had improved spindle position relative to the bim1Δ single mutant. This cross-suppression between bim1Δ and kar3Δ may be due to a net effect on cytoplasmic microtubule length, because bim1Δkar3Δ cells arrested in α-factor had microtubule lengths similar to BIM1KAR3 cells and of intermediate length compared to each single mutant. Overall, the interaction between bim1Δ and kar3Δ is reminiscent of the interaction of kar3Δ with tub3Δ (the minor α-tubulin), and the suppression of the kar3Δ vegetative phenotypes by the microtubule destabilizing drug Benomyl. Additionally, the parameters of microtubule dynamicity were similar to those of the bim1Δ single mutant: slower shrinkage rate, greater pause time, and fewer transitions during G1 than wild-type controls. Dynamicity during G1 was 17 dimers/s in the bim1Δkar3Δ double mutant (similar to 12 dimers/s in the bim1Δ single mutant, compared to 44 dimers/s in wild-type cells). Presumably, Bim1p and Kar3p regulate microtubule length independently of each other. BIM1 is the sole budding yeast member of the family of EB1-related genes. Like human EB1 proteins ( 4 , 23 , 34 ), Bim1p localizes to spindle microtubules and cytoplasmic microtubule ends throughout the cell cycle. It will be interesting to see whether EB1 family members will promote microtubule dynamics in higher cells, and whether the G1 specific effect of Bim1p, presumably important for nuclear positioning, is retained among other EB1-related proteins. Human EB1 was identified as a binding partner of APC in cells derived from the polarized colonic epithelium ( 49 ), and human RP1 mRNA was found to be upregulated during T cell activation, a process during which the T cell becomes polarized ( 39 ). One could imagine that similar to increases in spindle microtubule dynamics which facilitate capture of the kinetochores during mitosis, changes in cell polarity may require increases in cytoplasmic microtubule dynamics to facilitate movements of the nucleus or MTOC during interphase. EB1 family members may mediate increases in microtubule dynamics during processes which require regulated cell polarization. | Study | biomedical | en | 0.999996 |
10352018 | Xenopus laevis keratocytes were isolated and cultured basically as described by Bereiter-Hahn and Vöth . In brief, tails were amputated from anesthetized tadpoles at stages 48–55 , cut into pieces with a razor blade, incubated in digestion solution (0.2% trypsin and 0.2% EDTA in PBS) for 5 min, and rigorously pipetted. Large tissue pieces were allowed to settle and the supernatant cell suspension was collected. Cells were centrifuged to remove digestion solution, resuspended in L-15 medium ( Sigma Chemical Co. ), diluted to 70% with distilled water, and supplemented with 20% FBS (HyClone Labs), 0.29 g/liter glutamine, and antibiotics plated onto glass coverslips and cultured at 27°C. After ∼1 h, cultures were washed free from unattached cells and debris, and used for experiments. A spontaneously immortalized cell line of Xenopus embryo fibroblasts obtained as in Daniolos et al. was provided by Dr. V.I. Rodionov (University of Wisconsin, Madison, WI) and cultured using the same medium and temperature as for Xenopus keratocytes. Human 356 fibroblasts, rat REF-52, mouse MFT-6, and Swiss 3T3 cell lines were cultured as described in Verkhovsky et al. and Svitkina et al. . Cytochalasin D (CD; Sigma Chemical Co. ), latrunculin A (LA; Molecular Probes), or staurosporine ( Sigma Chemical Co. ) were added to culture medium from concentrated stock solutions in DMSO. For recovery from serum starvation experiments, cells were kept in serum-free medium overnight and then returned to serum-containing medium. For energy starvation, cells were incubated for 30 min at 37°C in PBS supplemented with 1 mM MgCl 2 , 0.5 mM CaCl 2 , 10 mM NaN 3 , and 50 mM deoxyglucose ( Sigma Chemical Co. ). The following antibodies were used for immunofluorescence and immuno-EM. Arp2/3 complex: affinity-purified rabbit polyclonal antibodies prepared against mammalian Arp3, p34-Arc, and p21-Arc were kindly provided by Dr. M.D. Welch (University of California, Berkeley, CA); antibodies against mammalian p33-Arc were provided by Dr. L.M. Machesky (University of Birmingham, Edgbaston, UK). Each of these antibodies was tested against lysates of Xenopus fibroblasts by immunoblot analysis and gave a single band of the predicted mobility (results not shown). ADF/cofilin: affinity-purified rabbit polyclonal antibodies to Xenopus ADF/cofilin were kindly provided by Dr. J. Rosenblatt (University of California, San Francisco, CA) and by Dr. J.R. Bamburg (Colorado State University, Fort Collins, CO). We confirmed that these antibodies gave a single band by immunoblot analysis. ABP 280: mouse mAbs to human ABP-280 were generously provided by Dr. J. Hartwig and Dr. T. Stossel (Harvard Medical School, Boston, MA). This antibody did not cross-react with Xenopus and was used only with human fibroblasts. α-Actinin: mouse mAb to α-actinin was purchased from Sigma Chemical Co. Immunoblot analysis gave a single band with Xenopus lysates. Secondary antibodies: secondary TRITC-, FITC-, and 10-nm gold-conjugated antibodies were purchased from Sigma Chemical Co. 18-nm gold-conjugated anti–mouse IgM antibody was obtained from Jackson ImmunoResearch Laboratories. Procedures for detergent extraction, immunostaining, S1 decoration, light, and EM were described previously . In brief, cells were washed in PBS and extracted for 3 min at room temperature with 1% Triton X-100 in PEM buffer (100 mM Pipes, pH 6.9, 1 mM MgCl 2 , and 1 mM EGTA) containing 4% polyethylene glycol (PEG), mol wt 40,000 (Serva), and either 0.5 μM TRITC-phalloidin (for light microscopy) or 2 μM phalloidin (for EM; Sigma Chemical Co. ). Extracted cells were briefly washed with the phalloidin-containing PEM buffer and fixed with 2% glutaraldehyde. For some experiments, PEG and/or phalloidin were omitted from the extraction procedure. PEG without phalloidin preserved lamellipodial network similar to the regular extraction. Decrease in PEG concentration from 4 to 1% or less in the absence of phalloidin, produced gradual loss of actin filaments from lamellipodial rear (see Results). Omitting PEG in the presence of phalloidin resulted in loss of granular material from lamellipodia, which is usually present in minor amounts, yet actin filaments seemed to be completely preserved. This granular material became abundant after LA treatment, and to a lesser extent, after CD treatment. Presumably, it represented insoluble forms of G-actin and might be similar to G-actin structures revealed after introducing fluorescently labeled actin into living cells . To remove granular material obscuring F-actin distribution, we omitted PEG from the extraction solution for LA- and CD-treated cells. For some antibodies, we used modifications of the regular immunostaining procedure. The ABP-280 antibody, which only worked with unfixed antigen, was applied in the PEM buffer containing phalloidin to extracted cells before glutaraldehyde fixation. For immunolocalization of XAC, we excluded phalloidin from the extraction solution to prevent competitive displacement of XAC from actin filaments. The presence of PEG, as we mentioned earlier, provided as good a quality of cytoskeleton preservation as phalloidin. For immunofluorescence microscopy with antibodies to Arp2/3 complex components, which worked only with denatured proteins, glutaraldehyde-fixed cytoskeletons were treated with 100% methanol for 20 min at 37°C and rinsed with PBS before application of antibodies. This procedure provided the brightest staining, compared with lower concentrations of methanol, but at the electron microscopic level, it completely ruined filament structure. Therefore, for immuno-EM we used aqueous 33% methanol instead of pure methanol, which provided acceptable morphology and detectable immunoreactivity of samples. Comparable results were obtained when glutaraldehyde-fixed cells were treated with 0.5% SDS in Tris-buffered saline for 20 min at 37°C and rinsed with Tris-buffered saline before application of antibodies. All of the four Arp2/3 complex antibodies tested gave essentially identical immunofluorescence patterns of staining. The immuno-EM reaction was of varying intensity, with the p21-Arc antibody giving the strongest signal. In all cases, at both the light and electron microscopic levels, staining by secondary antibody reagents alone was negligible (not shown). Results of our previous study on fish keratocytes suggested that actin filaments were organized into a branched network similar to that proposed by the dendritic model . However, few antibodies are available which cross-react with components in fish cells. Consequently, to test the presence and determine the distribution of predicted molecular constituents, we focused on the lamellipodial network in Xenopus keratocytes and fibroblasts, systems better suited for immunolocalization studies. Xenopus keratocytes had similar cytoskeletal organization to fish keratocytes. Actin filaments in lamellipodia formed an extensive network, with the highest filament density at the leading edge which gradually decreased with distance from the edge. Assaying for actin filament polarity using myosin S1 decoration demonstrated that barbed ends faced forward (not shown) as in fish keratocytes . Although ultrastructural observation, per se, does not permit one to say that actin filaments do not terminate in capping protein, for our operational purposes, their ends are designated as free if the filament did not terminate at another filament or any other visible structure. With this criterion, the most peripheral zone (up to ∼1 μm from the leading edge) was highly enriched in apparently free barbed ends, but some barbed ends were also found deeper in the lamellipodium. Many Y-junctions between filaments , but no free pointed ends, were visualized within the lamellipodial network. As determined by quantification of visible Y-junctions per unit area on electron micrographs, Y-junctions were ∼2–3 times more abundant in the peripheral ∼1 μm zone, as compared with the zone immediately behind this region, namely, 1–2 μm from the leading edge. The real difference in Y-junction frequency between these zones may be even greater, since the number of Y-junctions near the edge was likely underestimated because of very high filament density in this region. Fibroblast lamellipodia had the same basic features of actin network organization, but had qualitatively lower network density and fewer free barbed ends at the leading edge, as compared with keratocytes. Thus, both Xenopus keratocytes and vertebrate fibroblasts confirmed the existence of abundant Y-junctions near the leading edge of motile cells. The high filament density in lamellipodia hindered observation of individual filaments for a significant distance and determination of the frequency of Y-junctions per unit length of filament. Occasionally, we could see several Y-junctions belonging to the same tree of actin filaments and they were usually closely spaced, suggesting frequent branching of individual filaments . To facilitate the visibility of branched filaments, we attempted to generate a sparser lamellipodial network by treatment with CD, which acts preferentially to cap barbed ends . At low concentrations (0.2–0.5 μM), CD gradually suppressed lamellipodial protrusion in keratocyte. TRITC-phalloidin staining of lamellipodia of CD-treated keratocytes revealed low and uniform actin density from front to rear (not shown) in contrast to control cells displaying a pronounced gradient of actin staining . EM demonstrated a significantly sparser actin network in lamellipodia of CD-treated cells, permitting observation of individual filaments for a significant length and improved visualization of Y-junctions. Depending upon the concentration of CD and time of treatment, as well as on the response of individual cells, it was possible to find the entire range of variation from an almost normal lamellipodial network to a sparse collection of branched filaments . The average angle subtended by a Y-junction was 67 ± 12° ( n = 212), similar to that reported for Arp2/3-nucleated branches in vitro . The spacing between adjacent Y-junctions was variable. However, many Y-junctions occurred within 20–50 nm of each other, indicating a high probability for branching near the leading edge. Some filaments appeared to have axial functions: they held numerous secondary filaments alongside. Assay for filament polarity (not shown) demonstrated that free ends were barbed, and pointed ends were involved in junction formation, as in untreated cells . As in control preparations, no free pointed ends were identified in CD-treated lamellipodia. Since the actin network in CD-treated cells was sufficiently sparse to visualize free pointed ends if they were abundant, this observation suggests that free pointed ends were virtually absent or very transient. The high frequency of branching seemed remarkable. To test the possibility that frequent branching was artifactually induced by CD, we examined lamellipodia in other situations, which also allowed for better visualization of branched filaments. They included: short term release from serum or energy starvation, which led to formation of nascent comparatively loose lamellipodia; treatment with the actin monomer sequestering agent, LA , which caused depolymerization of actin filaments in cells; and cell lysis under conditions allowing for actin filament depolymerization. In all three experimental conditions, we were able to visualize filaments with multiple branches and the spacing between branches was similar to that observed in CD-treated cells. Again, free pointed ends were not observed. Thus, actin filaments in lamellipodia displayed frequent branches, which engaged virtually all detectable pointed ends into Y-junctions and left numerous barbed ends apparently free. Extensive filament branching was a general feature of lamellipodia in different situations, including expanding lamellipodia in starvation-release experiments, steady state conditions in untreated keratocyte lamellipodia, or declining protrusions after drug treatment. Our next goal was to identify a molecule that localized to actin filament branch points. Arp2/3 complex seemed to be a primary candidate, but other cross-linking proteins which localize to lamellipodia, such as α-actinin or ABP-280/filamin, were also possibilities. We performed immunolocalization of these proteins in keratocytes and fibroblasts. Antibodies to various components of the mammalian Arp2/3 complex have been shown to stain lamellipodia in cultured cells of mammalian origin . We found that they also stained lamellipodia in Xenopus cells , but in fibroblasts, Arp2/3 complex was excluded from most filopodia . The overall pattern of Arp2/3 staining at the electron microscopic level in keratocytes and fibroblasts correlated with the results obtained by light microscopy. The gold label was distributed all over the dense lamellipodial network and gradually declined toward the lamellipodial rear. The high density of the actin network, however, did not allow us to routinely attribute gold particles to any specific filaments or branches. As before, we used CD treatment to visualize individual Y-junctions and were able to stain branch points with antibody to p21-Arc as a result . Distinctly labeled branches were seen clearly in regions with very sparse filament distribution. More commonly, clusters of gold particles with short filaments sticking out were observed (not shown). Such distribution of label would be predicted for multiple branches in close proximity to each other, as was observed (see above). Not all visible branches in the specimen were labeled, but incomplete labeling could be the result of a variety of factors, including suboptimal immunoreactivity because of the procedure used for structural preservation (Materials and Methods). To test whether a fraction of branches was mediated by other proteins, we performed immunolocalization of α-actinin and ABP-280, proteins which make side-to-side cross-links . Immunofluorescence of Xenopus fibroblasts and keratocytes with antibody to α-actinin, and of human fibroblasts with antibody to ABP-280 , revealed some lamellipodial staining, but it was not as prominent as staining of internally located actin structures. Compared with Arp2/3 complex, α-actinin and ABP-280 in lamellipodia were much less abundant with respect to actin content . A similar relationship between α-actinin and Arp2/3 complex distribution was found in Acanthamoeba . Immuno-EM of CD-treated cells with antibody to α-actinin and ABP-280 demonstrated negligible staining of branched filaments near the leading edge , but significant staining of internal actin networks, predominantly at points of filament crossovers or juxtapositions , which contrasted with the opposite pattern of Arp2/3 staining . Thus, our data demonstrate that Arp2/3 complex is located at Y-junctions, implying that it plays a role in their formation or maintenance. The structural evidence for ABP-280 and α-actinin suggests that they are unlikely to play a significant role in branching near the leading edge. Their impact on filament cross-linking is likely to be expressed more deeply in the cytoplasm. Involvement of pointed ends in Y-junction formation and localization of Arp2/3 complex at the same positions suggested that pointed end depolymerization in lamellipodia may be significantly blocked due to pointed end capping by the Arp2/3 complex . We tested depolymerization properties of the lamellipodial network using two approaches, cytoskeletal preparations and living cells. To allow for actin depolymerization in cytoskeletons, we omitted certain precautions in the process of detergent extraction, which are usually necessary to preserve the actin network in the lamellipodium. Our regular extracting solution contains two protective agents to stabilize the actin network: a nonspecific stabilizer, PEG; and a specific F-actin stabilizing drug, phalloidin. In the absence of these chemicals, significant loss of actin filaments was observed in the rear of lamellipodia, whereas the peripheral zone (∼1 μm) at the leading edge remained almost as dense as in control cytoskeletons . This zone frequently looked barely connected to the rest of the cytoskeleton and usually contained numerous free barbed ends rendering it a brush-like appearance. Only a minor proportion of actin filaments seemed to be lost from this actin brush. CD (1 μM), when added to the extraction solution to suppress depolymerization from the barbed ends, did not prevent loss of actin filaments in the rear network (not shown), although the density of the actin brush at the front was similar to that of untreated cells. These results demonstrated that loss of actin filaments at the lamellipodial rear occurred mostly via pointed end depolymerization, whereas loss from barbed ends had only minor impact. In living cells, we used LA under conditions which shifted the actin steady state toward depolymerization, but did not completely block motility. Observations on living keratocytes by phase-contrast microscopy demonstrated that at concentrations of 0.3 μM or higher, LA induced fast cessation of motility and collapse of lamellipodia (not shown). At lower concentrations (0.1–0.2 μM), protrusion of the keratocyte leading edge continued with a progressively slower rate and eventually ceased, after which the cell body continued to migrate forward for a short time approaching the leading edge, similar to what has been reported for CD-treated keratocytes . Pseudopodial activity at cell edges persisted for up to 40 min, but became progressively irregular and frequently ended up with the formation of phase dense beads along the cell periphery. Thus, low level LA treatment permitted analysis of the pathway whereby sequestration of actin subunits leads to cessation of leading edge protrusion. We studied actin filament organization in LA-treated keratocytes by TRITC-phalloidin staining and by EM. At the light microscopic level in most cells, which were still motile after LA treatment, the lamellipodium looked like a narrow band of phalloidin-stained actin at the cell edge, which was separated by a wide dark zone from internally located actin bundles, as if the lamellipodium ran away from the cell body . As the motility of the lamellipodium decreased and the cell body caught up, the distance between the lamellipodium and the rest of the cytoskeleton decreased. At late stages of LA treatment, actin was found only in bright spots along the cell periphery (not shown). At the electron microscopic level, the runaway lamellipodium of an LA-treated keratocyte was very similar to the actin brush revealed by unprotected extraction. It was a dense, narrow (0.5–1 μm) band of actin network containing numerous Y-junctions and free ends . In cells treated for a longer time and/or with higher concentrations of LA, discontinuous foci of the actin brush were found instead of a continuous brush. In fibroblasts, LA treatment gave basically the same results as in keratocytes. After ∼10 min in 0.1 μM LA, numerous runaway lamellipodia composed of dense actin network were formed . A possible mechanism for the formation of runaway lamellipodia is continued protrusion under nonsteady state conditions. Although the rate of actin polymerization is predicted to be decreased by LA, the rate of depolymerization would be unaffected, leading to an erosion of the brush from the rear. If actin filaments were free to depolymerize throughout the lamellipodium, such imbalance would result in fast and complete disassembly of the entire lamellipodial network. Thus, our results on actin depolymerization in cytoskeletons and in live cells demonstrated that actin filaments are protected from depolymerization within a narrow zone at the leading edge, but were susceptible to depolymerization farther away from the edge. The most likely mechanism of protection is capping of filament pointed ends by the Arp2/3 complex. In addition, our results with LA treatment revealed a possible minimal system for actin turnover. Under conditions of G-actin deficiency, cells progressively depolymerized actin from the rear of the lamellipodium retaining just a narrow, runaway actin brush. Progressive locomotion of such cells suggested that actin turnover can occur within this narrow zone. Differential depolymerization of the lamellipodial actin network suggested a spatially regulated mechanism for exposing the pointed ends, which would allow for actin depolymerization at the lamellipodial rear. Exposure of the pointed ends may occur by dissociation of the Arp2/3 complex or by filament severing. The mechanism of actin filament depolymerization in lamellipodia is likely to involve activity of ADF/cofilin. The exact mode of action of ADF/ cofilin in cells (pointed end depolymerization, severing, or both) is not clear. If we assume that ADF/cofilin severs actin filaments to expose pointed ends, then to explain local protection of the front actin brush from depolymerization we should expect ADF/cofilin to be excluded from the protected zone. Alternatively, if ADF/cofilin depolymerizes actin filaments only from pointed ends, then binding of ADF/cofilin to actin filaments within the protected area would not result in actin depolymerization until pointed ends were released. In this case, spatially regulated pointed end uncapping could be the rate-limiting mechanism for actin network disassembly. We performed immunolocalization of ADF/cofilin in keratocytes and fibroblasts to get insight into this problem. Since we found differences between the two cell types, we present results for keratocytes and fibroblasts separately. Immunofluorescence staining of keratocytes with antibody to XAC revealed XAC in lamellipodia , similar to what has been shown for other cells . However, upon double-staining with TRITC-phalloidin, we found a new feature of XAC distribution in keratocyte lamellipodia: exclusion of XAC from a narrow marginal zone. Immuno-EM demonstrated that XAC was absent from the most peripheral 0.3–0.7 μm of the lamellipodial actin network . The intensity of XAC staining gradually declined toward the cell center and was at a minimum at the rear of the lamellipodium. The absence of XAC from actin filaments at the leading edge is consistent with in vitro data which indicate that ADF/cofilin does not bind to actin filaments with bound ATP or ADP and Pi . If this were the explanation, the length of the XAC-free zone would be a measure of the polymerization velocity of actin filaments and hence, of the protrusive speed of the cell. To test for a possible relationship between the width of the XAC-free zone and the rate of keratocyte locomotion, we performed correlative light and immuno-EM of individual cells crawling at naturally varying speeds. To broaden the range of speed variations, we slowed cell locomotion by a protein kinase inhibitor, staurosporine (50 nM), or by decreasing serum concentration in the medium, and carried out a statistical analysis of the covariation of cell speed and width of XAC-free zone. Surprisingly, no correlation between these two parameters was evident (data not shown). All keratocytes in culture had XAC-free front zones, including cells locomoting at normal rate and round stationary cells, which were occasionally found in the culture (not shown). The lack of correlation of the XAC-free zone with cell speed suggests that additional or alternative factors influence the binding of XAC to actin filaments. Unprotected extraction and LA treatment had the effect of depolymerizing the bulk of cellular actin, although a lamellipodial brush survived. If ADF/cofilin binding was sufficient for actin depolymerization, surviving filaments would be predicted to be devoid of XAC. However, immunostaining with XAC antibody showed that the depolymerization-resistant brush obtained during unprotected extraction contained XAC at its rear . In LA-treated cells, XAC also localized to the posterior portion of the runaway lamellipodium leaving a narrow XAC-free zone at the front . Since in LA-treated cells, the brush continued to move, these data suggest that the brush effectively treadmills with assembly of XAC-free actin filaments at the front and disassembly of XAC-containing filaments at the rear. Immunofluorescence also revealed XAC localizing to lamellipodia of Xenopus fibroblasts . In contrast to keratocytes, no distinct XAC-free zone at fibroblast leading edges was revealed in double staining with TRITC-phalloidin: both proteins were found all the way to the cell edge . Only in rare cases was actin staining slightly extended beyond the XAC staining. Most filopodia were not stained with XAC antibody, but some of them were stained. Immuno-EM confirmed that XAC in fibroblast lamellipodia was distributed all the way to the periphery . The concentration of XAC was highest at the extreme outer margins of lamellipodia and gradually disappeared toward the rear. Since protrusion of lamellipodia in fibroblasts is not as persistent as in keratocytes and may frequently alternate with withdrawals, we performed correlative analysis of locomotory behavior and XAC staining of fibroblast lamellipodia. In 13 examined cells, the vast majority of protruding or stationary lamellipodia, as well as ruffling lamellipodia, had XAC distributed all the way to the edge or very close (within 0.1 μm of the edge). Thus, XAC in fibroblast lamellipodia was found essentially throughout the depolymerization-resistant actin brush, as well as in the more labile rear parts of the lamellipodium. The difference between keratocytes and fibroblasts cannot be attributed simply to speed of protrusion. Although the net speed of a fibroblast cell is slow compared with that of a keratocyte, the speed of many fibroblast protrusions is comparable, ∼5–6 μm/min. Again, the lack of evident correlation between protrusive speed and distribution of XAC suggests that factors in addition to ATP or ADP-Pi regulate the binding of ADF/cofilin to actin filaments. Our results indicate that the actin network at the leading edge of crawling cells, the dendritic brush, is distinctive in its structural organization, dynamics, and biochemical composition. Structurally, the brush is characterized by an extensively branched organization of actin filaments, with barbed ends facing approximately forward and pointed ends essentially all involved in Y-junctions. That barbed ends are enriched in the brush as compared with the rest of the lamellipodium is consistent with the idea that actin assembly occurs primarily within the dendritic brush. Further, the depth of the actin brush matches well with the depth of the zone, which has been demonstrated to incorporate actin . Pointed ends are more evenly distributed throughout the lamellipodium as compared with barbed ends, but they are more abundant in the actin brush. Remarkably, individual Y-junctions in the brush are frequently spaced by as little as several tens of nanometers. As a result, the dendritic brush contains numerous short filaments incorporated into the actin array by Y-junctions, as well as a proportion of longer filaments which continue into the more internal lamellipodial regions. Dynamically, differential depolymerization experiments indicate that the dendritic brush is highly protected from disassembly. The fact that capping of barbed ends by CD during extraction did not significantly affect the differential depolymerization of the lamellipodial network implies that depolymerization indeed occurs from pointed ends and that the status of pointed ends (capped or uncapped) is responsible for the differential behavior of the actin brush and the internal actin network. Remarkably, protection of the actin brush from depolymerization does not interfere with its dynamic behavior. Indeed, cells which retained just the actin brush (runaway lamellipodium) and lost almost all the internal actin network were still able to locomote and therefore, to maintain continuous actin turnover within the actin brush. Thus, pointed end capping as a putative mechanism for protection of the actin brush from depolymerization is dynamic and probably regulated. Protection of newly formed barbed ends from capping, recently demonstrated in neutrophil extracts stimulated by Cdc42 , may also have an impact for dynamic persistence of the actin brush in addition to protection of pointed ends from depolymerization. Biochemically, the dendritic actin brush contains significant amounts of the Arp2/3 complex, which localizes specifically to Y-junctions, whereas other possible cross-linking proteins, α-actinin and ABP-280, have their predominant association with X-junctions deeper in the cytoplasm. These results support the idea that the Arp2/3 complex is the major cross-linker in the actin brush and that it plays a role in stabilization of the actin brush by capping pointed ends . Another lamellipodium-specific protein, ADF/cofilin, which plays a role in actin depolymerization and thus has an antagonistic activity to the Arp2/3 complex, is not a uniform component of the actin brush. Depending on cell type, it may associate with actin filaments throughout the brush, but it may also localize just to the brush's posterior portion. We propose that ADF/cofilin is functionally regulated in the actin brush and performs actin depolymerization after dissociation of the Arp2/3 complex, predominantly in the lamellipodial network behind the brush region. The central problem of dendritic brush formation is the origin of side branches: actin filaments which have pointed ends associated with Arp2/3 molecules at sides of other filaments. Two different, although nonexclusive, possibilities may be considered for the location of the Arp2/3 complex at Y-junctions: Arp2/3 complex nucleates filaments de novo or Arp2/3 complex captures pointed ends of pre-existing filaments nucleated elsewhere. Filament nucleation mediated by the Arp2/3 complex residing on pre-existing filaments is proposed by the dendritic nucleation model and is supported by biochemical data indicating that binding of the Arp2/3 complex to preformed filaments significantly increases its nucleating activity . However, filament nucleation by the Arp2/3 complex near the leading edge, followed by docking via the Arp2/3 complex onto pre-existing filaments have not been formally excluded. In the second mechanism, the Arp2/3 complex residing on pre-existing filaments captures the pointed ends of filaments formed elsewhere, either new filaments nucleated near the leading edge by an Arp2/3-independent mechanism or older filaments in the process of depolymerization. Constitutive locomotion similar to that expressed by keratocytes or Listeria theoretically could be maintained primarily by continuous elongation of pre-existing filaments. However, in many other systems actin-based motility is characterized by frequent protrusion–withdrawal cycles, like in locomoting fibroblasts, or by explosive actin polymerization in response to external stimuli, e.g., during chemotaxis. In such cases, generation of new sites for actin polymerization is unavoidable. In addition to nucleation, barbed end uncapping and filament severing have been proposed as mechanisms. Our data, together with a growing mass of evidence indicating a role for the Arp2/3 complex in filament nucleation , are most consistent with the de novo nucleation mechanism mediated by Arp2/3 complex resulting in the formation of the dendritic brush. However, uncapping and severing mechanisms may also work in other systems or along with de novo nucleation . Dendritic nucleation of actin filaments may require a mechanism to ensure that most face forward. Preferential growth of barbed ends, perhaps by involvement of plasma membrane-associated factors, is one possibility. Recent data showing a role for the Ena/VASP protein family in directional motility of Listeria , supposedly by keeping growing barbed ends in a correct position , is an example of such function. Additional possibilities include membrane-dependent regulation of nucleation or capping. The high frequency of branches in the dendritic brush carries implications for its dynamics and regulation. If each Y-junction in the brush indeed represents an individual nucleation event, frequent branching is predicted to result in rapid, exponential growth of filament number, which may occur in expanding protrusions. However, extensively branched filaments also were observed in apparently steady state protrusions, such as keratocyte lamellipodia, suggesting that continuous de novo nucleation may be a constitutive mechanism for generating protrusions. One way to maintain steady state would be for most of the nucleated filaments to be capped soon after nucleation and only a small proportion of them continue to elongate and branch. This assumption is consistent with data showing that capping protein is localized at the leading edge , the vast majority of free barbed ends are capped , and stimulation of actin polymerization leads to association of capping protein with the cytoskeleton . Although we have referred to free barbed ends in the dendritic brush, our structural observations do not distinguish between ends free to grow and ends terminated by capping protein. Frequent nucleation followed by capping should generate a significant number of short filaments, which is indeed what we observed in the actin brush. Increase in filament number, but not in their length during stimulated actin polymerization , is also consistent with frequent nucleation as a pathway for actin polymerization. Estimates of a low average length of filaments in vivo are hard to explain otherwise. A nucleation-capping mechanism is attractive because it offers considerable flexibility in terms of organization and level of actin polymerization, which could be controlled via activity of the Arp2/3 complex and/or capping protein. Our working hypothesis is that the Arp2/3 complex protects actin filaments in the dendritic brush from depolymerization, whereas ADF/cofilin acts to promote disassembly of the actin network. Two mechanisms of ADF/ cofilin action in actin disassembly have been described in vitro, facilitated release of actin subunits from the pointed end and severing . Which mechanism ADF/cofilin uses in vivo and how antagonistic activities of ADF/cofilin and Arp2/3 complex are coordinated in cells are important questions for understanding actin turnover in lamellipodia. The extreme distal zone of keratocyte lamellipodia remains free of XAC, whereas the actin network toward the rear of lamellipodia is intensively stained by XAC antibody. The mechanism for XAC exclusion from the keratocyte leading edge is not clear. One possibility is preferential association of ADF/cofilin to ADP-bound forms of actin . Because of the lag of ATP hydrolysis and Pi release following actin filament elongation, ADP-actin filament domains would not be present or would be less frequent at the extreme front. Such a mechanism implies almost synchronous growth of actin filaments at the keratocyte leading edge to account for the observed pattern of XAC staining. A different possibility suggests that some regulatory factors affecting XAC may inhibit its binding to actin filaments at the cell front. Spatially confined phosphorylation of XAC, production of inhibitory phosphoinositides at the leading edge, and/or a pH gradient may restrain XAC from binding to actin filaments. Finally, we cannot exclude the possibility that XAC was lost from the leading edge of keratocytes during permeabilization. In contrast to keratocytes, XAC in fibroblast lamellipodia was found even at the extreme leading edge. The different pattern of XAC distribution in keratocytes and fibroblasts seems not to be related to the difference in the instantaneous rate of protrusion, as we showed by correlating rate of protrusion and localization of XAC for individual lamellipodia. Asynchronous growth of actin filaments at the fibroblast leading edge or a different mode of regulation may permit XAC binding at the fibroblast leading edge. Despite apparently different mechanisms for determining localization of XAC in two cell types, the presence of XAC within the actin brush in fibroblasts suggests that XAC binding to actin filaments is not sufficient for filament disassembly. The same conclusion can be drawn from XAC staining of actin brush in keratocytes after unprotected extraction or after LA treatment. In these preparations, XAC binding to posterior parts of the depolymerization-resistant actin brush demonstrates that the XAC-containing network and the network susceptible to depolymerization do not completely coincide. An XAC-independent step is consistent with the results of Rosenblatt et al. who showed that excess of XAC in cytoplasmic extracts was unable to shorten Listeria tails below a certain limit. These findings are hard to reconcile with a pure severing activity of XAC because rapid depolymerization would be expected to follow severing. The results more readily fit with the idea that severing activity of XAC is not significantly expressed in the actin brush in vivo. We suggest that XAC binds actin filaments within the actin brush and waits for the release of Arp2/3 complex to get a chance to facilitate subunit dissociation from pointed ends. In this framework, the XAC-independent step would be Arp2/3 dissociation from pointed ends, the signals for which remain to be determined. Our data on the structural organization of the actin network at the leading edge and localization of the Arp2/3 complex at Y-junctions in vivo are fully consistent with the dendritic nucleation model concerning cross-linking and pointed end capping activity of the Arp2/3 complex in vitro. Consequences of the dendritic model suggest a novel concept of actin turnover in lamellipodia in which the actin array as a whole treadmills, but not individual actin filaments, per se . All polymer turnover mechanisms require a balance in the steady state of formation and disassembly. In the treadmilling filament model, each actin filament in the actin network simultaneously adds subunits at its barbed end and releases subunits from its pointed end, thus continuously reproducing itself by balanced growth and disassembly. Treadmilling of individual filaments collectively results in the treadmilling of the lamellipodial network. In contrast, in the array treadmilling model, an individual filament does not treadmill, but rather first grows at the barbed end and later shrinks at the pointed end. However, the actin filament array as a whole treadmills, reproducing itself at the cell front and dismantling itself at the lamellipodial rear. Formation of new filaments occurs by Arp2/3-mediated nucleation, within a narrow zone at the leading edge, the dendritic actin brush. Newborn filaments become incorporated immediately into the actin array as branches of pre-existing filaments. Some of these nascent filaments continue to grow and branch, whereas others are predicted to be capped after a short period of elongation to prevent exponential increase in filament mass. Thus, in contrast to the individual filament treadmilling model which is characterized by low nucleation frequency and extensive filament growth, the array treadmilling model is characterized by high frequency of nucleation and limited filament growth. In the array treadmilling model, a debranching reaction must exist to balance the branching reaction. The dendritic brush assembled at the leading edge is protected from depolymerization due to pointed-end capping activity of the Arp2/3 complex. Disassembly of actin filaments is favored farther away from the leading edge through abrogation of the protection mechanism. Debranching of filaments would result from release of Arp2/3 complex from Y-junctions. Subsequently, depolymerization would result from ADF/cofilin-mediated dissociation of actin subunits from their pointed ends. Thus, the life cycle of an actin filament would consist of steps of nucleation with pointed-end capping, elongation, barbed-end capping, pointed-end uncapping, and disassembly. Depending on the rate constants and probabilities of the individual steps, an actin filament could undergo repeated reactions of capping and uncapping, growth and shortening, branching and debranching. Alternatively, it could arise as a new branch and undergo a single episode of growth balanced by a single episode of shortening at a later time. In contrast to the stochastic life cycle of an individual filament, the array, consisting of a large number of filaments, on average would add polymer continuously at its leading edge and disassemble polymer continuously toward the rear, resulting in a uniform treadmilling of the array as a whole. From the functional point of view, a dendritic brush of actin filaments at the leading edge of locomoting cells seems well designed for lamellipodial protrusion. First, the brush naturally can support massive actin polymerization because of the presence of numerous nucleating sites and the level of polymerization, in principle, can be readily controlled by regulation of the activity of the Arp2/3 complex and/or capping protein. Second, the brush is mechanically organized for efficient polymerization-driven force generation because of its high filament density, extensive cross-linking of actin filaments, and the angular orientation of actin filaments . Finally, the brush is dynamically regulated so as to generate polymerization-driven protrusion by an array treadmilling mechanism which may play a role in the persistence of lamellipodial protrusion and in the ability to adapt to change in direction. | Study | biomedical | en | 0.999999 |
10352019 | Collagenase A from Clostridium histoliticum and DNase-I were obtained from Boehringer Mannheim . Trypsin was purchased from Difco Laboratories. MEM was obtained from GIBCO BRL . Percoll was purchased from Pharmacia . ET-1 and big ET-1 were obtained from Peninsula Laboratories, Inc.; other reagents, when not specified, were purchased from Sigma Chemical Co. Plastic culture dishes and multiwell plates were from Falcon. The animals used were adult and three-week-old Wistar rats (Charles River), fed ad libitum until killed by CO 2 asphyxia or cervical disarticulation. Animals were kept in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Selective myoid cell identification through alkaline phosphatase cytochemistry was performed as previously described , based on the method of Ackermann . In brief, the fixed cells were incubated in an alkaline solution containing 0.5 mg/ml Fast Blue RR in water and 40 μl/ml α-naphtol phosphate (0.25% solution, pH 8.6). After 30 min incubation in the dark, a purple-blue precipitate appeared specifically on the surface of myoid cells . Primary Sertoli cell cultures from 18–20-d-old Wistar rats were prepared as previously described . Seminiferous tubules obtained by trypsin dispersion of testicular parenchyma were subjected to collagenase digestion to remove the peritubulum. The resulting fragments of seminiferous epithelium, mainly composed of Sertoli cells, were cultured at 32°C in a humidified atmosphere of 5% CO 2 and 95% air in a chemically defined medium (MEM). After 3 d in culture, germ cells contaminating the Sertoli cell monolayer were selectively removed through hypotonic shock ; the cells were used one day after the treatment. The supernatant-mixed cell population resulting from the collagenase treatment of seminiferous tubules (see above) was centrifuged at 40 g , yielding mostly minute fragments of tubular wall (Sertoli cells and myoid cells): culturing of this preparation in MEM for 3 d at 37°C results in a mixed monolayer in which myoid cells can be identified by differences in their morphology in phase contrast and through alkaline phosphatase cytochemistry after fixation. For pure myoid cell cultures, the tubular wall fragments were digested in trypsin and EDTA to a single cell suspension, subsequently fractionated on a discontinuous Percoll density gradient . Percoll-purified myoid cells were cultured under serum-free conditions at 37°C. The assessment of myoid cell purity, performed routinely for each preparation on the basis of the presence of alkaline phosphatase activity, was never below 96%. Seminiferous tubules from 35–60-d-old rats were freed from interstitial tissue by collagenase treatment and dispersed into single cells, as previously described . The resulting cell suspension, highly enriched in germ cells, was used as such (“mixed germ cells”) or fractionated into several cell classes by velocity sedimentation at unit gravity in an albumin gradient . The two cellular fractions, composed, respectively, of middle-late pachytene spermatocytes and of round spermatids (steps 1–8 of spermiogenesis), were found to be ∼90% pure; the fraction composed of intermediate spermatids (steps 9–14) was ∼60% pure, also containing late spermatids (∼10%) and residual bodies (∼30%). RNA was extracted from testicular cells and different organs using the acid guanidine thiocyanate-phenol-chloroform extraction method . Sertoli cell poly(A) + RNA was prepared by means of a Quick Prep mRNA purification kit ( Pharmacia Biotech ). Total RNA (10 μg) and mRNA (4 μg) were separated in a formaldehyde and 1.1% agarose gel, transferred to a nitrocellulose membrane Gene Screen Plus, and then hybridized in QuikHyb solution, as recommended by the manufacturer (Stratagene). Random-primed 32 P-labeled cDNA inserts (∼4.7 and ∼2 kb) encoding bovine ECE-1 and ECE-2 were used as probes . The membranes were washed in 2× SSC and 0.1% SDS at 55°C and were exposed to an x-ray film for 3 d at −80°C. [Ca 2+ ]i was measured by dual wavelength fluorescence in single cells loaded with the Ca 2+ -sensitive indicator fura-2 . Testicular myoid cells were plated onto coverslips in serum-free MEM. After 4 d in culture, the cells were incubated in MEM containing 3 mM fura-2-acetoxymethylester for 1 h at 37°C. The cells were then rinsed with Krebs-Henseleit-Hepes (KHH) buffer (140.7 mM Na + , 5.3 mM K + , 132.4 mM Cl − , 0.98 mM PO 4 2− , 1.25 mM Ca 2+ , 0.81 mM Mg 2+ , 5.5 mM glucose, and 20.3 mM Hepes) supplemented with 0.2% fatty acid–free BSA. Measurements were performed in single cells, at 340- and 380-nm excitation wavelengths, with an AR-Sm microfluorimeter (Spex Industries) connected to a Diaphot TMD inverted microscope ( Nikon Corp. ) equipped with a CF ×40 objective. Emission was collected by a photomultiplier carrying a 510-nm cut-off filter and recorded by an ASEM Desk 2010 computer (ASEM SpA), which automatically calculated real-time 340/380 ratios. Calibration of the signal was obtained at the end of each observation by adding 5 μM ionomycin to saturate the dye maximal fluorescence, followed by 7.5 mM EGTA plus 60 mM Tris-HCl, pH 10.5, to release Ca 2+ from fura-2 and obtain minimal fluorescence. [Ca 2+ ]i was calculated according to previously described formulas . Seminiferous tubules were prepared as previously described . In brief, testes from 2-mo-old rats were decapsulated and digested under gentle shaking at room temperature in MEM containing 1 mg/ml collagenase. After dispersion of the interstitium, the tubular mass was rinsed in MEM, then stretches of tubules were dissected by means of sharp needles and carefully transferred to 35-mm culture dishes in 300 μl of medium. For the dissection of homogeneous samples at precise stages of the seminiferous epithelium, the tubular segments were identified under transillumination . The tubules were incubated for 10 min at 32°C in a humidified chamber under an atmosphere containing 5% CO 2 . At the end of the incubation time, the medium was replaced by 600 μl of medium to be tested at different experimental times, as detailed in figure legends. Samples were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO 4 , dehydrated and critical point dried in ethanol, coated with gold, and then viewed in a Hitachi S-570 scanning electron microscope. ECE activity in Sertoli cell cultures was assayed through estimation of exogenous big-endothelin conversion . The culture medium, conditioned for 30 min to 3 h in the presence of big ET (with or without ECE inhibitors), was purified on a Sep-Pak C18 solid phase cartridge (Waters). After drying by vacuum centrifugation and reconstitution in buffer, the samples were assayed for endothelin content by means of a commercial enzyme immunoassay (EIA) kit (Cayman Chemical Co.) according to the manufacturer's instructions. Adult and 18-d-old Wistar rat testes were fixed in 4% paraformaldehyde in PBS at 4°C overnight. The fixed testes were dehydrated with ethanol and embedded in paraffin by standard procedures. 5-μm-thick paraffin sections were placed on slides pretreated with 3-amino-propyltriethoxysilane. Sections were analyzed by in situ hybridization using the procedure described by Davidson et al. . In brief, before hybridization, sections were deparaffinized, rehydrated, partially digested with proteinase-K (20 μg/ml), and then treated with acetic anhydride. These last two steps were necessary to improve access of the probe to the mRNA and reduce nonspecific binding of the nucleic acid probes. Sections were dehydrated and then incubated at 55°C for ∼18 h with 35 S-labeled RNA probes. For generation of RNA probes, the 0.5 Kb 5′ PstI-PstI fragment of bovine ECE-1 cDNA, nucleotides 214–751, was subcloned in pBluescript vector and transcribed in vitro with T7 (anti-sense) and T3 (sense) RNA polymerases. Unbound cRNA probe was removed by incubation in RNase solution (40 μg/ml) for 30 min at 37°C in 0.5 M NaCl, TE buffer and by two 20-min washes at 65°C in 2× saline sodium citrate (SSC). Autoradiography was performed with Ilford K2 liquid emulsion (Ilford). After exposure for the time periods indicated, sections were stained with carmalum and examined under a Zeiss microscope using dark- or brightfield illumination. The stages of the seminiferous epithelium were identified from adjacent sections using the criteria of Leblond and Clermont . Data are presented as the mean ± SE of results from at least three independent experiments. Student's t test was used for statistical comparison between means where applicable. RNA blot analysis with the bovine ECE-1 cDNA as probe showed that a ∼4.7 kb ECE-1 mRNA is expressed abundantly in cultured Sertoli cells . As shown, the expression of ECE-1 mRNA is much higher in the testis from 20-d-old rats than in the adult. Since the increase in weight of the testis depends mostly on germ cell multiplication, the evidence that homogenous preparations of specific types of germ cells exhibit no expression of ECE-1 indicates that the reported high expression of ECE-1 mRNA in the testis could be attributed prevalently to Sertoli cells. Peritubular myoid cells express much lower levels than Sertoli cells . In addition, a 3.1-kb mRNA is expressed at a lower level. Since only the 4.7-kb ECE-1 mRNA is present in Northern blots of poly(A) + RNA from cultured rat Sertoli cells, the two different sizes of mRNA are presumably generated by alternative poly(A) + addition in the 3′ noncoding region . Conversely, Northern blot analysis of testicular cells with the bovine ECE-2 cDNA as probe revealed no expression of ECE-2 mRNA in the seminiferous epithelium cells, while a 3.3-kb mRNA was detected in the control neural tissue and adrenal gland (not shown). To analyze ECE activity, we examined whether cultured Sertoli cells can convert synthetic rat big ET-1 exogenously added to the culture medium. We therefore assayed the generation of mature ET-1 by means of a competitive enzyme immunoassay (EIA) that does not cross-react with the substrate big ET-1. As shown in Fig. 2 , big ET-1 was efficiently converted into ET-1 by intact Sertoli cells in a time-dependent fashion. At a substrate concentration of 1 μM, up to 69% of the added big ET-1 was converted into ET-1. The Sertoli cell ECE was more efficient in converting big ET-1 than big ET-3. When the metalloprotease inhibitor phosphoramidon (PR), known to specifically inhibit ECE activity , was present during incubation, it completely inhibited the production of mature ET-1 . The analogue of big ET-1, [D-Val 22 ]big ET-1 , an inhibitor of ECE , strongly inhibited ECE activity and was as effective as PR in completely inhibiting the production of ET-1 by Sertoli cells incubated with big ET-1. We have previously demonstrated that ET-1 is able to induce PI turnover and rapidly increase [Ca 2+ ]i in testicular myoid cells . Cytofluorimetric analysis of intracellular calcium mobilization, measured by dual wavelength fluorescence in single cells loaded with the Ca 2+ -sensitive indicator fura-2, indicate that the inactive precursor of ET-1, big ET-1, does not induce calcium mobilization in myoid cells; however, the same cells are able to respond to the addition of ET-1 with an increase in calcium levels , which confirms the total biological inactivity of big ET-1. Conversely, when myoid cells were stimulated with medium conditioned by Sertoli cells for 30 min in the presence of 100 nM big ET-1 (SCMbig), a rapid [Ca 2+ ]i transient comparable to that induced by ET-1 was observed; subsequent stimulation with ET-1 was ineffective . Therefore, medium conditioned by Sertoli cells in the presence of big ET-1 desensitizes myoid cells to the actions of ET-1, which clearly indicates that the biologically active molecule in SCMbig is ET-1 itself, converted from big ET-1 by ECE expressed in Sertoli cells. The observed slow calcium response is comparable to that obtained in response to 0.5–1 nM ET-1 which is below EC 50 , but sufficient to desensitize to 100 nM ET-1 (not shown). When SCMbig were conditioned in presence of phosphoramidon (PR), an inhibitor of ECE, no effect on calcium response was observed even though myoid cells were still responsive to ET-1 . Fig. 3 d shows the levels of ET-1–, SCMbig– (treated or untreated with phosphoramidon), and big ET-1–dependent [Ca 2+ ]i increases (both peak and plateau). To corroborate the presence of fully functional ECE activity from intact Sertoli cells, we have treated cultured myoid cells with SCMbig one day after plating. Treatment at this culture time with SCMbig resulted in an immediate rounding-up with retraction of cytoplasm, which could be directly followed in an inverted microscope (not shown). To assess whether the observed myoid cell contraction was specific for this cell type and whether Sertoli cells express significant activity of ECE, which is able to process big ET-1 into an amount of ET-1 sufficient to determine the contraction of myoid cells, we used mixed cultures containing fragments of seminiferous epithelium and patches of myoid cells. This mixed population of tubular and peritubular tissue was treated with 100 nM big ET-1 and shape changes which occurred 10–20 min after treatment were photographically recorded . In these cultures, myoid cells patches were observed to undergo contraction in response to big ET-1, while the morphology of adjacent Sertoli cells remained unmodified. We processed the same sample for the detection of alkaline phosphatase activity, a specific marker for testicular myoid cells , and found that the cells that contracted are stained for alkaline phosphatase . Inhibition of ECE activity by 2 mM PR resulted in the block of contractile response to big ET-1 . Since PR is a metabolically stable phosphorylated sugar derivative and is unlikely to enter cells at an appreciable rate within a short incubation time, our observation indicates that the conversion of big ET into ET is a plasma membrane event that occurs on the extracellular side, analogous to the production of the vasoconstrictor angiotensin II from angiotensin I. Fig. 6 a shows the surface of a seminiferous tubule as viewed in the scanning electron microscope. In the adult testis, the myoid cells appear arranged in a continuous monolayer of ephithelioid polygonal cells, particularly flat and wide and with bulging central nucleus. Addition of either 100 nM ET-1 or 100 nM big ET-1 results in dramatic contraction of the myoid peritubular cells, which display enhanced bulging of the central area and reduced distance between cell centers in most areas. From a morphological point of view treatment with either ET-1 or with its inactive precursor, big ET-1, induces a basically equal contraction of myoid cells; the only difference between these two treatments is in the timing required to achieve this effect. In fact, we observed myoid cell contraction within 15 s of ET-1 treatment, but only ∼10 min after big ET-1 addition, presumably because more time is required for a sufficient amount of big ET-1 to be converted into biologically active ET-1 by ECE-1 expressed by adjacent Sertoli cells in the seminiferous tubule. When seminiferous tubules were challenged with big ET-1 in the presence of PR, we did not observe any contraction of myoid cells, which appeared as flat as in the control sample . Furthermore, as a further control, we challenged seminiferous tubules with SCMbig. In this case, strong contraction of the myoid peritubular cells was observed within a few seconds . When the seminiferous tubules were stimulated only with Sertoli cell– conditioned medium, the surface of myoid cells appeared to be unaffected, as in the control samples . To explore the possibility that the production of ET within the seminiferous epithelium is a discontinuous, cyclically regulated process, we studied the transcription of ECE-1 by in situ hybridization. Sense and antisense RNA for ECE-1 mRNA was prepared as detailed in Materials and Methods. Interestingly, the ECE-1 probe showed striking regional differences in the level of signal . The density of the grains was maximal at stages IX-X of the cycle and at the background level in all other stages. The control samples, hybridized with sense ECE-1 probe, displayed a low level of background labeling, with no appreciable differences in grain density between seminiferous tubule profiles and interstitium, thus confirming the specificity of the hybridization signals. In tubules at stages IX-X, the bovine ECE-1 probe hybridized to a basal columnar region surrounding the germ cells . This indicates that the ECE-1 gene is expressed above all in Sertoli cells prevalently in the basal region. These findings are in agreement with and extend the above Northern blot analysis, indicating that ECE-1 is expressed in Sertoli cells from adult animal in a cyclical fashion during the seminiferous cycle in stages IX-X soon after spermiation. By contrast, testis from 20-d-old rats exhibits homogeneous labeling intensity in all seminiferous tubules (not shown). To investigate whether the restricted expression of ECE-1 could be functionally related to the regulation of peritubular contractility induced by big ET-1, seminiferous tubule segments from adult testis were microdissected to isolate specific “stages” of the seminiferous tubules and their ability to respond to either ET-1 or big ET-1 was studied at the scanning electron microscope. Fig. 9 a shows a transilluminated tubular segment in which the transition from stage VIII to stage IX is very apparent. The hatched line indicates the level at which the tubules were dissected. Two groups of specific stages of the seminiferous tubule were tested: VII-VIII and IX-XI, which showed low and high ECE expression, respectively. Fig. 9 shows that treatment with big ET-1 is able to induce a strong contraction of seminiferous tubules at stages IX-X in 10 min ; by contrast, the seminiferous tubule fragments containing stages VII-VIII are totally unaffected by the treatment with big ET-1 . Furthermore, ET-1 was still active in inducing an immediate contraction of the myoid peritubular cells in both groups of seminiferous tubule fragments . These data suggest a direct correlation between the restricted expression of ECE-1 and the functional regulation of seminiferous tubule contraction. Seminiferous tubule contractility is fundamental for sperm progression towards the rete testis and its regulation represents, therefore, a key point in male fertility. In this study, we focused on the paracrine communication between Sertoli cells and peritubular myoid cells as it represents an interesting model of cell–cell interactions between epithelial cells of the seminiferous tubule (which play a crucial role during spermatogenesis) and a particular class of nonvascular smooth muscle cells (responsible for seminiferous tubule contraction). Peritubular myoid cells express α-smooth muscle actin and desmin , and specifically respond to endothelin undergoing cell contraction both in cell culture and in peritubular tissue . Given the cyclicity that characterizes seminiferous epithelium activity, we wondered whether endothelin production might be cyclically regulated at the level of the maturation of its precursor by ECE. In this report we describe the distribution of ECE-1 during the seminiferous epithelium cycle and present evidence that differential expression of ECE-1 in the Sertoli cells during spermatogenesis results in specific and regional seminiferous tubule contraction. It has been shown that cultured Sertoli cells exhibit a basal production of ET-1 in the media . Preliminary observations, which showed that Sertoli cells incubated with ECE-1 specific inhibitors strongly reduced the secretion of ET-1 while increasing the accumulation of big ET-1 (not shown), prompted us to hypothesize a role for ECE-1 as a local regulator of ET-1 actions. Furthermore, the occurrence of phosphoramidon–sensitive ECE activity on Sertoli cells suggests that some processing of secreted big ET-1 may occur on the surface of ET-1–producing cells, adjacent to myoid cells. Since big ET-1 appears to be much more stable than ET-1 to generic proteolytic degradation , this targeted conversion may allow more effective delivery of the active product in intact form to its receptors on myoid cells. Since the prediction of its existence , ECE has been considered to be a potential site of regulation of endothelin production as well as a plausible target for therapeutic intervention in the endothelin system. Recently, the existence of three distinct ECE-1 isoforms has been demonstrated . These three isoforms (ECE-1a, ECE-1b, and ECE-1c) differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. The three isoforms show similar kinetic rate constants, processing big ETs with similar velocities and have all been found to cleave the three big endothelins, but with a clear preference for big ET-1, which is in agreement with our results showing that intact Sertoli cell ECE-1 converts big ET-1 more efficiently than big ET-2 or big ET-3. Recently, Yanagisawa et al. clearly demonstrated that the activity of ECE-1 is essential and that a physiologically relevant endothelin-converting enzyme exists for both big ET-1 and big ET-3 in vivo. In fact, ECE-1−/− mice (which all died within 30 min of birth) reproduced the phenotype resulting from the defects in both ET-1/ ET A – and ET-3/ET B –mediated signaling pathways, which clearly shows that mature ET-1 and ET-3 are not synthesized in the relevant microenviroments without ECE-1 activity. Furthermore, a significant amount of mature ET-1/ ET-2 still existed in the serum of ECE-1−/− embryos despite the absence of ECE-1, which suggests that other peptidases are responsible for the production of mature ETs. Intriguingly however, these remaining mature ETs completely failed to rescue the developmental phenotype of ECE-1−/− mice, which indicates that defined mature ETs must be produced at specific microenviroments in order to achieve a biological effect. The present study provides evidence that the restricted expression of ECE-1 might play a pivotal role in the control of peritubular contractility by providing a fine local modulation of biologically active ET levels. If ET acts as a local regulator of seminiferous tubule contractility, it is conceivable that ECE is localized on the basal side of the Sertoli cells. In fact, Northern blot analysis showed ECE-1 mRNA in cell extracts from purified Sertoli cells. Our in situ hybridization studies indicate that ECE-1 is predominantly localized in tubular areas where Sertoli cell bodies reside, particularly in the basal region. Sertoli cells are the only somatic cell type in the seminiferous epithelium; along the side of these elongated perennial elements, it is possible to observe, at any given time, several generations of germ cells, which flow radially to be eventually released as mature sperm into the tubular lumen. It has long been known that activities of the Sertoli cell, among which FSH responsiveness, vary according to the specific subset of differentiating germ cells with which it is associated (“stages” of the seminiferous epithelium) . In the prepuberal rat, in which the cyclicity of the epithelium has not been established yet, uniform expression of ECE was observed; in the adult, by contrast, ECE expression appears to be regulated in a temporal and spatial manner during spermatogenesis and the seminiferous epithelium cycle. Interestingly, expression of ECE-1 is exclusively restricted, in the adult rat, to stages IX-X of the cycle. These stages are characterized by the fact that they immediately follow spermiation and represent ∼5% of the entire cycle length, which may explain why ECE expression was overlooked in a previous study . When segments of seminiferous tubule at precise stages of the seminiferous epithelium cycle were dissected and individually exposed to the inactive precursor big ET-1 to test their ability to induce myoid cell contraction through the generation of active ET-1, fragments containing stages preceding IX were found to be unresponsive to the precursor. By contrast, in segments from stages after spermiation, normal contraction of myoid cells was observed in response to the inactive precursor, which indicates efficient processing of big ET-1. In parallel samples, directly stimulated with ET, no difference in responsiveness to the active peptide was observed, which suggests that myoid cells are constantly capable of responding. These experiments demonstrate a direct correlation between a restricted expression of ECE-1 and its biological function. A perspective that warrants exploration is the mechanisms that regulate the expression of ECE-1 and the developmental transition from the diffuse to the restricted pattern of distribution of ECE-1, which may be connected to the known cyclic and developmental changes in hormonal sensitivity Sertoli cells undergo . Moreover, alterations in the pattern of ECE-1 and ET production might be involved in the pathogenesis of peritubular hyalinization, given the well-known role played by ET in fibrosis and matrix overproduction in a number of tissues . In conclusion, our data could be used to outline a simplified model concerning the regulation of seminiferous tubule contractility, according to which the restricted expression pattern of ECE-1 would finely modulate local endothelin levels. In this model, ET-1 precursors produced by Sertoli cells are processed to biologically active ET-1 only in restricted areas of seminiferous tubule according to the spatiotemporal control of ECE-1 expression on the Sertoli cells (in turn, presumably dependent upon the spermatogenic cycle). Thus, seminiferous tubule contraction may originate in the specific tubular segments adjacent to those at which spermiation has just occurred, to be propagated as effective peristaltic waves by additional mechanisms that have yet to be identified. | Other | biomedical | en | 0.999997 |
10352020 | Male hemizygotes and wild-type littermates derived from the crossing (>3 generations) of heterozygous synapsin I 129/Sv/C57Bl/6 mice were used for this study. The animals were housed under a 12-h light/12-h dark cycle with free access to water and food. All animal procedures were approved by the Institutional Animal Care and Use Committee. The electrophysiological and culture techniques used have been described earlier . Data acquisition and analyses were performed without knowledge of the genotype of the mouse under study. In brief, low density primary cultures of hippocampal neurons were prepared from 16.5-d-old embryonic mice and used for the electrophysiological experiments at 8 d in vitro . All the electrophysiological experiments were performed at room temperature (24–28°C). Whole-cell currents were recorded from pyramidal cells at the holding potential of 0 mV for IPSCs, or −70 mV for excitatory postsynaptic currents (EPSCs). The bathing solution contained 137 mM NaCl, 3.5 mM KCl, 10 mM Hepes, 10 mM glucose, 0.7 mM CaCl 2 , 2 mM MgCl 2 , 10 −4 mM tetrodotoxin (TTX), and 0.5 × 10 −3 mM strychnine, pH 7.2. 2 mM kynurenic acid was added for recording IPSCs and 0.1 mM picrotoxin for EPSCs. A small region of the dendritic tree was superfused with a hypertonic solution (800 mosM with sucrose) of the same aforementioned ionic composition except for the presence of TTX at the concentration of 10 −3 mM. The pipette solution contained the following: 122.5 cesium gluconate, 17.5 mM CsCl, 8 mM NaCl, 10 mM Hepes, 5 mM EGTA, 2 mM Mg ATP, and 0.3 mM Na 3 GTP, pH 7.2, for IPSC recordings, and 120 mM potassium gluconate, 12 mM KCl, 5 mM NaCl, 2 mM MgCl 2 , 1 mM CaCl 2 , 10 mM Hepes, 5 mM EGTA, and 2 mM Na 2 ATP, pH 7.2, for EPSC recordings. We determined the parameters of vesicle turnover according to a previously described model . The model assumes two pools of synaptic vesicles: the readily releasable pool and the infinite reservoir pool. The vesicle turnover rate of the readily releasable pool at time t can be described as, 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}d{\alpha}({\mathit{t}})/d{\mathit{t}}={\mathit{K}}_{doc}{\cdot}(1-{\alpha}({\mathit{t}}))-{\mathit{K}}_{exo}{\cdot}{\alpha}({\mathit{t}}),\end{equation*}\end{document} where α( t ) designates the probability of the synaptic vesicle existence at a docking site in the readily releasable pool at time t (0 < α( t ) < 1, α(0) ∼1), K doc , the rate of replenishing one vesicle from the infinite reservoir pool to a docking site, and K exo , the rate of releasing one vesicle for transmitter release from a docking site. Hence, 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\alpha}({\mathit{t}})={\tau}{\cdot}(e^{-{\mathit{t}}/{\tau}}-1.0)+{\alpha}(0),\end{equation*}\end{document} where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}=1/({\mathit{K}}_{doc}+{\mathit{K}}_{exo})\end{equation*}\end{document} and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\alpha}(0)=1.\end{equation*}\end{document} The cumulative number of events of miniature EPSCs or IPSCs (mEPSCs of mIPSCs) at time t (C( t )) can be described as, 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}C({\mathit{t}})={\int _{0}^{{\mathit{t}}}}{\mathit{N}}{\cdot}{\mathit{K}}_{exo}{\cdot}{\alpha}({\mathit{x}})d{\mathit{x}}+B{\cdot}{\mathit{t}},\end{equation*}\end{document} where B represents the background frequency of events derived from unperfused synapses, N is the total number of docking sites at synapses in a small dendritic region superfused with the hypertonic solution (800 mosM). K doc , K exo , and N can be determined by fitting the time course (0– 2.5 s) of cumulative mIPSCs or mEPSCs during depletion with equation 3. Transverse hippocampal slices (300-μm-thick) were prepared from mice (10–14-d-old) after decapitation under halothane anesthesia . Slices were maintained in an incubation chamber for 1 h at 37°C and at room temperature thereafter. The external solution contained the following: 119 mM NaCl, 2.5 mM KCl, 1.0 mM NaH 2 PO 4 , 26.2 mM NaHCO 3 , 11 mM glucose, 2.0 mM CaCl 2 , 1.0 mM MgCl 2 , 0.5 × 10 −3 mM strychnine, 2 mM kynurenic acid, pH 7.2, equilibrated with 95% O 2 : 5% CO 2 . A stimulating electrode filled with the external solution was placed close to an interneuron in the striatum oriens of CA3. IPSCs evoked by short (100 μs) current pulses (3–10 V) at 0.05 Hz were recorded from a CA3 pyramidal neuron by whole-cell recording at a holding potential of 0 mV. The IPSCs were blocked reversibly by bicuculline (10 μM), indicating that they were mediated by γ-aminobutyric acid (GABA)- gated Cl − channels. Whole-cell currents were recorded by a patch-clamp amplifier (EPC-7; List), filtered at 5 kHz, and digitized at 10 kHz for further analysis. Cultured neurons were incubated in medium containing 10 −3 mM TTX for 5 min, stimulated with the above mentioned 800-mosM hypertonic solution for 10 s, washed out with medium for 1 min, and fixed with 0.1 M cacodylate buffer, pH 7.2, containing freshly prepared 4% paraformaldehyde and 0.1% glutaraldehyde for 15 min. The samples were quenched with 100 mM glycine for 5 min, permeabilized, and blocked in 0.5% BSA, 0.1% gelatin, 0.05% Tween 20, 20 μM digitonin, 1% skim milk, and 500 mM NaCl for 30 min; both solutions were prepared in PBS, pH 7.2. Subsequently, they were incubated for 1 h with a polyclonal anti-GABA antibody (diluted 1:450 with the above mentioned blocking solution) and blocked again for 30 min. Up to here all procedures were carried out at 37°C, thereafter the samples were incubated at room temperature for 10 min with 10 μg/ml biotin-labeled goat anti–rabbit IgG antibody (Nichirei), treated with 100 μg/ml HRP-labeled streptavidin (Nichirei) for 5 min, and processed for DAB staining. Subsequently, they were postfixed by dipping in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, for 10 min on ice, stained with 1% uranyl acetate for 1 h at room temperature, dehydrated in a graded series of ethanol concentrations, and embedded in Quetol-812 (Nisshin EM). Ultrathin sections were cut on a conventional ultramicrotome (Ultratome Nova; LKB Bromma), stained with uranyl acetate and lead citrate, and examined and photographed under a transmission electron microscope at an accelerating voltage of 80 kV. The number of synaptic vesicles in presynaptic nerve terminals was determined by direct counting from the micrographs. These numbers were converted into the number of vesicles per unit area (μm 2 ) by dividing the vesicle count by the presynaptic nerve terminal area as described before . We tested the possibility of the inhibitory synapses becoming easily fatigued and recovering slowly in synapsin mutants. Spontaneous mIPSCs were recorded from cultured hippocampal neurons in the presence of 1 μM TTX, and a hypertonic sucrose solution (800 mosM) was applied locally to a neurite for 5 s, followed by application for 2 s every 5 min to monitor the size of the readily releasable pool of synaptic vesicles. Most of the readily releasable vesicles were released during the first 2 s of application, and the size of released quanta measured by second 2 s applications recovered in parallel with the amplitude of the evoked EPSCs . Assuming that the replenishing time constant of the readily releasable pool is ∼ 10 s, the 5-min intervals would be expected to minimize the effect of replenishment. When the hypertonic solution was applied for 5 s, the frequency of mIPSCs declined exponentially , presumably because of a depletion of available transmitter quanta or synaptic vesicles . After a 5-min interval, the second application of hypertonic solution (2 s) caused a similar magnitude of increase in the frequency of mIPSCs in cells from the wild-type mice, whereas a much smaller increase was noted in the cells from synapsin I mutant mice . The total amount of quanta released during the first 2 s of hypertonic application in the second sucrose application was 43 ± 14% ( n = 6) in the case of mutant mice, whereas it remained at 97 ± 5% ( n = 5) in the case of wild-type mice . Subsequent applications of the hypertonic solution at 5-min intervals did not further reduce the amount of quantal release, either in the mutant or in wild-type mice. In contrast to the result at inhibitory synapses, no such depression was observed at the excitatory synapses in the mutant mice (95 ± 8% at the second application, n = 6) . We analyzed the changes in the kinetics of synaptic vesicle turnover. Paired-pulse application of the hypertonic solution at various intervals commonly is used to assess the recovery time following depletion. But in our case, repeated stimulation with the hypertonic solution suppressed the subsequent mIPSCs in mutants that suggested the possibility that either the replenishing time might be altered on the order of tens of minutes or that the vesicular recycling process, including endocytosis, might be severely retarded. If the latter were the case, it would not be appropriate to apply the paired superfusion method to inhibitory synapses in mutants. We applied the model described by Stevens and Tsujimoto to estimate the replenishing time constant after a single 5-s hypertonic application. Assuming an infinite reservoir pool and a readily releasable pool of synaptic vesicles, the vesicle turnover rate in the readily releasable pool can be described by a simple model equation (see Materials and Methods.). As shown in Fig. 2 a, because the mIPSCs in a mutant fail to respond to hypertonic stimulation after 3 s from the beginning of superfusion, we calculated the parameters for the synaptic vesicle turnover by equation 3 to the cumulative events of mIPSCs during 0–2.5 s of hypertonic application. (For these examples, the calculated replenishing time [1/ K doc ] was 10.1 s [wild type], 5.7 s [mutant]. The rate of releasing one vesicle from a docking site was 0.56 s −1 [wild type], 1.83 s −1 [mutant].) At 11 inhibitory synapses from 4 synapsin I–deficient mice and at 5 inhibitory synapses from 3 wild-type mice, the mean time to replenish vacant release sites (1/ K doc ) was 5.5 ± 3.7 s and 10.7 ± 3.7 s, respectively . Thus, the replenishing time of synaptic vesicles from a reservoir pool to a readily releasable site was reduced in synapsin I mutants (* P < 0.05, t test; the asterisk indicates that the related probability demonstrates statistical significance). In contrast, at the excitatory synapses, no significant difference was observed in the replenishing time between synapsin I–deficient mice (8.3 ± 2.3 s, n = 12) and wild-type mice (10.4 ± 2.6 s, n = 12, P > 0.5) . The releasing rate from a readily releasable pool ( K exo ) of mIPSCs was higher by a mean of 36% for the mutants (1.5 ± 0.8 s −1 , n = 11) compared with the wild-type mice (1.1 ± 0.5 s −1 , n = 5). However, because of a large variance, the difference was not statistically significant ( P = 0.18). The releasing rate from a readily releasable pool of mEPSCs was unaltered between mutants (1.2 ± 0.4 s −1 , n = 12) and wild-type mice (0.9 ± 0.2 s −1 , n = 12, P > 0.5) . It is suggested that the accelerated replenishment from the reservoir pool to the readily releasable pool in mutant inhibitory presynaptic terminals accelerates the vesicular depletion of the reservoir pool, especially after rigorous hypertonic stimulation, at mutant inhibitory synapses. To obtain morphological correlates, we performed electron microscopic analysis of the presynaptic terminals of hippocampal neurons in culture. From a morphological perspective, at the 8-d stage in vitro, the synapses of cultured hippocampal neurons were still not fully mature, although functionally, their mIPSCs or mEPSCs corresponded to that of fully mature synapses. The presynaptic terminals were smaller, the synapses often lacked postsynaptic densities, and synaptic vesicles seemed less apparent. However on immunoelectron microscopy using the anti-GABA antibody, we could visualize inhibitory presynaptic terminals because they were filled with fuzzy DAB staining, especially intense just around the synaptic vesicles . Without hypertonic stimulation, there was no difference between the two genotypes with respect to synaptic vesicle densities at the inhibitory presynaptic terminals as identified by positive staining with anti-GABA antibody . However, after rigorous hypertonic stimulation, a clear loss of synaptic vesicles was observed at the inhibitory presynaptic terminals in synapsin I mutant mice but not in the wild-type mice . No such morphological difference was observed between the mutant and wild-type mice at the excitatory presynaptic terminals as identified by the absence of anti-GABA staining . We performed morphometrical analyses on the synaptic vesicle densities at the presynaptic terminals of each genotype and, as shown in Table I , the density was significantly reduced only in the stimulated inhibitory synapses from mutants (* P < 0.05, t test). To determine whether this reduction affects the distribution of synaptic vesicles, we further counted the number of synaptic vesicles within a distance of 200 nm from the synaptic clefts. This revealed that the vesicle density near the synaptic clefts was not statistically different between wild-type and mutant mice. Thus, the physiological and morphological results consistently suggest that the reservoir pool of inhibitory presynaptic terminals could be depleted after hypertonic stimulation in synapsin I–deficient mice. To assess the physiological role of synapsin I in synaptic transmission, we evoked IPSCs in pyramidal cells in the CA3 region of mouse hippocampal slices . IPSCs in mutant mice showed a wider scatter in amplitude than those of wild-type mice . Also, the mean amplitude of the evoked IPSCs was smaller in the case of mutants (159.0 ± 21.3 pA, n = 10) than in wild-type littermates (219.6 ± 31.1 pA, n = 9) . The coefficient of variation estimated from the regression line for the data points in the relationship between mean amplitude and SD of IPSCs was larger in mutants (0.18 ± 0.02, n = 10) than in the wild-type mice (0.12 ± 0.02, n = 9) . There was no significant difference between mutant and wild-type mice with respect to the rise time (10–90%) (wild-type, n = 9, 2.6 ± 0.3 ms; mutant, n = 10, 2.8 ± 0.3 ms) and biexponential decay time constants (wild-type, n = 9, 21.1 ± 0.9 ms and 39.9 ± 6.3 ms; mutant, n = 10, 22.5 ± 1.6 ms and 31.6 ± 3.3 ms) of evoked IPSCs. To examine whether the quantal size may be altered in synapsin I mutant mice, we recorded the spontaneous mIPSCs from hippocampal CA3 neurons in the presence of TTX . No significant difference was observed in the amplitude of mIPSCs between the case of mutant (18.6 ± 3.7, n = 6) and wild-type mice (17.6 ± 4.0, n = 4). The mIPSC amplitude distributions of the wild-type and mutant cells are not significantly different (Kolmogorov-Smirnov test; P > 0.1). These results suggest that synapsin I deficiency reduces the inhibitory synaptic efficacy by reducing the quantal content (i.e., the number of synaptic vesicles released by a single presynaptic action potential). Synaptic vesicles normally are anchored to actin filaments by synapsins . As the association and dissociation of synapsins to synaptic vesicles are regulated by protein kinases, it is believed that synapsins may play an important role in the regulation of synaptic transmission by the following mechanisms. First, synapsins may modulate the vesicular traffic from the reservoir pool to the readily releasable pool (predocking mechanism) . The fluorescence resonance energy transfer experiment revealed that the association and dissociation kinetics of synapsin I and synaptic vesicles is the same order of magnitude as the kinetics of synaptic vesicle recycling . It has been reported that domain E of synapsins is responsible for maintaining the reservoir pool of synaptic vesicles . In cultured synapses, the number of vesicles exocytosed during action potential trains and the total recycling vesicle pools are reduced in synapsin I knockout mice . Also, synapsins have a binding activity to ATP and are predicted to transfer phosphate to an unidentified substrate that may suggest the possibility that synapsin I may be involved in the priming process . Second, synapsins may inhibit synaptic vesicle fusion for exocytosis . Presynaptic injection of the synapsin domain E peptide reduced the size of EPSCs, accompanied by retarded kinetics of exocytosis . Recently, it has been postulated that the efficacy of neurotransmitter release may be regulated by the size of the readily releasable pool as supported by the results of the following experiments. The time courses of paired-pulse inhibition of action potentials and hypertonic solution– evoked release are correlated with each other at individual interpulse intervals when the exocytosis is evoked by action potentials followed by hypertonic solution application . The release probability as measured by the minimal stimulation technique is related directly to the size of the readily releasable pool as measured by repetitive nerve fiber stimulation . The replenishment of the readily releasable pool of giant presynaptic terminals in brainstem slices was accelerated by preceding high frequency action potentials in a calcium-dependent manner . The replenishing time of the readily releasable pool in our case was 10.4 ± 2.6 s (wild-type EPSC) and 10.7 ± 3.7 s (wild-type IPSC), comparable with values reported previously . This variation may arise from variations in the intraterminal environment (e.g., the concentration of Ca 2+ and protein kinases). Indeed, it is suggested that the replenishment process is accelerated by an elevation in the concentration of intracellular Ca 2+ in hippocampal synapses in culture , retinal bipolar cells , and brainstem giant synapses . Activation of protein kinase C by phorbol ester also has been reported to reduce the replenishing time of the readily releasable pool . Our results in synapsin I mutants suggested a significant reduction in the replenishing time of inhibitory synaptic vesicles from the reservoir pool to a readily releasable site in synapsin I mutants. Synapsin I deficiency had no statistically significant ( P = 0.18) effect on the rate of release from a readily releasable pool ( K exo ). Presumably, this was due to a large variance between the two populations, although the mean value was 36% higher in the case of mutants compared with the wild-type mice. Thus, our results are consistent with the proposed predocking mechanism of synapsin I in the inhibitory presynaptic terminals. However, we cannot exclude the possibility that synapsin I gene knockout may accelerate the release rate of synaptic vesicles during hypertonic superfusion. Both synapsin I and II are contained in the excitatory mossy fiber terminals in the hippocampus, whereas the inhibitory terminals of cerebellar Purkinje cells lack synapsin IIa and express only a low level of synapsin IIb . Also in the rat retina, glutamic acid decarboxylase-positive terminals lack synapsin II . Although there is no direct evidence that hippocampal inhibitory synapses lack synapsin II, our results suggest at least that synapsin I plays a very specific role that cannot be compensated for by the presence of synapsin II. In addition, previous studies on synapsin mutants have shown that synapsin II knockouts, but not synapsin I knockouts, exhibit decreased posttetanic potentiation and severe synaptic depression on repetitive stimulation at the excitatory synapses . Considering together, we suppose that synapsin II may compensate for the absence of synapsin I at the excitatory synapses, whereas the deficiency of synapsin I exerted a more serious effect at the inhibitory synapses, presumably because of the poorer compensation. During the baseline synaptic transmission, the apparent velocity to replenish the readily releasable pool is determined by the true replenishment velocity (the speed of transferring vesicles from the reservoir pool to the readily releasable pool), if the size of the reservoir pool is large enough. Alternatively, when the reservoir pool size reduces small enough because of the prolonged consumption of synaptic vesicles by repetitive stimulation, the apparent replenishing velocity will be limited by the velocity of refilling the reservoir pool. Indeed, our results indicated that the mutant inhibitory synapses in culture could not maintain the capability of subsequent transmitter release because of the exhaustion of reservoir pool after the first massive transmitter release. We consider that the size of reservoir vesicular pool is progressively reduced in the mutants in an activity-dependent manner, maybe by the accelerated exocytosis/replenishment, the impaired endocytosis, retarded incorporation of endocytosed vesicles into the reservoir pool, or slow uptake of neurotransmitter into the vesicles. At the equilibrated state, the exhausted reservoir pool can no longer support full replenishment of the readily releasable pool, causing reduction in amplitude of evoked IPSCs. If this is also the case in vivo, one could picture use-dependent changes in the reliability of synaptic transmission as follows. Briefly, in the mutant inhibitory synapse after a long enough quiescent period, synaptic vesicles are fully loaded to the reservoir pool and to the readily releasable pool. When bursts of action potentials reach the nerve terminal at this state, a larger amount of neurotransmitter (GABA) is released because of the accelerated replenishment of the readily releasable pool. This causes rapid exhaustion of the reservoir pool and the nerve terminal can no longer release GABA although the action potentials reach the terminal. This would be one of the underlying mechanisms for epilepsy observed in the mutant mice. Primary epilepsy in humans is largely known to be unassociated with mental retardation . Similarly, synapsin I mutants exhibiting epileptic seizures are normal in terms of conditioning and spatial learning , whereas most rodents with epileptic phenotypes are characterized by learning disabilities. In this respect, the synapsin I mutants may well be a model of epilepsy in humans. The cellular mechanisms underlying epilepsy are as follows: (1) an increased electrical excitability of neurons capable of firing at a high frequency ; (2) frequency potentiation of excitatory synaptic transmission ; and (3) use-dependent depression of inhibitory synaptic transmission resulting in the removal of GABAergic inhibition. In an animal model of temporal lobe epilepsy, GABAergic inhibitory synaptic transmission from basket cells to CA1 pyramidal cells is known to be suppressed because of the reduced synaptic input to the inhibitory cells . Our results suggest that synaptic vesicle turnover at the inhibitory synaptic terminals can be altered by synapsin I deficiency. A subtle change in the turnover rate at inhibitory synaptic vesicles may reduce the inhibitory synaptic efficacy, thereby possibly causing epilepsy in synapsin I mutant mice. Any defect at the inhibitory synapses potentially can produce disintegration of the central nervous system such as epilepsy and in this respect, much attention must be paid to inhibitory synaptic transmission in various mutants with their molecules genetically ablated. | Study | biomedical | en | 0.999997 |
10352021 | Heterozygous c-jun +/− male and female mice of C57BL/129 background were mated and the appearance of the vaginal plug was taken as E0.5. Pregnant mothers were killed at 11.5–14.5 d of gestation by cervical dislocation and the fetuses were isolated. Genotypes of fetuses were determined by PCR using the primers c-jun 1430 and c-jun 2287 (Table I ). Mouse fetuses were fixed in 10% phosphate-buffered formaldehyde, paraffin-embedded, and then 4-μm sections were stained with hematoxylin-eosin (HE). Apoptotic cells were analyzed in paraffin sections by in situ DNA end labeling , and labeled DNA was detected with the ABC procedure (DAKO). For double-label immunofluorescence analysis fetuses were snap-frozen in isopentane at the temperature of liquid nitrogen, and sections (4 μm thick, fixed in acetone at −20°C for 10 min) were sequentially incubated with the following antibodies: monoclonal mouse antibodies against desmoplakin I and II ( Boehringer Mannheim ), E-cadherin (ZYMED), connexin 43 (Cx 43; Transduction Laboratories), and monoclonal rat antibodies TER119, CD71, CD11b, and CD34 ( PharMingen ) against hematopoietic cells or polyclonal rabbit antibodies against desmin (Chemicon) or keratins 8 and 18 . Primary antibodies were detected with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated antibodies directed against mouse and rat or rabbit immunoglobulins, respectively. For negative control, primary antibodies were omitted or replaced by unrelated isotype-matched immunoglobulins. Specimens were analyzed with a MRC600 (Bio Rad Laboratories) laser-scanning confocal device attached to a Zeiss Axiophot microscope. Alternatively, sections of paraffin-embedded liver samples were stained with the antibodies to keratins 8 and 18 or the antibody TER119 after pretreatment with pronase E, and bound antibodies were detected by the APAAP procedure (DAKO). Liver cells isolated from C57BL/129 E12.5 c-jun +/− and c-jun −/− fetuses were resuspended in PBS, and 10 6 cells were injected intravenously into adult C57BL/129 wild-type recipient mice that were lethally irradiated (9.5 Gy). All control mice injected with PBS died after 2 wk. After 8 mo, reconstitution of the various hematopoietic lineages was analyzed by FACScan ® with the following cell surface markers: B220, CD43, GR1, MAC1, CD4, CD8, TER119, and HSA as described . For generation of exogenous artificial RNA standards that served as competitors in quantitative RT-PCR, sequences corresponding to the mRNA or heterologous (unrelated) sequences were cloned into the expression vector p CRI I (Invitrogen; Table I ). For restriction standards, a cleavage site of a restriction enzyme between the primer binding sites was mutated by filling it with Klenow fragment ( Promega Corp. ). Standard plasmids with a deletion between the primer binding sites were constructed either by Bal 31 exonuclease digest or by loop out mutagenesis. For heterologous standards, a heterologous sequence, flanked by the specific primer binding sequences, was generated by amplification with hybrid primers and cloned into p CRI I. Exogenous artificial RNA transcripts were generated from linearized standard plasmids by in vitro transcription with the corresponding RNA polymerases (T3, T7, and SP6 were obtained from Boehringer Mannheim ). First strand cDNA synthesis for quantitative RT-PCR was performed in a 20-μl reaction mixture containing 0.1 μg of total RNA , 0.5 units Inhibit-ACE (5′→ 3′ Inc.), 1×AMV reverse transcription buffer (100 mM Tris-HCl, pH 8.3 at 42°C, 40 mM KCl, 10 mM MgCl 2 , and 0.5 mM spermidine), 5 mM dNTPs (1.25 mM each), 4 mM sodium pyrophosphate, 5 units AMV reverse transcriptase ( Boehringer Mannheim ), 0.5 μM lower primer, and a certain number of exogenous artificial RNA molecules. For PCR optimal buffers for each primer pair were selected using the PCR optimizer kit (Invitrogen; Table I ). PCR was performed with one-tenth of the cDNA products amplified in a 50-μl reaction containing 1× PCR buffer, 5 μl DMSO, 1 μM of each primer (except 0.5 μM for quantitation of hepatocyte growth factor mRNA), 0.25 μM of each dNTP, and 2.5 units AmpliTaq DNA Polymerase ( Perkin Elmer Cetus). The reaction was heated to 94°C, then Taq Polymerase was added, and subsequently cycled for 45 cycles at 94°C, 1 min, 55°C (except 50°C for albumin and transferrin, 52°C for erythropoietin), 1 min, and 72°C, 1 min. At the end of the last cycle a final extension step of 4 min at 72°C was added. PCR products were separated on ethidium bromide-stained agarose gels and band intensities were estimated by video densitometry (Docu Gel V densitometer and Rflp-Scan or ONE-Dscan software; Scanalytics ). mRNA copy numbers were calculated from the differences in the band intensities which were corrected by application of standard curves as described . c-jun −/− ES cells were injected into C57BL/6 blastocysts. Tissues of chimeric mice were analyzed for glucose phosphate isomerase (GPI) isoenzyme distribution as described . The contribution of c-jun −/− ES cells to fetal liver tissue was analyzed in short-term cultures of fetal liver cells. Fetal livers were dissected from staged fetuses and after a brief rinse in PBS subjected to 5-min subsequent incubations in solution A (EBSS without Ca 2+ and Mg 2+ containing 0.5 mM EGTA), solution B (EBSS containing Ca 2+ , Mg 2+ , and 10 mM Hepes, pH 7.4), and solution C (EBSS containing Ca 2+ , Mg 2+ , 10 mM Hepes, pH 7.4, and 0.3 mg/ml collagenase). Fetal livers were washed once in PBS and liver cells were dispersed by pipetting several times with a 1-ml glass pipette. After centrifugation, cells were cultured in plastic dishes (Falcon Primaria, Becton Dickinson ) in DMEM containing 10% fetal calf serum for 2–3 d and nonadherent hematopoietic cells were removed by repeated washings twice daily. GPI analysis was then performed with cultured fetal liver cells and with the residual fetal tissues, which remained after dissection of the liver, to calculate the relative contribution of c-jun −/− ES cells to the liver in relation to the average chimerism. The contribution of c-jun −/− ES cells to the various tissues of chimeric mice at various age after birth was furthermore determined by PCR using the primers c-jun 1430 and c-jun 2287 (Table I ). Hepatocytes were isolated from livers of 8-wk-old chimeric mice by collagenase liver perfusion essentially as described by Seglen except that perfusion was performed via the left ventricle . This technique yields a hepatocyte preparation with ∼90% purity (as estimated by immunohistochemical detection of keratin), and, based on trypan blue exclusion, >85% viability. For preparation of bile ducts, sections of snap-frozen livers were stained with methylene blue, and bile ducts were microdissected under a stereo-microscope using a 25-G needle attached to an 1-ml syringe. The microdissected tissue was collected in an Eppendorf tube and DNA was isolated for PCR analysis. PCR products were separated on ethidium bromide–stained agarose gels and band intensities were estimated by video densitometry. Nonlinearity of band intensity ratios was corrected with a standard curve . Livers from E12.5 mouse fetuses were mechanically dissociated and plated onto plastic chamber slides (NUNC, Kamstrup, DK) in DME containing 10% FCS. Liver cells were cultured for 2 wk in order to remove hematopoietic cells. Purity of cultured hepatoblasts was controlled by keratin immunostaining using rabbit antibodies to keratins 8 and 18, showing that more than 95% of the cells were keratin positive. Primary fibroblasts from E12.5 fetuses were cultured as described . [ 3 H]thymidine labeling was performed with 300 kBq methyl [ 3 H]thymidine/ml medium ( Amersham ). Primary hepatocytes were labeled for 2 h, fibroblasts for 1 h at 37°C followed by an incubation step in medium without [ 3 H]thymidine for 30 min. Thereafter cells were rinsed twice with medium and PBS and fixed with 4% paraformaldehyde in PBS, pH 7.4, for 30 min at room temperature. Apoptotic cells were stained with the in situ cell death detection kit ( Boehringer Mannheim ). After dehydration with increasing concentrations of ethanol [ 3 H]thymidine-labeled cells were visualized with Kodak NTB2 photoemulsion and detected with Kodak D19 developer. Previous studies revealed no readily detectable differences in development or morphology between wild-type and c-jun −/− fetuses at least up to E11.5 . Between E11.5 and E14.5, in some c-jun −/− fetuses rounded hepatoblasts, which were detached from each other and scattered cells with characteristic features of apoptosis and necrosis, were detected . However, the cell types that are affected primarily in the absence of c-Jun had not been determined yet and the developmental stage at which c-Jun becomes essential for liver development was unclear. To address these questions, detailed histological analyses of fetal livers from E12.5 and E13.0 c-jun −/− fetuses were performed. At E12.5, no morphologic abnormalities were observed in c-jun −/− livers. At E13.0, however, 8 out of 19 c-jun −/− fetuses showed increased apoptosis in the livers when compared with wild-type littermates . In two of these c-jun −/− fetuses, apoptosis was very extensive, affecting up to one-third of all liver cells. Staining by in situ DNA end labeling (TUNEL) revealed fragmented DNA present in the condensed nuclei of cells undergoing apoptosis . Characterization of the affected cell type by immunohistochemical staining with antibodies against keratins 8 and 18, which is expressed in hepatoblasts, and the erythroid-specific antibody TER119 confirmed, that condensed and fragmented nuclei typical of early apoptosis were present in hepatoblasts and in erythroid cells . However, cells in advanced stages of apoptosis could not be classified by this method. But from the high number and distribution of apoptotic cells it is likely that apoptosis affects hepatoblasts as well as erythroid cells. Until E12.5, hepatic erythropoiesis appeared normal since immunohistological analyses of livers using the erythroid-specific antibody TER119 and an antibody to CD71, a marker which is present in high amounts on erythroid cells, revealed no differences in the number and distribution of erythroid cells between c-jun +/+ and c-jun −/− E12.5 fetuses. Moreover, macrophages, as demonstrated by staining with antibody to CD11b (Mac-1) and CD34-positive cells, were also present in comparable amounts (data not shown). This suggests that c-Jun is not required for proper establishment of hepatic hematopoiesis but rather is essential for its maintenance. At around E11.5, the liver becomes the primary site of hematopoiesis and liver cells are necessary to provide the proper microenvironment to support survival of hematopoietic cells . The apoptosis observed in c-jun −/− erythroblasts could either be due to a cell-autonomous defect in this particular cell lineage or, alternatively, c-jun −/− fetal liver cells may be unable to provide the proper microenvironment for hematopoietic cells. To discriminate between these two possibilities and in order to investigate whether c-jun −/− hematopoietic cells were affected in a cell-autonomous manner, E12.5 c-jun −/− fetal liver cells were injected intravenously into wild-type lethally irradiated syngeneic adult mice. After six months, mice reconstituted with c-jun −/− cells were healthy and flow cytometric analysis of spleen, bone marrow, and thymus of two mice showed a similar distribution of myeloid and lymphoid cells as the controls . Analysis of peripheral blood showed no significant differences in hematocrits and red blood cell counts (data not shown). PCR analysis of genomic DNA isolated from bone marrow, spleen, and thymus of the reconstituted mice confirmed that these organs had been mostly colonized by c-jun −/− hematopoietic cells . These results indicate that hematopoietic cells of all lineages are present and functional in c-jun −/− fetal livers, which excludes an absolute cell-autonomous defect. Therefore, the observed apoptosis in the erythroid lineage might be caused by non–cell-autonomous alterations, such as a disturbance of the microenvironment in c-jun −/− fetal livers which is essential to sustain hematopoiesis. An altered hepatic microenvironment in c-jun −/− fetuses could be the consequence of deregulated gene expression in liver cells. Therefore, we analyzed the expression patterns of genes that are either known to be regulated by AP-1 or serve as indicators for hepatoblast differentiation and function. Since only small amounts of RNA can be obtained from E12.5 livers, we established and validated a variety of quantitative RT-PCR assays for estimation of mRNA concentrations . This technique allowed analysis of several mRNAs in single fetal livers of littermates. The expression of differentiation markers for hepatoblasts, like hepatocyte nuclear factor-1 (HNF-1), and keratins 8 and 18, as well as the expression of genes involved in growth regulation, namely, the mRNAs for hepatocyte growth factor (HGF), c-myc and cyclin D2 was unaffected in c-jun −/− livers . Moreover, the mRNA levels for secreted liver proteins as indicators for liver cell function, such as serum albumin and transferrin, did not differ between c-jun +/+ , c-jun +/− , and c-jun −/− livers . To see whether the ablation of c-Jun modified the expression of other Jun and Fos family members in order to compensate the loss of c-Jun functions we further analyzed the expression levels of junB , junD , c-fos , and fosB . None of these genes revealed altered expression in c-jun −/− mice . In addition to the analysis of possible alteration of gene expression in hepatoblasts, we investigated possible effects of the absence of c-Jun in erythroid cell. Because at around E12.0 there is the shift from embryonic ε-globin to adult β-globin expression, we analyzed the expression levels of β-globin and ε-globin mRNAs. The ratio as well as the total mRNA concentrations for both globins were similar in wild-type, c-jun +/− and c-jun −/− mice , which is in line with the immunofluorescence data demonstrating a proper establishment of hepatic hematopoiesis. We further analyzed the expression of erythropoietin, which is the most important growth and survival factor for erythroid cells. The liver is expected to be the major site of erythropoietin synthesis during late fetal development, and decreased amounts of this factor could be responsible for apoptosis of erythroid cells as observed in c-jun −/− livers. . Using quantitative RT-PCR we show here that erythropoietin is expressed already in E12.5 fetal livers, although at a very low level. Furthermore, the analyses revealed no difference in the erythropoietin mRNA copy numbers between c-jun +/+ and c-jun −/− livers, indicating that erythropoietin synthesis is not decreased in c-jun −/− mice. It is surprising that all mRNAs analyzed, including mRNAs of genes that are known to be regulated by AP-1, such as keratin 18 and β-globin , showed no significantly deregulated expression levels in E12.5 livers. One explanation is that at E12.5, when these analyses were performed, c-jun is expressed at a very low level (3 × 10 4 copies per 0.1 μg RNA) in the liver , and there is an up to threefold induction of c-jun in the liver at E13.5-E15.5 , which coincides with the observed apoptosis of hepatoblasts and erythroid cells as well as the death of c-jun −/− fetuses. This indicates that c-Jun gains significance in the liver around E13.5 possibly by exerting functions that are not essential in earlier phases of fetal development. Previous studies of chimeric mice that were generated by injection of ES cells lacking c-jun into wild-type mouse blastocysts showed that the ES cells contributed to all tissues except to the liver , suggesting that in the absence of c-Jun no mature hepatocytes can be generated. However, the morphologic as well as molecular characterization of liver differentiation and function revealed no striking differences between c-jun +/+ and c-jun −/− fetuses up to E12.5 , which poses the question up to which developmental stage c-jun −/− hepatoblasts are able to survive and differentiate properly in chimeric mice. Analysis of c-jun −/− ES cell contribution in E14.5-E17.5 chimeric mouse livers was performed in short-term fetal liver cell cultures. A short culturing period of the fetal livers allowed us to remove most of the hematopoietic cells, which adhere much less efficiently to the culture dishes than hepatoblasts. In these cultures similar amounts of c-jun +/+ and c-jun −/− hepatoblasts were detected by GPI assay . ES cell derivatives lacking c-jun were also present in chimeric mouse livers at several weeks after birth . Detailed analysis of the various tissues from an 8-wk-old chimeric mouse by PCR showed substantial contribution of c-jun −/− cells to the liver cell mass . These c-jun −/− cells were present among the hepatocyte cell population, which was isolated and enriched by collagenase liver perfusion, ensuring that the c-jun −/− cells did not reflect nonparenchymal cells, like sinusoidal endothelial or Kupffer cells. Moreover, bile duct epithelial cells were analyzed after enrichment by microdissection. Since we found substantial contribution of c-jun −/− cells to bile duct epithelia, the absence of c-Jun has no obvious adverse effect on the differentiation of hepatoblasts into the hepatocytic or the bile duct epithelial lineage. There was, however, a tendency of continuous loss of c-jun −/− hepatocytes in older chimeric mice. c-jun −/− hepatocytes were detectable up to 8 wk after birth but not in 3-mo-old or older mice. This points to an imbalance in the regulation of hepatocyte cell turnover in adult mice in that c-jun −/− hepatocytes have either a proliferation or a survival disadvantage over wild-type hepatocytes. A possible impact of the loss of c-Jun on hepatocyte cell turnover, namely, on proliferation capacity and apoptotic rate, was analyzed in primary cell cultures of E12.5 c-jun +/+ and c-jun −/− livers. The purity control of these cultures by immunohistochemical detection of keratin expression showed presence of >95% keratin positive cells in c-jun +/+ as well as in c-jun −/− cultures (data not shown). The growth rates and the achieved cell densities were markedly reduced in c-jun −/− cultures . Simultaneous staining of S-phase and apoptotic cells by combined [ 3 H]thymidine incorporation and TUNEL reaction revealed approximately four times increased apoptosis of c-jun −/− hepatoblasts . Concomitant with the increase in the number of apoptotic cells the number of S-phase cells was decreased to ∼50%. Analysis of fibroblast cultures established in parallel from E12.5 fetuses yielded essentially similar results. These findings show that c-Jun is an important proliferation regulator of hepatoblasts and fibroblasts, and that in addition to the reduced mitotic capacity the increase in apoptotic rates is a major factor contributing to the reduced growth potential of both cell types. The data obtained in vitro together with the observation that several c-jun −/− E13.0 fetuses showed increased apoptoses of erythroblasts and hepatoblasts in their livers, point to an essential role of c-Jun in the regulation of apoptosis in a diversity of cell types in vitro and in vivo. Deregulation of apoptosis in the absence of c-Jun was not evident at E12.5, whereas at E13.0 an increase of apoptosis was noted in 42% (8/19) of the c-jun −/− livers. It appears that apoptosis of liver cells occurs only shortly before death of the fetuses and, therefore, can be missed if fetuses are not examined at the appropriate time point. However, some of the fetuses died without substantial apoptotic rates in their livers. Thus, apoptosis of hepatoblasts and erythroid cells in the livers of c-jun −/− fetuses cannot explain fetal lethality in all cases. This prompted us to look for additional defects in c-jun −/− fetuses. Histological analysis of E12.5 fetuses by horizontal serial sections revealed defective development of the heart in all (19/19) of the investigated c-jun −/− fetuses . The animals showed a malformation of the outflow tract with a single outflow vessel that arose entirely from the right ventricle resembling the congenital human heart malformation of a persistent truncus arteriosus (incomplete separation of the aorta and the pulmonary artery). In addition to this anomaly of the outflow tract we found in some animals an abnormal remodeling of the aortic arch arteries resulting in a right-sided aortic arch. Moreover, the wall of the right ventricle was constantly thinner and the endocardial cushion material of the bulbus arteriosus appeared more prominent in c-Jun knockout mice as compared to wild-type mice. These alterations could, in principle, also reflect a delay in heart development rather than a true malformation. However, a mere developmental delay is very unlikely since we could analyze two c-jun −/− fetuses which survived until E14.5 and had heart defects. These fetuses showed a malformation of the outflow tract with only one common outflow vessel that arose from the right ventricle, had an abnormal positioning of the aortic arch, and revealed a wide connection between the left and right ventricle. In contrast, c-jun +/+ E14.5 littermates had a regularly developed aorta ascendens and pulmonary trunk as well as showed complete septation of the left and right ventricles . Since there is a general concept that malformations of the heart outflow tract are due to neural crest cell defects, and similar malformations were described in mice defective in genes necessary for neural crest cell development or migration , we analyzed possible alterations of the contribution of neural crest cells to the peritracheal and cardiac mesenchyme. Neural crest cells were detected by immunohistochemical staining of Cx 43 . The analysis showed that neural crest cells expressing the marker Cx 43 were present in c-jun −/− mice and had properly migrated from the spinal cord to the mesenchyme surrounding the trachea. There was, however, a difference between wild-type and c-jun −/− mice in that fewer Cx 43 positive cells were detected in the outflow tract of the right ventricle of mutant mice. Although the direct comparison of Cx 43– expressing cells in wild-type and c-jun −/− mice is hampered because of the differences in the anatomy of the outflow tracts, the immunofluorescence data are in good accordance with the observed spectrum of heart malformations that are typical for a neural crest cell defect. c-jun is one of the earliest genes that is transcribed after stimulation of cells with growth factors and during tissue regeneration. A central role of c-Jun in the regulation of cell proliferation has been underlined by previous findings with cultured c-jun −/− fibroblasts that had markedly reduced proliferation capacities . In vivo, however, lack of c-Jun did not lead to growth defects in fetuses but caused mid-gestational lethality that was suggested to be due to a liver defect . Until E12.5 of fetal development, no obvious liver defects were observed in the absence of c-Jun and the expression levels of 15 different mRNAs were similar in wild-type and c-jun mutant mice. At E13.0, however, numerous apoptoses were seen in the liver of c-jun −/− fetuses and lethality occurred. These alterations coincided with the time of c-jun mRNA induction in the liver indicating that c-Jun exerts a specific function in liver development at this stage, and that in the absence of c-Jun, erythroblasts and hepatoblasts undergo apoptosis. A cell-autonomous defect as the primary and only cause of erythroblast apoptosis was ruled out by the ability of hematopoietic precursor cells isolated from E12.5 c-jun −/− livers to fully reconstitute all hematopoietic lineages in lethally irradiated mice. Thus apoptoses of erythroblasts in homozygous c-jun deleted livers cannot be exclusively the consequence of the absence of c-Jun in erythroblasts themselves, but most likely involves additional exogenous factors, such as an altered microenvironment in c-jun −/− fetal livers, that do not allow maintenance of hematopoiesis. This microenvironment that has to be provided by the cells of the fetal liver is yet poorly characterized. It might resemble the microenvironment generated by stromal cells of the bone marrow in adult animals. Stromal cells secrete a variety of growth factors that stimulate proliferation and differentiation of hematopoietic cells as well as protect them from apoptosis. One of these growth factors is erythropoietin, which is an essential, and probably the most important survival factor for erythroid cells. Mice carrying loss of function mutations of erythropoietin or the erythropoietin receptor suffered from anemia and died at around E13.5, revealing numerous apoptoses of erythroid cells in their livers, thus resembling in part the alterations seen in c-jun knockout mice . Because the liver is expected to be the major site of erythropoietin synthesis during fetal development, functional deficiencies in c-jun −/− livers could result in reduced erythropoietin levels. However, RT-PCR results revealed that in E12.5 c-jun −/− fetuses, the erythropoietin mRNA concentrations were not altered, so apoptoses of erythroid cells cannot be explained by decreased erythropoietin synthesis. Moreover, a general impairment of erythropoietin signaling in the absence of c-Jun was excluded by the reconstitution experiments in which cells without functional c-Jun properly differentiated into erythrocytes. So far, we could not further specify the mechanism that caused apoptosis of erythroblasts in c-jun −/− mice, and besides deficiencies in erythropoietin, alterations in the expression or function of a variety of other growth factors as well as changes in cell–cell and cell–matrix interactions must be considered. The effect of c-Jun inactivation was not restricted to erythroid cells but also affected other cell types, such as hepatoblasts and fibroblasts. Primary liver cell as well as fibroblast cultures established from E12.5 c-jun −/− fetuses and their corresponding wild-type littermates showed markedly increased apoptotic rates in the absence of c-Jun. This was surprising, since in contrast to our findings, previous studies reported that overexpression of c-Jun forced fibroblasts into apoptosis, and functional inhibition of c-Jun prevented cell death in some cell types . These controversial observations indicate that the role of c-Jun in apoptosis depends on the cellular context and mode of treatment. It is interesting that in c-jun −/− mice no obvious alteration of apoptosis was noted during fetal development until E11.5, whereas primary cell cultures derived from E12.5 c-jun −/− mice had markedly increased apoptotic rates. One possible explanation for this difference between the in vivo and in vitro situation is that apoptosis is preferentially triggered under enforced stimulation of proliferation as it is the case under cell culture conditions. It is possible that the absence of c-Jun modulates the intracellular signals induced by growth factors so that cells respond with apoptosis instead of mitosis. In addition to the alterations in apoptosis, we noted reduced mitotic rates in primary cultures of c-jun −/− fibroblasts and hepatoblasts. One mechanism by which lack of c-Jun could result in impaired proliferation was recently shown by Schreiber et al., 1999 . The proliferation defect in c-jun −/− fibroblast cell lines was found to be p53-dependent, indicating that the alterations of proliferation, and probably also the increased propensity of cells to undergo apoptosis may involve p53-dependent pathways. The altered regulation of cell proliferation and apoptosis in the absence of c-Jun could be an explanation for the occurrence of massive apoptosis of erythroblasts and hepatoblasts in c-jun −/− E13.0 fetuses. Moreover, the proliferation defect as well as the increase in apoptosis could lead to the loss of c-jun −/− cells in livers of adult chimeric mice because of a lower capacity of c-jun −/− hepatocytes to contribute to the cell turnover. However, it is unlikely that the deregulation of proliferation and apoptosis is the only cause of fetal lethality since massive apoptosis was seen in only 2 livers out of 19 c-jun −/− fetuses, and we have analyzed some c-jun −/− fetuses that had died in utero without showing severe morphological liver alterations. It is known from other gene knockout mice, that in addition to the liver, defects in other organ systems, especially the cardiovascular system, have to be considered as causes of mid-gestational lethality . Detailed investigation of hearts of c-jun −/− fetuses revealed a novel function for c-Jun in fetal heart development. Lack of c-Jun led to several anomalies of the heart outflow tracts. All of the c-jun −/− fetuses had compared with wild-type littermates anomalies of the aorta ascendens and pulmonary artery in that in mutant mice there was a single outflow vessel arising from the right ventricle resembling a truncus arteriosus persistens. In addition to the outflow tract alteration, some mice showed a right-sided aortic arch. These anomalies are typical for a neural crest cell defect . It has been shown that the ectomesenchymal cells of cardiac neural crest, which extends from the midotic placode to the caudal limit of somite 3, migrate into the outflow tract of the heart where they contribute to the aorticopulmonary septum. Moreover, cardiac neural crest cells differentiate into smooth muscle cells of the aortic arch and contribute to the stroma of other derivatives of the pharyngeal arches such as thymus, parathyroid, and thyroid gland . Experimental ablation of neural crest cells in chick embryos led to various defects of which a persistent truncus arteriosus was a common denominator. These lesions were always combined with a ventricular septal defect . Furthermore, a right-sided aortic arch or anomalies of the other great arteries were seen after partial neural crest cell ablation. The cardiovascular defects observed by us in c-Jun knockout mice were almost identical to those found after neural crest cell ablation in chicken. Similar to the observations in chicken, a variety of gene mutant mice with impaired neural crest cell development showed anomalies of the outflow tract. For instance, the mouse mutant Splotch, which harbors a mutation in the homeobox gene pax3 , exhibits conotruncal and aortic arch defects . In contrast to the situation in chicken and in the above-mentioned mouse mutant, where cardiac neural crest cell were either absent or had a general defect, we have observed no difference in neural crest cell distribution in c-jun knockout mice except for a reduced number of Cx 43–expressing cells in the outflow tract of the right ventricle. This observation suggests that the consequences of c-Jun inactivation primarily affects neural crest cell function in the heart and does not result in a general neural crest cell defect. As known from neural crest cell ablation experiments in animals as well as from human diseases with a truncus arteriosus persistens (e.g., DiGeorge syndrome), the malformations are not restricted to the heart but also affect other neural crest cell derivatives, such as the thymus or parathyroid. In c-jun mutant fetuses, however, morphologic analysis provided no evidence for a thymus defect, and thus for a general alteration of the cardiac neural crest . Nevertheless, the cardiac malformations observed are highly reminiscent for a disturbance of neural crest function, which may be restricted to the heart and great vessels. Moreover, as shown by neural crest cell transplantation experiments, the mere presence of neural crest cells does not exclude functional deficiencies . The cardiac malformation of a truncus arteriosus persistens may be a major factor contributing to the lethal phenotype. In principle, occurrence of a truncus arteriosus persistens is compatible with survival because of compensation of the hemodynamic imbalance. This compensation may not occur in c-jun mutant fetuses and fetal lethality might be due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis. | Study | biomedical | en | 0.999993 |
10352022 | The Df(2R)47A deficiency in the bkh gene region was issued from a cross between AQ65 (a P-element (P[ w + , lac Z]) enhancer trap line in 47A) homozygous males which were γ-irradiated (4,000 rad) and ( w; Sp/Cyo ) females. Males and females from the F1 generation which were Cyo , but not Sp , were crossed together to obtain the strain. Among the 5,000 chromosomes which were screened, only one line affected the bkh mRNA expression: Df(2R)47A . Three P-lines P[ w, lacZ ] l(2)k06915, l(2)k11003, and l(2)k07810, inserted on the second chromosome, had been located in the 47A region and were homozygous lethal. PCR analysis of the P-lines' genomic DNA was used for mapping these insertions. The primers were the 31-bp terminal sequence of the P-element (5′-CGACGGGACCACCTTATGTTATTTCATCATG-3′) and bkh specific primers: ( bkh 6 : 5′-CAGCCCATTGTGGTTGGG-3′; bkh 3 : 5′-AAGTCCTCAGCAGTGAAGCCGCTCTCG-3′). P07810 was localized 1.5 kb upstream of the class II cDNA ATG and the 1.5-kb PCR fragment obtained was sequenced by Genome Express. P06915 and P11003 were localized 800 bp upstream of the class I cDNA ATG. All the mutant stocks were balanced with Cyo ( wg , lac Z) to identify the homozygous mutant embryos. A genomic region in the 5′ end of the bkh gene was identified with the aid of the DS01583 P1 phage obtained from the Drosophila Genome Project. The 5′ end of the DS01583 P1 phage was located 4.5 kb downstream of the class II cDNA ATG. Amplifications of genomic sequences from this region were performed with the pair of specific primers bkh 6 and bkh 16 (bkh16 5′-CTGAACTTCGAGCGTCGTC-3′). Sequences were made by Genome Express and analyzed with Gene Finder to identify transcription units. The bkh 007 mutation was generated by P-element mobilization in the P07810 line and a cross with flies bearing Δ(2-3) as a source of transposase and of w; Sp/Cyo; Δ(2-3),Sb/TM6B genotype. Germline transformation was performed using standard procedures as described by Rubin and Spradling . The bkh cDNA (class II) was subcloned in the pUAST vector and injected at a concentration of 300 ng/μl with the Δ(2-3) helper plasmid (100 ng/μl) . Several independent transformants were obtained and mapped, and lines of interest were made homozygous with the help of a double balancer stock w; Sp/CyO; MKRS/TM2 . To rescue the Df(2R)47A phenotype at the embryonic stage, we used the yeast GAL4-directed transcription system . Females homozygous for a UAS- 8 . 31bkh class II cDNA on the X chromosome (UAS- bkh ) and bearing the Df(2R)47A deficiency were crossed to homozygous males bearing the homozygous 24B-GAL4 driver inserted on the third chromosome and the Df(2R)47A deficiency on the second chromosome. Homozygous animals were sorted out with the aid of a labeled CyO balancer and scored by observation of their phenotypes. Digoxigenin (DIG) 1 -labeled antisense or sense RNA probes were generated from DNA with T3 or T7 RNA polymerase ( Promega ) and DIG-UTP ( Boehringer ). They were used for whole mount in situ hybridization of fixed staged embryos prepared as described in Zaffran et al. . The DIG-labeled RNA probes were detected with the aid of a preadsorbed anti-DIG antibody coupled to alkaline phosphatase ( Boehringer ) and NBT/BCIP as substrate. The embryos were mounted in Geltol medium (Immunotech) for further observation. A specific probe for class I bkh cDNA was obtained by PCR amplification of genomic DNA with the oligonucleotides bkh 4 and bkh 7 . A probe for class II cDNA was prepared by amplification of the cDNA inserted in pBlueScript with the T7 primer and the bkh6 oligonucleotide ( bkh 4 : 5′-GCCCATGGTGCGTATTGCCTCGACGC-3′; bkh 7 : 5′-CCGCTAGCGGTCAAATCGGGTATCCT-3′; bkh 6 , see above). For immunohistochemistry experiments, embryos were fixed and stained with antibodies according to the protocol described by Ashburner . β-Galactosidase in embryos was detected by using a mouse anti– β-galactosidase ( Promega ) antibody diluted 1,000-fold. The following primary antibodies were used: anti-BKH ; anti-DMEF2 ; anti-EC11 ; anti–Fas II, anti–Fas III, anti-Eve and anti-En (1:10, 1:2, 1:500, and 1:10 dilutions, respectively; Developmental Studies Hybridoma Bank); anti–α-spectrin ; anti-Nrt and anti-Tin . Affinity-purified secondary antibodies (Jackson ImmunoResearch Laboratories) were either coupled to alkaline phosphatase or Biotin and used at a 1:1,000 dilution or conjugated to TRITC or FITC and used at a 1:100 dilution. The stained embryos were mounted in Geltol medium (Immunotech) for further observation under an Axiophot Zeiss microscope or, when fluorescent, in Permafluor (Immunotech) for observation under an Axiophot or a LSM 410 Zeiss confocal microscope. In double labeling experiments, the conditions were the same as above. In situ hybridizations were performed before staining with antibodies. After dehydration of the embryos labeled with antisense RNA probes, they were rehydrated in PBS containing 0.3% Triton X-100 for at least 1 h and saturated in the same buffer containing 10% FCS before incubation with the primary antibodies. The organization of the gene brokenheart ( bkh ) encoding the α subunit of G o , as described by Yoon et al. , is schematized in Fig. 1 . Two classes of cDNAs (class I and class II) have been characterized . They arise from an alternative splicing in a unique gene and differ only in the ATG-containing first exon. Three developmentally regulated transcripts (3.4 kb of maternal origin, 4.2 and 6 kb) were revealed and by using specific probes, we have shown that the class I cDNA corresponded to the 6-kb mRNA appearing after 12 h of development whereas the class II cDNA corresponded to the 4.2-kb mRNA more abundant in earlier stages (data not shown). Both cDNAs encoded proteins of the same size (354 amino acids) that diverged only by 7 amino acids among their 21 NH 2 -terminal residues and for which it is not known whether they exert different functions. By using the class II cDNA as probe, a strong expression of bkh was observed in preblastoderm embryos due to the presence of the 3.4-kb maternal transcript (not shown). In early stage 11 embryos, the zygotic transcript could be detected for the first time in clusters of cells within 11 segments . The cells appear to be cardial and visceral muscle progenitor cells since, based on bkh staining patterns in later stage 12 embryos, the cells became integrated into the conspicuous monocellular layer of cardial and visceral mesoderm cells on each side of the embryo . This assumption was further supported by bkh expression in embryonic tissues which were unambiguously constituted of such cells and, also, in tinman ( tin ) loss of function mutants. In tin mutants, neither heart nor visceral muscles are formed and, correlatively, bkh expression was completely abolished in the territories from which the precursor cells for these two tissues originated . From the middle of stage 11 onwards, neuroblasts of the central nervous system (CNS) became labeled and bkh expression persisted in the neurons of the CNS in later stages of embryogenesis . In a similar way, all the neurons of the peripheral nervous system (PNS) expressed the bkh mRNA from stage 12 onwards, slightly before the onset of axonogenesis . Probes for either cDNA gave identical spatial patterns of expression although class I transcripts were quantitatively less abundant and were expressed later than the class II transcripts (not shown). Antibodies directed against a COOH-terminal peptide whose sequence was conserved in the α subunit of all G o proteins showed that the pattern of expression of the protein was superimposable to that of the mRNA during embryogenesis . However, probably due to the presence of the protein of maternal origin, these antibodies were poorly efficient in detecting a significant signal in the cardial cells as early as did mRNA probes. In stage 11 embryos, the zygotic bkh mRNA was detected at first in the precursors of the cardioblasts in a repeated pattern of 11 clusters in the dorsal mesoderm . These clusters, constituted initially of two to four cells, were located in the anterior compartment of each mesodermal parasegment in a position anterior to the domain of En ectodermal stripes and closely neighboring the Wg-expressing cells in parasegments 2–12 of the overlying ectoderm. These cardioblast precursors belonged to the functional domain of Ladybird (Lbd) and of Evenskipped (Eve) expressing cells in the mesoderm which are the precursors of a fraction of the pericardial cells and of a dorsal muscle . Within the cluster, the expression of the bkh mRNA was initially very intense in only two of the cells and then it became detectable in the four cells with an almost uniform intensity. Due to their position relative to the parasegment boundary, we call these cells A (for anterior) cardioblasts. Soon after the onset of bkh expression in the first cluster, a second cluster of stained cells appeared posterior to the first one and not in continuity with it . This group of cells called P (for posterior) cardioblasts was located in the posterior domain of the next parasegment and it never contained more than two bkh -expressing cells. Their position coincided with that of an area posterior to En stripes in the ectoderm . During germ-band retraction (late stage 11 and stage 12), the cardioblast progenitors that are mesenchymal in nature at that stage began to establish contacts between themselves, to extend filopodes , and to reorganize their shape to form a continuous layer on each side of the dorsal opening . The origin of this process was the result of the encounter of the A and P groups of bkh -expressing cells in each parasegmental domain . Later in development and due to the shortening of the segment during germ-band retraction, the segmentally repeated six cell clusters spread along the anterior-posterior axis to finally join as a monolayer of polarized epithelial cells . Still further in the process, the two rows of cardial cells, together with the pericardial cells that are attached to their basal membrane, fused at the dorsal midline to form the heart tube. Our observations led to the conclusion that a mature heart at the end of embryogenesis was constituted of 52 cardial cells per hemiembryo partitioned in six cells in every segment from T3 to A7 and in only four cells in the T2 segment. The results described above suggest the existence in each segment of two distinct populations of cells composed of four and two cardial cells that derived from mesodermal cells and that were located in domains corresponding, respectively, to the anterior and posterior compartment of each parasegment . None of the cardioblasts present in parasegments 2 and 3 and only a part of those found in PS4 participated in the mature dorsal vessel, but it was, however, difficult to follow their fate since the expression of bkh vanished rapidly from these cells during development. Similarly, by using the EC11 antibody specific for the pericardial cells , we counted 36 such cells distributed in 4 cells per segment from T2 to A7. Eve-expressing cells from the labial segment and the segment T1 did not contribute to the pool of pericardial cells and they probably gave rise to other structures that we were unable to recognize. The cardial cells, which are also muscular cells, were determined as epithelial , based on the expression of several markers for polarity (see below). For example, antibodies directed against α-spectrin specifically labeled the basal-lateral membrane of the epithelium and the staining by anti-Neurotactin antibodies (Nrt) was restricted to the lateral and apical domains of the cell surface . In contrast, phosphotyrosyl proteins and Armadillo were weakly expressed in the heart cell membranes and had no polarized localization (not shown), in good agreement with the lack of authentic Zonulae adherens in Drosophila secondary epithelia . Finally, the basal membrane of the epithelium was visualized with the mAb EC11 . This antibody recognized an antigen probably secreted by the pericardial cells which was detected on the basal membrane of the cardial cells and around the pericardial cells . In the visceral mesoderm that also undergoes a mesenchymal-epithelial transition before its differentiation into the visceral musculature, bkh was expressed in a repeated pattern of clusters of cells in the same 11 parasegments as the precursors of the cardial cells and anterior to them . These precursor cells in parasegments 4–12 were ultimately transformed in a polarized epithelial ribbon of one cell in width , whose polarity was assessed by the localization of Fasciclin III to cell-cell contacts . The embryonic expression of bkh suggests a function of the protein G o in the formation of the cardial and visceral mesoderm epithelia as well as in axonal growth. A deficiency in the 47A region in which is localized the bkh gene was obtained by using as a starting tool the homozygous viable AQ65 line whose lac Z reporter gene was specifically expressed in the entire nervous system. Among the 5,000 chromosomes mutagenized by x-ray irradiation and screened for the loss of the phenotypic white marker, a lethal deficiency Df(2R)47A was obtained. It was shown by cytology that it had lost half of each of the A and B bands in 47 (not shown). Quantitative analyses of Western blots revealed that in animals heterozygous for that deficiency, half the amount of the BKH protein was produced as compared with its production in the initial AQ65 line (not shown). Moreover, no mRNA could be detected in homozygous mutant embryos . Three P-insertion lines, issued from the systematic screen of lethal P-insertions on the second chromosome , have been located within the cytological region of the bkh gene. In the P07810 insertion, the lac Z reporter gene expression was superimposable to that of the bkh mRNA , whereas, in the P06915 and P11003 insertions, the β-galactosidase activity was restricted to the visceral mesoderm . The different insertions were mapped by PCR in the 5′ region of the class II and class I cDNAs, respectively . The lethality in these lines was not consecutive to the insertion of the transposon but was rather due to a secondary mutation since the homozygous lethal lines were perfectly viable in trans of Df(2R)47A and of other deficiencies uncovering the same region [Df(2R)E3363, Df(2R)X1, Df(2R)Stan2, Df(2R)12]. In contrast, in the P07810 line, mobilization of the P-element has resulted in the obtention of several viable revertants and of one lethal bkh 007 white − revertant in trans of Df(2R)47A. Homozygous bkh 007 embryos had the same heart and visceral muscle phenotype as that displayed by the homozygous Df(2R)47A-deficient animals (see below). To analyze the molecular lesions resulting from the imprecise excision of the P07810 transposon, the 5′ region of the gene in which P07810 lies has been further characterized. A P1 phage covering the whole length of the bkh gene contained 4.5 kb of genomic sequence upstream of the class II cDNA ATG. This fragment was cloned, fully sequenced, and analyzed for a more detailed definition of the class II transcription unit. Three canonical potential TATA boxes were located within a 510-bp sequence downstream of the P07810 insertion site. The analysis of the bkh 007 allele showed that a segment of 839 bp was deleted that contained the three potential TATA boxes together with 311 bp of the 5′ end of the transcription unit. As a consequence, no class II mRNA was produced in the homozygous bkh 007 embryos. Homozygous Df(2R)47A embryos (recognized by the lack of a labeled CyO balancer) harbored defects in the three tissues in which bkh was expressed with a total penetrance meaning that all homozygous Df(2R)47A embryos had defects in the heart, the visceral mesoderm, and the nervous system. When probed with anti-EC11 antibodies , the mutant embryos showed interruptions in their dorsal vessel that were also visible when DMef 2 expression was investigated in all the muscle cells including the cardial cells . The cardial cells were unambiguously present and they had migrated properly in dorsal position, but, in some places, they were no longer arranged as a continuous layer but rather as unorganized clusters of cells . Fas III expression revealed the same types of defects in the visceral mesoderm . Similar alterations were detected in bkh 007 mutants with the same total penetrance (not shown). In the nervous system, longitudinal axons were often missing and important modifications were observed in the guidance and axonal growth of motoneurons (not shown). It should be recalled here that the lola gene was also deleted in the Df(2R)47A deficiency and that mutations in this gene result in missing longitudinal axons in the CNS . Since the Df(2R)47A phenotype was slightly stronger (in terms of penetrance of the phenotype) than that provoked by a loss of function of lola , this suggests a function per se for G o in axonal growth or guidance. This was confirmed by the analysis of the bkh 007 mutation which fully complemented mutations in the lola gene but continued to display a neuronal phenotype. In homozygous bkh 007 mutants, axons of the motoneurons were clearly misrouted (not shown). However, in contrast to the Df(2R)47A mutation, the longitudinal axons in the CNS were missing with a lower frequency but were often pinched . The heart and visceral mesoderm phenotypes could be rescued at least partially by expressing the class II cDNA under the control of 24B-GAL4 that drives the expression of UAS-cDNAs in the myogenic lineage including the cardioblasts and the visceral mesoderm. In >65% ( n = 250 observed embryos) of homozygous Df(2R)47A embryos, a normal development of these tissues had been restored . In contrast, the phenotype in the nervous system was not abolished as expected, first because the deficiency uncovers the lola gene, and second because of the restricted specificity of expression of the 24B-GAL4 line. The bkh 007 mutation that affects only the bkh gene could also be rescued by a bkh transgene (>80% of homozygous bkh 007 embryos were rescued over a total of 147 embryos examined). The loss of bkh function produced heart and visceral mesoderm phenotypes that suggest a participation of the G o protein in epithelia formation. During the mesenchymal-epithelial transition, subsets of membrane and cytoskeletal proteins localize to distinct regions of the cell surface to create the apical and basal-lateral membrane domains that confer to the cells their epithelial polarity. The bkh mutant phenotype was more accurately described with the aid of markers localized specifically to these different domains. We focused our analysis on regions of the mutant heart in which the cells formed a continuous uninterrupted layer but which displayed abnormal marker expression. As shown in Fig. 8 , b and d, the localization of two polarity markers was affected in several places in the heart epithelium of bkh mutant embryos. α-Spectrin, specific for the basal-lateral membrane of epithelial cells, was now expressed on the entire surface of the mutant cells, however with a somewhat lower intensity than in wild-type cells. In addition, these mutant cells remained round, had no signs of shape remodeling, and failed to express the EC11 antigen in their basal membrane. The pericardial cells which are normally attached to the basal membrane of the cardioblasts were absent from these same regions and were rather often associated in clusters in the domains of high EC11 antigen expression (not shown). Similarly, Nrt which was localized to the apical and lateral membranes of wild-type cardial cells was, in bkh embryos, scattered in several locations on the entire surface of those cells which were round and in which the epithelial array was disorganized . Finally, Fas III expression also revealed phenotypes in the visceral mesoderm characterized in some regions by a uniform and lower Fas III staining on the surface of still round cells with a partially destroyed epithelial structure . In a deficiency, Df(2R)47A, which completely deleted the brokenheart gene encoding the α subunit of the heterotrimeric G o protein, the morphogenesis of the heart and of the visceral mesoderm was impaired. The defects associated to the nervous system have not been studied in detail, essentially because this deficiency also uncovered the longitudinal absent gene ( lola ) whose requirement for axonogenesis of longitudinal fascicles is known . However, several arguments strongly suggest that the phenotypes associated to the heart morphogenesis and to the visceral musculature were a consequence of a loss of the function of bkh . First, in deficient animals, alterations were restricted to the tissues that expressed bkh and they appeared in synchrony with the temporal expression of bkh . Second, P-element mobilization in enhancer trap lines located within the bkh gene and imprecise excision of the P-transposon led to the isolation of mutants with phenotypes identical to those observed in homozygous Df(2R)47A embryos. Finally, we were able to rescue the heart and visceral mesoderm embryonic phenotypes in homozygous bkh mutants by expressing the cDNA encoding bkh in transgenic flies. The failure to totally rescue the phenotype probably resulted from the fact that 24B-GAL4 did not drive the expression of the UAS- bkh cDNA in mesodermal cells early enough to be totally efficient . Although the heart was perturbed in the totality of the mutant embryos (100% penetrance), defects were observed only in some of the cells that constituted the dorsal vessel. This result does not support well the contention that bkh is an essential partner in the acquisition of polarity for the epithelial cardial cells since, if this were the case, all the cells would be equally affected. The maternal protein which is abundant and stable until mid-embryogenesis could have partially compensated the loss of zygotic transcript. Production of germline clones lacking bkh activity could help to elucidate this point. Moreover, another heterotrimeric G i protein is expressed in the cardial cells and functional redundancy might have been at work. The myoendothelial heart tube is considered as a secondary epithelium that forms by a mesenchymal-epithelial transition . The mesenchymal cardial precursor cells, after their migration from the ventral site of gastrulation in the direction of their final dorsal position, reorganize their plasma membrane to acquire their cellular polarity and establish cell junctions to build up the cardial epithelium. A signal must be received by the mesenchymal cells to create the first asymmetry on their surface . Interactions with localized extracellular matrix components are believed to be largely responsible for this first event and Drosophila mutants, for example, in Laminin A or in an integrin subunit , present severe disruptions in their dorsal vessel. The initiating signal is probably emitted in response to inductive interactions between the mesenchymal cells and the overlying dorsal ectoderm. The newly created asymmetry could then trigger a reorganization of the cytoskeleton. Finally, the different membrane domains (apical and basal-lateral) will be established by the acquisition of different combinatorials of membrane proteins that have been specifically routed towards them; this sorting-out step relies on an absolute specificity of the vesicular traffic . In the absence of bkh function, the mesenchymal cardial cells did not remodel their shape and, in the case of the most extreme phenotype, they failed to form a continuous row of cells. In addition, whenever such a layer was eventually formed, in several places some but not all cells neither acquired nor maintained a proper polarity. Based on the timetable of bkh expression, its participation in the first step creating the asymmetry of the cardial cells is not very likely. It might rather be required for the subsequent steps that concern cell shape change and polarization by addressing membrane proteins to their respective domains. It has been suggested that heterotrimeric G proteins could contribute to the vesicular protein traffic by regulating early steps in the secretory pathway . This hypothesis stems from the observation that AlF4 − , an activator of heterotrimeric but not of monomeric G proteins, inhibits ER to Golgi and intra-Golgi transport as well as vesicle budding from the trans-Golgi network. In particular, G o proteins have been implicated in granule exocytosis from chromaffin cells , insulin secretion , and transcytosis . It has been shown that the secretion of the protease Nexin-1 by glioma cells was under the control of G o 1 . The G o α1 protein was detected on the membrane of small intracellular vesicles and the secretion of Nexin-1 was stimulated by G o α1 overexpression and by activators of G o proteins such as mastoparan. It was further suggested that the GTPase activity of the G o α1 protein could be stimulated in the absence of a classical serpentine receptor . Thus, we are tempted to predict that the Drosophila G o protein has a function in a particular type of vesicular traffic responsible for the acquisition or maintenance of cell polarity in the cardial and visceral mesoderm cells. Preliminary observations on the subcellular localization of Bkh in embryonic cells were consistent with this prediction. In the totality of the cells examined, Bkh was located to the cytoplasm rather than associated to the cell membrane and the staining pattern revealed a typical granular appearance (data not shown). Since the exportation and the localization to the plasma membrane of the protein markers we have used were all equally affected in bkh mutants, we conclude that bkh is involved in a general aspect of vesicular traffic rather than in the specific process of the sorting out of membrane proteins. bkh might also be required for the reorganization of the cytoskeleton. The protein G encoded by the concertina gene participates in cell shape changes taking place at gastrulation , probably via a modulation of the invaginating blastoderm cell cytoskeleton resulting from the activation of RhoA . The early role of the G o protein in the formation of the heart epithelium does not exclude a function in later events leading to the formation of the heart or to the acquisition of its function. It has been shown recently that knocking-out the G o α gene in the mouse resulted in heart dysfunction. G o -deficient mice had lost the muscarinic inhibition of isoproterenol-stimulated cardiac L-type Ca 2+ currents . It will be interesting to investigate whether the Drosophila G o protein could also be involved in such a process. Before the determination of cardiac precursors in the mesoderm, the overlying ectoderm is subdivided in segmentally repeated units partitioned into an anterior compartment (A-compartment) and a posterior compartment (P-compartment). Analysis of the expression of genes involved in the specification of mesodermal derivatives and other observations lend support to the idea that, after gastrulation, the mesoderm also becomes subdivided into segmentally repeated units, each of which consists of two separate domains . The domains that are located below the ectodermal P-compartments are subject to influences from the striped regulators eve and hh and have been termed “P-domains” or “ eve -domains.” By contrast, the development of the metameric domains that are located below the A-compartments depends largely on the striped regulators wg and slp ( sloppy-paired ) and these domains have been termed “A-domains” or “ slp -domains.” In that scheme, three basic groups of genes are at work to pattern the mesoderm either along the dorso-ventral axis ( dpp and tinman ) to specify the dorsal mesoderm, or along the anterior-posterior axis ( wg and slp ) to subdivide it into segmental units, or at defined positions to control tissue specification. For example, recent evidence suggests that wg , whose expression is restricted to striped domains in each of the A-compartments and which is required for a variety of inductive signaling events during embryonic development, is directly involved in heart formation, in that it is necessary for further subdividing of the dorsal mesoderm and for specifying cardial cell fates. Elimination of the wg function shortly after gastrulation, at a time when tin becomes restricted to the dorsal mesoderm, results in the selective loss of heart progenitor cells with little effect on segmental patterning of the cuticle or other mesodermal derivatives . From these and other observations, a picture has emerged in which specification of precardiac and dorsal somatic muscle precursors requires intersections of the dorsal domains of dpp expression with the transverse stripes of the dorsal expression of wg . However, the results presented herein are merely consistent with some precursors of the cardial cells originating from the P-domains and subjected to the influence of hh signaling rather than to that of wg . This hypothesis is somewhat difficult to verify because wg expression after gastrulation requires hh and vice versa , but, later in embryogenesis, the two signals become independent. Therefore, we have investigated the expression of Tin in temperature-sensitive hh ts2 mutant embryos submitted to a temperature shift ∼5 h after egg laying (stage 10) . In these embryos, the wild-type expression of Tin in four cardial and four pericardial cells was expanded to the two cardial cells located in the P-domains of each segment. We predict that these two tin -expressing cells are the same as the two bkh -expressing cells in this same domain. The observation that the cardial progenitor cells can be divided into two cell subpopulations is consistent with the situation in the mature heart tube in which two genetically distinct populations of cardial cells have been described . For example, tin as well as β3-tubulin and several P-lacZ reporter genes from enhancer trap lines are expressed in only four cell pairs per segment among the six pairs present. In the same line, a D-mef2 enhancer element directed lacZ transcription in four cardial cell pairs per segment consistent with a direct regulation by tin which is expressed in these same cells . Interestingly, these two subpopulations of cardial cells, respectively, reside below the anterior and the posterior ectodermal parasegmental domains . These different observations could mean that the two P-cardioblasts were specified by the inductive instruction of hh rather than by that of wg . However, it is not known whether the hh pathway provides a direct late cardiogenic signal or exerts its effect via suppressing the wg function in the posterior domain. This hypothesis is unlikely in that, at that stage, reducing the function of hh in the epidermis does not lead to any visible effect on the wg signaling pathway . Indeed, wg expression in the dorsal epidermis was not expanded in a hh mutant (not shown). We predict then that hh might behave as a repressive signal for tin expression in the two P-cardioblasts. Cell heterogeneity in terms of gene expression could then be achieved along the anterior-posterior axis by an efficient cooperativity of wg and hh signals in the specification of the cardial cells. | Study | biomedical | en | 0.999997 |
10352023 | T47D cells (American Type Culture Collection) were transfected with constitutively active constructs for R-Ras(87L) or (38V), TC21(72L), N-Ras(12D), or K-Ras(12V), or with dominant negative constructs for R-Ras(41A) or (43N), or TC21(26A) that were expressed in pZIP. Cells were selected in G418 as described and expanded as pools of stably transfected cells. Control cells were transfected with pZIP alone and selected in parallel with the other cells. Expression of constructs was determined by immunoblotting. Because these different molecules could not be probed by the same antibody on the same blot, it was impossible to determine absolute expression levels of R-Ras compared with TC21, N-Ras, or K-Ras. However, a comparison of each molecule to endogenous levels was made: R-Ras(87L) was 8.8-fold above endogenous R-Ras (or 8.8×), R-Ras(43N) = 10.7×, R-Ras(41A) = 1.8×, TC21(72L) = 4.0×, TC21(26A) = 1.0×, N-Ras(12D) = 0.9×, and K-Ras(12V) = 2.1×. For double transfectants to create X4 chimeric cell lines, cells were cotransfected with X4 chimeras expressed in pSFneo and pCGN (20:1), and selected in hygromycin-containing growth medium as described . Expression of α4 integrin on the surface of the cell, indicating expression of the X4 chimeras, was determined by flow cytometry (see below). X4 chimeras were a gift of Dr. Martin Hemler (Dana-Farber Cancer Institute). Migration and invasion across transwells were performed as described previously . Transwells were coated from the underside with 6 μg/ml collagen I or 25 μg/ml fibronectin (Collaborative Biomedical Products). For studies with X4 chimeras, transwells were coated with specific α4 ligands: 6 μg/ml of the 40-kD chymotryptic heparin binding fragment of fibronectin ( GIBCO BRL ) or 50 μg/ml of the CS-1 peptide (EILDVPST; Peninsula Labs Inc.) . CS-1 peptide was coupled to ovalbumin as described and the best coating concentration for cell migration was determined. Cell migration was performed in the absence of serum or growth factors for 16 h. For inhibitor assays, cells were pretreated for 15 min with DMSO (control), Wortmannin (30 nM), LY294002 (25 μM), bisindolylmalemide (1 μM) all from Calbiochem-Novabiochem Corp. , or PD98059 (25 μM; Alexis Biochemicals), and allowed to migrate across collagen in the continued presence of the inhibitor for 5 h. For antibody inhibition assays, cells were preincubated with control IgG or anti–α2 integrin antibody, P1E6 ( GIBCO BRL ) at a final concentration of 1:1,000. Migration was performed in the continued presence of the antibody for 16 h. Invasion was assessed on serum starved cells that were seeded onto the top of 2-mm collagen gels and allowed to migrate in response to 10% serum placed in the lower chamber. Invasion assays were allowed to proceed for 26–36 h. Unless otherwise noted, values presented are the mean ± SEM of at least three experiments normalized to control values. Data were analyzed by performing ANOVA using Microsoft Excel software. Morphogenesis in three-dimensional collagen gels was assessed after 7 and 16 d as described . Adhesion to Immobilon plates (Dynatech) coated with various concentrations of collagen I or fibronectin was performed and adherent cells quantitated by hexosaminidase activity as described . Values given represent the average of two separate experiments, each of triplicate determinations, ± SEM. Cells were detached in versene, washed in PBS containing 5 mg/ml BSA, and incubated with primary antibody on ice. To determine cell surface integrin expression, cells were incubated with the following mAbs: P4C2 against the α4, P1D5 against the α5, P1E6 against the α2, or P1B5 against the α3 subunits (all from GIBCO BRL ), or TS2-7 against the α1, or 4F10 against the α6 subunits (Serotec). Primary antibody was diluted 1:1000. After a 20-min incubation, cells were washed once in PBS/BSA, and incubated with FITC-conjugated donkey anti–mouse secondary antibody at 1:100 ( Jackson Labs ) for 20 min on ice. Cells were washed again and analyzed on a Beckman FACScan ® . For conformation studies, cells were either treated with EDTA or with manganese at 2 mM before incubation with 12G10 antibody (Serotec), which detects conformational changes in the β1 integrin subunit. After this primary antibody incubation, analysis proceeded as above. Cells were pretreated with DMSO or bisindolylmaleimide (1 μM) for 20 min, lysed in buffer containing 0.05% Triton-X 100, and the in vitro activity of PKC was determined on crude lysates using the PKC assay system (SignaTECT; Promega Corp. ), according to manufacturer's directions. In brief, a single purification step over a DEAE cellulose column was used to generate a crude PKC-containing fraction. Background activity (nonspecific) was determined in the absence of phosphatidylserine and diacylglycerol, and compared with PKC activity determined in the presence of phosphatidylserine and diacylglycerol. Cells expressing R-Ras(87L), R-Ras(38V), TC21(72L), K-Ras(12V), N-Ras(12D), or control vectors were cultured overnight either in the presence or absence of serum, treated for 20 min with DMSO (control) or PD98059 (25 μM), and lysed. Lysates were electrophoresed, immunoblotted with anti-p42/44 ERK antibody ( Santa Cruz Biotech ), and the activation of MAPK was determined by gel-shift . Determination of the mean image density was made using NIH image software. Well differentiated human breast carcinoma T47D cells maintain the ability to polarize into tubule and ductlike structures when cultured in three-dimensional collagen gels . This differentiation is dependent on the α2β1 integrin . One of the characteristics of T47D differentiation into tubules is the multicellular nature of the structure, such that individual cell–cell boundaries become more difficult to distinguish, as can be noted in Fig. 1 A. Stable expression of activated R-Ras(87L) in T47D cells disrupted the ability of these cells to differentiate and polarize in collagen gel culture, instead the cells grew as disorganized clumps and sheets of individual, nonpolarized cells . Similar results were obtained with cells stably expressing another activated R-Ras isoform, R-Ras(38V) (not shown). Thus, activation of R-Ras results in a less differentiated, more transformed phenotype. In contrast, stable expression of dominant negative R-Ras(43N) enhanced differentiation; tubule structures developed earlier and were more extensive . This suggests that endogenous R-Ras may play a negative role in regulating the rate or extent of normal epithelial differentiation. Like R-Ras, expression of constitutively active TC21(72L) disrupted differentiation in collagen gels , whereas expression of dominant negative TC21(26A) enhanced differentiation in a qualitative manner . This suggested that TC21, like R-Ras, is a negative regulator of breast cell differentiation. Breast carcinoma progression is accompanied by the loss of polarization and differentiation, and the acquisition of a migratory invasive phenotype. Migration was determined using a Boyden chamber haptotactic assay in which transwells were coated on the underside with collagen. Expression of activated R-Ras isoforms, R-Ras(87L) or R-Ras(38V), significantly enhanced cell migration relative to control cells , indicating that R-Ras activation promotes a more migratory phenotype. Dominant negative R-Ras(41A) inhibited migration by ∼45–50% , suggesting a contribution of R-Ras to basal migratory mechanisms. Activated TC21(72L) also enhanced cell migration, albeit to a somewhat lesser extent than R-Ras . Expression of activated isoforms of other Ras family members, N-Ras(12D) and K-Ras(12V), also enhanced cell migration across collagen, but to a lesser extent than R-Ras . The migration induced by R-Ras(87L) across collagen-coated filters was dependent on the α2β1 integrin, since it could be completely inhibited by anti-α2β1 blocking antibodies . Similarly, TC21(72L)-induced cell migration was also α2β1 integrin-dependent . These results are consistent with the finding that the α2β1 integrin is a major collagen receptor in breast epithelial cells that mediates many of their responses to collagenous matrices . To examine whether the effect of R-Ras was specific for collagen, cell migration across fibronectin was determined. Surprisingly, expression of R-Ras(87L) or R-Ras(38V) did not enhance and, in fact, partially inhibited migration across fibronectin by ∼40% . R-Ras(87L) did not enhance or inhibit migration of cells across laminin (not shown). Similar results were obtained for TC21(72L), which also inhibited migration across fibronectin by 25%, although this difference was not statistically significant . The difference between migration across collagen and fibronectin is not due to dramatically different levels of migration, since similar numbers of control cells migrate across each substratum, as shown by a representative experiment in Table I . In contrast to the results with R-Ras and TC21, expression of either N-Ras(12D) or K-Ras(12V) enhanced migration across fibronectin . Thus, R-Ras and TC21 differ from N- and K-Ras in the specificity of substrata on which they enhance cell migration. To metastasize, cells must not only migrate but must also invade through extracellular matrices. Therefore, we examined the ability of transfected cells to invade through a 2-mm-thick collagen gel in response to a serum gradient. Consistent with their more migratory phenotype, cells expressing activated R-Ras(87L) or R-Ras(38V) were significantly more invasive through collagen than control cells . Similarly, activated TC21(72L), N-Ras(12D), and K-Ras(12V) induced cell invasion . Because basal invasion levels of T47D cells are already so low, we did not further investigate the effect of dominant negative R-Ras or TC21 on invasion. Therefore, we find that expression of activated R-Ras or TC21 converts cells from a polarized, differentiated phenotype into a migratory and invasive phenotype. Next, we examined the effect of R-Ras on adhesion of cells to collagen and fibronectin since R-Ras activation specifically enhanced cell migration across collagen, but decreased migration across fibronectin. This was of particular interest since it has been shown that expression of activated R-Ras(38V) increases the adhesion of myeloid cells to fibronectin and vitronectin through activation of integrin subunits . Similar to these results, we found that expression of activated R-Ras(87L) enhanced adhesion to collagen by a modest but consistent amount , demonstrating an effect of R-Ras activation on the avidity of cells for collagen. Dominant negative R-Ras(41A) decreased adhesion to collagen by a similar amount, demonstrating a contribution of endogenous R-Ras to cell adhesion. The other dominant negative isoform of R-Ras, R-Ras(43N), did not affect cell adhesion (not shown). Expression of TC21 isoforms had no consistent or significant effect on adhesion of cells to collagen (not shown). In contrast to the effect on cell adhesion to collagen, expression of R-Ras(87L) or R-Ras- (41A) did not affect cell adhesion to fibronectin . This is in contrast to the results of Zhang et al. who found increased adhesion of myeloid cells to fibronectin substrata. This suggests that there are cell-type specific differences in the effects of R-Ras on different integrin subunits. To determine whether changes in cell migration and adhesion could be explained by altered expression of endogenous integrin subunits, integrin α subunit expression was examined by flow cytometry. Cells expressing activated R-Ras(87L) or dominant negative R-Ras(41A) or R-Ras(43N) showed no change in the expression of integrin subunits α1, α2, α3, α5, or α6 (not shown). Additionally, there were no changes noted in expression of α4 (not shown), an integrin subunit not normally expressed in these cells. Therefore, R-Ras–induced changes in cell migration and adhesion are not due to changes in integrin subunit expression. To examine if an apparent change in integrin avidity was reflected in a detectable change in integrin conformation, we used a conformationally sensitive anti–β1 integrin antibody, 12G10. The 12G10 epitope is known to be expressed on integrins that are activated by manganese. We found no increase in 12G10 binding, as determined by flow cytometry, in cells expressing R-Ras(87L) compared with control cells (not shown). This result suggests that changes in apparent integrin avidity because of R-Ras activation either do not correlate to conformational changes, or that R-Ras changes integrin conformation in a manner not detected by this antibody. The R-Ras–enhanced migration and adhesion of cells to collagen via α2β1, but not fibronectin via α5β1, suggests that, in these cells, R-Ras affects specific integrin subunits. Since the β subunits are identical, we speculated that R-Ras specifically signals to the α2 cytoplasmic domain. To test this hypothesis, the effects of R-Ras on a chimeric integrin containing the α2 cytoplasmic domain were compared with its effects on a similar chimera containing the α5 cytoplasmic domain. Chimeric integrins with the extracellular domain of the α4 subunit and the cytoplasmic domain of either the α2 subunit (X4C2) or the α5 subunit (X4C5) were transfected into control and R-Ras(87L)– expressing cells. Some cells were instead transfected with the complete α 4 subunit (X4C4). We selected α4 integrin chimeras since T47D cells normally do not express the α4 subunit which allowed us to monitor the expression of the chimeras in our control and R-Ras–transfected cells. Each of the α4 chimeras was expressed at similar levels on the surface of control and R-Ras–expressing cells . Since individual integrin α subunits are not expressed on the cell surface, this indicates that α4β1 heterodimers were formed. Importantly, expression of α4 chimeras did not affect the expression of endogenous α2 or α5 subunits (not shown). The ligand for the α4β1 integrin is the CS-1 sequence found in the COOH-terminal 40-kD heparin-binding fragment of fibronectin . This 40-kD fragment does not contain the RGD sequence that is the ligand for the α5β1 integrin in fibronectin. Therefore, to specifically assess the effects of R-Ras expression on the X4 chimeras, cell migration across membranes coated with either the CS-1 peptide or the 40-kD fragment was determined, since untransfected T47D cells do not migrate across these substrata (not shown). We found that expression of the X4 chimeras in T47D cells conferred the ability to migrate on the 40-kD fragment and the CS-1 peptide . Interestingly, R-Ras(87L) enhanced migration of cells expressing X4C2 and decreased migration of cells expressing X4C5 . This is consistent with our finding that R-Ras enhances migration across collagen and decreases migration across fibronectin . R-Ras(87L) also enhanced migration of X4C4-expressing cells, suggesting that R-Ras not only signals to the α2, but also the α4 cytoplasmic domains in breast epithelial cells . Cells expressing an α4 chimera containing no α cytoplasmic domain, X4C0, were unable to migrate across the 40-kD and CS-1 substrata (not shown), indicating the importance of an intact α cytoplasmic domain for cell migration. These results suggest that R-Ras activation enhances α2β1 integrin function and diminishes α5β1 integrin function, at least with regard to cell migration. We also determined whether cells expressing α4 chimeras had altered migration across collagen. Since the α4 chimeras do not bind collagen, any effect will be due to pleiotropic effects on endogenous integrin subunits. Expression of X4C2, but not X4C4 or X4C0, blocked the increase in migration of cells across collagen that is induced by activated R-Ras(87L) , suggesting that there is some form of competition between the X4C2 subunit and the endogenous α2 subunit. Thus, the effects of R-Ras on endogenous α2 subunits can be antagonized specifically by de novo expression of α2 cytoplasmic domains. Such dominant negative effects of integrin cytoplasmic domains have been noted previously with expression of exogenous β cytoplasmic domains . However, this is the first demonstration of transdominant inhibition by a specific α integrin subunit. This suggests that there may be competition for a limited supply of α cytoplasmic domain binding factors. Conversely, X4C5 expression slightly enhanced the R-Ras(87L)–induced migration across collagen , although this enhancement is not statistically significant when compared to the results obtained with cells expressing X4C4 or X4C0. These results provide additional evidence that R-Ras signaling involves specific integrin subunits. We previously found that migration induced by the small GTPases, Rac and Cdc42, was dependent on PI3K . Like Rac and Cdc42, R-Ras also has been shown to activate PI3K . Therefore, we determined whether pharmacological inhibitors of PI3K, Wortmannin, and LY294002, could inhibit the migration induced by R-Ras. Wortmannin (30 nM) and LY294002 (25 μM) significantly inhibited the migration induced by activated R-Ras(87L) or R-Ras(38V) . However, unlike the total inhibition previously noted with Rac and Cdc42-expressing cells , the effect of PI3K inhibition on R-Ras–induced migration was only partial; migration was inhibited only by ∼50%. Wortmannin and LY294002 also only partially inhibited the migration induced by activated TC21(72L) . Thus, PI3K activity contributes to R-Ras–induced cell migration, but other signaling pathways are probably also involved. PKC has been implicated in the migration of some cell types . Pretreatment of cells with 1 μM bisindolylmalemide, an inhibitor of most isoforms of PKC, significantly inhibited the migration of R-Ras(87L) or R-Ras(38V)–expressing cells . Once again, inhibiting PKC produced only partial inhibition of R-Ras– induced cell migration. Bisindolylmalemide also partially inhibited the migration of cells expressing TC21(72L) and K-Ras(12V), albeit to an even lesser extent than the effect on R-Ras cells (not shown). The concentration of bisindolylmalemide used was adequate, since pretreatment of cells with this concentration inhibited PKC activity to background levels in an in vitro PKC kinase assay (data not shown). Since certain isoforms of PKC (δ, ε, and η) can be activated downstream of PI3K , we wished to determine if PKC and PI3K might be part of the same signaling pathway leading to cell migration, or separate pathways that both contribute to migration. Treatment of cells with Wortmannin and bisindolylmalemide had an additive effect and completely inhibited the increased migration induced by R-Ras expression , suggesting that PI3K and PKC each contribute to R-Ras–induced cell migration through separate pathways. Other pharmacological inhibitors of signaling pathways potentially downstream of R-Ras were also tested. The MEK inhibitor, PD98059 , had no effect on migration induced by activated R-Ras or TC21 , despite inhibiting MAPK activation 50–60% in cells expressing R-Ras(38V), R-Ras(87L), or TC21(72L) (data not shown). In sharp contrast, PD98059 significantly inhibited K-Ras–induced cell migration , demonstrating important differences in the mechanism by which R-Ras and K-Ras induce migration. Rapamycin, an inhibitor of p70 S6 kinase, also did not inhibit cell migration (not shown), which is consistent with previous results with Rac and Cdc42 . In this study, we show that activated R-Ras disrupts mammary epithelial polarization in three-dimensional collagen gels and induces migration and invasion across collagenous matrices. These results implicate a role for R-Ras in the transformation of mammary epithelial cells, a role that has not been described previously. We find similar results for a molecule closely related to R-Ras, TC21, which extends previous results by Clark et al. demonstrating that TC21 can alter breast epithelial morphology. We also find that R-Ras enhances cell adhesion to collagen, which is consistent with its previously described role in promoting increased integrin avidity . Importantly, R-Ras enhances cell migration and adhesion specifically on collagen, but not fibronectin. This substratum specificity reflects a difference in the effect of R-Ras on the α2 versus the α5 integrin cytoplasmic domains. Such specificity has not been described previously for the effects of R-Ras on integrin subunits, and could have important implications for inside-out integrin signaling events that lead to migration and invasion on collagenous matrices. Finally, migration induced by R-Ras is partially blocked by inhibitors of PI3K and PKC, but not by a MEK inhibitor, suggesting that unique combinations of signaling pathways are activated by R-Ras compared with other members of the Ras superfamily. Currently, it is not understood how different integrin subunits specifically link to different inside-out or outside-in signaling pathways. Here, we demonstrate that R-Ras can stimulate cellular migration that is dependent on the α2β1 or α4β1 integrin, but not the α5β1 integrin. In fact, R-Ras activation seems to have a slight inhibitory effect on α5β1-mediated cell migration. The effect of R-Ras is specifically on the α cytoplasmic domain since only this domain differs between the X4C2 and X4C5 chimeras. Our results are consistent with other observations that α subunits appear to regulate the specificity and appropriateness of integrin response. For example, swapping integrin α cytoplasmic domains on the α2 integrin changes the response of cells to collagen , whereas deletion of the α2 cytoplasmic domain causes unregulated recruitment of integrins into focal complexes, even when the cells are attached to fibronectin . Here we are able to link a specific signaling pathway involving R-Ras to the α2β1 integrin, which may help explain these previous observations regarding specific α2 subunit effects. Activation of MAPK by integrins also depends on specific integrin subunits . Specifically, α5β1 integrin, but not α2β1 integrin, associates with Shc and activates the MAPK pathway in osteosarcoma cells . The failure of α2β1 to couple to the MAPK pathway may partially explain why R-Ras–induced migration through α2β1 was unaffected by MEK inhibition. Our results imply that cell migration will differ on different matrices, depending in part on the signaling pathway that has been activated. Our finding that R-Ras enhances cell adhesion to collagen suggests that R-Ras enhances α2β1 integrin avidity for its ligand. An increase in integrin avidity is consistent with the findings of Zhang et al. . However, we find that R-Ras enhances α2β1- but not α5β1-mediated adhesion, whereas Zhang et al. noted that R-Ras enhances adhesion to fibronectin through α4β1, α5β1, and αvβ3 integrins . This difference could relate to the difference between the myeloid cells used in those studies and the epithelial cells used here. The R-Ras– induced increase in avidity determined by cell adhesion could not be correlated to conformational changes of the integrin as detected by the 12G10 anti-β1 antibody. Other investigators have also noted a lack of correlation between ligand binding capability and β1 conformation as detected by various conformationally sensitive antibodies . Our result could mean that R-Ras alters integrin conformation in a manner not detected by this antibody. Alternatively, changes in integrin avidity after R-Ras activation may not be due to obvious conformational changes but to alterations in integrin clustering, adhesion strengthening, or cytoskeletal attachments. It is not clear at this point how R-Ras stimulation differentiates between the α2 and α5 integrin subunits. An attractive model invokes proteins or factors that bind specifically to either the α2 or α5 integrin subunits. Stimulation of R-Ras may alter the binding properties of such molecules, perhaps through phosphorylation/dephosphorylation events or membrane targeting. These molecules could bind to specific integrin subunits and activate their ligand binding properties, or could alter integrin association with the cytoskeleton. Alternatively, these factors could be inhibitory, such that R-Ras activation releases the integrin subunit from inhibition by these factors. Our finding that the X4C2 chimera blocks the R-Ras–induced migration of cells on collagen, which is not a ligand for X4C2, suggests that X4C2 acts as a dominant negative molecule in this case, presumably through competition for these putative integrin binding molecules. Such transdominant inhibition has been suggested to explain how one integrin β subunit can inhibit the function of another or how chimeras expressing the cytoplasmic tail of the β subunit suppress integrin function . Interestingly, transdominant β1 integrin suppression can be overcome by expression of CD98 , lending support to the notion that accessory binding molecules regulate integrin function. To our knowledge, ours is the first demonstration of transdominant inhibition on α integrin subunits and implies that factors bind not only to the β, but also the α cytoplasmic domains. Future studies should identify factors specific for the α2 cytoplasmic domain that may play a role in R-Ras–enhanced integrin adhesion and migration. The fact that R-Ras specifically enhances α2β1 integrin– mediated migration and disrupts tubulogenesis is interesting in light of previous work specifically implicating the α2β1 integrin in these events in mammary epithelial cells . Thus affecting α2β1 function, either through changes in expression levels or by inside-out signaling events, has important consequences for the phenotype of these cells. It is intriguing that apparent increased function of the α2β1 integrin by R-Ras results in the same outcome of increased migration and decreased cell polarization that is noted when α2β1 levels are decreased . This could indicate that R-Ras activation fundamentally changes the signaling pathways used by the α2β1 integrin, disrupting the normal signaling events that cause the cell to differentiate and polarize in response to the extracellular matrix. These same normal signaling events could be disrupted or diminished when α2β1 levels are decreased, as is noted in breast carcinomas . It will be interesting in future studies to determine exactly how cross-talk between the α2β1 integrin and R-Ras affects cellular differentiation, migration, and invasion. We find a new role for endogenous R-Ras and TC21 in the differentiation and migration of breast epithelial cells. Dominant negative R-Ras inhibits basal cell migration, suggesting that endogenous R-Ras plays a role in the migration of breast epithelial cells. Additionally, we find that inhibitors of PI3K and PKC partially inhibit basal migration, consistent with our model that these molecules are part of the mechanism by which R-Ras contributes to cell migration, whether basal or induced. This further suggests that the signaling pathways under investigation here are likely relevant to understanding the migration of spontaneously occurring breast carcinomas. Dominant negative isoforms of R-Ras or TC21 also enhance tubulogenesis in three-dimensional collagen culture, indicating that antagonizing activated R-Ras or TC21 promotes differentiation. This suggests that activated R-Ras and TC21 are negative regulators of breast cell differentiation, consistent with the finding that expression of activated R-Ras or TC21 isoforms disrupt tubulogenesis. These results could suggest that activation of R-Ras and TC21 turns on signaling pathways that are incompatible with the decision of a cell to slow proliferation and differentiate. Thus, inappropriate activation of R-Ras or TC21 could result in a cell with a more migratory and less differentiated phenotype, which is consistent with a role in transformation. Our results add to the growing body of evidence pointing to an important role for PI3K in cellular migration events . The effect of R-Ras on PI3K possibly is direct since it has been shown that R-Ras binds to PI3K in vitro and activates PI3K in Cos-7 cells . Since R-Ras has direct effects on integrin avidity, it is interesting that a role for PI3K in affecting integrin activation has been shown in lymphocytes and platelets . Other Ras superfamily members, including H-Ras, Cdc42, and Rac, also bind and activate PI3K , which in the case of H-Ras plays an important role in cellular transformation . We previously found a role for PI3K in migration induced by Rac and Cdc42 as well as by N- and K-Ras (Keely, P., unpublished observation). Consistent with these observations, expression of activated PI3K promotes migration and invasion . Although R-Ras is similar to Ras, Rac, and Cdc42 in its use of a PI3K signaling pathway, it differs in its use of other signaling pathways. Migration induced by R-Ras or TC21 was only partially blocked by PI3K inhibitors; this effect was much less dramatic than for migration induced by Rac and Cdc42, which could be completely blocked by the same concentrations of these PI3K inhibitors . This suggests that PI3K-independent signaling pathways also contribute to R-Ras– stimulated migration in these cells. Although MEK and MAPK activation have been implicated in cell migration in various cells , we found that MEK inhibition did not affect migration induced by R-Ras or TC21. In contrast, MEK inhibition completely abolished migration induced by K- and N-Ras. These results are consistent with observations that R-Ras activates PI3K but not Raf , unlike H-, N-, and K-Ras, which activate both PI3K and the Raf-MAPK pathway . Since a recent report suggests that the Raf-MAPK pathway might be involved in transformation by TC21 , our results suggest that the mechanisms by which TC21 induces migration and transformation differ. Moreover, it appears that different small GTPases stimulate cell migration by activating different combinations of downstream signaling pathways. The use of different signaling pathways may explain why R-Ras differs from N- and K-Ras in stimulating migration across fibronectin. We find a role for PKC, in addition to PI3K, in migration induced by R-Ras. Although certain isoforms of PKC (δ, ε, and η) can be activated downstream of PI3K and contribute to cell migration , the additive effect of PI3K and PKC inhibitors in our assays suggests that PKC is on a separate pathway from PI3K in these cells. Whether PKC is downstream of R-Ras, or part of an independent obligate pathway remains to be determined. To this end, we did not observe an increase in in vitro PKC activity in cells expressing R-Ras in preliminary experiments (our unpublished observation), although translocation of PKC to the membrane and subsequent activation in R-Ras–expressing cells cannot be ruled out. Dominant negative R-Ras was unable to block cell migration induced by PMA (our unpublished results), suggesting PKC is not upstream of R-Ras in our system. In addition to R-Ras, we find a role for PKC in migration induced by K-Ras. Others also have noted synergy between PKC and small GTPases. The Rac exchange factor, Tiam-1, can be activated by PKC , which would place PKC upstream of Rac activation. Similarly, PKC is upstream of Ras activation in platelets . Additionally, PKC activation synergizes with Rho to induce focal adhesion kinase phosphorylation, cell spreading, and actin assembly . Interestingly, activation of the α2β1 integrin in monocytes by ligand binding to α5β1 requires PKC , which would be consistent with a model in which PKC is part of the pathway by which R-Ras enhances avidity of the α2β1 integrin. Moreover, PKC activation is involved in activation of the αIIbβ3 integrin by a number of agonists in platelets . Our results are also consistent with other observations that PKC activity contributes to cell migration . In summary, we find that activation of R-Ras stimulates unique combinations of downstream signaling pathways compared with other GTPases of the Ras superfamily. These unique signaling combinations lead to specific effects on certain integrin α subunits to alter cellular responses to collagen such as polarization, migration, and invasion. Such specificity will decide how a cell might respond to different extracellular environments, depending on which Ras family member is activated, and could ultimately determine if a given carcinoma is metastatic or not. These differences have important implications for targeting signaling pathways in antimetastatic therapies since affecting certain signaling pathways may not have the desired effects on all neoplastic cells. | Study | biomedical | en | 0.999999 |
10352024 | Male Sprague-Dawley rats (125–150 g) were supplied by Charles River Breeding Laboratories. Wortmannin, cycloheximide, cytochalasin D, and nocodazole were purchased from Sigma Chemical Co. Wortmannin, cytochalasin D, and nocodazole were stored at −20°C as 10-, 1-, or 16.5-mM stock solutions, respectively, in DMSO. Cycloheximide was prepared as a 10-mg/ml stock solution in 5% ethanol and used directly. LY294002 was purchased from Calbiochem Corp. and stored at −20°C as a 10-mM solution in DMSO. Cell culture media and FBS were purchased from GIBCO BRL . Fluorescently labeled secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Anti–β-tubulin antibodies were obtained from Sigma Chemical Co. Texas red–conjugated phalloidin was purchased from Molecular Probes Inc., and stored at −20°C as a 200-U/ml stock in methanol. The antibodies recognizing the 120-kD lysosomal glycoprotein (LGP-120), mannose 6-phosphate receptor (M6P-R), 5′nucleotidase (5′NT), and endogenous canine plasma membrane antigens (3F2 and G12) were kindly provided by W. Dunn (University of Florida, Gainesville, FL), Peter Nissley (National Institutes of Health, Bethesda, MD), Paul Luzio (Cambridge University, Cambridge, UK), and George Ojakian (State University of New York, Oswego, New York), respectively. Antibodies against aminopeptidase N (APN), CE9, HA4, asialoglycoprotein receptor (ASGP-R), HA321, syntaxin 3, and endolyn-78 were all prepared by the Hubbard laboratory and have been described elsewhere . WIF-B cells were grown in a humidified 7% CO 2 incubator at 37°C as described . In brief, cells were grown in modified Ham's F12 medium, pH 7.0, supplemented with HAT (10 μM hypoxanthine, 40 nM aminoterpin, 1.6 μM thymidine) and 5% FBS. MDCK cells were grown at 37°C in a 5% CO 2 humidified incubator as described . For indirect immunofluorescence experiments, cells were seeded onto glass coverslips at 1.3 × 10 4 cells/cm 2 and cultured for 8–12 d (WIF-B) or 4–5 d (MDCK) until they reached maximal density and polarity . To examine the effects of wortmannin or LY294002 on the steady state distributions of various proteins, cells were incubated at 37°C up to 3 h in their respective serum-free culture medium buffered with either 20 mM Hepes, pH 7.0 (for WIF-B cells), or 44 mM NaHCO 3 , pH 7.0 (for MDCK cells), in the presence or absence of either agent . After treatment, cells were rinsed briefly in PBS and placed on ice, fixed with chilled PBS containing 4% paraformaldehyde for 1 min, and permeabilized with methanol (also chilled) for 10 min . Cells were rehydrated in PBS by three washes of 5 min each. Cells were further processed for single- or double-labeled indirect immunofluorescence according to previously published methods with the following primary antibodies: anti–HA321, –LGP-120 and –APN (rabbit polyclonals, 1:100, 1:200, and 1:300, respectively), anti– 5′NT, –endolyn and –HA4 (mouse monoclonal ascites, 1:300, 1:500, and 1:100, respectively). MDCK cells were processed for indirect immunofluorescence using anti–3F2 and –G12 (hybridoma culture supernatants, 1:10). The secondary antibodies (FITC or Cy3 goat anti–rabbit or anti–mouse) were used at 5–10 μg/ml. To assess the effects of microtubule, actin, or protein synthesis disruption on redistribution, cells were pretreated for 1 h at 37°C with nocodazole (33 μM), cytochalasin D (1 μM), or cycloheximide (25 μg/ml), respectively. Cells were incubated an additional 3 h at 37°C in the presence of wortmannin, and the continued presence of either nocodazole, cytochalasin D, or cycloheximide. The treatments were stopped by fixation and the cells were processed for indirect immunofluorescence. Anti–β-tubulin antibodies (mouse monoclonal) were diluted to 1:500. Texas red–conjugated phalloidin was diluted to 5 U/ml. Livers from fasted (12–18 h) rats (125–200 g) were surgically removed and perfused in a recirculating balanced salt solution as described in the absence or presence of 2 μM wortmannin and 50 μM leupeptin. Biopsies (0.25–1.0 g) were removed at the indicated times and fixed by immersion in Krebs buffer containing 4% PFA for at least 1 h at room temperature. The intact liver was fixed by perfusion with 200–300 ml of the same fixative. After fixation, the biopsies and intact liver were processed as described and semithin sections (0.5 μm) cut with a Reichert Jung Ultracut E microtome. The liver sections were processed for immunofluorescence according to previously published methods using the following primary antibodies: anti–APN, –HA321 (rabbit polyclonals, 1:300 and 1:100, respectively), anti–HA4 and –endolyn (mouse monoclonal ascites, 1:100 and 1:300, respectively). The secondary antibodies (FITC or Cy3 goat anti–rabbit or anti–mouse) were used at 5–10 μg/ml. The transcytosis assays were performed as described . WIF-B cells were placed in Hepes-buffered, serum-free medium and treated at 37°C with the agents as specified in Results or the figure legends. The cells were placed on ice for 5 min to cool to 4°C. Antigens present at the basolateral cell surface were labeled (those also present at the apical plasma membrane are excluded from labeling) at 4°C for 15 min with anti–APN (rabbit polyclonal, 1:50) or purified mouse monoclonal antibodies against 5′NT (purified IgG fraction, 20–50 μg/ml) or CE9 (mouse monoclonal ascites, 1:25). Total IgG was recovered from mouse ascites using EZ Sep ( Pharmacia LKB Biotechnology Inc. ) according to the instructions from the manufacturer. All antibodies were diluted in buffered, serum-free medium containing 2 mg/ml BSA in the absence or presence of the various agents. After labeling, the cells were washed extensively in buffered, serum-free medium containing 2 mg/ml BSA to get rid of excess and nonspecifically associated antibodies. Cells were then returned to 37°C and incubated for the desired times of chase in the absence or presence of the different agents . Treatments were stopped by fixation and permeabilization as described above. The trafficked antibodies were labeled with Cy3- or FITC-conjugated goat anti–rabbit or anti–mouse secondary antibodies (5–10 μg/ml) and visualized by indirect immunofluorescence. The transcytosis assay was performed as described above with anti–5′NT antibodies (20–50 μg/ml) directly conjugated to 5-nM gold particles . The cells were fixed and processed in situ (on the glass coverslip) using standard Epon embedding techniques . Ultrathin sections were cut parallel to the growth substrate, stained with lead citrate, visualized, and photographed with an EM10 transmission electron microscope ( Carl Zeiss, Inc. ). The cells and perfused liver sections were visualized by epifluorescence (Axioplan Universal Microscope; Carl Zeiss, Inc. ). Pictures were taken and processed using standard photographic techniques. Scanned images of the photographic prints were compiled, and figures prepared using Adobe Photoshop and Microsoft PowerPoint software. For Fig. 4 , WIF-B immunofluorescence was analyzed using a laser scanning confocal microscope (LSCM 410; Carl Zeiss, Inc. ). Our initial approach to examine the effects of the selective inhibitors, wortmannin and LY294002, on apical plasma membrane dynamics was to determine the steady state distributions of apical membrane proteins in WIF-B cells in treated cells. As observed for other cell types, these agents led to extensive vacuolation . By indirect immunofluorescence, we observed that the apical membrane proteins, 5′NT and APN, accumulated in vacuoles after treatment with 100 nM wortmannin or 200 μM LY294002 . Similar patterns of redistribution were observed for dipeptidyl peptidase IV, another apical membrane protein (data not shown). Vacuole size in cells treated with LY294002 was consistently smaller than observed in wortmannin-treated cells for all markers examined. This may reflect differences in the inhibitory mechanisms of these agents or the decreased potency of LY294002 . Vacuole formation and accumulation of the apical proteins into vacuoles were both time dependent, with vacuole formation preceding redistribution . By phase microscopy, vacuoles were observed within 15 min of treatment and, by 45 min, all cells contained numerous, large vacuoles . In contrast, apical proteins were first faintly detected in vacuoles after 45 min of treatment and did not reach their peak redistribution until after 120–180 min, when all cells were positive for vacuole staining . The vacuoles containing apical proteins increased in diameter as wortmannin treatment was prolonged. At 45 min, apical protein-positive vacuoles were ∼1.9 ± 0.5 μm in diameter, whereas by 180 min, the diameter had increased to an average of 3.1 ± 0.9 μm (Table I ). Both the increase in vacuole size and gradual accumulation of apical protein staining suggest the inhibitors allowed forward traffic into vacuoles, but blocked transport from them. Alternatively, the inhibitors may have altered the relative kinetics of membrane traffic to and from the vacuole. The wortmannin concentration used in this and many other published studies was 100 nM. At higher concentrations, wortmannin can inhibit other enzymes, including myosin light chain kinase and PI 4-kinase . To determine whether the effects described here were due to specific inhibition of PI 3-kinase isoforms, we incubated WIF-B cells with varying concentrations of wortmannin. The extents and rates of accumulation of the apical proteins and vacuole formation were similar in cells treated with wortmannin concentrations ranging from 10 nM to 6 μM (data not shown). Also, increased times of treatment with either agent (up to 10 h) did not lead to visibly greater extents of accumulation (see Discussion). Not all apical membrane proteins accumulated in vacuoles. Although the cells were heavily vacuolated after 180 min of treatment, both HA4 and syntaxin 3 (data not shown) maintained their apical distributions. This suggests that wortmannin and LY294002 were selectively perturbing protein trafficking rather than generally disrupting apical membrane integrity. Interestingly, the distributions of the resident basolateral plasma membrane proteins HA321 and CE9 (data not shown) did not change in the presence of either inhibitor, indicating the effect was specific to proteins at the apical surface. In addition, the staining patterns of the tight junction component, ZO-1, were not changed in the presence of wortmannin (data not shown), suggesting tight junctions remained intact during treatment. Together, these data indicate that plasma membrane polarity was not compromised in treated cells. We next determined what other molecules were present in the vacuoles containing apical proteins. The distributions of TGN-38, p115, and albumin (a major hepatic secretory protein) were unaltered by wortmannin treatment (data not shown). This is in agreement with findings from other cell types that wortmannin does not affect organelles or transport steps of the biosynthetic pathway . However, disruption of endocytic compartments has been reported in nonpolarized cells. In particular, the formation of large intracellular vacuoles derived from early and late endosomes and lysosomes has been observed . We examined whether wortmannin induced similar morphological changes in WIF-B cells. For these experiments, cells were treated for 180 min with 100 nM wortmannin and the steady state distributions of three recycling membrane proteins, ASGP-R (an early endosomal membrane protein), M6P-R (a late endosomal membrane protein) and endolyn-78 (a lysosomal membrane protein), were examined by confocal microscopy. The most notable vacuolation was observed when cells were stained for endolyn-78 or LGP-120 (data not shown). Unlike reports from other cell types, few vacuoles contained early or late endosomal markers . Rather, these markers were primarily found in intracellular structures that did not significantly overlap with the endolyn-78 positive structures. These results indicate that wortmannin was disrupting mainly lysosomal compartments in WIF-B cells. Comparison of APN and endolyn-78 staining patterns revealed striking colocalization . After 180 min of wortmannin treatment, all cells contained vacuoles that were positive for both APN and endolyn-78 . Similar results were obtained when 5′NT distributions were compared with those of another resident lysosomal membrane protein, LGP-120 (data not shown). The kinetics of appearance of endolyn or LGP-120 into vacuoles generally paralleled those of vacuole formation itself and preceded the appearance of apical plasma membrane proteins . By 45 min, the majority of cells examined contained vacuoles that were positive for endolyn-78 and LGP-120 and, by 120 min, redistribution was complete. The appearance of LGP-120 in vacuoles consistently trailed the appearance of endolyn-78. Whether this reflects differences in their cellular itineraries or kinetics is not known. From these data, we conclude that the apical plasma membrane proteins accumulated in lysosomally derived vacuoles in the presence of PI 3-kinase inhibitors. The apical proteins found in vacuoles could have originated from three possible sources: from the biosynthetic pathway, the transcytotic pathway, or from the apical plasma membrane itself. To rule out a biosynthetic origin, WIF-B cells were pretreated in the absence or presence of 25 μg/ml cycloheximide for 60 min to inhibit protein synthesis. Cells were further incubated for 180 min with 100 nM wortmannin in the continued absence or presence of cycloheximide. Cells treated with wortmannin alone or with cycloheximide and wortmannin achieved the same levels of vacuole formation (100% positive cells) and 5′NT vacuole redistribution (92 and 93% positive cells, respectively). Cells treated with cycloheximide alone were not observed to vacuolate, nor did apical plasma membrane protein distributions change. Similar results were obtained for APN, further indicating that newly synthesized apical proteins were not the source of apical molecules accumulating in vacuoles. We next investigated the possibility that the apical membrane proteins observed in vacuoles originated from the transcytotic pathway. For these experiments, WIF-B cells were pretreated in the absence or presence of 100 nM wortmannin for 15 min at 37°C. Transcytosis of apical proteins was examined after their labeling with specific antibodies at 4°C. Since the antibodies were excluded from the apical plasma membrane by tight junctions, only those molecules en route from the basolateral plasma membrane were labeled . After extensive washing, the cells were warmed to 37°C and the antibody–antigen complexes chased for the indicated times. The cells were fixed and permeabilized and the trafficked antibody–antigen complexes were visualized with secondary antibodies. After 90 min in control cells, the majority of 5′NT staining was observed at or near the apical plasma membrane and, after 180 min, nearly all transcytosing 5′NT was at the apical surface signaling its successful delivery . Similarly, in wortmannin-treated cells, 5′NT was also successfully delivered to the apical plasma membrane by 180 min. However, we observed more intracellular accumulation of the trafficked molecule after 90 min . Similar results were obtained for APN and HA4 in both control and wortmannin- or LY294002-treated cells (data not shown). We also monitored the dynamics of the resident basolateral protein, CE9, using the antibody-labeling and trafficking approach. In wortmannin-treated cells, CE9 maintained its basolateral distributions (data not shown), further indicating that PI 3-kinases differentially regulate resident proteins at each domain. Because only a 15-min wortmannin pretreatment was used in these experiments, we needed to rule out the possibility that transcytosing molecules were transported past a potential point of diversion before vacuoles were fully formed. Therefore, we pretreated cells for 120 min with wortmannin before antibody labeling of live cells. As shown in Fig. 6 , transcytosis remained fully functional. We observed no further impairment of 5′NT delivery to the apical surface and no increased intracellular staining relative to cells pretreated for only 15 min. Importantly, we also determined the steady state distributions of APN in the same cells to determine if transcytosing 5′NT molecules appeared in vacuoles. Comparison of the staining patterns in Fig. 6 , a and b, and c and d, reveals that the structures containing trafficking 5′NT were distinct from vacuoles. Some 5′NT molecules were found in discrete structures surrounding the APN-positive vacuoles, but the patterns never overlapped. These results revealed that basolateral-to-apical transcytosing molecules in wortmannin-treated cells did not accumulate in vacuoles, but seemed to traverse similar membrane compartments (albeit displaced or delayed) as in control cells. Therefore, we turned to ultrastructural analysis of antibody-antigen complexes in wortmannin-treated cells to further examine the compartments through which the molecules were traveling. In particular, we were interested in determining whether 5′NT traveled through the SAC, a known intermediate of the transcytotic pathway in hepatocytes . As shown in Fig. 5 , gold-conjugated anti–5′NT antibodies were detected at the apical surface in both control and treated cells after 90 min of trafficking. As predicted, gold particles were also observed just adjacent to the apical cell surface in the SAC. Examination of the SAC in control and treated cells revealed no significant differences in its placement, size, or number (Table II ). These data indicate that the SAC remained functional during treatment and further show that the intracellular structures containing trafficking 5′NT in wortmannin-treated cells were not an altered SAC. The identity of the compartment containing trafficking 5′NT in treated cells is not known, but may likely be an altered early endosomal compartment, another known intermediate of the transcytotic pathway in hepatocytes . Preliminary studies revealed that these structures stained positive for ASGP-R (data not shown). Also, when 5′NT and ASGP-R were trafficked together and visualized, both molecules traversed a similar compartment (data not shown), presumably an early endosome. To directly determine whether the resident apical plasma membrane proteins in vacuoles originated from the apical cell surface, we examined the trafficking of antigen–antibody complexes that were first delivered to the apical surface. For this analysis, APN or 5′NT molecules were labeled at the basolateral surface at 4°C, chased to the apical membrane for several hours at 37°C, chased an additional 2 h in the presence or absence of wortmannin, and labeled with secondary antibodies for immunofluorescent detection. As shown in Fig. 7 , b and h, nearly all the labeled APN or 5′NT molecules were at the apical surface in control cells. After the final 2 h chase in the presence of wortmannin, APN and 5′NT staining was observed in vacuoles . These data indicate that the apical membrane proteins found in vacuoles originate from the apical surface. This is the first direct evidence for internalization of membrane proteins from the hepatic apical cell surface. To further characterize the mechanism(s) by which resident apical membrane proteins were internalized and accumulated intracellularly, we examined the effect of cytoskeletal disruption on redistribution. Microtubules were depolymerized by addition of 33 μM nocodazole for 1 h at 37°C (as assayed by β-tubulin staining, data not shown). The cells were further incubated for 180 min with wortmannin in the continued absence or presence of nocodazole. As shown in Fig. 8 , the addition of nocodazole prevented vacuole formation, further implying that vacuolar compartments were enlarged due to accumulated cargo and membrane received from vesicular intermediates. Consistent with this conclusion, microtubule disruption also prevented any detectable intracellular accumulation of APN . Similar experiments were performed using 1 μM cytochalasin D to disrupt the actin cytoskeletal network. Although actin filaments were depolymerized, no changes in the kinetics of vacuole formation or apical protein redistribution were observed (data not shown). Because the effects of PI 3-kinase inhibitors on protein trafficking patterns and cellular morphology have been examined only in cultured cells, it was important to rule out the possibility of a tissue culture–induced effect by the inhibitors. Therefore, we treated hepatocytes in isolated perfused livers with 2 μM wortmannin for 180 min. We observed hepatocyte vacuolation with positive staining for APN and 5′NT (data not shown). As observed for WIF-B cells, the vacuoles also contained the resident lysosomal membrane proteins, endolyn-78 and LGP-120 (data not shown). HA321 and HA4 distributions did not change in treated livers, indicating that similar wortmannin-sensitive transport steps were operating in intact hepatocytes as in WIF-B cells . These results indicate that regulation of apical internalization by wortmannin-sensitive molecules occurs in the intact liver. To determine whether vacuolar accumulation of apical membrane proteins is a phenomenon specific to hepatocytes, we examined the effects of wortmannin and LY294002 on the distributions of membrane proteins in MDCK cells. As shown in Fig. 10 , wortmannin induced the formation of large vacuoles that also stained positive for the endogenous resident apical membrane protein, 3F2 . The distribution of the basolateral resident membrane protein, G12, remained unaltered by wortmannin treatment (data not shown). The kinetics of vacuolar formation and apical protein redistribution in MDCK cells were similar to those observed in WIF-B cells. Within 30 min, all cells contained numerous large vacuoles . Vacuolar 3F2 staining was observed after 30 min of treatment in ∼50% of cells and, by 120 min, nearly all cells examined contained vacuoles positive for 3F2 . These results indicate that PI 3-kinase is regulating the dynamics of resident plasma membrane proteins in MDCK cells and suggest that similar processes may be operating in other polarized epithelial cell types. Using a pharmacological approach, we have documented for the first time the internalization of apical membrane proteins from the canalicular surface of hepatocytes. When the PI 3-kinase inhibitors, wortmannin and LY294002, were applied to WIF-B cells or intact liver hepatocytes, large lysosomal vacuoles formed that contained apical plasma membrane proteins. Residents of other organelles, including the basolateral plasma membrane and early or late endosomes were completely or largely absent from these structures. The source of the apical membrane proteins was the apical plasma membrane itself. Our study has several important implications. First, our finding that PI 3-kinase inhibitors affect apical endocytosis differently from basolateral endocytosis in both MDCK and hepatic cells adds to the list of differences between endocytic properties of the two plasma membrane domains in epithelial cells. Secondly and unexpectedly, the SAC, which we identified both in vivo and in WIF-B cells as an intermediate in the basolateral-to-apical transcytosis of apical proteins, is not involved in apical endocytosis. We know this because the plasma membrane proteins found in vacuoles did not traverse the SAC. Furthermore, PI 3-kinase inhibitors have little or no effect on SAC's basolateral-to-apical function or morphology. Thus, the results of this study support our earlier hypothesis that apical plasma membrane proteins present in SAC are delivered exclusively to the apical membrane even though other SAC cargo (i.e., endolyn-78) can be sorted off to other destinations . Finally, since our results show that the life cycles of selected resident apical membrane proteins include delivery to lysosomes, we propose that degradation in this organelle is the normal fate of such membrane proteins. Mammalian cells, including hepatocytes, encode at least three different classes of PI 3-kinase isoforms . Class I includes the catalytic 110-kD subunits complexed with other accessory proteins. Class II PI 3-kinases include higher molecular weight kinases that contain C2 domains, and class III kinases share the highest sequence similarity with the sole isoform identified in yeast, Vps34p. All mammalian PI 3-kinase isoforms are sensitive to wortmannin and LY294002 at the concentrations used in the studies described here and elsewhere . This has led to ambiguity in distinguishing the roles that specific PI 3-kinases play in membrane transport. Despite the present uncertainties in a pharmacological approach, a picture emerging from studies in multiple cell types identifies the lysosomal system as the principal target of PI 3-kinase regulation. Yeast strains deficient in the sole PI 3-kinase isoform, Vps34p, missort lysosomal hydrolases from the trans-Golgi network to the extracellular medium . Recently, a mammalian homologue of this enzyme has been implicated in both transferrin recycling and delivery of activated PDGF receptor to lysosomes, pointing to its role in endosomal sorting/retrieval . However, others have reported that a p110p85 isoform (Class I), traditionally viewed as a mediator of signal transduction , also influences transferrin recycling and PDGF receptor delivery to lysosomes . Equally intriguing is the report that Vps34p, and by inference membrane transport, may play a role in mitogenesis . Clearly, we need specific probes to dissect these complex and interacting pathways. Our understanding of how PI 3-kinases regulate vesicle trafficking in mammalian cells is also in its infancy. Several proteins with roles in membrane transport have been shown to bind PI 3-kinase catalytic products (phosphatidylinositol 3 phosphate (PtdIns(3)P), PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 ) in vitro including dynamin, the clathrin adaptor protein, AP2, GRP1, cytohesin-1, EEA1 and ARNO . One possibility is that the inhibition of the formation of specific lipid species by wortmannin in turn inhibits the recruitment of these factors to cellular membranes where they function in membrane transport. In hepatocytes, the roles these proteins play (if any) in apical membrane internalization have not been identified. Alternatively, the lipids themselves may contribute to alterations in membrane physical properties that allow membrane transport to occur (e.g., fluidity, fusibility). Further experimentation is clearly needed to discriminate between these possibilities. Our finding that PI 3-kinase inhibitors differentially affect the behaviors of apical and basolateral membrane proteins in both WIF-B and MDCK cells is consistent with other pharmacological studies of endocytosis in polarized epithelial cells. For example, in MDCK cells, addition of calmodulin antagonists (W7 and trifluoperazine) or phorbol esters activated apical endocytosis of ricin, while no changes were seen at the basolateral domain . In Caco2 and MDCK cells, addition of cytochalasin D inhibited apical uptake of fluid phase markers and no changes were observed from the basolateral domain . These differences, as well as those observed with PI 3-kinase inhibitors, may reflect the specialized functions and/or environments of each membrane domain. Differential effects of wortmannin administration were also seen among apical plasma membrane proteins. APN and 5′NT (as reported here) and dipeptidyl peptidase IV and polymeric IgA receptor (Tuma, P.L., data not shown) all accumulated in lysosomal vacuoles in treated cells, whereas HA4 and syntaxin 3 maintained their apical distributions. No overt sequence or structural motifs are apparent that group the proteins in the two classes, nor are the half-lives of the proteins significantly different. Differences have been determined in the kinetics of delivery of newly synthesized proteins to the apical surface . In particular, HA4 was found to be the slowest molecule with only 15–20% detected at the apical membrane after 2.5 h. Whether slower delivery also implies slower rates of internalization from the apical membrane and, by extension, decreased vacuolar accumulation, is not known. Further examination of these molecules in terms of lipid binding properties and specific internalization mechanisms (e.g., vesicle types, signals) are required before we understand the reasons for the selective regulation by PI 3-kinases. Although wortmannin and LY294002 have been shown to impair transcytosis of dIgA and ricin in MDCK cells , the effect of these agents on cellular morphology or the distributions of apical membrane proteins had not been examined before this report. Thus, our finding that MDCK cells mirror WIF-B cells in the regulation by PI 3-kinases leads us to predict that many of the apical regulatory mechanisms described for other polarized epithelial cells may be operating at the hepatocyte canalicular domain. Based on the results of this study, we propose that apical membrane proteins are degraded in lysosomes as part of their normal life cycle. While such a fate might have been expected, this study is the first to document accumulation of resident apical membrane proteins in a lysosomal compartment of polarized hepatic cells. These results are consistent with limited examples from polarized intestinal cells . Our proposal raises several questions. For example, why are the levels of apical proteins in the vacuoles so low? From fluorescent micrographs, we estimate that <5% of the apical proteins are found there after 120 min treatment with wortmannin. The subtlety of the effect is probably not due to degradation of a portion of the population since addition of lysosomal proteinase inhibitors, such as leupeptin, together with longer exposure of cells to wortmannin, did not significantly increase the signal. Rather, we favor an explanation that takes into account the apical proteins' relatively long half-lives, which are between 3 and 6 d in vivo . That is, if such long-lived molecules remain at the apical membrane until they are retrieved for degradation, only 1–3% of the total APN or dipeptidyl peptidase IV would be sorted to lysosomes in 120 min, a value consistent with our estimations. Despite the good agreement, we cannot rule out the formal possibility that wortmannin induced endocytosis at the apical domain. Interestingly, this model assumes that the apical proteins do not recycle through an intracellular compartment(s) as part of their normal life cycle. Alternatively, the recycling compartment is not sensitive to wortmannin. Another question raised by our results is why there is any expression of apical proteins if the vacuoles are degradative. The ability to detect apical proteins in the vacuoles suggests that they have decreased degradative potential, consistent with their identification in other cells as prelysosomal intermediates. In treated WIF-B cells, cathepsin D (an acid hydrolase) staining was not observed in vacuoles, but in smaller, punctate structures, implying that mature lysosomes are not altered by wortmannin (Tuma, P.L., data not shown). Reports from NRK and human melanoma (Mel JuSo) cells have documented that dense core (and presumably end-stage) lysosomes were not changed by wortmannin with regard to their morphology or their ability to receive internalized proteins and lipid dyes . In addition, previous studies have shown that acid hydrolases are missorted to the constitutive secretory pathway in wortmannin-treated cells . Thus, similar missorting may occur in hepatic cells such that the vacuoles have decreased hydrolytic activity. A final puzzle focuses on the vacuoles themselves. If entry of apical membrane into this prelysosomal compartment continues but subsequent events are blocked by PI 3-kinase inhibition, resulting in an enlarged vacuole, what is the “normal” mechanism for maintaining the steady state dimensions of the compartment in untreated cells? A tempting hypothesis is that membrane microdomains invaginate into the luminal space and pinch off to form multivesicular endosomes/lysosomes. This scenario would accomplish both degradation of membrane components and maintenance of compartment size. However, there is limited experimental evidence for such an idea . Our recent studies of plasma membrane biogenesis have focused on the “indirect” route taken by newly synthesized apical membrane proteins and identified SAC as a subapical compartment in that route . It is important to emphasize that transcytosing apical proteins moved through the SAC and were successfully delivered to the apical surface in wortmannin-treated cells. Ultrastructural analysis confirmed these results and further revealed that the morphology, placement, and number of the subapical organelles were not changed by wortmannin. Thus, the function of the SAC as an intermediate in the transcytotic pathway was not changed. Most importantly, in the presence of wortmannin, the resident apical proteins did not travel through the SAC on their way to lysosomal vacuoles. This indicates that the SAC is not an intermediate of the apical endocytic pathway and is consistent with work in hepatocytes that biochemically identified the SAC and found it contained no recycling resident populations of apical membrane proteins . Previously, we proposed that the SAC is a “one-way” sorting station to the apical domain and the results presented here continue to support our hypothesis. What is the entry point for those apical membrane residents that were subsequently found in the lysosomal vacuoles? Endosomal structures have been identified in other polarized cells that receive proteins internalized from the apical surface . Morphologically, these compartments resemble the SAC in hepatic cells . Since we are limited in our ability to access the apical surface, we have been unable to directly test whether the hepatic SAC acts similarly. However, there are recent reports of a subapically located structure in HepG2 cells receiving fluorescently labeled glycosphingolipids (C6-NBD-glucosylceramide and C6-NBD-sphingomyelin) that have been first accumulated at the apical surface . Whether this compartment is identical to the SAC or if it receives apically internalized proteins is not currently known. Future experiments are needed to distinguish among the various possibilities and to identify the putative intracellular compartment that receives incoming cargo from the apical membrane. | Study | biomedical | en | 0.999997 |
10352025 | CF-1 mice were obtained from Sasco. Laminin, rabbit anti–mouse laminin, fibronectin, collagen type IV (Col IV), rabbit anti–mouse collagen type IV, Matrigel, Pln, human recombinant bFGF, TGF-β1, and rabbit antibody to human bFGF, which does not cross-react with mouse bFGF, were all obtained from Becton Dickinson Labware . Rat mAb against mouse Pln and mouse mAb to a Col II peptide sequence, recognizing both mouse and human epitopes, were purchased from Chemicon International, Inc. Rabbit polyclonal antibody against rat aggrecan, recognizing mouse and human aggrecan, was provided by Dr. Kurt Doege (Shriner's Hospital for Children Portland Unit). Mouse mAb to chicken Col II (antibody II-II6B3 used at 1:100 dilution, which recognizes mouse collagen type II) was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), and the Department of Biological Sciences, University of Iowa (Iowa City, IA) under contract N01-HD-6-2915 from the NICHD. HP, HP-BSA, bovine kidney HS (BK-HS), bovine intestinal mucosa HS (BIM-HS), CS, hyaluronidase, heparin sulfate proteoglycan, ascorbic acid, sodium pyruvate, and sodium citrate were obtained from Sigma Chemical Co. Species-specific, fluorescein-conjugated secondary antibodies were purchased from Amersham Corp. The mouse Pln cDNA (clone 5) was the generous gift of Dr. John Hassell (Shriners' Hospital, Tampa Bay, FL). BMP-2 and -4 and Balb-c 3T3 cells were the generous gifts of Drs. Randy Johnson and Benoit de Crombrugghe (University of Texas, M.D. Anderson Cancer Center, Houston, TX), respectively. Under the I.A.U.C.C. approved guidelines for animal use, CF-1 female mice were subjected to superovulation by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin followed by 5 IU human chorionic gonadotropin (hCG) 48 h later. After hCG injection, females were caged with stud males overnight. Females were inspected the next morning for vaginal plugs, indicating d 0.5 of pregnancy at noon. Uteri and embryos were collected on various days of pregnancy. The tissue was snap frozen in isopentane chilled by dry ice and stored at −70°C. 8-μm sections cut on a Reichert-Jung cryostat were allowed to air dry briefly and stored at −70°C until they were processed. Immunostaining was carried out as described previously . Sections were not decalcified before staining. In brief, sections or wells were fixed in 100% methanol for 10 min at room temperature, washed with Dulbecco's PBS without magnesium or calcium (D-PBS) twice for 5 min each, incubated with the primary antibody for 1 h at 37°C, washed with D-PBS three times for 5 min each, incubated with the secondary antibody for 45 min at 37°C, washed with D-PBS three times for 10 min, and mounted. Samples were stored in the dark at −20°C until they were photographed on a Leitz microscope equipped for epifluorescence. Some sections were pretreated with hyaluronidase to ensure no epitopes were masked. Frozen sections were washed in PBS three times for 4 min each before treatment with hyaluronidase (4 mg/ml in PBS, pH 5) at 37°C for 30 min. After treatment, sections were washed for 4 min in PBS and the standard staining procedure described above was followed, starting with fixation in methanol. Detection of HS chains in the tissue was performed as described previously . In brief, methanol fixed sections were rehydrated in 0.15 M NaCl, 20 mM EDTA, and 10 mM Tris, pH 8 (TEN), and incubated with human recombinant bFGF (0.05 μg/ml in TEN) for 2 h at 37°C in a humid chamber. Sections were then washed with TEN three times for 5 min each at room temperature, incubated with rabbit anti–human bFGF for 1 h at 37°C, washed again as before, incubated with FITC-conjugated donkey anti–rabbit antibody for 40 min at 37°C, washed three times for 10 min each, and mounted in glycerol/PBS (9:1, vol/vol) buffered to pH 8 with 0.5 M sodium carbonate buffer, pH 9, and containing 0.1% (wt/vol) p -phenylenediamine. Embryos were isolated on either d 14.5 or d 15.5 of pregnancy and frozen as described above. In situ hybridization was performed as described previously . The Pln probe was prepared as described previously . In brief, linearized Pln cDNA clone 5 was used in an in vitro transcription kit from Ambion Inc. Probes were purified using phenol/chloroform extraction and ethanol precipitation. Hydrolysis to reduce probe size was performed and afterwards probes were resuspended in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 10 mM dithiothreitol and stored at −70°C until use. 10-μm frozen sections were prepared as described above and used in in situ hybridization as described . Sections were not decalcified before use. In brief, sections were fixed in 4% (wt/vol) paraformaldehyde for 15 min after thawing. Each sample was incubated with 2 × 10 7 cpm/ml 35 S-labeled riboprobe after prehybridization and covered with a siliconized coverslip. After hybridization at 45°C for 4 h, sections were digested with RNase A (20 μg/ml) at 37°C for 15 min. After 2 wk of autoradiography with Kodak NTB-2 emulsion, hybridized probe was visualized after development. Sections were counterstained with hematoxylin and eosin. Tissue culture dishes of either 4 wells (Nunc) or 24 wells (Corning) were coated with 5 μg each of the following matrix components: Pln (9 nmol/ well), HP (330 nmol/well), HP-BSA (HP chains covalently linked to BSA, 41 nmol/well), BSA (75 nmol/well), bovine intestinal mucosa HS (660 nmol/well), collagen type IV (16 nmol/well), laminin (5 nmol/well), fibronectin (11 nmol/well), or Matrigel. For coating wells, 5 μg of the matrix component was added to the well followed by D-PBS to attain a total volume of 200 μl. The area of wells in either the 4- or 24-well dishes is 1.76 cm 2 . Matrigel was applied undiluted to the well as a thin layer and excess was removed immediately. The plate was allowed to dry overnight at 37°C. In all cases, on the following day wells were washed twice with D-PBS before adding cells. Coating efficiency was determined by including 125 I-iodine labeled Pln at the time of coating and counting material rinsed from the wells. Bound Pln gave a coating density of 1.5 μg Pln/cm 2 . Murine 10T1/2 cells obtained from ATCC were maintained as subconfluent cultures in tissue culture flasks with Dulbecco's modified Eagle's media/Ham's F12 (1:1) media containing 100 mg/ml streptomycin sulfate, 100 U/ml penicillin, 1.2 g/liter NaHCO 3 , and 15 mM Hepes (pH 7.2) supplemented with 10% (vol/vol) heat-inactivated FBS. For matrix assays, cells were detached with trypsin/EDTA (Irvine Scientific) and plated on matrix-coated wells in CMRL-1066 media supplemented with antibiotics and either high (15% [vol/vol]) or low (2.5% [vol/vol]) FBS for preliminary experiments. In later experiments, the media was supplemented with 15% (vol/vol) FBS, pyruvate (50 μg/mg), citrate (50 μg/ml), and ascorbic acid (50 μg/ml). Plating density was 2 × 10 5 cells per well (114,600 cells per cm 2 ). Media was added to bring the total volume in a well to 500 μl. Media was changed every other day during assays. For growth factor assays, growth factors were added at 1 ng/ml (bFGF, BMP-2 at 0.055 nM, TGF-β1 at 0.2 nM, and BMP-4 at 0.063 nM) to CMRL-1066 supplemented with 15% (vol/vol) FBS, citrate, ascorbate, and pyruvate. Medium was changed every other day and a fresh aliquot of growth factor added. Human costochondral cartilage samples were acquired from pectus excavatum surgeries after appropriate consent was obtained and with institutional IRB approval. Cartilage samples were minced and digested in collagenase, 2 mg/ml, overnight at 37°C. The chondrocytes were plated and grown in DMEM with 15% FBS. All chondrocytes were expanded in monolayer cultures, and used at passage 3. Formation of aggregates was assessed by visual examination using a microscope. Cells that had drawn together into dense, multi-layered regions reminiscent of condensing mesenchyme of developing cartilage, leaving areas of the well bare, were scored as aggregate positive. Also, cells in these regions were highly rounded compared with the fibroblastic morphology typical of 10T1/2 cells. To determine whether expression of chondrocyte markers was uniform throughout the aggregate mass, aggregates formed on Pln were fixed in paraformaldehyde, embedded in cryoprotectant, and sectioned. Alcian blue staining was performed before embedding. Sections were processed for immunostaining as described for tissue sections. Cells grown on plastic were similarly treated and scraped off the plate before embedding. Wells were coated with Pln, as described above. Before plating of 10T1/2 cells, wells were subjected to digestion with chondroitinase ABC ( Sigma Chemical Co. ) or a mix of heparinases I, II, and III ( Sigma Chemical Co. ). To each well, 0.25 ml of enzyme mix containing 1 U/ml enzymes, 0.5 mM Mg 2+ /Ca 2+ , a protease inhibitor mixture in D-PBS was added. Plates were incubated at 37°C for 4 h. After digestion, wells were rinsed with D-PBS before plating cells. As a control, 0.5 ml of CS or HP at 1 mg/ml was treated with enzyme or with buffer lacking enzyme for the same time. After digestion of control solutions, 1/10 volume 10% (wt/vol) cetylpyridinium chloride was added to precipitate and visualize GAGs to confirm enzyme activity. A successful digest resulted in at least 50% reduction of precipitated GAGs in the digested control compared with undigested. To determine if digestion removed Pln from wells, digested and undigested Pln wells were subjected to an ELISA binding assay. No loss of Pln protein core through digestion was detected by this assay. 10T1/2 cells were cultured for 10 d on various matrices, washed twice with D-PBS, and fixed for 15 min in 10% (wt/vol) paraformaldehyde in PBS. After fixation, cells were washed twice with ultrapure water before Alcian blue (1% [wt/vol]) in 0.1 N HCl was added to the cells for 30 min. Cells were washed twice with 0.1 N HCl and allowed to dry. Photography was performed on a Nikon inverted microscope using bright-field conditions. After culture for various times on different matrices, RNA was isolated from ∼4 × 10 5 10T1/2 cells per treatment using the method of Chomczynski and Sacchi . After determining the concentration of RNA for each sample, reverse transcription and polymerase chain reactions (RT-PCR) were performed. PCR primers for various chondrocyte markers were acquired from GIBCO-BRL . Collagen type I α2: forward 5′ GAACGGTCCACGATTGCATG 3′, reverse 5′ GGCATGTTGCTAGGCACGAAG 3′; collagen type II α1: forward 5′ CACACTGGTAAGTGGGGCAAGACCG 3′, reverse 5′ GGATTGTGTTGTTTCAGGGTTCGGG 3′ ; aggrecan: forward 5′ CTACGACGCCATCTGCTACA 3′, reverse 5′ ACGAGGTCCTCACTGGTGAA 3′ ; Pln: forward 5′ CCTACGATGGCCTTTCCCT 3′, reverse 5′ TTGGCACTTGCATCCTCCA 3′ . First-strand cDNA was synthesized with total RNA extracted from cultured cells by random hexamer priming using an RNA PCR kit ( Perkin Elmer ). In brief, purified total RNA (840 ng) was incubated at 42°C for 60 min with a mixture of 1 U of RNase inhibitor, 2.5 U of MuLV reverse transcriptase, 2.5 μM of random hexamers, 5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 1 mM each of dGTP, dATP, dTTP, and dCTP in a volume of 20 μl. Aliquots of 1/10 (2 μl) of the cDNA were used to amplify coding sequences from type I and type II collagen, Pln, and aggrecan. The PCR conditions for collagen types I and II were 94°C for 30 s, 60°C for 1 min, 72° for 1 min for 25 cycles, and final extension at 72°C for 5 min. The PCR conditions for Pln and aggrecan were 94°C for 30 s, 55°C for 30 s, 72°C for 1.5 min for 25 cycles, and 94°C for 30 s, 68°C for 30 s, 72°C for 1.5 min for 25 cycles, respectively. The products were separated by electrophoresis through 4% Nusieve 3:1 agarose gels (FMC Bio Products) along with molecular weight markers and detected by ethidium bromide. PCR products were subsequently sequenced and determined to be specific for either Col I or Col II. The expression pattern of Pln protein at various stages of postimplantation development was analyzed by immunohistochemistry . During early postimplantation stages, Pln was consistently detected in muscular elements such as the uterine myometrium and the developing heart in the embryo. In the d 7.5 uterus Pln was expressed in basal lamina of vascular elements, basement membrane of the epithelial cells of the residual lumen, in the amnion surrounding the embryo, and the embryo itself. At d 8.5, Pln protein was detected surrounding vascular elements in the decidua of the uterus and had accumulated to high levels in the developing lacunae as well as in the extracellular matrix of the decidua itself. At d 8.5, staining of the embryo persisted while extraembryonic tissue was negative. At later stages of development Pln protein is detected in basal lamina throughout the embryo . Staining within the liver is seen, again defining vascular elements. The amnion , consisting of two distinct layers separated by a basal lamina, stains intensely with anti-Pln. Muscular tissue such as the tongue and heart are positive, as are regions of the brain such as the choroid plexus. Notably, by d 15.5 of development, Pln staining was particularly intense in developing cartilage including the ribs and the cartilage primordia . Membranous bone regions were negative for Pln staining. To further examine the temporal expression of Pln in developing cartilage primordia, longitudinal sections containing dorsal regions from d 12.5, d 13.5, and d 14.5 were stained with Pln antibody . While at d 12.5 Pln is essentially undetectable in the condensing intervertebral disc, by d 13.5 an initial accumulation of Pln was detectable. By d 14.5 , Pln signal was clear and discretely localized within the discs, as well as in basal lamina of surrounding tissues. In situ hybridization confirmed that Pln mRNA accumulation also occurred in developing cartilage primordia . At d 14.5, the level of Pln mRNA in cartilage primordia was higher than the surrounding tissue . Sense cRNA probes demonstrated negligible background hybridization signal . After examining Pln expression in cartilage primordia, immunohistochemistry was used to determine if other basal lamina proteins, namely collagen type IV and laminin, also accumulated in these regions. As shown in Fig. 4 , in the d 15.5 embryo, Pln protein levels were high , while neither collagen type IV nor laminin accumulated in the discs, although these proteins were readily detectable in the surrounding tissue. Predigestion of sections with hyaluronidase had no effect on Pln staining and did not reveal collagen type IV or laminin staining (data not shown). Thus, these observations indicated that general accumulation of basal lamina components did not occur in developing cartilage. Immunostaining with antibodies to collagen type II demonstrated that accumulation of this early cartilage marker protein preceded that of Pln. As shown in Fig. 5 , at d 11.5 of development cartilaginous condensations are moderately positive for collagen type II , but negative for Pln . By d 12.5, a similar region is strongly positive for collagen type II accompanied by low levels of Pln . These observations suggested that while Pln expression is a component of the program of ongoing chondrogenesis, it is not an initial marker of this process. At d 15.5 Pln accumulation persisted in the cartilage primordia regions . As expected, these same structures strongly stained with Alcian blue , consistent with the cartilaginous nature of this tissue. These structures also stained positively for HS using a bFGF- dependent HS detection system . Staining by bFGF was completely blocked by inclusion of soluble HP , thus indicating that HS chains rather than FGF protein receptors were the major ligands detected in the tissue. In contrast to the pattern observed at d 14.5, in situ hybridization performed at d 15.5 did not show a preferential accumulation of Pln mRNA in cartilage primordia relative to the surrounding tissue . This finding indicated that persistent, high-level Pln mRNA expression was not required for persistent, high-level Pln protein retention in developing cartilage. Given the dramatic accumulation of Pln in developing cartilage, it was considered that Pln might potentiate the chondrogenic process. Initially, 10T1/2 cells were plated on a variety of matrices and visually inspected for morphological changes typical of chondrogenesis, i.e., aggregate formation. When plated on tissue culture plastic , 10T1/2 cells maintained their fibroblastic phenotype. If plated on Pln, cells became rounded and aggregated into nodules that stained positively with Alcian blue . A scoring index was developed to describe different behaviors observed during in vitro culture and is described in Table I . Of a variety of extracellular matrix components studied, only Pln and, to a lesser extent, the Pln-containing complexes in Matrigel were able to induce nodules (Table I ). While heparinase digestion severely reduced Pln's activity in this regard, HP, HS, or HP-BSA displayed very little activity, indicating that both the protein and HS chains of Pln are required. 10T1/2 cells plated on a variety of other matrices or tissue culture plastic maintained a fibroblastic phenotype. Some Pln preparations from sources other than Becton Dickinson did not display this activity, presumably due to differences in HS chain composition (see below). Various potential chondrogenic growth factors, including BMP-2, BMP-4, bFGF, and TGF-β1, were added to the culture medium of cells plated on inactive Pln preparations and failed to induce aggregate formation (Table II ). Moreover, extraction of Pln-coated surfaces with high salt, a procedure expected to remove HS-bound growth factors, had no effect on aggregate formation (Table I ). Thus, contamination of “active” Pln preparations with these growth factors did not account for its chondrogenic activity. Interestingly, Balb-c 3T3 cells, plated on active Pln, form aggregates, but many of these aggregates remained Alcian blue negative. Thus, while other embryonic fibroblastic cell lines respond to Pln, there appear to be differences in the degree of this response (see below). 10T1/2 cell aggregates also accumulated large amounts of collagen type II as well as aggrecan (data not shown). In contrast, 10T1/2 cells cultured on tissue culture plastic failed to accumulate collagen type II . Aggregates sectioned and probed for chondrocyte markers were positive for matrix molecules such as collagen type II and aggrecan . Alcian blue staining was also present in the aggregate . Hematoxylin and eosin staining show distinctive nonfibroblastic morphology when cultured on Pln compared with 10T1/2 cells cultured on plastic . Cells cultured on plastic again express little collagen type II . RT-PCR analyses of RNA isolated from 10T1/2 cells cultured on Pln also demonstrated increased expression of collagen type II mRNA . Plating of 10T1/2 cells on Pln digested with heparinase, to remove HS chains, resulted in delayed and incomplete aggregation into nodules . In contrast, nodule formation was both rapid and complete on chondroitinase-digested Pln and was indistinguishable from undigested Pln . ELISA demonstrated that neither heparinase nor chondroitinase digestion reduced the amount of Pln protein core bound to the tissue culture surfaces (data not shown). The kinetics and extent of differentiation on Pln is described in Fig. 11 . 10T1/2 cells efficiently aggregated within 1 d of culture on Pln and became uniformly Alcian blue positive within 4 d. Chondroitinase digestion had no effect on these parameters while heparitinase digestion severely and persistently retarded all aspects of this process. Collectively, these in vitro studies indicated that while the protein core of Pln participates in promotion of chondrogenesis, the HS chains also are required for maximal activity. Furthermore, different embryonic fibroblast cell lines display different degrees of Pln responsiveness. To examine the behavior of stable chondrocytes on Pln, human chondrocytes were plated on tissue culture plastic or Pln and examined for the expression of aggrecan or collagen type II. On plastic, human chondrocytes appeared fibroblastic and expressed lower levels of aggrecan . In contrast, chondrocytes on Pln formed aggregates maintaining their rounded morphology, and accumulated high levels of aggrecan . Similar to murine 10T1/2 cells, human chondrocytes cultured on Pln also accumulated high levels of collagen type II . When human fibroblasts are cultured on Pln, they fail to attach . As a component of extracellular matrix and basal lamina, Pln is found in various embryonic and adult tissues. The major cartilage proteoglycans are of the chondroitin/dermatan sulfate variety; however, expression of Pln has been reported recently . While the Pln core protein has the ability to carry CS chains, it has been demonstrated that Pln in cartilage also carries HS chains . Consistent with these findings, we detected both Pln protein and mRNA as well as HS chains in developing mouse cartilage. One description of Pln expression during embryonic development has been reported , with similar results to those presented here. An examination of Pln expression throughout murine development demonstrated a relationship with chondrogenesis. During this process, Pln protein was found to accumulate in the cartilage primordia progenitor cells. This does not reflect a general expression of basal lamina components by the cells, since neither laminin nor collagen type IV was detected in these tissues. A hallmark of chondrocyte differentiation, collagen II expression, was apparent by d 12.5, a time when Pln was only faintly detected. Thus, these cells appear to be committed to initial aspects of the chondrocytic pathway before displaying high level Pln expression. It is possible that low levels of Pln accumulate before collagen type II and trigger further chondrocytic differentiation in vivo. In this regard, 10T1/2 cells and human chondrocytes also respond to culture on Pln by expressing high levels of Pln themselves (data not shown). Alternatively, Pln expression may be independent of collagen type II or Pln may promote more complete differentiation of cells already poised to undergo chondrogenesis. 10T1/2 cells respond more rapidly and completely than Balb-c 3T3 cells. Similarly, human chondrocytes, but not human fibroblasts, can respond to Pln in vitro. It is not clear what factors, e.g., cell surface receptors, transcription factors, etc., predispose cells to Pln responsiveness. Initial assays determined that 10T1/2 plated on Pln and, to a lesser extent, the Pln-containing matrix, Matrigel, formed aggregates and stained positively with Alcian blue. The ability of 10T1/2 cells to undergo chondrogenic differentiation in vitro may be enhanced by the chondrogenic transcription factor, SOX9 . In developing cartilage, SOX9 expression precedes that of Pln . Interestingly, 10T1/2 cells express much higher levels of SOX9 than Balb-c 3T3 cells . This may account, in part, for the ability of 10T1/2, but not 3T3 cells to differentiate more completely in response to Pln; however, 3T3 cells stably transfected with SOX9 form aggregates, but fail to stain with Alcian blue as completely as 10T1/2 cells (data not shown). Collectively, these data suggest that while SOX9 may be necessary, it is not sufficient to promote chondrogenesis either alone or in combination with Pln. As described previously, other extracellular matrix molecules and glycosaminoglycans fail to induce differentiation. Heparinase-treated Pln displayed inductive ability, albeit at a greatly reduced rate compared with intact Pln. These experiments indicate that cooperation between the Pln core protein and its constituent HS chains occurs in the potentiation of chondrogenesis. To exclude action of potential contaminating growth factors, certain inactive Pln preps, obtained from either commercial sources or prepared in our laboratory, were supplemented with growth factors previously proposed as chondrogenic agents, i.e., bFGF, BMP-2, BMP-4, and TGF-β1. These potential contaminants failed to stimulate nodule formation on inactive Pln preparations. It was surprising that neither BMPs nor TGF-β1 stimulated chondrogenesis on surfaces coated with inactive Pln since these growth factors do so when 10T1/2 cells are cultured under other conditions . The assay developed here relies on the response of the cells to a matrix molecule presented in a solid phase, presumably similar to the state found in vivo. The features differentiating inactive Pln from the active preparations have not yet been clarified. However, it is clear that both the GAG chains and the protein core participate in this activity. Damage to the protein core or HS chain alteration, e.g., heparanase digestion or undersulfation, might result in a lack of activity in the 10T1/2 assay. We have found that chondrogenic activity maps to particular recombinant Pln domains and requires glycosaminoglycans (French, M., M. Hook, R. Timpl, and D.D. Carson, unpublished observations). It is possible that, in the absence of appropriate glycosaminoglycan chains, portions of cell surface receptors or binding sites become occupied that require glycosaminoglycan interactions to transmit signals fully. FGF receptors require such interactions with HS chains in complexes with FGFs . In the absence of appropriate glycosaminoglycan complexes at these sites, cells may become locked in a state preventing them from responding to other signals. In addition, the observation that the Pln that is active in solid phase assays is inactive in soluble form also indicates that the context of Pln presentation is an important aspect of this response. The receptors involved in Pln recognition by 10T1/2 cells must be identified to understand these responses in molecular detail. Rinsing of Pln-coated surfaces with high salt concentrations, a treatment that would be expected to release HS-bound growth factors had no effect on this inductive activity. From these assays, inductive activity appears to be dependent in part on both the Pln core protein and the HS chains. This may account for the lack of inductive activity in some Pln preparations. If cells require interaction with both the HS chains and the core protein to elicit a full response, one or the other interaction might act to block stimulation from another source. Thus, contact with the core protein of inactive Pln primes the 10T1/2 cells to respond to stimulation from a linked HS chain. In the absence of the chain, the cell may be unable to respond to TGF-β or BMP-4 signals. Variations in isolation protocols or the actions of tissue heparanases during isolation could account for a depletion of specific classes of HS structures in these preparations. A preference for more highly negative molecules containing l -iduronic acid by differentiating chondrocytes in micromass culture has been demonstrated by SanAntonio et al. . If larger and more negatively charged HS chains were selected against in the isolation process, or were degraded by tissue heparanases during isolation, the stimulatory effect of Pln might be lost or reduced. Another possibility is the large Pln core protein may become partially denatured or otherwise modified at subtle, yet critical, sites during isolation. A simple model for the action of Pln on chondrogenesis focuses on attachment of the cells to a matrix. If cell– matrix interactions are impaired, then the cells would remain more rounded and attach to each other rather than the matrix, forming aggregates. When cultures from chick limb buds were supplemented with soluble HS or HP, increased aggregate formation and sulfate incorporation, i.e., glycosaminoglycan synthesis, was observed . In these studies, it was suggested that a possible effect of the HS/HP was to free the cells from their attachment to the matrix, allowing them to round up and promoting cell–cell interaction. The induction of chondrogenesis observed in the present studies is more complex. Induction of chondrogenesis in vitro appears to require some specific interaction and binding to Pln. 10T1/2 cells attach, but do not spread, on Pln. Also, Pln can maintain human chondrocytes in their differentiated form in vitro. In contrast, human fibroblasts do not attach to or differentiate on Pln-coated surfaces. It remains unclear which receptor systems trigger chondrogenesis in vitro. Such systems appear to involve both HS- and Pln-recognizing events. Complexes containing integrin subunits α v , β 3 , and β 1 have been reported to bind Pln, although Pln lacking HS chains appeared to be more effectively recognized . These integrin subunits also have been detected in developing cartilage . While several potential chondrogenic factors have been identified, none of these factors have been examined in the context of interactions with the extracellular matrix surrounding the cells in vivo. In this study, examination of temporal and spatial patterns of the extracellular matrix protein, Pln, supports a role for this protein in the program of maturation or maintenance of chondrocytes. The combination of appropriate extracellular matrix molecules with various growth factors, secreted either by chondrocytes or surrounding cells, may prove to be an essential mix for development of cartilage in vivo. | Other | biomedical | en | 0.999997 |
10352027 | In 1955, Alan Hodgkin and Richard Keynes published a study whose intent was to answer a rather technical question, but whose result triggered the modern analysis of ion channel permeation . They aimed to determine whether the K + fluxes in giant axons followed the “Ussing flux ratio criterion,” which was then considered to indicate whether flux through a membrane occurred passively through pores. The Ussing ratio states that passive unidirectional flux should be proportional to the activity of the compound on the side from which the flux occurs. Therefore, the ratio of inward:outward unidirectional K + fluxes in the axon was expected to equal the ratio of external:internal K + electrochemical activities. The K + fluxes in axons clearly failed the Ussing test, as Hodgkin and Keynes noted that the ratio of fluxes varied with the activity ratio raised to the 2.5th power. However, they argued that this indicated a different kind of pore: a long one that could hold multiple ions traversing the pore in single file. To corroborate their mathematical arguments, they mimicked their observations with the mechanical model shown in Fig. 1 A. Now, another dramatic paper has directly demonstrated multiple ions arranged in single file within a crystallized potassium channel pore . A 12-Å length of this pore is so narrow that it provides a tight fit to a K + that is stripped of water molecules. This must be the channel's selectivity filter. Two ions were found within the filter 7.5-Å apart. Thus, the multi-ion, single-file nature of the business region of the potassium channel pore is confirmed absolutely. The filter is electronegative, and the sources of the negativity are backbone carbonyl oxygens rather than amino acid side chains. An array of nearby α-helices appear poised to rigidly hold the selectivity filter structure so that it perfectly fits a K + ion (∼3 Å), but cannot collapse to fit a Na + ion (∼2 Å). This supports the classic “perfect fit” hypothesis for potassium channel selectivity , which suggested that a 3–4-Å pore, deduced from permeation experiments, could fully solvate K + ions, but fail to do so for the smaller Na + ions. In contrast to potassium channels, permeation measurements of sodium channels and calcium channels suggest that their selectivity filters are far wider than their preferred ion. Sodium and calcium channels excel at distinguishing Na + from Ca 2+ , two ions of identical diameter. These facts suggest that a rigid, perfect fit mechanism should not explain selectivity in sodium and calcium channels. Four decades apart, Hodgkin and Keynes and Doyle et al. form fundamental pillars to the field of ion channel permeation. They unequivocally demonstrate multi-ion, single-file permeation. Theoretical work between these two landmarks has explored the functional consequences of this mechanism. The crux of permeation and selectivity is to understand how channels can be highly selective and still pass millions of ions per second. Quantitative models of flux through multi-ion, single-file pores suggest possible solutions to this paradox. Fig. 2 illustrates the paradigm for calculating selective flux through a single-file pore using elementary chemical kinetics. Fig. 2 A represents the hills and valleys of potential energy experienced by one kind of ion as it traverses the pore. Fig. 2 B indicates the various ways that the two energy minima of the model might be occupied (or unoccupied, 0 ) by two different ions, A and B . The lines show the allowed transitions between occupancy states. For example, A0 (state 2) has ion A in the left site and nothing in the right; the transition to 0A (state 4) involves the ion moving from left to right. Two rules determine which transitions can occur: (a) an ion cannot enter a site that is already occupied by another, and (b) an ion cannot pass over a site without binding to it. Transitions between sites are considered to be instantaneous. These rules—hopping without bumping or jumping— define single-file premeation models. The rules are analogous to the physics of ion movement in crystals and semiconductors, in which electrons hop between “holes” or vacancies in the crystal lattice. Ion flux in semiconductors has been modeled best with drift diffusion equations, which are the underpinning of the “PNP” description of ion channel permeation (see Perspective by Nonner et al.). Thus, single-file models and PNP models have the same basic process in mind, although the first uses discrete and the second continuous mathematics to describe the process. If the point is to debate discrete versus continuous descriptions of permeation, these Perspectives may lack perspective. Diffusion in homogeneous media is correctly described by both approaches , and this must also be true for ion channel permeation. Debate about this fundamental fact arises only because present models of permeation, both discrete and continuous, are but crude approximations of real ion channels. The rule that transitions are instantaneous is analogous to “collision theory,” the most simple-minded description of chemical reactions. Accordingly, flux of an ion over an energy barrier diminishes exponentially as the barrier height increases and is described by the rate constant: r = ν(exp[− G / kT ]), where [ G / kT ] is the height of the energy barrier ( G ) relative to the atom's thermal energy ( kT ). The frequency factor, ν, is the maximum possible value of a rate constant, the value when there is no energy barrier. In single-file models, the frequency generally is set to kT / h (∼6 × 10 12 s −1 at biological temperatures; k and h are the Boltzmann and Planck constants, respectively, and T is absolute temperature). The rationale for this and the difficulties with it are discussed below ( Limitations of Single-File Models ). The touchstone of single-file modeling studies is Hille and Schwarz . The paper successfully mimicked a variety of unintuitive potassium channel permeation properties, including the failure of unidirectional fluxes to obey the Ussing ratio, a minimum in conductance when the relative fractions of two permeant ions are varied (the “anomalous mole fraction effect”), and rectification due to blocking ions. Hille and Schwarz found that repulsion between intrapore ions had profound effects on the calculated flux and concluded that two properties of the model were necessary to reproduce physiological phenomena. First, external energy barriers had to be high compared with internal ones. This allows ions to equilibrate readily between intrapore sites—to rattle about the pore more easily than they can exit it. Second, the pore had to have at least three sites and contain at least two ions most of the time. Now, Doyle et al. find two ions in the potassium channel selectivity filter sitting at opposite extremes of the filter's length. This image is consistent with repulsion pushing the two ions to the extremes of a pore from which exit is more difficult than is intrapore mobility. Thus, the image is consistent with the physical principles suggested by Hille and Schwarz , as well as their number of ions. This is the value of single-file calculations. Although the models must be grossly simplistic representations of permeation through ion channels, they still provide a means to detect and test the basic principles involved. Other important single-file papers have described permeation and selectivity in gramicidin channels , sodium channels , and calcium channels (below). Calcium channels have been useful for studying permeation for two reasons: (a) they are extraordinarily selective, and (b) they are the clearest case of a channel that selects its preferred ion through intrapore binding. The most elementary question about selectivity is whether a channel acts like a molecular sieve to reject certain ions, or whether it preferentially binds its chosen ion. Calcium channels select Ca 2+ over Na + at a ratio of 1,000:1, even though the two naked ions have identical diameters; no sieve could do this. Instead, the pore binds Ca 2+ with an apparent K d of ∼1 μM. When the high affinity site is unoccupied by Ca 2+ , Na + is freely permeant; when Ca 2+ occupies the site, as it must at physiological Ca 2+ concentrations, Na + current through the channel is blocked. Thus, Ca 2+ confers selectivity on calcium channels because it blocks permeation of competing ions. Similarly, Na + current through potassium channels is blocked by K + . Discovery of high affinity binding in the calcium channel raised a fundamental issue. Since K d = k off / k on , and since the maximum k on is the diffusion limit of 10 9 /M −1 s −1 , the maximum off rate from a 1-μM binding site should be only 1,000/s. However, picoampere currents (millions of ions per second) pass through calcium channels. A goal of permeation models has been to suggest mechanisms for solving this 1,000-fold discrepancy between expected and observed flux. This “sticky pore problem” is diagrammed in Fig. 3 A. Binding affinity is determined by the difference in energy between the outside environment and the well (arrow 1); flux is limited by the height of the greatest barrier to exit from the pore (arrow 2). In such a single site pore, tighter binding (a longer arrow 1) necessarily causes slower flux (a longer arrow 2); this makes selectivity by selective binding impossible. Models of multi-ion, single-file calcium channels have deduced two separate solutions to this problem, although the two mechanisms might well work together in a real pore. The repulsion model imagined two high affinity sites within the pore and allowed ions to repel each other when they occupied the pore simultaneously . Foreign ions are blocked at micromolar Ca 2+ concentrations when a single Ca 2+ ion occupies the pore (left). When two Ca 2+ ions occupy the pore, they repel each other, and this effectively diminishes the pore's affinity for Ca 2+ (apparent K d changes from ∼1 μM to 10 mM, right). Once the pore is in this low affinity state, Ca 2+ flux occurs readily. Recent mutation studies (see next section) cleanly disprove one aspect of this model because they demonstrate only a single high affinity site in the calcium channel. Nevertheless, the more fundamental point—that flux occurs because multiple occupancy lowers the affinity of the pore for Ca 2+ —remains reasonable. The step model has just a single high affinity site, but this is flanked by low affinity sites on either side . Although the pore can be occupied by multiple ions, the pore's affinity for Ca 2+ does not change with multiple occupancy; nevertheless, the model fits Ca 2+ channel data almost identically to the repulsion model. High Ca 2+ flux occurs because the flanking sites provide steps of potential energy. Stepwise conquest of potential energy barriers is a general mechanism for speeding chemical reactions; it is closely analogous to the way stairsteps increase the fraction of people who can surpass gravitational potential energy barriers. In essence, a Ca 2+ ion takes the stairs to exit the deep binding well in the step model. The PNP model of calcium channels provides another major, and unexpected, insight. The model's pore is an electronegative tube that is 10-Å long and 6-Å wide. Despite the absence of an explicit high affinity binding site, the model exhibits high affinity Ca 2+ block of Na + current as well as high Ca 2+ flux. This discovery is very timely: it shows that a rigid, diffusely negative tube is sufficient to generate selectivity through high affinity binding. The pore binds Ca 2+ without ligands that wrap around the ion, as is generally assumed necessary for high affinity binding; surely, this has great significance for ion channel permeation. In an important sense, the differences between the single-file and PNP models have been overstated. The PNP/calcium channel demonstrates an unexpected mechanism for intrapore, high affinity binding. Single-file models do not, and cannot, address the mechanism or structures of binding (see Limitations of Single-File Modeling ). Rather, they focus on the forces that allow rapid exit from the high affinity site. Two forces were described and here the models agree: both repulsion and potential energy steps contribute to rapid efflux from the PNP/calcium channel pore according to the authors. It is the mechanism of flux that seems so different in the two models, but this may largely be a matter of mathematical appearances. As discussed above, both types of models envision ion movement to be similar to that through crystals, but one describes this with discrete steps and the other with continuous functions. If the models are ever complete (and neither pretends to be), the two approaches must give identical results just as diffusion is described equally well through random walk or continuum equations . For the time being, it is encouraging that such different approaches agree in the description of forces that underlay high flux out of high affinity pores. Like every other model, the PNP/calcium channel comes with ad hoc assumptions. A critical one necessary to allow Ca 2+ to block Na + current is that the pore has an excess chemical potential that is negative for Ca 2+ and positive for Na + ; in other words, some undefined chemistry of the model pore is attractive for Ca 2+ but repulsive for Na + . What chemistry could attract one positive ion while repelling another of the same size is unclear. Another issue, discussed below, is an explicit disagreement with observations made in mutation studies. Such failings confirm only that the PNP model is an incomplete approximation of the calcium channel. But, by definition, all models are incomplete. In my view, such specific inconsistencies should not detract from the more general discovery inherent in the PNP/ calcium channel: that selectivity by high affinity binding can occur in a rigid pore. The most fundamental facts known about the structural basis of Ca 2+ selectivity had their birth in a sodium channel paper. Heinemann et al. noted four conspicuous glutamate residues in the structure of calcium channels. Though each is separated by ∼500 amino acids of sequence, these glutamates are in the putative pore-lining region of the four internally conserved repeats. In the analogous sodium channel sequence, two of the negative charges are present, but a neutral and a positive residue substitute at the other two sites. Mutation of these two residues to glutamates made the sodium channel behave like a calcium channel in that Ca 2+ could block Na + current at low concentrations. The four glutamate residues in calcium channels are called the EEEE locus. Yang et al. mutated the EEEE locus in L-type calcium channels, and thereby demonstrated the role of these glutamates in high affinity Ca 2+ binding . Neutralizing any of the glutamates to a glutamine shifted the block of monovalent current to higher Ca 2+ concentrations. The extent of the shift differed for glutamates in the different repeats, and neutralizing multiple residues shifted the blocking concentration by as much as 1,000-fold . Because a single point mutation can shift the dissociation constant for Ca 2+ , there must not be two independent high affinity sites in the pore. Moreover, pairwise mutations of the glutamates rigorously refute the two sites postulated by the repulsion model . Yang et al. suggested that the four glutamate side chains form a binding site for Ca 2+ in much the same way that the four carboxyls in EGTA wrap around Ca 2+ . When Ca 2+ is absent from the site at low, submicromolar concentrations, Na + can permeate. As its concentration rises above a micromolar, Ca 2+ stably occupies the site and Na + flux is blocked. When Ca 2+ rises to millimolar concentrations, two Ca 2+ ions likely occupy the pore simultaneously. The two compete with each other for the glutamate residues, thereby lowering the affinity of the pore for Ca 2+ and allowing Ca 2+ permeation. In an important sense, the idea is like the repulsion model because a decrease in binding affinity during multiple occupancy is what allows high Ca 2+ flux. However, competition for binding ligands drives the affinity change rather than the electrostatic repulsion originally suggested by the model. These mutation studies potently argue against the PNP model for the calcium channel. The PNP model pore contains four carboxyl groups that are meant to mimic the EEEE locus. However, there is no difference in the concentration at which Ca 2+ blocks Na + current in the PNP/calcium channel when the number of intrapore carboxyl residues is set to 1, 2, 3, or 4 . Thus, the PNP model starkly fails to reproduce the data in Fig. 4 A. Whatever may be the actual structure of the EEEE locus, the four carboxyls in the PNP model do not mimic it. The present goal of permeation studies is to understand how specific pore structures control flux and selectivity. In general, single-file models are unsuitable for this because they do not rely on known structure. More specifically, three problems diminish the value of the single-file approach to structure–function studies. First, the models do not define physical distances. The “electrical distance” along which the ion travels through these models is the fraction of the electric field experienced at each step; the relation between this and real distance is unaddressed and unaddressable in the single-file paradigm. Second, the relation between energy minima and protein structures is ambiguous. A minimum of energy is a place where the ion likely pauses longer than at adjacent spots, but this need not correspond to a specific binding site built into the protein. For example, an ion is relatively stable in the central cavity in the potassium channel of Doyle et al. ; this is likely not due to interaction of the ion with the protein, but to a more stable degree of hydration than in the narrower adjacent regions. The third problem is that the models are rigid, but proteins are not. In cases where dynamic protein structures have been described, substrates generally induce a fit of themselves into their binding sites, rather than fitting like a key in a rigid lock. Accumulating evidence argues the same for channels. For example, Immke et al. show that a K + ion must be bound to the potassium channel selectivity filter for tetraethylammonium to effectively block the pore. Tetraethylammonium is about the size of a K + ion with a single shell of attached water and its binding site is in the vestibule. Thus it appears that the presence of a naked K + ion in the filter confers on the vestibule the shape appropriate for binding an incoming hydrated K + ion. This agrees with earlier arguments that flexibility of the pore entrance is essential for rapid and efficient dehydration of entering ions . Although clearly critical, such flexibility cannot be addressed with single-file models (or the PNP system). Ultimately, dynamic structure-based calculations are necessary for describing ion permeation. The Appendix in Nonner and Eisenberg takes a long step further in criticizing the single-file approach. It uses the framework of the PNP model to recalculate flux through the step model and finds that: (a) Ca 2+ flux through this step-PNP model is many hundred-fold less than experimental observations, and (b) the pore fails to select Ca 2+ over Na + . The results are not shown for other models, but it is stated that similar PNP recalculations of the repulsion models are “even less consistent” with experimental observations, and that “the reasons for failure are generic for rate theory models in the tradition of Hille and Schwarz .” The problem, it says, is that single-file models use a frequency factor for calculating rate constants that is 10,000-fold too high. If so, all single-file models are incorrect by many orders of magnitude, implying that their insights are based on false physics and should be abandoned. The primary problem with the Appendix in Nonner and Eisenberg is that it fails to recognize that frequency factors and barrier heights are not treated as separable entities in single-file models. For example, our models set the external barriers (the values that limit entry and exit of the pore and are therefore the most critical) so that they agree with experimental observation. Lansman et al. demonstrated that Ca 2+ enters the pore at a rate of ∼10 9 M −1 s −1 , the value expected from diffusion-limited access. Assuming a prefactor of 6 × 10 12 s −1 , the external barriers are set no lower than 8.7 kT, thereby creating an entry rate of 10 9 M −1 s −1 . If we had used a lower prefactor, we would have set the external barrier energies lower, always keeping the rate constant (the product of the prefactor and the exponential) consistent with experimental observations. If one diminishes the prefactor by 10,000 while keeping the barriers the same, the entry rate will be 10,000-fold less than experimental observation. In this trivial way, calculated flux would be unphysiologically low. This is the essence of the error in the Appendix in Nonner and Eisenberg : it uses precise energies from one calculation scheme while applying assumptions from another. Although the appendix's calculation is artificial , the point about frequency factors is important. To understand the flaw in setting the frequency factor, ν, equal to kT / h , one should understand its basis. kT / h is an atom's theoretical vibration frequency, obtained by assuming that all thermal energy ( kT ) becomes kinetic, or vibrational, energy ( h ν). The assumption confers a simple physical interpretation on the equation for a rate constant: the atom can attempt to surpass the reaction barrier each time that it vibrates, but the probability of a successful attempt is very low. The attempt rate is kT / h , about six times per picosecond, and the probability of success is exp[− G / kT ]. The problem with this is nicely explained by Andersen and Koeppe , and by Andersen's introduction to these Perspectives. In short, the frequency of vibration is only legitimate to use if the distance to surpass the barrier is similar to the length of a vibration, and this is just a small fraction of an angstrom. To traverse a selectivity filter that is many angstroms long, many such individual steps are necessary. Assuming that movement within the filter is diffusive, these individual steps would have low energy barriers and transit would be fast compared with exit from the pore. Therefore, most single-file models simply lump these many intrapore steps into one or two individual steps that are not rate limiting. The models thereby focus on the high barriers that limit exit from the pore; these external barriers, because their size is determined by experimental measurements (see above), are handled appropriately by the models. However, there is no denying that lumping the internal steps together represents the pore in a physically inaccurate manner. A perspective on the insights from single-file models arises from considering the ultimate aims of the permeation field. A major one is to construct an accurate map of the chemical potential energy of an ion as it traverses a pore of precisely known structure. This is analogous to converting photographs of a hillside into a topographic map, which describes your gravitational potential energy as you hike the hill. Single-file models have skipped directly to the topographic map without having the photographs. Unlinked to real structures, these energy diagrams are nothing more than quantitative cartoons, but the quantitation has been valuable. The models do not pretend to tell us about protein structure, but, by finding which forces allow calculated fluxes to mimic real experiments, they suggest general principles relevant to permeation. These principles include: the importance of having multiple ions moving in a coordinated fashion through a pore, that ions likely equilibrate within the pore far more easily than they exit it, that channels can select their ions through binding sites and still have high flux, and that exit from a tightly binding pore is promoted through either ion– ion interactions in a multiply occupied pore or by a series of lower affinity sites. First, there is a question that is not real: it is irrelevant to ask which of the earlier models is “correct.” Each provides insight, but each is incorrect. These models are crude approximations that cannot explicitly show how specific protein structures control permeation. However, their insights might help to guide the questions posed in future, structure-based modeling. Answers to the following three questions should provide a fundamental understanding of calcium channel permeation and selectivity. (1) How can Ca 2+ have diffusion-limited access to the pore? Three decades ago, Eigen and Winkler noted that ions enter carriers at the diffusion limit; Lansman et al. demonstrated the same for calcium channels. The high rate is unexpected: ions must completely dehydrate to enter pores and dehydration requires so much energy that it should occur only rarely. In addition to replacing an ion's hydrating waters with solvating intrapore ligands, channels and carriers must also rapidly catalyze the dehydration process. How is this done? Eigen and Winkler argue that rapid dehydration requires systematic, stepwise replacement of water molecules by substituting ligands, rather than a loss of all waters at once. Andersen and Koeppe note that this requires a pore entrance that is not rigid. What structures at the mouth of the pore are responsible for stepwise dehydration, and over how long a distance does this process occur? (2) Is the strong binding of the selectivity filter of the calcium channel due to a flexible binding site analogous to Ca 2+ chelators? The calcium channel mutation studies of Yang et al. and Ellinor et al. are most easily explained this way. However, the potassium channel selectivity filter described by Doyle et al. is a rigid pore and the PNP model for calcium channels shows that high affinity binding can occur in such a structure. Is the selectivity filter of calcium channels rigid or flexible? Is it lined by backbone carbonyl oxygens or by amino acid side chains? (3) What forces allow ions to exit this high affinity binding area so much faster than expected? Two have been suggested in various models: ion–ion interactions and stepwise increases in energy during exit. What might cause ion–ion interactions in the real pore— competition for a binding site and/or electrostatic repulsion? What might cause stairsteps of energy in the real pore—specific binding structures of successively lower affinity and/or stepwise rehydration? What is the relative importance of these two forces, and are there others that have not previously been considered? | Study | biomedical | en | 0.999997 |
10352028 | Physical laws are written as mathematical equations, and for good reason. Verbal formulations of physical laws are not specific and do not permit direct comparison with experiments (which usually have numbers as their output). Verbal formulations lead different people to different conclusions, depending on how they subjectively weigh different effects: verbal theories are rarely objective and often lead to interminable argument. Mathematical statements of physical laws are less subject to these human distortions, but they are hardly perfect either. Mathematical laws lead to mathematical complexities and computational problems quite independent of their physical meaning, and the necessary approximations are often illogical and can be distorted as well. These generalities are easily replaced with specifics when theories of channels are considered. Verbal models of permeation cannot link structure and function because permeation usually involves competing effects. If one effect increases current and another decreases it, the net effect can only be determined quantitatively. It is natural then to turn to computational models of channels that promise to be complete and rigorous. Simulations of molecular dynamics promise to directly compute properties of biological interest, starting with fundamental physical laws and decent representations of the properties of atoms. Unfortunately, the simulations of molecular dynamics cannot provide a quantitative theory of ions moving through a channel, driven by a gradient of concentration and electrical potential, even though the underlying equations of motion of ions and atoms in channels are reasonably well known. Direct simulations are not possible because the simulated system needs to be large enough and computed long enough to define the variables that are measured and controlled experimentally; e.g., current, transmembrane potential, and concentration. Direct simulation of atomic motions use a discrete time step of 10 −16 s to resolve atomic vibrations. Calculations with (substantially) longer time steps than 10 −16 s are no longer direct simulations, but depend on theory and assumptions to fill their gaps. A single ion takes some 100 ns to cross a channel (10 9 time steps), and calculations of thousands to billions of crossings must be made to estimate measured currents. Direct integration of differential equations of molecular dynamics (i.e., the differential equations that are Newton's laws) is not reliable over times larger than several picoseconds because the calculated trajectories exponentially diverge and are exquisitely sensitive to initial conditions and round-off error as can easily be verified by running any of the codes widely available, some without cost. It is not known mathematically whether calculated trajectories (after a few picoseconds of calculation) are reliable, unbiased, or even useful estimates of the trajectories of real atoms (“unbiased estimate” here is defined as in probability theory and statistics). Simulated trajectories may not sample some of the phenomena of biological interest at all, particularly those phenomena that appear slowly (i.e., after microseconds). Trajectories of chaotic systems, starting from particular initial locations, are often confined to limited regions of “phase space” and do not explore other regions at all. Even within an accessible region of phase space, simulated trajectories may not be reliable or unbiased estimates of systems. After all, while the trajectories of real atoms are not subject to round off error, the computation is, and how the round off error influences the computation is not known . Simulated systems of proteins and channels must also be large enough to define concentrations of ions in the bath, if those concentrations influence biological phenomena of interest (as they almost always do). Since errors tend to vary with the square root of the number of particles studied, something like 1,000 ions need be present in a simulation to define a concentration with 3% precision. Those thousand ions are solvated by a very much larger number of water molecules, since in biological systems the mole fraction of ions is small (ranging from, say, 0.150/55 for typical external sodium concentrations to 10 −5 /55 for typical internal calcium concentrations). Simulations need to involve some 3 × 10 5 molecules (nearly one million atoms) to define a typical extracellular Na + concentration in the solutions surrounding a channel and 2 × 10 9 atoms to define a typical intracellular Ca 2+ concentration. Short duration simulations of small systems are often extrapolated to biological sizes and times in a sensible attempt to describe biological phenomena, but then they are indirect calculations, no better than the theory used to extrapolate them. Such indirect simulations are not a priori any more reliable than those made with a theory of lower resolution. Finally, a technical difficulty is important when considering proteins like channels that act as devices in the engineering sense of the word. Devices and channels almost always function away from equilibrium, but simulations of molecular dynamics of channels are nearly always done at equilibrium and with treatments of the electric field that do not allow transmembrane potentials at all . The mathematical difficulties involved in computing nonequilibrium biological and chemical systems with transmembrane potentials have not been overcome , and the methods used by physicists to perform such calculations have not, to the best of our knowledge, been tried in channels, proteins, or ionic solutions. The best portal to these methods seems to be the Dacmocles website ( www.research.ibm.com/0.1um/laux/dam.html ). Given the difficulties of direct simulations of molecular dynamics, it seems that some of their resolution must be abandoned if one wishes to actually fit biologically relevant data. The choice is only what to give up. The choice is made by using a series of simplified theories and picking the simplest that describes the experiments and behavior of interest, while retaining as much structural meaning as possible. Here we will show that a simplified model that considers (mostly) the electrical properties of the open channel (the fixed charge on its walls and the mobile charge in its contents) does surprisingly well in understanding and predicting the currents that flow through several open channels, for a range of potential and concentrations of several ions. An electrical model seems a good place to begin a theory of channels, since the function of channels directly involves currents and transmembrane potentials and the structure of channels involves highly charged molecules lining tiny volumes. (One charge in a selectivity filter 7 × 10 Å is some 5 M.) It does not take much charge to produce huge forces . The model we will consider forms a working hypothesis, to be tested and then revised as it fails. In its simplest and original form, the working hypothesis was that a channel could be described as a one-dimensional distribution of fixed charge, corresponding in a crude way to the fixed charges on the atoms that line the wall of the channel's pore. In the crudest form, the effective charge would be the total charge on the wall of the channel in some cross-sectional slice. In more refined versions of the model, other estimates of the effective charge would be used. The only contribution of the channel protein to the permeation properties of mobile ions was supposed to be the electric field created by these charges, and the concomitant distribution of the probability of location (“concentration”) of the permeating ion. Single filing effects, and specific chemical interactions (not described by Coulomb's law) were purposely omitted from the first version of the model, so the entire force field on the permeating ions could be computed unambiguously (at this level of resolution) once the charges on the channel protein were specified (along with the bath concentrations, transmembrane potential, geometry, diffusion, and dielectric coefficients). The electric field in this model is computed by the Poisson equation, which is the differential form of Coulomb's law. Details are described in Eisenberg , and the code that solves these equations is available in various forms at an anonymous ftp site ( ftp.rush.edu in directory /users/Eisenberg). In the initial working hypothesis, the electric field is supposed to influence current only according to electrodiffusion as described by the Nernst-Planck equation. The model is specified by the Poisson-Nernst-Planck (PNP) equations, which, as it happens, are nearly identical to the drift diffusion equations that have been used for a very long time to describe the motion of charged quasi-particles like the holes, electrons, and superparticles of semiconductors or the hydrated ions of electrolyte solutions . We have suggested that the correlated motion of an ion, water, and atoms of the channel protein might act as a quasi-particle or super-particle, a permion , with the permion rather than the individual ions satisfying the conservation laws of PNP. PNP depends on the same approximations and representations of underlying atomic variables as the Gouy-Chapman model of an electrified interface, the (nonlinear version of) the Debye-Hückel [DH] theory of ionic solutions), and the (nonlinear) Poisson-Boltzmann theory of proteins. Although the physics underlying PNP and these other models are similar, the actual behavior, mathematics, and computational properties of the systems are quite different because PNP includes flux in its every calculation, never requiring equilibrium . The Nernst-Planck equations have much more resolution than a crude continuum description of ionic concentration and movement. Theory and four types of simulations show that the Nernst-Planck equation with constrained potential describes the concentration and flux of discrete particles diffusing over barriers of arbitrary shape and size from a bath of one concentration and potential to another. The Nernst-Planck equations use one parameter to describe frictional forces that limit the two components of flux, diffusion and drift, assuming that the Nernst-Einstein relation between diffusion coefficient and mobility is valid under all conditions of interest. Less elementary versions of PNP do not require that assumption. The diffusion coefficient of the ion in fact also depends on the properties of the channel wall, both because of steric effects and (probably much more importantly) because of dielectric friction produced by motions of the protein's atoms induced by the electric field of the permeating ion. But PNP does not explicity invoke this important physical mechanism . The PNP model contains much less atomic detail than most models of channels or proteins. PNP neglects correlation effects of individual atoms. It contains only those effects that are mediated by the mean field. For example, PNP is rich with binding phenomena because it often predicts localized maxima in the concentration of permeating ions, but it does not predict the phenomena of single filing that depend on the correlated motion of ions. We have considered correlation effects to be important for a long time, and so it seemed highly unlikely to us (as we derived the model and as Duan Chen solved its equations) that PNP would actually fit data. Indeed, that is why we worked so hard on higher resolution models. However, the low resolution PNP model does fit a wide range of data, using a different distribution of fixed charge for each type of channel (corresponding presumably to the different structure of each type of channel) and different diffusion coefficients for each type of ion. Note that no parameters are changed as solutions or transmembrane potentials are changed other than the concentrations and potentials themselves. Thus far, the simple PNP model fits the highly rectifying (sublinear) I–V relations measured in a wide range of solutions, symmetrical and asymmetrical, from the synthetic cation-conducting leucine serine (LS) channel . It fits most (but not all) of the highly rectifying, superlinear I–V relations in the neuronal background anion (NBAC) channel . It fits the rectifying sublinear I–V relations from porin , a channel of known structure, and its mutant, G119D, also of known structure. It fits I–V relations from both cardiac and skeletal forms of the calcium release channel (CRC) of sarcoplasmic reticulum , all of which are quite linear; and it fits I–V relations from a number of other channels (most notably gramicidin, see below). So far, PNP has fit the I–V relations of every channel for which we have data, but we continually expect it to fail on the next attempt. The simplest form of PNP fits most of the data but, in some cases, an extra parameter (a constant) is needed that describes chemical interaction, as described below. In most cases, PNP is the only theory that fits the entire data set, including asymmetrical solutions. The fact that PNP fits these data sets contradicts the general view, which we certainly shared until PNP showed us otherwise, that a theory of permeation must explicitly include much more than Coulomb's law. Specifically, it contradicts the view that a successful theory must have separate components and parameters that describe dehydration/ resolvation, obligatory single filing, or chemical interactions of the permeating ion with the channel protein. Chemical interactions are seen in some cases. Binding must be included as an extra parameter in PNP when describing permeation of Li + and Na + in the cardiac CRC channel , or the anomalous mole fraction property of K + channels or the interactions of Na + , Ca 2+ , and pH in the L-type calcium channel . It is enough (at the present level of experimental and structural resolution) to add a single constant for each ion that describes the excess chemical potential of that ion in the channel. That constant is the same for a given type of ion in a given type of channel and does not vary with transmembrane potential or concentration of any of the ions. This constant is the only representation of dehydration/resolvation, nonelectrostatic binding, and single filing needed to fit these data sets. Indeed, it is possible that this constant arises entirely from Coulomb's law, as a correction for effects of the three-dimensional field not correctly described in the one dimensional model. As we consider more complex channels and mixed solutions containing many different ionic species, it seems likely that more specific chemical effects will be seen. We propose to deal with these (in the first place) in the Mean Spherical Approximation (MSA) that has proven so successful in bulk solution, as discussed later in this paper. If a theory is to serve as a link between structure and function of an open channel, it must do more than fit I–V relations. It must bear a well defined and close relation to the structure of the channel. The crudest form of PNP is a one-dimensional theory, and so the relation of its parameters and those of a three-dimensional structure are not immediately obvious even though the one-dimensional theory was derived by explicit mathematics; e.g., averaging of the structurally based three-dimensional (3-D) theory. The question remains what is the meaning of the parameters estimated by fitting 1-D PNP to data? The answer to that question is not completely known, but we note that all of the parameter estimates are reasonable, and so it is possible that they may be meaningful and reliable estimates of the underlying physical properties, although that certainly has not been proven to be the case. For example, typical diffusion coefficients are some 10–50× smaller than in bulk solution; the fixed charge densities correspond to only one or two fixed charges in the “selectivity filter” (narrow part) of the channel, although those one or two charges induce a concentration of mobile charge in the channel of 5–10 M, since they attract one or two counter ions into the channel's pore, and the pore volume is very small. In fact, the fixed charge of the CRC channel turns out to be very close to −1 , and this is an invariant of the curve fitting procedure (changing all sorts of parameters in the curve fitting does not change the estimate of the total fixed charge, although it changes the estimates of the diffusion coefficient), suggesting that the selectivity filter of this channel (in the form studied here) is dominated by a single fully ionized acidic amino acid, although of course the PNP results do not prove this. The validity of 1-D PNP can be probed by studying the effects of mutations on its estimates of fixed charge. Tang et al. compared the estimates of charge in a wild-type porin and its aspartate-substituted mutant, G119D, which has one additional negative charge and known crystallographic structure. The estimated difference −0.93, found in those and many subsequent experiments, is surprisingly close to the value expected. A three-dimensional version of the PNP theory has recently been developed and calculated (3DPNP), in which all atoms are assigned the charges used in a standard molecular dynamics program and all atoms are assigned positions known from nuclear magnetic resonance. Unlike 1DPNP, 3DPNP is a structural theory: the charges are estimated from the chemical literature, not from curve fitting. In 3DPNP, only the diffusion coefficient has to be specified by experimental measurements of I–V curves. Only the diffusion coefficient is left unspecified by a priori structural and physical data. In the first fairly low resolution finite difference calculation , 3DPNP shows qualitative agreement between the theory and experimental data. A high resolution analysis using spectral elements shows quantitative agreement. It seems then that for a range of open channels, a simple electrostatic model is able to fit all the I–V data available in a range of solutions, sometimes with the addition of a single extra parameter to describe the excess chemical potential of a given ion in a given channel. There are physical reasons why a mean field theory of a tiny, highly charged, (nearly) one-dimensional system is a good approximation. Generally, one would expect low resolution–averaged theories to be a reasonable approximation because of the long duration of channel currents (on an atomic time scale). Very little temporal resolution is needed to describe single channel currents. One would also expect low resolution–spatially averaged theories to be a decent initial description of (tiny) channels because of the large number of atoms outside the channel involved in determining concentrations of ions, transmembrane potential, and the reaction field to the ions in the pore. The reaction field for a permeating ion is largely in the surrounding baths where a mean field theory is likely to be more than adequate. Specifically, it is known (and not just as a matter of speculation) that mean field terms dominate correlation terms when charge is distributed along a narrow cylinder or when systems are highly charged . Despite these arguments, a derivation of PNP is needed to understand its theoretical limitations. Deriving PNP requires a superior theory of higher resolution that describes single file motion of ions while still computing the electric field from the charges in and near the channel. Such a Langevin-Poisson theory is being worked on, by us and others, but is not yet available. It seems idle to us to spend much further effort discussing models of permeation that do not fit data, when a simple model is available that does, particularly given our discussions in recent papers . Nonetheless, it is necessary, given the purpose and arena of this paper, to reiterate some of the things we have said previously about traditional barrier models of open channels that have been so widely used to study permeation. It should be clearly understood that barrier models rarely are able to actually fit I–V curves measured over a range of transmembrane potentials and solutions, including asymmetrical solutions, even when using many adjustable parameters, including an incorrect or adjustable prefactor (see equation in Scheme I). As a rule of thumb, I–V relations observed in asymmetrical solutions are more linear, often much more linear, than predicted by barrier models. Traditional barrier models are not very useful for relating structure and function, because they involve states and transitions only vaguely related to the actual structure. The models do not contain spatial coordinates as variables. The positions of individual components like ions are not within the scope of the model and so the Heckmann level of description is a priori incapable of relating ion flow to the geometrical structure of a channel. Traditional barrier models also contain two large errors, each factors ≈10 4 , that act in opposite directions and so more or less balance each other in the limited sense that they allow the model to fit the current measured at one transmembrane potential in one symmetrical solution. Most glaring is the choice of prefactor in the expression for the current over a high barrier. For historical reasons, the prefactor used in nearly all barrier models of open channels is k B T / h , even though that prefactor leaves out friction altogether (i.e., it contains no diffusion coefficient or variable to describe friction). Friction is a dominant determinant of atomic movement in condensed phases (like ionic solutions, proteins, and channels) because condensed phases contain (almost) no empty space. Workers on channels pointed out this problem some time ago and, indeed, Eyring clearly states that k B T / h is not to be used by itself as a prefactor in condensed phases . There is general agreement among workers on condensed phases that the expression k B T / h is inappropriate . When reading these classic papers, it is important to be aware that the value of the transmission factor is now known in condensed phases dominated by friction . The correct expression for the rate constant k j = J f /ℓ C l in a condensed phase for one-dimensional (unidirectional) flux J f from a solution of concentration C l over a high barrier was apparently first published by Kramers, 1940 . The rate constant and unidirectional flux can be written in a particularly neat form if the potential profile is a large symmetrical parabolic barrier spanning the whole length ℓ of the channel, with peak height φ max ( x max ), at location x max = ℓ/2, much larger than the transmembrane potential and k B T / e . The prefactor can be viewed as a measure of the entropy of activation and thus a measure of the effective volume available for ions to diffuse in the channel compared with the volume available in the surrounding solution . It seems natural that the effective volume should involve the length and height of the potential barrier, and Hill discusses the role of diffusion velocity in the prefactor . In this equation, D j is the diffusion coefficient in the channel of ion j of valence z j , e is the charge on the proton, k B is the Boltzmann constant, and T is the absolute temperature. Now, if the barrier is, say, k B T / e high and 1-nm long, and the diffusion coefficient is some 1.3 × 10 −6 cm 2 /s, the numerical value of the prefactor is ∼2.8 × 10 8 s −1 . The numerical value of the usual prefactor in barrier theory is k B T / h , which is ∼2.2 × 10 4 times larger, ∼6.3 × 10 12 s −1 . As one might expect, ions hopping over barriers experience much less friction than ions diffusing over them, and the amount of the friction will depend on the identity of the ion. Numerical errors of this size have serious qualitative as well as quantitative consequences, as was pointed out some time ago . The error in barrier models that more or less balances the error in the prefactor involves the potential barrier. The Heckmann model does not detail electrical interactions among its internal components. The spatial coordinates needed to specify an electric field are not present in the model as originally specified, but are put in by an artificial concept of electrical distance. The model postulates discrete sites when none need be present. It is not surprising that the Heckmann model does not predict the same electric field as Poisson's equation (see below). In barrier models, and all other models of permeation familiar to us (that actually predict current), except PNP, the potential barrier is assumed, not computed. Potentials in channels arise, however, from the fixed charges on the protein, the mobile charges inside the channel's pores, and the charges in the solutions and electrodes outside the channel. These produce a potential profile that changes as conditions change; e.g., as transmembrane potential, bath concentration, or fixed charge on the channel protein changes. In general, the resulting potential profiles do not have large barriers, and so currents are much larger (indeed, exponentially larger) than they would be in otherwise similar theories with large barriers. The currents predicted are very different from those of traditional barrier models. Having been raised in the barrier tradition, we are often surprised by the electrostatic effects predicted by PNP, although they are easy to compute and to understand once they are computed. An example may be useful. Nonner and Eisenberg compute the electric field of a barrier model of the L-type calcium channel using Poisson's equation instead of repulsion factors. The energy profile of Dang and McCleskey was included as a spatially varying excess chemical potential μ 0 Ca ( x ) to describe the binding properties of the channel. Cl − was excluded from their calculation, so, to be fair and comparable, we also excluded Cl − by applying a large repulsive energy, specifically ∼12 k B T / e, just for Cl − , which we thought would reduce Cl − occupancy by ∼e 12 ≈ 1.6 × 10 5 . The repulsive potential did not act on the cations, and diffusion coefficients of all ions were set equal for illustrative purposes in this calculation. Surprisingly, the PNP calculation using this profile predicted a reversal potential close to zero for external calcium concentrations <10 mM. That is, the “calcium” channel became a nonselective channel when Ca 2+ was <10 mM. When Ca 2+ in the external solution was 100 mM, the selectivity reversed (i.e., the reversal potential became −20 mV). The calcium channel had become a chloride channel, if we use common lab jargon. The change in selectivity of the channel was produced without invoking any specific chemical effects at all, it was produced by electrostatic repulsion, computed from PNP, just as the anomalous mole fraction effect was a purely electrostatic effect. Neither single filing nor definite occupancy were involved. When placed in a 100 mM Ca 2+ solution, 0.2 Ca ion was found in each pore in the calculation. This bound calcium produced a (small positive) local excess in net charge and that produced a large positive potential of nearly 110 mV . Of course, that potential was a severe barrier for cation movement. This potential barrier (produced by calcium binding) reduced cation movement (particularly divalent cation movement like calcium flux) so effectively that the residual conductance was dominated by chloride, even though chloride was subject to a repulsive potential of 12 k B T / e. By explicitly using Poisson's equation, we had found that binding of calcium dramatically reduces the calcium current, not because of “interference” by single filing, or an effect on diffusion constant, but because of an electrostatic effect. Of course, the details of this effect depend on the size of the repulsive potential that we chose. If we had chosen a repulsive potential smaller in magnitude, the channel would have become a chloride channel at lower external calcium concentration. If we had chosen a repulsive potential larger in magnitude, the chloride “selectivity” would have not appeared in the 100 mM Ca 2+ solution. But the electrostatic effects of the binding would have been profound in any case. Such effects are absent in traditional barrier models or other models that do not use Poisson's equation or Coulomb's law to go from charge to potential. Binding invariably has a large effect on potential because of the accumulation of charge that binding necessarily entails (that is what the word “binding” means!). That charge changes potential, and the change in potential is large because the system is so small (i.e., its capacitance is tiny). Bound ions repel nearby ions and thus have large effects, creating, for example, depletion layers that can dominate channel properties. These electrostatic effects of binding will be seen no matter what the details of the calculation or choice of repulsion potential, and the existence of these effects is the main point of our discussion. Barrier models miss the electrostatic effects of binding altogether. Rather than solving Poisson's equation, barrier models use fixed profiles of free energy to characterize the interactions of permeating ion and channel protein independent of charge; they use repulsion factors to characterize interactions between permeating ions as if the ions were always separated by a fixed distance, producing a fixed repulsive energy independent of the fixed charge of the protein and the screening of this fixed charge by nearby ions (in the pore and in the baths). Screening has been known to dominate the properties of electrolyte solutions and interfaces since the work of Debye-Hückel and Gouy and Chapman and it seems unwise to ignore it in channels. Using fixed profiles of free energy to characterize the interactions of permeating ion and channel protein is inaccurate because the potential profile inside the channel depends strongly on the concentration of ions in the bath, as is easily verified in a Poisson Boltzmann or 3DPNP calculation, because of the long range nature of the electric field: the ionic atmosphere of the fixed charge lining the channel's wall extends into the surrounding baths. Or, to put the same thing another way, the dielectric charge in the protein and lipid and the mobile charges in the channel's pore do not screen the fixed charge of the protein from the ions in the bath. Thus, the interactions of ions within the channel cannot be described by a theory that ignores the concentration of ions in the bath. The ions in the bath help determine the interaction between ions in the channel's pore. Using fixed interionic distances in traditional barrier models is inaccurate because the distance between ions is in fact quite variable. The distance depends on screening that varies with the concentration of ions in the bath, the transmembrane potential, and the charges and shape of the channel protein itself. In fact, to maintain a fixed average distance between permeating ions (as conditions are changed), large amounts of energy would have to be injected directly into the channel's pore by a deus ex machina, always the theorists' most helping hand. We suggest that the fundamentally flawed treatments of electrostatics in barrier models are likely to produce a qualitative misunderstanding of the role of occupancy and quantitative errors of at least one order of magnitude. Our calculation shows occupancy predicted by Poisson's equation is very different from that predicted with barrier models. Our calculations show that if a channel is to hold a certain number of mobile charges, it must have balancing structural charges, and the interactions of these mobile and fixed charges cannot be described by a fixed free energy. The wide variations in ionic occupancy that typically occur in barrier models are likely to be in severe conflict with the electrostatics of Poisson's equation customarily used to describe the electric field. That is to say, if such variations in occupancy actually occurred, the electrical potential would vary wildly because of the severe violations of local electrical neutrality. If a channel is lined by a fixed structural charge, electrostatic effects tend to maintain a (nearly equal) number of mobile ions within the pore, thereby maintaining approximate electroneutrality. Wide variations in ionic occupancy are buffered by the need for approximate electrical neutrality; i.e., wide variations in occupancy can only be produced by large energies not typically available to channels. Even though ionic occupancy is buffered, screening depends on bath concentration because the electric field generated by the fixed charge lining the wall of channels reaches through the protein and lipid into the surrounding baths: the electric field is long range. Electroneutrality in the channel is approximate, not exact, and the residual (“unneutralized”) charge is large enough to have profound effects. We conclude that traditional barrier models overestimate current because they neglect friction and underestimate current because they compute electrostatics incorrectly. These errors of course do not balance precisely and that is why barrier models fail to fit reasonably large data sets, particularly if the data sets include I–V relations measured in asymmetrical solutions. These errors are fundamental to the whole class of barrier models, and so we believe such models must be abandoned. When a model fails as badly as barrier models do, its qualitative features cannot be considered a reliable indicator of underlying mechanism. It is not useful for our main purpose. To us, abandoning barrier models seems no great loss. Those models do not fit the data anyway (if the data is taken over a reasonable range of conditions). But abandoning barrier models is a great loss, in a more human sense, because so many gratifying insights into mechanism have been developed using them, often at great effort. Abandoning barrier models means calling these insights into question. It means these insights must be reexamined using theories that fit data and have some physical basis. It means that much more experimentation is needed to reexamine issues already thought to be settled. Reexamination of settled issues is bound to be unsettling. The outstanding problems in permeation are to understand the role of electrostatics, chemistry, and geometry in determining the movement of ions (in our view). So far, the role of electrostatics and geometry seems approachable by 3DPNP. We suspect, but have not proven, that the effective one-dimensional profile of charge we call P ( x ) will be approximated by derivatives of the solution Φ 3DPNP ( x , r ,θ) of the three-dimensional PNP equation (specifically, the divergence of the three-dimensional electric field in the radial direction and equivalently its derivative along the path of permeation, with the concentration of permeating ions subtracted off). Further analysis will tell whether the excess free energy needed to fit some of our data sets is an expression of the three-dimensional field, of actual chemical interactions, or of some other effect. Knowledge of permeation is limited by a surprising lack of published I–V curves in asymmetrical solutions. These are important because they are often much more linear than expected from traditional models. Knowledge is also limited because we have so little structural information, particularly of the eukaryotic channels of greatest anthropomorphic interest; e.g., the voltage-gated Na + and K + channels of nerve fibers. As we turn to these specialized channels, it seems likely that the simplest version of PNP will need to be supplemented by models containing more explicit chemistry; i.e., binding energies in binding regions. The question is how to introduce chemistry into a model without requiring analysis of uncomputable trajectories. We are following the chemists, taking a most successful theory of ions in bulk solution, namely the MSA and applying it inside a channel: it is comforting to work with a theory and people who have solved the problem of selectivity in bulk solution. The MSA has a long history , and recent versions have been remarkably successful at predicting the properties of solutions from infinite dilution to saturation, even molten salts . The MSA is a mean field theory that in essence reworks Debye-Hückel analysis, now treating ions as spheres. The distribution of point charges around a central sphere (as assumed in DH) is quite different from the distribution of spherical charges around a sphere (MSA), particularly at concentrations more than a millimole or so, because finite ion diameters create exclusion zones around ionic charges. The resulting charge distributions produce quite different electric fields, according to Poisson's equation, and this difference allows MSA to fit much data that DH cannot. Fortunately, the MSA is hardly more complex than DH because both theories express thermodynamic functions in terms of one quantity, a characteristic length of screening κ −1 that is given by algebraic formulas, albeit more complex formulas in the MSA than in DH. MSA in its primitive form treats water as a continuous ideal dielectric, whereas “nonprimitive” versions include the solvent as a polar molecular species (up to octopolar, as is needed to model hydrogen bonding). More accurate theories are available , but they are more elaborate to compute and do not lead to algebraic expressions for activity coefficients. Interestingly, most current theories of bulk solution and narrow spaces (DH, HNC, and MSA) are mean field theories; they do not try to follow or explicitly average individual ionic trajectories. These theories deal self-consistently with the average effects of excluded volume, including diameter-constrained electrostatic interactions. To be applied rigorously to channels, these theories need to be reexamined and rederived for the geometry of channels and the specific properties (e.g., excluded volume) of the amino acids that line the wall of the channel, taking note of its high surface charge density. Even better, density-functional theory should be applied to channels and proteins. We are trying, thanks to Laura Frink of Sandia National Laboratory (Albuquerque, NM). Even before these rigorous treatments are available, it is already clear that many of the distinguishing characteristics of K + , Na + , and Ca 2+ channels can arise naturally from the two main (antagonistic) effects in MSA: electrostatic attraction between permeating cations and the groups forming the selectivity filter, and “chemical” repulsion arising from the effects of the finite volume of ions . In the analysis of these three types of channels, no other specific chemical interactions are needed to describe selectivity (beyond those resulting from finite volume of ions as described by the MSA). Finally, we address the issue of obligatory single filing, an important property of ionic channels that has concerned us for many years. Nonner et al. shows that one of the experimental phenomena (the anomalous mole fraction effect) thought to require an explanation involving obligatory single filing can in fact be explained without single filing. The effect appears (in a mean field theory without obligatory single filing) as a necessary consequence of (a small amount of) localized excess chemical potential; i.e., binding or repulsion. Nonetheless, it is clear that PNP in its several forms does not predict the ratio of unidirectional fluxes observed in K + channels, or expected in single file systems. The paradoxical fact is that PNP predicts net fluxes (i.e., currents) in a wide range of channels and conditions, while it does not fit the ratio of the component unidirectional fluxes correctly, at least if we assume the flux ratio of all channels is rather like that of the K + channel. Resolution of the paradox requires analysis or simulation of a system with both obligatory single filing and with an electric field computed from the charges present. Wolfgang Nonner has constructed a mean field model of single filing, by extending PNP to include convection. This Navier-Stokes extension of PNP is clearly able to predict the appropriate ratios of unidirectional flux and I–V curves that PNP itself has not fit. We have also done much work (with Schuss and his students, available by anonymous FTP from ftp.rush.edu in directory /pub/Eisenberg/Schuss) to formulate a self-consistent model of Brownian motion; i.e., one in which ions move according to a Langevin equation in an electric field determined by all charges present, computed by a Poisson equation updated at each time step. The mean field arises naturally in the model because of the long range nature of the electric field. The fixed charge lining the wall of the channel is “neutralized” by ions in the bath (in large measure), and those ions can be described by a mean field theory under biological conditions. That is to say, the reaction field of the fixed charge on the wall of the channel is the mean field, even in a model constructed in atomic detail. It is clear from this work that a well-posed mathematical model can be constructed, and can predict flux ratios, but the model has not been solved in general. Interestingly, the analysis with Schuss shows that nonindependent flux ratios can arise without changing net flux. In that analysis, a “single file term” is found in both the influx and efflux, and so the net flux is not changed by single filing, but the flux ratio is. Perhaps this is how PNP manages to fit net flux data so well in the K + channel, while it gives ratios of unidirectional flux not expected in single file systems. We, along with others, are also trying to simulate such a single file Langevin-Poisson system. Until this simulation is actually performed, it is wise to be prudent and not guess its outcome. Rather, we will trust the work, particularly the resulting numbers. | Other | other | en | 0.999997 |
10352029 | The subject we will look upon is calcium channel permeation, models of which have been described in lucid detail . This case serves to bring out essential differences in a palpable and physiologically important context. I deal with just one small corner of this subject—the key observation that launched calcium channel permeation as a rich area of investigation. This is the remarkable fact that under physiological conditions the channel is strongly selective for Ca 2+ , but when bath [Ca 2+ ] is reduced below 1 μM this selectivity is lost and monovalent cations easily permeate. In the well-known classical experiment, inward current through calcium channels is measured as a function of external Ca 2+ concentration in a conventional physiological bath medium at a fixed holding voltage of, say, −30 mV. At very low Ca 2+ (<0.1 μM) there is a large inward current carried by Na + . As [Ca 2+ ] is raised up to ∼100 μM, the current decreases to near zero. But then, as [Ca 2+ ] increases farther into the 1–10 mM range, inward current rises again, with Ca 2+ as the charge carrier, and the reversal potential shifts positive towards E Ca . This nonmonotonic variation in current with external [Ca 2+ ], sometimes termed the “anomalous mole-fraction effect” (AMFE), is explained in vastly different ways by the two opposing viewpoints. According to the canonical model, the observed AMFE is a direct reflection of the binding of two Ca 2+ ions in a single-filing pore. The idea is simple, proceeding from the postulate that the channel is designed to coordinate Ca 2+ at specific anionic sites. In the absence of Ca 2+ , when these are electrostatically hungry, the pore is merely charge selective, allowing virtually any monovalent cation to permeate as long as it is physically small enough to squeak through. Thus, at low [Ca 2+ ], the Na + conductance is high. In the presence of micromolar Ca 2+ concentrations, the pore's selectivity region now becomes occupied by a single Ca 2+ a significant fraction of the time (which varies according to the bath concentration). Because of its intimate coordination by protein groups, this bound ion's dissociation rate from the channel is low, ∼10 3 s −1 , some three to four orders of magnitude slower than the throughput of Na + ions. Under these conditions, Na + roars through the pore when Ca 2+ is absent; but whenever a Ca 2+ binds, the flow of Na + current is fully blocked. This block lasts on the order of 0.1–1 ms, and it is due directly to the single-filing property: the impossibility of a Na + ion diffusing “around” a bound Ca 2+ . Only after the Ca 2+ vacates the binding site can the flow of Na + through the channel resume. This effect, averaged over many channels in a macroscopic experiment (or over time in a single-channel experiment), leads to the “falling phase” of the AMFE; i.e., the decrease of inward current as Ca 2+ increases through the micromolar range. If Ca 2+ concentration is pushed up into the millimolar range, a new phenomenon appears. Now a second Ca 2+ can bind and, as a result of this double occupancy, the exit rate of Ca 2+ from the pore increases ∼1,000-fold. This huge increase in Ca 2+ off rate is usually explained by invoking electrostatic repulsion between the two ions, but other mechanisms could be involved . In any case, as a result of this double occupancy, Ca 2+ now flows through the channel at rates high enough to show up as current, which increases with Ca 2+ concentration to produce the “rising phase” of the AMFE. To quantify these effects, the chemical-kinetic approach makes an explicit distinction between four different occupancy forms of the channel: a Na + -conducting form with no bound Ca 2+ [O, O], two nonconducting forms, each with one Ca 2+ bound on either side [O, X] and [X, O], and a Ca 2+ -conducting form with two Ca 2+ bound [X, X]. The average current and Ca 2+ /Na + selectivity are given by the kinetic transitions among these various forms of the channel; i.e., by a set of rate constants between explicit chemical intermediates, exactly as in any conventional chemical kinetic problem. The values of the rate constants cannot be estimated from first principles, but must be derived by fitting experimental data to a kinetic model, a straightforward but rarely unambiguous procedure. Much of the present controversy centers on the use of Poisson-Nernst-Planck electrodiffusion models in biological channels. Such models have been in use for a long time, both as qualitative handles for the classical squid axon channels and as more intricate frameworks for permeation in ion-selective channels with firm structural foundations . For this discussion, I will focus on a recent application to calcium channels termed PNP2 , which views the pore as a continuum containing several negatively charged groups smeared out over a reasonable pore volume; this represents a very high concentration of fixed charge (∼10 M). Both Ca 2+ and Na + have free access to this forest of negative charge, where they act as gegenions, cations that are not chemically coordinated by the fixed charge, but rather are held nonspecifically by the demands of electroneutrality (or, more properly, the Poisson equation), as in an ion- exchange resin. The system is described by the simultaneous solutions of three equations in which the three crucial variables, ion concentration, electrical potential, and distance along the pore, are nonlinearly entwined. The solutions lead to mathematically self-consistent predictions of ionic current as a function of transmembrane voltage and bath ion concentrations. In modeling the AMFE, PNP2 is a theory of ionic cleansing. With no Ca 2+ present, the pore conducts well because Na + ions dwell there at high concentration, this being the only cation available for electroneutrality. But when a little Ca 2+ is added to the bath, these divalent intruders, with their heavy artillery in the form of a +2 valence, take over, displacing the numerous but poorly armed Na + ions. In electrostatics, divalents always beat monovalents. Thus, as Ca 2+ is increased, the pore, initially Na + rich, becomes loaded with Ca 2+ , and the conductance goes down because the Ca 2+ diffusion coefficient is assumed to be lower than that of Na + . By the time bath Ca 2+ concentration reaches, say, 100 μM, all the Na + has been expelled from the pore, and the current is carried solely by Ca 2+ . Thus, the falling phase of the AMFE. But why does the channel conductance rise again as Ca 2+ is raised further? The surprising answer provided by the PNP2 treatment is that it doesn't! The conductance is predicted to remain essentially flat as Ca 2+ rises to high levels because electroneutrality forbids admittance to additional Ca 2+ over and above the fixed negative charge; but the current measured at a given voltage (e.g., −40 mV) does increase to produce the AMFE for a simple reason: the reversal potential keeps moving positive as external Ca 2+ is increased. It's the driving force that goes up, not the conductance. In other words, this analysis asserts, everyone in the field has been dunderheaded all these years on a most elementary point, having apparently forgotten that current equals the product of conductance and driving force! (I am oversimplifying a little here; a small rise in conductance with [Ca 2+ ] is predicted by PNP2, but this is a second- order effect having to do with surface polarization.) So here we have two very different ways of interpreting a fundamental set of facts about ion permeation in calcium channels. I will state my opinion bluntly. First, no theory, however mathematically sophisticated, that rejects specific ionic coordination by protein moieties, dismisses the finite size of ions, and ignores the single-filing effects necessarily arising from the small spaces in the molecular structures of ion channels can have much worthwhile to say about selective ion permeation. Second, a ubiquitous feature of continuum theory— the mean-field assumption—invalidates, or at least greatly vitiates, its application to channels in which only a small number of ions reside at any one time. Third, PNP2 is inadequate to understand the particular calcium channel problem under examination here. Fourth, the undoubted quantitative weaknesses of the chemical-kinetic approach do not undercut its value in capturing the mechanistic essence of permeation in ion- selective channels. For many years, indirect experiments have suggested that ions permeate selective channels by binding to localized sites at which protein functional groups replace waters of hydration, and that ion selectivity mainly reflects the energetics of the switch from water solvation to protein coordination . In soluble proteins, it is hardly a radical notion that binding of dehydrated inorganic ions lies at the basis of a multitude of functions , and now, with the structure of KcsA , this idea has been confirmed directly for a strongly selective ion channel. The KcsA structure dramatically confirms for a K + channel the multi-ion single-filing assumption, long known also to be valid for the peptide channel gramicidin A . For permeation, the qualitative consequences of localized, structured binding sites and single-filing are profound; they lead naturally and necessarily to familiar phenomena seen in many channels: strong, concentration- dependent selectivity, discrete ionic block of permeation, and anomalously high ratios of unidirectional ionic fluxes. It is not surprising that the continuum theories presently under discussion have been unable to satisfactorily reproduce these “enzyme-like” phenomena, since they (a) disregard the close-up chemistry of ionic coordination, (b) explicitly permit ions to move through one another within the pore, and (c) treat permeation mainly in terms of electric fields acting at a distance. This approach asserts the virtue of pristine, mathematically tractable physical principles, but it commits the vice of ignoring the messy parts: the prominent, obvious structural characteristics of channel proteins. To be sure, continuum theories have traditionally endeavored to include chemistry by superimposing upon the electrical potential a position-dependent free energy profile that may differ for different ions or by assigning to each ion its own diffusion coefficient. These are worthy additions to otherwise “featureless” electrodiffusion theories, but they are simply not enough; nobody has yet figured out how to weld single-file arrangements of binding sites to continuum theories in a general manner, although Levitt has achieved impressive success in incorporating these features in particular cases, and Nonner et al. similarly have modeled a subset of K + channel behaviors with a selective binding “region.” Conquest of this analytical impediment would represent a major advance in modeling permeation; in such a case, the entire channel field would unhesitatingly embrace continuum electrodiffusion as the preferred approach to the problem. To obtain solutions for the ionic fluxes, continuum treatments must use concentration and electrical potential as continuous spatial variables in the coupled differential equations. The concentration at a given position determines the net charge density, which in turn influences the value of potential at that and nearby positions. Concentration is an intrinsically probabilistic quantity—the average number of ions per unit volume. For a macroscopic object such as a worm of ion-exchange gel, the number of ions present is sufficiently large that this average can be taken within each cross-sectional slab of the object at each moment in time. The concentration at each position will fluctuate with time, but, if the object is large enough, these fluctuations will be negligible and the concentration, and therefore the potential, will be a time-invariant spatial average. This is the “mean-field” assumption: this average potential may be validly used in the three crucial equations. In an object of molecular dimensions, however, a huge problem arises. For something the size of a calcium channel selectivity filter, a concentration of 10 M represents on average only one or two ions in the entire volume. “Concentration” is still defined as a statistical average, but in this case the average must be taken over time; i.e., by sitting at a given position in the pore and asking what fraction of the time an ion is present. This is a perfectly good stochastic definition of concentration, but when you try to use it to relate concentration to potential via the Poisson equation, a fundamental difficulty asserts itself. A channel containing, say, one Ca 2+ on average will be fluctuating in occupancy among 0, 1, or 2 ions (0, 5, and 10 M concentration). The mathematics represents the channel as having a time-invariant potential equivalent to the average situation: single Ca 2+ occupancy. But this is a severe misrepresentation of the potential that a Ca 2+ approaching an empty channel, or a Ca 2+ about to leave a doubly occupied channel, actually sees, and these events are often rate determining for permeation. It is as if the I.R.S. applied to every taxpayer a uniform exemption calculated for 2.6 children, the mean number of children per American family. Described another way, a Ca 2+ aspiring to enter an empty channel at a given moment is treated by the electrodiffusion equations as though it experiences the repulsive electric field that existed, say, a microsecond before this moment, when the channel had one ion in residence; since occupancy-dependent changes in field are enormous and are established instantaneously, large errors in predicted behavior will arise from using an average potential. Thus, while valid for macroscopic objects and large, wide channels, the mean-field assumption applied to physically small channels yields solutions to the electrodiffusion equations that are mathematically chaste but physically debauched. The chemical-kinetic treatment avoids this problem by explicitly assigning distinct properties to the different occupancy forms of the channel. It asserts, for example, that the probability per unit time (i.e., the rate constant) of a Ca 2+ entering an unoccupied channel is very different from the probability of entering a singly or doubly occupied channel precisely because of the very different electric fields in the three situations. This description deliberately avoids doing what continuum theory, for mathematical reasons, must do: treating the channel as a single entity with properties averaged over the different occupancy forms. The single example in the literature of a continuum treatment of calcium channel behavior, PNP2 , does not achieve the goal it sets for itself. The analysis is very similar to that of the classical macroscopic ion-exchange membrane, where electrodiffusion is well understood . Emphasized in the analysis is that only the channel current at a fixed voltage, and not the conductance, is expected to show AMFE. Nonner and Eisenberg claim that published calcium channel experiments have demonstrated an AMFE only in current at fixed voltage, and that proponents of the standard view have merely assumed without evidence that the AMFE also applies to conductance, as chemical-kinetic theory says it must. This claim, if correct, would be a deadly criticism of the chemical-kinetic approach. But the claim is false. The original papers on calcium channel permeation reported strong AMFE in current at fixed voltage as well as in conductance, based on macroscopic I–V curves over a [Ca 2+ ] range from 60 nM to 10 mM. The conductance minimum is unambiguously observed at the single-channel level as well, in both Ca 2+ and Ba 2+ . These elementary facts, well-known to the channel community, contributed mightily to the swift and widespread acceptance of the chemical-kinetic view of permeation. The PNP2 analysis proceeds as though these facts do not exist, and it accordingly fails to explain the most basic hallmark of calcium channel permeation. This incorrect prediction of a Ca 2+ -independent conductance at physiological concentrations illustrates how badly a continuum theory that uses the mean-field assumption and ignores coordination chemistry can falter. As for the weaknesses of the chemical-kinetic view, they are certainly prominent and well-appreciated . It is impossible to predict a priori what the absolute values of the rate constants should be or how to relate rate constants to transition-state free energies. Likewise, the use of Eyring-like exponential voltage dependence to the rate constants is theoretically unjustified and always leads to incorrect I–V curve shapes. And physical space inside of channels is in fact continuous, not a lattice of sites. But so what? Most channel researchers don't really care about predicting absolute values of currents, just as enzymologists don't feel the need to calculate the k cat of an ATPase from quantum mechanics; it's the patterns of permeation behavior that count, not the absolute rates. As for the precise shapes of open-channel I–V curves, this is not a particularly compelling issue in channel physiology; the examples of unusual I–V shapes encountered in biologically meaningful contexts are invariably due not to intrinsic ionic diffusion properties, but rather to specific block (on discrete binding sites) by exogenous molecules (e.g., polyamine-induced inward rectification in K ir channels, or Mg 2+ -induced outward rectification by NMDA-receptor channels). And chemical kineticists don't believe that ions leap over tens of angstroms of pore length in a single bound; we do posit, however, in analogy to chemical reaction mechanisms, that sojourns on binding sites represent the preponderance of time the ion spends within the pore, and thus define the important rate- determining steps of ion permeation. Finally, there is a particularly compelling reason not to reject chemical kinetics in spite of its formal flaws: when used with an understanding of its limitations, it works. Its track record is excellent. It is primarily by chemical-kinetic analysis of ionic permeation over the past two decades that we have achieved physical pictures of ion channel proteins in the complete absence of direct structural information. It was chemical-kinetic analysis that told us that channels are built as axially symmetric structures with discrete selectivity filters and ion-binding sites at which ions are largely dehydrated, with narrow regions where ions and water lie in single file, with wide vestibules where drugs bind, and with enzymologically unprecedented regions where multiple ions bind simultaneously in close proximity. All of these features, which underpin the mechanisms by which ion channels achieve their paradoxical combination of selectivity and high transport rate, have now been observed directly in the first structure of a selective channel protein. | Study | biomedical | en | 0.999998 |
10352030 | This approach is made possible by the availability of channel structures that are known to atomic resolution. Until recently, the only channel that met this condition was the relatively simple gramicidin channel. However, with structures now available for the porin channel , potassium channel , iron transporting channel , and a mechanosensitive channel (all from bacteria), this approach now has the potential to be applied to these biologically interesting channels. The basic idea is quite simple. First, one assembles an initial atomic model of the channel protein, the channel water, a nearby region of the channel lipid membrane, and samples of the bulk water at both ends of the channel. One then places some ions in the water, sets the temperature, and applies a voltage or concentration gradient and directly measures the ion flux as a function of time as the exact atomic dynamics of the model are simulated on the computer. At each time step in the simulation, all the forces on each atom are calculated (covalent, fixed charges, dipolar, etc.) and the atom moves under this force for a time period short enough that the forces should remain constant. Unfortunately (or, fortunately, if you want to justify the use of other models), this direct approach is still beyond our current computational limits. The simulation must be run for a time period that is at least long enough to observe an ion crossing the channel. In fact, since the ion can take a large variety of paths through the channel and since the specific interactions with the channel and the other ions will vary randomly during the crossing, one should sample a minimum of 10 such crossings to estimate the channel conductance. For a channel with a conductance of 50 pS and an applied voltage of 100 mV, 10 crossings would take about 0.4 μs. For comparison, a recent simulation of the porin trimer channel OmpF (consisting of 1,020 amino acid residues, 300 phosphatidyl choline molecules, 12,992 water molecules, and 27 sodium atoms) required 2 h of computer time for 1 ps of real time . Thus, the 0.4 μs of real time needed to simulate the 10 ion crossings would require ∼100 yr of computer time. This calculation is for the ideal case where the entire channel and part of the lipid are included in the simulation. Computer times could be reduced by simulating only the protein residues lining the channel and replacing the rest of the protein and lipid by some continuum approximation. For example, in the molecular dynamic (MD) simulation of the acetylcholine channel , only the M2 helix bundles were directly simulated and the computer time was reduced to ∼1 yr for a single ion crossing. Because porin has such a high conductance, Suenaga et al. were able to observe a single Na + ion crossing the channel during a 1.3-ns MD simulation of a reduced porin model. Given the projected improvements in computational speed, such direct MD simulations should become possible within a few years. These numerical calculations also have some other limitations, beyond that of the computer time. The most serious problem is a fundamental limitation in the accuracy of the atomic force constants. For example, if one wants to estimate an ion channel conductance accurate to within a factor of 7, one needs to be able to determine the interaction energy of the ion and channel to within a factor of ∼2 kT. This is less than half the energy in a single hydrogen bond! Second and third order effects (polarization, etc.) that are usually neglected in molecular dynamic calculations can easily lead to errors many times this size. In addition, although the models of the water molecules that are used in these calculations have been carefully refined and tested, they still are not likely to be accurate enough for the purposes of calculating ion channel flux. These fundamental limitations are clearly illustrated by the recent MD studies of gramicidin. Because of its small size and known atomic structure, gramicidin has, historically, been the proving ground for ion channel models. It is the channel for which any new MD approach or extension is first tested. It presents a particularly rigorous test of these models because the small uniform channel radius (≈3 Å) means that the ion directly interacts with the channel wall along its entire length (≈30 Å). Most importantly, there is direct experimental NMR information about the specific localized binding sites of ions in the channel. Woolf and Roux attempted to predict these NMR results using MD simulations. They used a fully solvated gramicidin-dimyristoyl phosphatidylcholine model and modified the standard CHARMM force field to include first and second order polarization effects of the ion on the peptide. Despite the fact that this MD model was optimized to fit gramicidin, the theoretical predictions of the ion binding sites still differed significantly from the experimental results. Accurate predictions of the ion binding locations require that the ion free energy as a function of position be calculated to an accuracy of a few tenths of a kilocalorie per mole , and this is beyond the limits of current MD calculations. Despite these current limitations, MD simulations still have an important function in modeling ion channels. From a cursory survey of the current literature, they are now the most popular approach to the theoretical investigation of ion channels. Simulations for short times (10 ps) can be used to obtain information about the local potential energy and diffusion coefficient of the ion as a function of position in the channel . This information can then be used in combination with Brownian dynamics or Poisson-Nernst-Planck theory (see below) to estimate the channel flux. Molecular dynamics is becoming a relatively routine procedure thanks to the availability of extremely sophisticated software (AMBER, GROMOS, CHARMM, XPLOR). Although MD calculations of flux in ion channels now require some special modifications of these routines, it is likely that some user-friendly interface will soon be developed to bring these calculations to the masses. The above discussion makes it clear that some simplifying assumptions are required. In the approach described in this section, it is assumed that the protein structure is held fixed and the water molecules are replaced by a continuum. With these assumptions, the three-dimensional (3-D) movement of ion i can be described by the following simple equation: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}m_{i}\frac{dv_{i}}{dt}=m_{i}\;f_{i}v_{i}+F_{R}(t)+q_{i}E_{i},\end{equation*}\end{document} where m i , v i , q i and f i are the mass, velocity, charge, and frictional coefficient on the ith ion, respectively. F R is a random thermal force representing the effects of collisions with the water and channel wall. E i is the total electrical field on the ion, including the partial charges in the protein, all the other ions in the system, and the induced charges from the variation in the dielectric constant at the boundaries between the protein, water, and lipid. It is assumed in Eq. 1 that the only significant forces on the ion are long range electrical forces plus some kind of short range scattering condition when the ion contacts the hard sphere radius of the channel wall. Although one could always add other short-range specific force terms, this would, in effect, be adding an empirical term that did not arise directly from the known protein structure. The solution for this approach proceeds as in the above molecular dynamics method. The channel boundaries are defined, all the ions in the channel and attached bulk reservoirs are positioned, and then, for each ion i, Eq. 1 is integrated in discrete time steps (Brownian dynamics). Because the dynamics of the water and protein are no longer included and relatively long time steps can be taken for the ion motion, this approach is many orders of magnitude faster than the exact molecular dynamics approach (see below for a specific example). The ability to accurately account for the interaction between ions in the channel system is one of the most difficult and critical aspects of modeling ion channels. In the absence of such interactions, the channel conductance will vary linearly with the ion concentration. Since almost all ion channels show some nonlinearity (e.g., saturation) in the physiological concentration range, it is essential that this interaction be accurately modeled. A major advantage of this Brownian dynamic approach is that it allows a direct simulation of this ion–ion interaction. At each step in the dynamics, the position of all the ions in the channel system are determined and their interaction energy ( E i ) is calculated for the next time step. One difficulty with this approach is that, because of the induced charges at the membrane and channel water interface, the calculation of the electrostatic energy ( E i ) at each step requires an involved, time-consuming calculation. S.H. Chung's group has recently described two three-dimensional Brownian dynamics (BD) simulations that use exact 3-D electrostatic potentials . In the first calculation, Li et al. used an idealized, unrealistic channel model geometry that allowed an analytical solution for E i . It provides an interesting analysis of the effects of allowing the ion to wander over the entire mouth and pore region of the channel area, rather then be constrained to the channel axis as in the standard 1-D solutions. The second calculation was for an idealized acetylcholine receptor channel (ACHR) . For this case, it was necessary to first use a numerical procedure to solve for the electric field and potential on a grid. This data was then stored in a lookup table that was used during the BD simulation of a channel that was in contact with reservoirs large enough to hold 52 ions. A 1-μs real time simulation of this ACHR channel required only 18 h of computer time, making this approach four to five orders of magnitude faster than exact MD simulations. It is now generally recognized that a combination of MD and BD provides the best available approach to modeling the ion conductance of channels whose atomic structure is known. In this combination approach, local MD simulations are carried out for different positions of the ion. These calculations are then used to determine the local diffusion coefficient and the perturbation of the local channel structure (e.g., main chain and carbonyl shifts) induced by the ion. This local value for the diffusion coefficient is then used to determine the frictional coefficient ( f i , Eq. 1 ) and the perturbed structure is used for the fixed channel structure and both are varied as a function of the position of the ion in the BD calculations. The next simplifying approximation is to keep Eq. 1 , but replace the exact expression for E i by a mean field approximation E i that represents a sort of average over all the possible positions of the other ions in the system. This E is calculated using Poisson's equation. This combination of random thermal motion of the ion combined with a Poisson solution for E is referred to as the Poisson-Nernst-Plank solution (PNP). Although most PNP solutions have been for the 1-D case (see below), a general 3-D PNP solver has recently been described . The 3-D steady state Nernst-Planck equation is given by: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}0={\nabla}{\cdot}[{\nabla}c_{i}(R)+{\beta}{\nabla}V_{i}(R)c_{i}(R)],\;\end{equation*}\end{document} where R is the 3-D position vector, c i is the concentration of the ith ion, β = 1/ kT and V i is total potential energy of the ith ion and is described by: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}V_{i}(R)=U(R)+z_{i}e{\phi}(R),\end{equation*}\end{document} where U ( R ) is the potential due to nonelectrostatic forces, φ is the electrostatic potential, z i is the valence of the ith ion, and e is the electron charge. The value of φ is then determined from the solution to the 3-D Poisson equation. Given the concentration and potential on the boundaries in the bulk solution, these equations have a unique interior solution for c i from which the channel flux of ion i can be obtained. PNP is much faster than BD because it replaces the simulation of the ion movement at a sequence of time steps by a global numerical solution of a differential equation. A unique feature of the approach of Kurnikova et al. is that the solution to Eqs. 2 and 3 is combined with the standard Poisson-Boltzmann Equation solver Delphi , which is routinely used to solve for the electrostatic potentials in proteins. This solver was tested using the gramicidin channel . A long standing question about gramicidin has been the origin of its cation selectivity. Since the channel is uncharged, this selectivity presumably arises from partial dipolar charges in the channel. This is now a classical problem that has been studied by a large number of investigators and it has been a challenge to obtain theoretical results that are in good agreement with experiment without imposing some arbitrary adjustable parameters. For example, in the molecular dynamics study of Roux et al. and the reduced dynamic model of Dorman et al. , special care and adjustments had to be made to get the models to agree with experimental results. Both of these models are significantly more complicated than the PNP model of Kurnikova et al. , which was applied to the gramicidin channel without any modifications. . In general, the results of this 3-D PNP solution for gramicidin were in remarkably good agreement with the experimental results except, possibly, for the location of one of the high affinity sites in the channel. This PNP result, which uses just the fixed, ion-free gramicidin structure, is surprising because the molecular dynamic calculations indicated that it was essential to include local ion-induced peptide (carbonyl) perturbations in channel structure to explain the qualitative features of the gramicidin channel. In any case, this PNP result is very impressive considering that it represents a direct application of this general “off the shelf” model. There are clearly some limitations to this 3-D PNP approach. The use of a mean field approximation (Poisson equation) is clearly a major simplification. It reduces the specific ion–ion interaction to an interaction between the ion and this mean field. It is difficult to quantitate the accuracy of this approximation, and its range of validity is the subject of considerable debate . Mean field theories have a habit of working better than one might expect (e.g., the Debye-Huckel theory), and the only way to test them is by comparison with theoretical models of higher accuracy; e.g., BD. Despite its obvious limitations, the 3-D PNP solver of Kurnikova et al. has the potential to become the routine, quick first approach for estimating the conductance of a channel whose atomic structure is known or can be estimated. All the user of this software would need to do is to input the atom coordinates, the bulk concentrations and applied potential, and the program would then output the fluxes of the different ions. The next major simplification is to replace the 3-D concentration function used above by a 1-D concentration averaged over the radial cross section of the channel. As with the use of the mean field approximation, it is difficult to quantitate this 1-D assumption, and its range of validity is not known. In general, with each additional assumption that has been described here, the model results become more empirical and less related to the detailed knowledge of the channel structure. However, if the channel structure is not known to atomic resolution, which is still the usual case, then the error introduced by these approximations is probably of the same order as the uncertainty in the structure, and there is no advantage in using more complicated models. As with the 3-D case, the 1-D BD approach has the major advantage that it allows for accurate modeling of ion–ion interactions. Despite this apparent theoretical advantage, there are not many examples of the use of this approach. One of the most detailed is the simulation of a multiply occupied cation channel by Bek and Jakobsson . These authors were only interested in the qualitative features of the solution, and did not try to model the real channel electrostatic potential terms. One problem that limits the accuracy of this approach is the difficulty of simulating the 3-D bulk solutions by an equivalent 1-D model. The most complete application of the 1-D PNP model to ion channels is the modeling of the acetylcholine receptor channel by Levitt . In this solution, realistic channel geometry and electrostatics were used and the cation and anion flux as a function of varying channel partial charges was obtained. This solution is appealing because just the fundamental structural features of the channel are used, and there is a minimum of adjustable parameters. The resulting ion flux was in good agreement with the experimental data. Recently, Nonner and Eisenberg have used this approach to model L-type calcium channels. Their solution does not attempt to rigorously model the channel geometry or electrostatics, but rather is used to illustrate the general features that are required to fit the flux data for these calcium channels. It had previously been assumed that calcium channel kinetics required direct interactions between two or more ion binding sites. The results of Nonner and Eisenberg demonstrated that a simple PNP model could qualitatively reproduce many of the experimental calcium channel properties. An essential feature of this solution is the addition of a local, nonelectrostatic ion– channel interaction . For example, to fit the calcium channel data, the channel was assumed to have a relatively high affinity site for calcium relative to sodium that could not be explained in terms of the partial charges in the channel. This is a semi-empirical correction factor and has the disadvantage that the channel behavior can no longer be understood in terms of its well understood long range electrostatic properties. Of course, it is possible that some nonelectrostatic ion–channel interactions are important in determining channel behavior. One potential problem in using a large local attractive potential is that there is nothing in the standard PNP theory to prevent the local ion concentration from reaching the unphysical concentration of more than one ion per hard sphere ion volume. In the approach of Levitt , a simple modification of the PNP theory was introduced to correct for this problem. As with the 3-D PNP approach, the 1-D PNP solution uses an approximation to the direct ion–ion interaction whose validity is difficult to evaluate. It certainly can reproduce some aspects of this interaction, as is evidenced by its ability to mimic the concentration dependence of the conductance for the calcium and acetylcholine receptor channels . Levitt has described a modification of the PNP solution to allow for direct interaction between two ions. Although the approach seems to provide a more accurate way to treat ion–ion interaction, it does so at the cost of a large increase in mathematical complexity. In this final simplification, it is assumed that the ions are localized in specific regions of the channel, and the kinetics are represented by the rate constants for jumping between these regions and between these regions and the bulk solution. This approximation obviously represents a gross simplification that reduces the channel kinetics to its bare fundamentals. The reaction-rate (RR) model should be classified as a simplification of BD rather than PNP because it does not use the mean field assumption. In fact, the RR approximation is ideally suited for modeling strong ion–ion interactions. There has been a long-standing debate centered on the issue of reaction-rate “versus” continuum theory (where continuum theory refers either to 1-D NP or PNP approximations). The answer depends on what one is trying to model. If one wants to interpret the channel conductance in terms of the actual channel geometry and electrostatics, then some sort of continuum model is essential since it provides a first order approximation to the actual channel structure. On the other hand, if one is simply trying to parameterize the channel or one knows that strong ion–ion interactions are important, then the RR model may be preferable . The problem with the RR model occurs when the investigator over-interprets the results and attempts to relate the rate constants to real energy barriers . For example, the rate of going from the bulk solution to a binding site in the channel may simply be limited by the ion diffusion rate and there may not be any physical energy barrier. The problems become particularly severe if one tries to explain the experimental current–voltage curves using the voltage dependence of the RR energy barriers. For example, most biological ion channels have relatively linear current–voltage relations, while the voltage dependence of a single RR energy barrier is highly nonlinear . To fit a linear I–V curve with the RR model, it is necessary to add multiple energy barriers that probably have no relation to the actual physical energy barriers in the channel. As illustrated in this brief review, there is a hierarchy of approaches to modeling ion channels ranging from the exact molecular dynamics simulation down to reaction-rate theory. Each new simplification introduces a new limitation or uncertainty in the result. In the past, there was little reason to use the most exact solutions because of the computational limitations and, more importantly, without atomic resolution channel structures, these solutions were basically empty exercises. That situation is clearly about to change and we are at the beginning of a new era in ion channel modeling. The obvious first candidate for this new approach is the Streptomyces lividans potassium channel, whose structure has been recently solved by x-ray diffraction . This channel should become a major testing ground for checking and calibrating this new generation of ion channel models. There is no question that strong ion– ion interactions are important for this channel since at least two ions are directly observed near the selectivity filter. Thus, an accurate simulation of this ion–ion simulation should require the use of the 3-D Brownian dynamic approach and rules out some sort of Poisson mean field approximation. One will probably need to use molecular dynamics to estimate the cation diffusion coefficient and potential function in the region of the narrow selectivity filter. A stringent test of the competing models will be provided by their ability to predict the conductance changes that occur for specific channel mutations. For most channels, the structure is not known, and the old fashioned use of channel models to guide the interpretation of flux measurements in terms of structure is still relevant. This is illustrated by the current debate about the selectivity mechanism of the calcium channel . Since the two protagonists in this debate are represented by separate articles in this issue, no more will be added here. Suffice it to say, the excitement that is generated by this issue is the best illustration that these old fashioned, semi-empirical models still have a role to play in our understanding of ion channel function. | Study | biomedical | en | 0.999997 |
10352032 | The essential functions of ion-conducting channels, selectivity and gating, have historically been described as independent processes, each determined by different features of the molecular structure of the channels . Direct investigation of cloned channels, however, has demonstrated that these functions may be linked. In voltage-gated K + channels, for example, the mutation of a single amino acid can change both the ion selectivity and the voltage dependence of activation . In N -methyl- d -aspartate channels, ion selectivity changes with the state of channel activity ; this is also the case in some voltage-gated K + channels . Because of structural similarities, cGMP-gated ion channels are recognized as members of the superfamily of voltage-gated ion channels . In cGMP-gated channels, there is no direct evidence that ion selectivity is linked to the state of channel activity. However, Cervetto et al. have reported that the relative ability of various divalent cations to carry current through these channels in intact rod photoreceptors appears to change as a function of cGMP concentration. The activity of cGMP-gated channels underlies the light-dependent conductance of rod and cone photoreceptors in the vertebrate retina. These channels select divalent over monovalent cations , although they select poorly among monovalent cations . The relative selectivity of Ca 2+ over Na + (PCa/ PNa) is higher in cone than in rod channels, both in native membranes and in recombinant channels formed from α subunits alone . This difference may be important in understanding the difference in phototransduction signals between the two receptor types, because the Ca 2+ influx through these channels, and its balance with efflux via a Na + /Ca 2+ ,K + exchanger, helps maintain the cytoplasmic free Ca 2+ concentration in these cells . The differences in PCa/ PNa between cones and rods make it likely that light-dependent changes in cytoplasmic Ca 2+ caused by the same light intensity will be larger and faster in cones than in rods . Studies of ion selectivity in cGMP-gated channels of photoreceptor membranes, however, have only been conducted at ligand concentrations that fully activate the channels. Yet, in intact photoreceptors and under physiological conditions, at most 1–5% of the cGMP-gated channels are open , which indicates that the highest cytoplasmic concentration of cGMP in the cells is about four- to five-fold smaller than K 1/2 , the nucleotide concentration that half-saturates current amplitude. If ion selectivity were linked to gating, then the channel attributes in the intact photoreceptor might differ from those known from studies under saturating agonist concentrations. We investigated the ion selectivity of cGMP-gated channels from both rods and cones as a function of cGMP concentration in membrane patches detached from intact outer segments. Contrary to the traditional view, we have found that the selectivity is indeed linked to gating: the selectivity for Ca 2+ over Na + increases continuously as the probability of channel opening rises. This proportionality is steeper in channels of rods than in those of cones. Under physiological cGMP concentrations, PCa/PNa in cone channels is ∼7.5-fold larger than that in rod channels, significantly larger than had been previously measured at saturating concentrations of cGMP . Cyclic nucleotide–gated channels in rod photoreceptors are heteromeric assemblies composed of at least two structural subunits, α and β . By homology, it is likely that channels in cones also comprise α and β subunits, but such subunits have not been identified to date. α subunits of bovine rod and cone channels expressed in Xenopus oocytes preserve the differences in Ca 2+ selectivity characteristic of native channels, but the ability of Ca 2+ to block the channel and the absolute values of PCa/PNa differ from those of native channels . This suggests that the selectivity and interaction of Ca 2+ with cGMP-gated channels may depend on the interaction of α and β subunits. Using Xenopus oocytes as an expression system, we determined that the cGMP-dependent shift in divalent cation selectivity is a property of heteromeric αβ channels and not of homomeric channels formed from α subunits alone. Striped bass ( Morone saxitilis ) were obtained from Professional Aquaculture Services and maintained in the laboratory for up to 6 wk under 10:14-h dark:light cycles. Tiger salamanders ( Ambystoma tigrinum ) were received from Charles Sullivan and maintained in the laboratory in an aquarium at 6°C under 12:12-h dark:light cycles. The UCSF Committee on Animal Research approved protocols for the upkeep and killing of the animals. l-cis-diltiazem was the kind gift of Tanabe Seiyako Co. Ltd. Under infrared illumination and with the aid of a TV camera and monitor, retinas were isolated from dark-adapted animals and photoreceptors were dissociated as described in detail elsewhere . Single cones were isolated by mechanical dissociation of fish retinas briefly treated with collagenase and hyaluronidase. Solitary cones were maintained in a Ringer's solution consisting of (mM): 143 NaCl, 2.5 KCl, 5 NaHCO 3 , 1 Na 2 HPO 4 , 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, pH 7.5, osmotic pressure 309 mOsM. Rod outer segments were isolated by mechanical dissociation of tiger salamander retinas and were maintained in a Ringer's solution composed of (mM): 100 NaCl, 2 KCl, 5 NaHCO 3 , 1 Na 2 HPO 4 , 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES, pH 7.5, osmotic pressure 227 mOsM. Solitary photoreceptors were firmly attached to a glass coverslip derivatized with wheat germ agglutinin . The coverslip formed the bottom of a recording chamber held on the fixed stage of an upright microscope equipped with DIC optics and operated under visible light. A suspension of photoreceptors in Ringer's in which glucose was replaced with 5 mM pyruvate was added to the recording chamber and cells were allowed to settle and attach to the coverslip. After 5 min, the bath solution was exchanged with the normal, glucose-containing Ringer's. The recording chamber consisted of two side-by-side compartments. Cells were held in one compartment that was continuously perfused with glucose containing Ringer's. The second, smaller compartment was continuous with the first one, but a movable barrier could be used to separate them . We used tight-seal electrodes to obtain inside-out membrane fragments detached from the side of the outer segments of either cones or rods. Electrodes were produced from aluminosilicate glass . After forming a giga-seal and detaching the membrane fragment, the electrode was moved under the solution surface from the compartment containing the cells to the smaller compartment. The barrier was moved to isolate the two compartments and the electrode tip was placed within 100 μm from the opening of a 300-μm diameter glass capillary that delivered test solutions onto the cytoplasmic (outside) surface of the membrane patch. In a significant fraction of patches, we initially failed to observe cGMP-activated currents. We assumed these were closed vesicles since we frequently succeeded in eliciting currents after rapidly crossing the air–water interface. Plasmids containing either the α or β subunits of the bovine rod cGMP-gated channel flanked by 5′ and 3′ untranslated regions of the Xenopus β-globin gene were kindly provided by the laboratories of W. Zagotta (University of Washington, Seattle, WA) and R. Molday (University of British Columbia, Vancouver, British Columbia, Canada), respectively. Capped RNA was transcribed from the linearized plasmid with T7 RNA polymerase . RNA was purified by extractions with phenol/chloroform, recovered by ethanol precipitation and dissolved in RNAase-free water at a concentration of 2 μg/μl. Xenopus laevis oocytes, generously provided by the lab of L.Y. Jan (University of California at San Francisco) were each injected with ∼45 nl of RNA (90 ng). When α and β subunits were coinjected, RNA was mixed at a weight ratio of 4:1 (β:α). Injected oocytes were gently rocked at 18°C in ND96 media supplemented with 2.5 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Oocytes were suitable for electrical studies 3–5 d after injection. Immediately before patch clamping, each oocyte was incubated for 5 min in a hypertonic solution composed of (mM): 200 NaCl, 10 HEPES, 1 CaCl2, 1 MgCl2, pH 7.5, and its vitelline membrane removed. Denuded oocytes were attached to clean glass coverslips in the recording chamber described above and bathed in ND96. We used tight-seal electrodes to obtain inside-out detached membrane patches. The electrodes were produced from aluminosilicate glass (1.5 × 1.0 mm o.d. × i.d.) with large tip openings (∼2 μm). In studies of both rod and cone photoreceptor membranes, we filled the tight-seal electrodes with the same, standard solution (mM): 150 NaCl, 5 BAPTA, 10 HEPES, adjusted with tetramethylammonium hydroxide (TMA-OH) to pH 7.5, osmotic pressure 300 mOsM. TMA-OH was used to titrate pH in all solutions because TMA does not permeate the channels of rods or cones . Free Ca 2+ concentration in this solution was <10 −10 M. After detachment, all membrane patches were first exposed for at least 2 min to a standard solution composed of (mM): 150 NaCl, 1 EDTA, 1 EGTA, and 10 HEPES, adjusted with TMA-OH to pH 7.5. This solution thoroughly removed any endogenous modulator that might remain associated with the channels . Ionic concentrations were calibrated by measuring the osmotic pressure of the solutions and comparing it with published standards . The solution bathing the cytoplasmic membrane surface was selected among four possible test conditions, depending on the objective of the experiment: (a) the standard solution defined above; (b) the standard solution containing varying concentrations of cGMP; (c) the standard solution containing varying concentrations of Ca 2+ or other divalent cations, with or without cGMP; and (d) solutions containing 150 mM of various monovalent cations replacing Na + in the standard solution, with or without cGMP. We began every experiment by measuring current– voltage (I–V) 1 curves under symmetric NaCl solutions first in the absence, and then in the presence, of 1 mM cGMP. The point at which these two curves intersect defined the origin (0,0) in the I–V plane for all measurements in that membrane patch. At the end of experimental manipulations, these curves were again measured. We only analyzed data from membrane patches in which the origin did not shift and in which the maximum cGMP-dependent conductance changed by ≤10%. Internal and external solutions used to study membrane patches detached from Xenopus oocytes were similar to those used in studies of photoreceptor membranes, except that Cl − was replaced with methanesulfonate to eliminate the Ca 2+ -activated Cl − current characteristic of the oocyte membranes . To use a Ag/AgCl electrode in the absence of Cl − ions, we modified the tight-seal electrode holder to incorporate a 1-M KCl/agar bridge between the electrode-filling solution and the Ag/AgCl half-cell. We measured membrane currents under voltage clamp at room temperature with a patch clamp amplifier . Analogue signals were low-pass filtered below 1 kHz with an eight-pole Bessel filter (Frequency Devices Inc.) and were digitized on line at 3 kHz (FastLab; Indec). Membrane voltage was normally held at 0 mV and membrane currents were activated with continuous voltage ramps from −70 to +70 mV (over 1 s) or from −30 to +30 mV (over 0.5 s). In experiments with biionic solutions of monovalent cations, the holding voltage was set at the reversal potential corresponding to the ionic condition under investigation. We did so to avoid current flow at the holding voltage in order to minimize errors due to ion accumulation (or depletion) within the electrode's tip. Before initiating the voltage ramp, the voltage was held for 200 ms at either −70 or −30 mV (depending on the ramp range). This interval was sufficient to attain a steady current at −70 or −30 mV, following the time- dependent changes due to the relief by voltage of divalent cation channel block . A Ag/AgCl reference electrode was connected to the bath through a 1-M KCl agar bridge to avoid shifts in electrode potential as solutions changed. As is conventional, outward currents are positive and the extracellular membrane surface was defined as ground. We calculated permeability ratios from reversal potentials using the Goldman-Hodgkin-Katz constant field equation. To determine reversal potentials of cGMP-activated currents, a straight line was fit to their I–V curve between −15 and +10 mV. Reversal was that potential at which this straight line intercepted the I–V curve measured in the same patch and under the same ionic gradient, but in the absence of cGMP (the leak current). Under biionic monovalent cation solutions, ion selectivity was expressed as a permeability ratio defined by the equation: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{PX}{PNa}=\frac{[Na]_{o}}{[X]_{i}}e^{-\frac{FV_{rev}}{RT}},\end{equation*}\end{document} where PX/PNa is the permeability ratio of the cation X relative to Na + , V rev is the measured reversal potential, [Na] o is the extracellular Na + activity, and [X] i is the intracellular activity of cation X. F, R , and T have their usual thermodynamic meanings. Activity coefficients were taken from Robinson and Stokes . Under conditions of symmetric Na + with Ca 2+ (or other divalent cation) added only to the intracellular side of the membrane, we used the following equation derived from a more general equation given by Lewis : 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{PCa}{PNa}=\frac{[Na]_{o}}{4[CA]_{i}} \left( e^{-\frac{FV_{rev}}{RT}}-1 \right) ,\end{equation*}\end{document} where [Na] is the Na + activity on both sides of the membrane, and [Ca] i is the intracellular Ca 2+ activity. Activity coefficients for Ca 2+ in the presence of monovalent cations were taken from Butler , and then squared in accordance with the Guggenheim convention . Activity coefficients for Sr 2+ were assumed to be the same as for Ca 2+ . Mathematical functions were fit to experimental data using nonlinear, least square minimization algorithms (Origin; Microcal Software, Inc.). Statistical errors are presented throughout as mean ± SD. We examined cGMP-dependent currents in inside-out membrane patches detached from the outer segment of rods isolated from the tiger salamander retina. In the presence of 300 μM cGMP, a concentration at which the probability of channel opening is at its maximum value, the currents under symmetric Na + solutions reversed direction at ∼0 mV and their I–V curves were nearly linear . Addition of Ca 2+ to the cytoplasmic membrane surface shifted the reversal potential to a more negative value and changed the shape of the I–V curve , as has been previously reported . The shift in reversal potential reveals that the channels are more permeable to Ca 2+ than to Na + and the change in the I–V curve reflects a voltage-dependent block of the channels by Ca 2+ . The average shift in reversal potential with 10 mM Ca 2+ was −6.3 ± 0.27 mV (range −8.5 to −5.1, n = 7). This indicates (Eq. 2 , using ion activities and assuming the Guggenheim convention, see materials and methods ) that the average value of PCa/PNa is 6.48 ± 0.35. We investigated whether the value of PCa/PNa changed with cGMP concentration. Under symmetric Na + solutions with 10 mM Ca 2+ added to the cytoplasmic membrane surface, we measured membrane currents generated by voltage ramps in the presence of various cGMP concentrations . As has been repeatedly shown before , the amplitude of the current, at a fixed voltage, increased with cGMP in a manner well described by the Hill equation . 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I=I_{max}\frac{[cGMP]^{n}}{[cGMP]^{n}+K_{1/2}^{n}},\end{equation*}\end{document} where I is the amplitude of the cGMP-dependent membrane current, I max is its maximum value, [cGMP] is the concentration of cGMP, K 1/2 is that concentration necessary to reach one half the I max value, and n is a parameter that reflects the cooperative interaction of cGMP molecules in activating the membrane current. Remarkably, the reversal potential of the I–V curves shifted as the cGMP concentration changed , suggesting that PCa/PNa is not constant, but changes as a function of channel gating. For experimental convenience and to reduce uncertainties due to potential changes in the leakage of the tight electrode seal, we used an alternative method to rapidly change cGMP concentration, which we will refer to as a cGMP concentration ramp. In this protocol, the patch was continuously superfused. In the presence of symmetric Na + with 10 mM Ca 2+ on the cytoplasmic membrane surface, we first measured I–V curves activated by fixed concentrations of cGMP up to 300 μM . We used these data to generate a current amplitude–concentration function at a fixed voltage, +15 mV, for that patch . Next, we imposed a step change in cGMP concentration to 300 μM and waited until currents reached a maximum, stationary value (typically 30 s). We then switched to a solution free of cGMP and continued superfusing, while repeatedly measuring I–V curves with rapid voltage ramps (−30 to +30 mV/0.5 s) delivered at 2-s intervals. As the cGMP diffused away from the patch, currents decreased in amplitude . As expected from simple diffusion between two compartments, the time course of cGMP loss was well described by a single exponential . We limited our analysis to patches in which the time constant of this exponential was ≥12 s. Under these conditions, we assume that individual I–V curves (each measured in 500 ms) are measured at constant cGMP. We measured current amplitude at +15 mV in each ramp I–V curve and, using the calibration data first generated in the same patch, we established the cGMP concentration at which each ramp I–V curve was measured. Using cGMP concentration ramps, we confirmed that the reversal potential measured under a Ca 2+ concentration gradient changes with cGMP concentration. The reversal potential shifted progressively towards less negative values as cGMP concentration decreased, revealing that the channels become less selective for Ca 2+ over Na + as their probability of opening decreases . The dependence of reversal potential, and therefore PCa/PNa (Eq. 2 ), on [cGMP] was well described by a Hill function modified in the following form : 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{P_{Ca}}{P_{Na}}(cGMP)=\frac{P_{Ca}}{P_{Na}} \left[ min+(max-min)\frac{[cGMP]^{n}}{[cGMP]^{n}+K_{1/2}^{n}} \right] ,\end{equation*}\end{document} where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{PCa_{min}}{PNa}\;and\;\frac{PCa_{max}}{PNa}\end{equation*}\end{document} are the asymptotic minimum and maximum values attained by PCa/PNa, K 1/2 is the concentration at which PCa/PNa has a value midway between its maximum and minimum values, and n is an adjustable parameter that reflects ligand cooperativity. The average value of the parameters that best fit our data were K 1/2 = 17.2 ± 8.6 μM, n = 2.43 ± 0.37, PCa/PNa max = 6.48 ± 0.35, and PCa/PNa min = 1.89 ± 0.52 ( n = 14). In every patch tested ( n = 14), the values of K 1/2 and n that best described the cGMP dependence of PCa/PNa were exactly the same as those that best described the dependence of membrane conductance on nucleotide concentration (Eq. 3 ). This is illustrated in Fig. 3 , where the change in conductance is plotted as the value of (1 − I/I max ) at +15 mV, where I is the current at a given cGMP concentration and I max is its maximum value. The plot is scaled to have its minimum and maximum values, 0 and 1, respectively, match the minimum and maximum values of PCa/PNa. While plotting conductance this way is unconventional, since the function decreases as cGMP concentration increases, it allows us to compare directly the cGMP dependence of conductance and PCa/PNa. Membrane conductance is a direct measure of the average probability of channel opening . In rods, therefore, PCa/PNa changes 3.42 ± 0.95-fold ( n = 14) between its minimum and maximum values in a manner that is directly proportional to the probability of channel activation. Patches excised from tiger salamander rod outer segments often exhibit cGMP-activated currents as large as 1–2 nA at −70 mV under symmetric Na + solutions. These large currents, when sustained over a long time period, display a slow exponential decline in amplitude caused by the accumulation of Na + ions at the electrode tip and a consequent, slow shift in reversal potential . It was important, therefore, to ascertain that the rapid changes in reversal potential we observed in the presence of symmetric Na + and a Ca 2+ gradient did not arise from Na + accumulation at the electrode tip. Using cGMP concentration ramps, we measured the reversal potential under symmetric Na + solutions as a function of cGMP . If Na + accumulation occurred to a significant extent under our experimental protocol, then the reversal potential should shift in time. In fact, we found that the reversal potential was time invariant and independent of [cGMP]. The same results were obtained in every patch we studied with this protocol ( n = 21). In solutions containing 10 mM Ca 2+ , Na + accumulation should occur to an even lesser extent since ionic currents are much smaller due to Ca 2+ -dependent block of the pore. Furthermore, accumulation of Ca 2+ ions on the extracellular side of the patch cannot occur due to the presence of 5 mM BAPTA in the patch pipette solution. Thus, the cGMP-dependent shift in reversal potential under Ca 2+ concentration gradients reflects changes in PCa/PNa, and not the generation of an asymmetry in Na + or Ca 2+ concentrations. Our results reveal the complex interactions between gating and ion selectivity for Ca 2+ ions. Previous reports have documented that the blocking effect of Ca 2+ on the channels is also a function of cGMP . Fig. 5 illustrates this phenomenon in our experiments. The extent of conductance block by 5 mM Ca 2+ in the voltage range between −80 and +80 mV was measured in the same patch at various cGMP concentrations (5, 10, 20, 40, 300 μM). The I–V curves under symmetric Na + solutions (150 mM) were first measured in the presence of the various cGMP concentrations using a voltage ramp. The same curves were then measured again at each of the cGMP concentrations, but now with 5 mM Ca 2+ added to the cytoplasmic surface. The extent of Ca 2+ -dependent conductance block, g Ca (v)/ g (v) was determined by dividing, for each cGMP concentration tested, the current ramp measured in the presence of 5 mM cytoplasmic Ca 2+ by the current ramp measured in its absence . At all cGMP concentrations, the conductance block was maximal near the reversal potential, and it was relieved by either depolarization or hyperpolarization. At any given voltage, the extent of block was a function of cGMP, and this dependence was well described by the same Hill function that describes the cGMP dependence of current amplitude . We obtained the same results in every patch we tested ( n = 4). Thus, changing the probability of channel opening affects not only the ion selectivity of the channel for Ca 2+ , but also the interaction between Ca 2+ and the pore. We explored whether the effect of cGMP on Ca 2+ selectivity was specific for this ion or was a feature common to other divalent cations. We elected to study the selectivity properties of Sr 2+ , rather than Mg 2+ , because this cation permeates the channels, but is a less effective channel blocker than Ca 2+ or Mg 2+ . Using cGMP concentration ramps, we measured the reversal potential under symmetric Na + solutions with 20 mM Sr 2+ added to the cytoplasmic membrane surface of the patch . In the presence of saturating cGMP (300 μM), the reversal potential was −5.3 ± 0.21 mV ( n = 8), which indicates that PSr/PNa = 2.71 ± 0.13. As with Ca 2+ , the reversal potential shifted to less negative values as cGMP decreased . We calculated PSr/PNa from the reversal voltage (Eq. 2 ) and found that the dependence on cGMP of this selectivity ratio was well described by the modified Hill equation . On average, the values of the parameters that best fit our data were: K 1/2 = 14.8 ± 6.2 μM, n = 2.6 ± 0.32, PSr/PNa min = 0.64 ± 0.27, and PSr/PNa max = 2.71 ± 0.13 ( n = 8). Again, in each instance, the values of K 1/2 and n were the same as those that best described the dependence of current amplitude on cGMP in the same patch (Eq. 3 ). Thus, the effects of channel gating on selectivity are not specific for Ca 2+ . While the selectivity among monovalent cations is poor in the rod channel, there are, nonetheless systematic differences in selectivity among these ions . We explored whether channel gating affected the selectivity among inorganic monovalent cations. We elected to test the effects of cGMP on the selectivity between Cs + and Na + or NH 4 + and Na + . Cs + is less permeable than Na + , while NH 4 + is more permeable . Using cGMP concentration ramps, we measured the reversal potential under biionic conditions of either 150 mM Cs + /150 mM Na + or 150 mM NH 4 + /150 mM Na + . At 300 μM cGMP, the reversal potential was 18.1 ± 1.6 mV ( n = 8) for Cs + and −22.2 ± 2.2 mV for NH 4 + ( n = 6). These results reproduce those previously reported by others . From Eq. 1 , we find that PCs/PNa = 0.50 ± 0.03 and PNH 4 /PNa = 2.44 ± 0.21. Unlike our findings with divalent cations, the reversal potential under monovalent biionic conditions was constant and independent of cGMP . Thus, the effect of gating on ion selectivity is exclusive for divalent cations. The relative selectivity of organic cations through the cGMP-gated channels has been used to asses the steric hindrance imposed on ion flux by the selectivity filter in the channel . To determine whether gating affects steric hindrance, we investigated the effects of cGMP on the selectivity between methylammonium (MA + ) and Na + or dimethylammonium (DMA + ) and Na + . Using cGMP concentration ramps, we measured the reversal potential under biionic conditions of either 150 mM MA + /150 mM Na + or 150 mM DMA + / 150 mM Na + . At 500 μM cGMP, the reversal potential was 16.5 ± 1.5 mV ( n = 4) for MA + and 52.0 ± 2.2 mV ( n = 5) for DMA + . Eq. 1 yields relative selectivities of PMA/PNa = 0.53 ± 0.03 and PDMA/ PNa = 0.13 ± 0.011, in agreement with previous measurements . As in the case of inorganic cations, we found no changes in the channel selectivity for organic monovalent cations as a function of cGMP . Thus the apparent pore radius of the selectivity filter does not appear to change as a function of channel gating. Cyclic nucleotide–gated channels in rod photoreceptors and olfactory neurons are heteromeric assemblies of at least two structural subunits, α and β . We investigated whether the cGMP dependence of ion selectivity of the native channel is a feature of the α subunits or requires the coexpression of α and β subunits. We expressed bovine rod α or αβ channels in Xenopus oocytes and measured cGMP- dependent currents in inside-out, detached membrane patches. As discussed in materials and methods , we took precautions to eliminate contamination of the recorded currents by Ca 2+ -dependent Cl − currents native to the oocyte. In experiments with αβ channels, we verified that the channels were indeed formed from both α and β subunits by testing the action of l-cis-diltiazem (10 μM), which effectively blocks only αβ heteromeric channels, and not α homomeric channels . Also, our data confirms that homomeric α channels are more sensitive to block by Ca 2+ than αβ channels . We measured I–V curves of cGMP-dependent currents in the presence of symmetric Na + with 10 mM Ca 2+ added to the cytoplasmic surface of the membrane containing either α or αβ channels . At saturating cGMP, the reversal potential for α channels was −4.8 ± 1.2 mV ( n = 7), which implies that PCa/ PNa = 4.6 ± 0.79, but for αβ channels the reversal potential was −8.4 ± 1.1 mV ( n = 12), which implies that PCa/PNa = 9.4 ± 1.1. Thus the heteromeric αβ channel is about twice more selective for Ca 2+ over Na + than the α homomeric channel. More remarkable, however, is the difference between the channels on the effect of gating on ion selectivity. In α channels, PCa/PNa was invariant with cGMP concentration in every patch we tested ( n = 8) . In αβ channels, as in native channels, the divalent cation selectivity decreased as cGMP concentration was lowered . The dependence of PCa/PNa on cGMP was well described by the modified Hill equation, (Eq. 4 ). The average value of the parameters in the equation that best fit our data were: K 1/2 = 45.2 ± 15.6 μM, n = 2.6 ± 0.24, PCa/PNa max = 9.4 ± 1.1, PCa/PNa min = 4.4 ± 1.4 ( n = 12). To determine whether the shifts in selectivity for the αβ channel were specific for divalent cations, we tested the selectivity for Cs + and NH 4 + in both α and αβ channels. In Fig. 11 , we illustrate I–V curves of cGMP-dependent currents measured in the presence of biionic solutions of Cs + /Na + or NH 4 + /Na + . The I–V curves were generally similar to those recorded from native rod channels under comparable conditions , except that α channels in the presence of biionic solutions of Cs + /Na + display an unusual nonlinearity at potentials more negative than the reversal potential. In the presence of saturating cGMP, the selectivity sequence of αβ was the same as α, although the absolute values of PX/ PNa differed between the two channels. Mean values of V rev for the α channel were Cs + , 28.6 ± 2.5 mV ( n = 8); NH 4 + , −31.2 ± 1.8 mV ( n = 8), which yields PCs/PNa = 0.33 ± 0.03 and PNH 4 /PNa = 3.49 ± 0.25. Mean values of V rev for the αβ channel were Cs + , 21.9 ± 1.9 mV ( n = 6); NH 4 + , −24.5 ± 2.2 mV ( n = 6), which indicate that PCs/PNa = 0.43 ± 0.32 and PNH 4 /PNa = 2.67 ± 0.23. The reversal potentials were the same at all cGMP concentrations tested . Thus, recombinant α or αβ channels, just like native channels, select poorly among monovalent cations, and this selectivity is unaffected by gating. However, the absolute value of the selectivity among monovalent cations, just as among divalents, differs in homomeric and heteromeric channels. cGMP-gated channels of cone photoreceptors are significantly more permeable to Ca 2+ than those of rods . However, past measurements were conducted under saturating cGMP concentrations. If channels in cones, like those of rods, change divalent cation selectivity as a function of cGMP, then differences observed under high cGMP concentrations might not occur under lower concentrations, such as those expected in the photoreceptor cells under physiological conditions. We investigated the effects of cGMP on the value of PCa/PNa in cGMP-gated channels from the striped bass single cone. Using cGMP concentration ramps, we measured the reversal potential under symmetric Na + solutions with 5 mM Ca 2+ added to the cytoplasmic membrane surface . At saturating cGMP, the reversal potential was −9.4 ± 0.5 mV ( n = 12), which implies that, in cones, PCa/PNa = 21.7 ± 1.65 ( n = 12), consistent with earlier measurements . As in rods, the reversal potential shifted to less negative values as cGMP concentration declined . Again, the dependence on cGMP of PCa/PNa was well described by the modified Hill equation, . On average, we found that K 1/2 = 43 ± 14 μM, n = 2.8 ± 0.4, PCa/PNa min = 14.0 ± 1.6 and PCa/PNa max = 21.7 ± 1.7 ( n = 12). The extent of shift in divalent cation selectivity with cGMP, however, was less in cones than in rods. The decrease in Ca 2+ permeability between saturating and zero cGMP (PCa/ PNa max and PCa/PNa min ) was 1.55 ± 0.21 ( n = 12). Thus, in intact, dark-adapted photoreceptor cells, where only ∼3% of the channels are open, PCa/PNa in cones can be expected to be 7.41 ± 0.85-fold larger than in rods. We tested whether in channels of cones, like those of rods, the selectivity among monovalent inorganic cations was independent of cGMP. We studied Cs + and NH 4 + selectivities relative to Na + using cGMP concentration ramps under biionic solutions of 150 mM Cs + / Na + or 150 mM NH 4 + /Na + . At saturating cGMP concentrations, the reversal potentials were 4.2 ± 0.53 ( n = 5) for Cs + and −16.4 ± 1.4 mV ( n = 6) for NH 4 + . Eq. 1 yields PCs/PNa = 0.87 ± 0.02 and PNH 4 / PNa = 1.94 ± 0.11, similar values to those previously reported by others . We found no changes in the reversal potentials at different cGMP concentrations. Thus, in cone channels, gating modifies the selectivity for divalent but not monovalent cations. We report that cyclic nucleotide–gated (CNG) channels in both rod and cone retinal photoreceptors change their relative Ca 2+ to Na + selectivity as a function of cGMP concentration. The linkage between selectivity and channel activity is specific for divalent cations and is not observed when the selectivity among inorganic or organic monovalent cations is explored. The dependence on cGMP concentration of the changes in relative ion selectivity is well described by a Hill equation, the same one that describes the dependence of probability of channel opening on the nucleotide concentration. The coupling of ion selectivity and gating is a feature of heteromeric recombinant rod channels formed by α and β subunits, but is absent in homomeric channels formed by α subunits alone. Two alternative molecular mechanisms could explain our macroscopic findings of the effect of cGMP on ion selectivity: (a) two or more types of CNG channels exist that differ in their ion selectivity or (b) only one type of channel exists that exhibits two or more conductance states, each of different ion selectivity. If two distinct channels exist, then one must have a high sensitivity to cGMP and low PCa/PNa, while the other must have a lower sensitivity to cGMP and higher PCa/PNa. Thus, at saturating cGMP concentrations, both channel types would be open and the selectivity would be the weighted sum of the selectivities of each type. As cGMP concentration declines, the low sensitivity channel would close, and the selectivity of the high sensitivity channel would define the selectivity observed experimentally. If a single molecular type of channel exists with multiple conductance states, than the probability of occupying different conductance states must be cGMP dependent, and the conductance states that exist primarily at low cGMP concentrations should have a much lower Ca 2+ permeability than states that exist at high cGMP concentrations. In intact rod photoreceptors, there exist two molecularly distinct types of cGMP-gated channels that differ in their kinetic properties . In the inner segment membrane, ∼1 of 20 CNG channels exhibit slow kinetics that are similar to those of recombinant channels composed of α subunits alone . The remaining channels, in contrast, exhibit rapid flickering similar to that observed in recombinant αβ channels and in channels of the outer segment . We do not think that the existence of these two kinetically distinct types of channels underlie the macroscopic behavior we report here since: (a) the channels are located in the inner segment of the cell and their copy number is variable, yet we observe cGMP-dependent permeability changes on every patch isolated from the outer segment alone; and (b) the two known channel types have identical sensitivity to cGMP, yet our observations demand that the channel types differ in their sensitivity to cGMP. Biochemical experiments have previously suggested that two types of cGMP-gated channels might exist in rod outer segments. Experiments on purified bovine rod outer segment membrane vesicles revealed two cGMP-dependent components with different Ca 2+ efflux kinetics . In these findings, however, the high Ca 2+ permeability component has a high affinity for cGMP. This behavior is contrary to our electrophysiological findings. Moreover, additional evidence suggests that the two kinetic components observed in the biochemical studies likely reflect the existence of two types of membrane vesicles, those with and those without Na + -Ca 2+ ,K + exchangers, rather than two different types of cGMP-gated channels . Modulation might give rise to the presence of populations of functionally distinct channels in the same patch. Recordings of cGMP-dependent currents in both photoreceptor and oocyte membranes show a high variability in the absolute value of K 1/2 from patch to patch and over time in the same patch, a fact that may reflect modulation, perhaps due to channel phosphorylation . Also, an endogenous modulator, partially mimicked by calmodulin, is known to modify K 1/2 in both rods and cones . To explain the data presented here, known modulation of the CNG channels would have to affect both the affinity for cGMP and the divalent cation selectivity. In the case of calmodulin or the calmodulin-like endogenous modulator, PCa/PNa of the channels in the presence and absence of the modulator is the same , and thus such modulation cannot explain the cGMP- dependent changes in selectivity. Any form of modulation that changes both the cGMP affinity and divalent cation selectivity should give rise to two observable features in our data. (a) The extent of modulation should be measurable by observing either the maximal PCa/PNa or K 1/2 . Thus, maximal PCa/ PNa (that measured at saturating cGMP) should vary from patch to patch, and over time in the same patch, just as K 1/2 varies. (b) Patches with low K 1/2 would be expected to have low maximal PCa/PNa values and vice versa. While K 1/2 , and to a lesser extent maximal PCa/ PNa, vary from patch to patch, we did not find any correlation between K 1/2 and maximal PCa/PNa, making it unlikely that modulation or in fact any multiple channel mechanism could explain our findings. If multiple populations of channels with different cGMP sensitivity and ionic selectivity do not coexist in patches of CNG channels, than there must be a more direct mechanism involved. A direct functional linkage between ion selectivity and gating has been previously observed in both voltage- and ligand-gated channels. In the presence of biionic solutions of monovalent cations, N -methyl- d -aspartate–gated currents do not exhibit a single reversal potential, and current fluctuations do not disappear at the reversal voltage . Single channel recordings demonstrate that this macroscopic behavior reflects the existence of at least two subconductance states that differ in their ion selectivity . Shaker K + channels exhibit at least two subconductance states that differ in their monovalent cation selectivity . Our observations could be explained if the open pore of a single channel had several possible structural states that differ in divalent cation permeability. Each of these open states might also correspond to the well documented subconductance states of the CNG channels. The presence of multiple conductance states in CNG channels was recognized in the first published single channel recordings , but only later analysis focused on these features. In studies of bovine rod membrane vesicles incorporated into lipid bilayers, Ildefonse and Bennett observed several single channel conductance states and proposed that sequential binding of four cGMP molecules correspond to the opening of four discrete conductance levels. Taylor and Baylor observed subconductance levels in single channel recordings from tiger salamander rods and reported that the fraction of time spent in the subconductance level decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening of partially liganded channels. Since cGMP-gated channels are tetrameric with four cyclic nucleotide binding sites , the subconductance levels have generally been interpreted as representing distinct states of one, two, or three bound cGMP molecules. Ruiz and Karpen , however, have suggested that this interpretation is incorrect. Using a photocross-linkable cGMP analogue, they locked single channels formed from α subunits in a specific ligand-bound state. Their results indicate that these channels do not have significant probability of opening until at least three ligands are bound and that the number of bound cGMP molecules alters the probability of occupying a particular open state, but does not define which state is occupied. Triply liganded channels display two strong subconductance states in addition to the fully open state, while the fully liganded channel mainly occupies only the fully open conductance state. Consistent with these findings, we suggest that the macroscopic effect of cGMP on ion selectivity may reflect the existence of at least two conductance states each of distinct ion selectivity. At low cGMP, the prevalent state is predicted to be of lower PCa/PNa than the state prevalent at high cGMP concentration. What types of pore structural changes might give rise to the subconductance states and the cGMP-dependent permeability changes observed here? Sun et al. have shown in a series of cysteine accessibility studies that the CNG channel pore may undergo large structural changes during gating and may in fact be the gate itself. Particularly intriguing is the finding that tetracaine binds to the pore, depending on whether it is open or closed, by forming a salt bridge with the E363 residue within the pore . The fact that tetracaine does not bind to the open channel may indicate that a conformational change within the pore alters either the position of E363 or its accessibility to tetracaine. Since E363 is critical in determining the divalent cation block and permeation in the cGMP-gated channel , and since this residue may change its position or accessibility in the course of gating , it is then possible that this structural change may also contribute to linking changes in gating with changes in divalent cation selectivity. If changes in open pore structure occur, these are not reflected in the steric dimensions of the pore since the selectivity for monovalent organic cations, a simple test of steric hindrance in these channels , is unaffected by cGMP. Changes not within, but around the mouth of the pore might also affect selectivity . The proposition that cGMP controls the prevalence of conducting state of different ion selectivity might be tested experimentally in single channel studies, but not with channels formed from recombinant alpha subunits alone since these channels do not exhibit cGMP-dependent changes in selectivity. On theoretical grounds, however, a simple model of two conducting states (triply liganded and fully saturated), each with different divalent cation selectivities, cannot fully explain our results. This is because a model of two conducting states with four cGMP binding sites can be shown to predict that changes in divalent cation selectivity as a function of cGMP should show little, if any, cooperativity when compared with the cooperativity of the current–cGMP relationship. Contrary to this expectation, we have found that the dependence of changes of PCa/PNa on cGMP is the same as the dependence of current on cGMP. Thus, a more complex mechanism must be at play. Resolving whether cGMP changes ion selectivity by affecting the structure of the pore or by changing the probability of opening of subconductance states of differing ion selectivity must await further experimental work. The stoichiometry of α and β subunits in the cyclic nucleotide–gated channels appears to be 2:2 in channels of olfactory neurons . The photoreceptor β subunit, while itself unable to form functional channels , confers several functional properties to the channel that are absent in channels formed from α subunits alone. These properties include: (a) the ability to flicker rapidly , (b) the Ca 2+ -dependent modulation of sensitivity to cGMP mediated by calmodulin , (c) increased sensitivity to diltiazem , and (d) reduced sensitivity to Ca 2+ block . We now add the dependence on cGMP of divalent cation selectivity and channel block. Under physiological conditions, in both rods and cones , the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1–5% to essentially zero under continuous, bright illumination. That is, in the intact photoreceptor, channels spend nearly all their time exposed to very low concentrations of cGMP. Since the Ca 2+ permeability reaches its minimum value at low cGMP concentrations, we would not expect the Ca 2+ permeability to change significantly in the course of the normal photoresponse. However, if the channel's Ca 2+ selectivity changes with cGMP in other cells, where the operating range of changes in cytoplasmic is larger than in photoreceptors, then modulation of Ca 2+ fluxes by cGMP (or cAMP) should be considered as a potentially important physiological modulation. Cone cGMP-gated channels are more permeable to Ca 2+ than those in rods . At saturating cGMP concentrations, we find that channels from striped bass cones are ∼3.3-fold more permeable to Ca 2+ than those of tiger salamander rods, a result consistent with previous measurements . This difference is even larger at physiologically relevant cGMP concentrations. At the cytoplasmic cGMP concentrations expected in dark adapted cells, PCa/PNa in cone channels is ∼7.4-fold greater than that in rods. The physiologically significant parameter, however, is not the difference in the values of PCa/PNa, but in the fraction of the ionic current carried by Ca 2+ in rods and cones. | Study | biomedical | en | 0.999998 |
10352033 | In the cloned potassium (K + ) channel, Kv2.1, current block by external tetraethylammonium (TEA) 1 is cation dependent. In the presence of physiological [K + ], TEA blocks Kv2.1 with an IC 50 of ∼5 mM . When currents are carried by Na + in the complete absence of K + , TEA at a concentration of 30 mM is completely without effect . Inhibition by TEA could be titrated back with relatively low [K + ]. These data indicated that the conformation of the external vestibule differed depending on what cation occupied the pore, and that occupancy of a site in the channel by K + put the pore into a conformation that allowed TEA to block . Although these data did not directly address the issue, they also suggested the converse possibility that, as K + exited the pore, the channel might change conformation to a TEA-insensitive state. It has been well-demonstrated that the conformation of the external vestibule, near the external TEA binding site and selectivity filter, changes during slow inactivation . In channels that exhibit classical “C-type” inactivation, the rate of the conformational change that underlies inactivation is determined largely by the exit rate of K + from the pore . The observation that occupancy of the selectivity filter slows the rate of inactivation suggests that the conformational change that underlies inactivation proceeds as K + comes off of the selectivity filter. Shaker residue 424, located at the outer edge of the external vestibule, also changes orientation during inactivation . These results suggested that the movement that occurs during inactivation produces wide-ranging changes in conformation of the outer vestibule. The mechanism of slow inactivation in Kv2.1 is less clearly understood than that of classical C-type inactivation. . Several arguments have been made, based on the voltage dependence of inactivation and the sensitivity of slow inactivation to external TEA and K + , that inactivation in Kv2.1 differs from that of C-type inactivating channels . However, as with Shaker and other C-type inactivating channels, the mechanism that underlies slow inactivation in Kv2.1 involves a change in conformation of the selectivity filter . Furthermore, data were recently presented that suggested that a slow inactivation mechanism that differs from C-type inactivation, called U-type inactivation, occurs in both Kv2.1 and Shaker . This suggests that slow inactivation in Kv2.1 shares some common mechanistic features with Shaker -like channels. To better understand the K + -dependent conformational changes that occur in the Kv2.1 pore, we examined the mechanism that accounted for loss of TEA block upon removal of K + . Two possibilities could account for the cation-dependent changes in TEA potency. One possibility was that the removal of K + produced a local disruption of the TEA binding site. An alternative possibility was that a conformational change occurred at a location external to and remote from the TEA binding site. This conformational difference in the permeation pathway leading to the TEA binding site could create a steric or electrostatic hindrance to the ability of the cationic TEA to reach and/or bind to its binding site. Two lysines in the outer vestibule of the Kv2.1 pore (K356 and K382) were candidates for this latter effect. These lysines, which are at positions equivalent to Shaker residues 425 and 451, respectively, impede the access of agitoxin, a K + channel blocker, to its binding site in the external vestibule . Mutation of these two lysines to the neutral glycine and valine, respectively, permits agitoxin to gain access to its binding site . According to the observed crystal structure of the Streptomyces lividans K + channel and toxin mapping studies , the residue equivalent to Lys 356 is located at the external edge of the outer vestibule. Lys 382 is just external to the putative external TEA binding site . Consequently, a cation-dependent alteration in the position of one or both of these lysines in the conduction pathway might be the cause of the loss of TEA block upon removal of K + . We tested these possibilities by examining the effect of mutation of Lys 356 and Lys 382 on K + -dependent changes in TEA potency. Our results indicate that the K + -dependent alteration in pore conformation extended to locations near the outer edge of the external vestibule, where Lys 356 is located. A K + -dependent conformational rearrangement also occurred internal to the selectivity filter, as block by internal TEA was eliminated upon removal of K + . The alterations in pore conformation, both internal and external to the selectivity filter, were associated with occupancy of the selectivity filter by K + and could occur under conditions that produced a decrease in K + occupancy of the pore during K + conduction. Finally, the rate of slow inactivation in Kv2.1 was [K + ] dependent and was correlated to the alterations in pore conformation associated with changes in TEA potency. All experiments were done on four channels: wild-type Kv2.1, Kv2.1 K356G, Kv2.1 K382V, and Kv2.1 K356G, K382V. Oligonucleotide mutagenesis was performed using the dut − ung − selection scheme . For K356G and K382V mutations, primers were designed to remove restriction sites PinAI and BpuAI, respectively. Mutagenized plasmids were then distinguished from wild-type plasmids by restriction enzyme digest of plasmid cDNA. The double lysine mutant was made with the K382V cDNA as template and the K356G primer. Consequently, the double lysine mutant had both restriction sites removed. K + channel cDNA was subcloned into the pcDNA3 expression vector. Channels were expressed in the human embryonic kidney cell line, HEK293 (American Type Culture Collection). Cells were maintained in DMEM plus 10% fetal bovine serum ( GIBCO BRL ) with 1% penicillin/streptomycin (maintenance media). Cells (2 × 10 6 cells/ml) were cotransfected by electroporation (71 μF, 375 V, Electroporator II; Invitrogen Corp.) with K + channel expression plasmid (0.5–15 μg/0.2 ml) and CD8 antigen (1 μg/0.2 ml). After electroporation, cells were plated on protamine (1 mg/ml; Sigma Chemical Co. )-coated glass cover slips submerged in maintenance media. Electrophysiological recordings were made 18–48 h later. On the day of recording, cells were washed with fresh media and incubated with Dynabeads M450 conjugated with antibody to CD8 (1 μl/ml; Dynal ). Cells that expressed CD8 became coated with beads, which allowed visualization of transfected cells . Currents were recorded at room temperature using the standard whole cell patch clamp technique . Patch pipets were fabricated from N51A glass (Garner Glass Co.), coated with Sylgard and firepolished. Currents were collected with an Axopatch 1D or 200A patch clamp amplifier, pClamp 6 software, and a Digidata 1200 A/D board ( Axon Instruments ). Currents were filtered at 2 kHz and sampled at 250–10,000 μs/point. Series resistance ranged from 0.5 to 2.2 MΩ and was compensated 80–90%. The holding potential was −80 mV, and depolarizing stimuli were presented once every 5–25 s, depending on the experiment. Concentration–response curves were fit to the equation, Y = 100*{( X ) n / [( X ) n + (IC 50 ) n ]}, where X is the drug concentration, IC 50 is the drug concentration that produced half maximal inhibition, and n is the slope of the curve. Data were analyzed with Clampfit ( Axon Instruments ); curve fitting and significance testing (unpaired Student's t-test) were done with SigmaPlot 2.0. All plotted data are represented as mean ± SEM, with the number of data points denoted by n . For IC 50 values, a range of values is given for n . This range represents the number of cells used for each data point in the complete concentration–response curve from which the IC 50 was calculated. Currents were recorded in a constantly flowing, gravity fed bath. Solutions were placed in one of six reservoirs, each of which fed via plastic tubing into a single quartz tip (∼100 μm diameter; ALA Scientific Instruments). The tip was placed within 20 μm of the cell being recorded before the start of the experiment. One solution was always flowing, and solutions were changed by manual switching (complete solution changes took 5–10 s). Control internal solutions contained (mM): 140 XCl (X = K + , Na + , or N -methyl- d -glucamine [NMG + ]), 20 HEPES, 10 EGTA, 1 CaCl 2 , and 4 MgCl 2 , pH 7.3, osmolality 285. Control external solutions contained (mM): 165 XCl, 20 HEPES, 10 glucose, 2 CaCl 2 , and 1 MgCl 2 , pH 7.3, osmolality 325. External solutions that contained between 0 and 30 μM K + used Puratronic NaCl . In internal solutions that contained <140 mM K + and external solutions that contained <165 mM K + or Na + , osmotic balance was maintained with NMG + . Other additions and substitutions are listed in the figure legends. At concentrations up to 30 mM, external TEA (TEA o ) blocked K + currents but not Na + currents through Kv2.1 channels that were expressed in L cells . Fig. 1 illustrates an expansion of these data to complete concentration–response curves for both K + and Na + currents up to [TEA] o of 100 mM in HEK 293 cells. TEA o blocked outward K + currents in Kv2.1 with an IC 50 of 4.5 ± 0.1 ( n = 3, •), which is similar to that reported previously for Kv2.1 K + currents recorded in oocytes and identical to that of inward K + currents recorded in L cells . When the current through Kv2.1 was carried by Na + , [TEA] o as high as 100 mM had absolutely no influence on current magnitude . Identical results were obtained on both inward and outward currents. To test for involvement of Lys 382 and Lys 356 in the cation-dependent loss of TEA o block, we replaced them with the smaller, uncharged Val and Gly, respectively, which are the corresponding amino acids in this position in Kv1.3. After these mutations, channels remained highly selective in the presence of high [K + ], conducted Na + in the absence of K + , and activated with identical voltage dependence as wild-type Kv2.1 (data not shown). Lys 382 is external to the putative TEA o binding site (Y380) by just two residues. Fig. 2 A illustrates K + (top) and Na + (bottom) currents through Kv2.1 K382V in the presence and absence of TEA o at the concentrations indicated. TEA o blocked K + currents with an IC 50 of 0.8 ± 0.05 mM , which represents an approximately fivefold increase in TEA o potency compared with the wild-type Kv2.1. The K382V mutation also allowed TEA o to inhibit Na + currents . However, TEA o remained remarkably impotent at blocking currents in the absence of K + (extrapolated IC 50 = 265 mM). The increased TEA o potency for block of K + currents suggests that the lysine at position 382 interferes somewhat with the ability of TEA to bind to its external binding site, either by inhibiting access of TEA to the site or by destabilizing the binding of TEA. However, the observation that TEA potency remained extremely low in the absence of K + indicates that the loss of TEA block upon removal of K + did not result primarily from movement of Lys 382 relative to the conduction pathway or the TEA binding site. The crystal structure of the K + channel pore from Streptomyces lividans , together with toxin mapping experiments in voltage-gated channels, suggest that Lys 356 is near the outer edge of the external vestibule . Fig. 2 C illustrates inward K + (top) and Na + (bottom) currents recorded from the mutant channel, Kv2.1 K356G, before, during, and after application of external TEA. The complete concentration dependence of TEA o block of K + and Na + currents through K356G is shown in Fig. 2 D. TEA o blocked K + currents with an IC 50 of 1.1 ± 0.06 mM ( n = 3–6). This approximately fivefold increase in TEA potency compared with the wild-type channel demonstrates that, despite its exterior position in the outer vestibule, Lys 356 influenced the ability of TEA o to interact with its binding site. In contrast to the moderate effect of this mutation on TEA o block of K + currents, mutation of Lys 356 had a dramatic effect on TEA o block of Na + currents . In the complete absence of K + , TEA o blocked Na + currents with an IC 50 of 21.1 ± 1.3 mM ( n = 4–5). Although the TEA o potency for block of Na + currents was still ∼20-fold lower than that for block of K + currents, these data demonstrate two important points. First, the observation that TEA o blocked Na + currents with an IC 50 of 21 mM indicates that the external TEA binding site itself was not dramatically disrupted upon removal of K + . Second, these data suggest that the conformational change that inhibits TEA o block in the absence of K + largely involved Lys 356. Finally, we examined TEA o block of K + and Na + currents in Kv2.1 channels with both lysines replaced . TEA o blocked K + currents with an IC 50 of 0.3 ± 0.04 mM . Thus, whereas mutation of either individual lysine shifted the IC 50 for TEA block of K + currents similarly , mutation of both lysines produced an additional half-log increase in TEA potency. These data suggest that each lysine contributed some repulsive influence for access to or binding of TEA to its binding site, even in the presence of K + . The similar and additive effect of these mutations on TEA block of K + currents also argues against the possibility that either of these mutations significantly altered the TEA binding site itself. TEA o blocked Na + currents in the double mutant with an IC 50 of 11.6 ± 1.9 mM . Consequently, the ∼1.5 log unit difference in TEA o potency for block of K + and Na + currents that was observed with the single mutant, K356G, was maintained in the double mutant. TEA block of K + and Na + currents was completely voltage independent between 0 and +60 mV (data not shown). Consequently, the different TEA potencies in the presence of K + and Na + indicate that even in the double mutant, a conformational change occurred in the outer vestibule upon removal of K + . Our working hypothesis for the results in Figs. 1 – 3 is that the outer vestibule undergoes a cation-dependent conformational alteration such that, upon removal of K + , the positively charged lysine at position 356 changes its position relative to the conduction pathway or TEA binding site. According to this hypothesis, the mutations did not alter the fundamental nature of the cation-sensitive conformational alteration; residue 356 still changed position relative to the pore, but substitution of a small uncharged amino acid at this site resulted in less interference with TEA binding. An alternative explanation, however, was that the mutations fundamentally altered either the channel conformation or the ability of the channel to change conformations upon removal of K + . This possibility would suggest that the effects of the mutations reflected a qualitative change in the mechanistic operation of the channel. To address this issue, we compared slow inactivation in wild-type Kv2.1 and the double lysine mutant. Slow inactivation involves a conformational change in the outer vestibule, near both the external TEA binding site and the selectivity filter . We postulated that if the fundamental structure of this region, or the ability of this region to change conformation, were altered by the mutations, slow inactivation would be disrupted. Fig. 4 A illustrates superimposed K + currents from Kv2.1 and Kv2.1 K356G, K382V, evoked by 8-s depolarizations to 0 mV. Fig. 4 B illustrates superimposed inward Na + currents from the same two channels. Under these conditions, the two channels inactivated at identical rates, which suggests that the conformational change(s) that occur during slow inactivation were essentially unchanged by the mutations to residues 356 and 382. Thus, these data suggest that the mutations do not fundamentally alter the conformation of the outer vestibule or the ability of the outer vestibule to change conformation in response to depolarization. Internal TEA (TEA i ) blocked K + and Na + currents in Kv2.1 with different potency. Fig. 5 illustrates this difference. Inward currents carried by K + ([K + ] o = 30 mM) and Na + ([Na + ] o = 165 mM) were compared in the presence or absence of 20 mM TEA i . In the absence of TEA i , peak inward K + currents were, on average, 1.4× the magnitude of Na + currents in the same cell . In the presence of 20 mM TEA i , K + current magnitude was just 0.2× the magnitude of Na + currents measured in the same cells . These results demonstrate that TEA i blocked K + currents more potently than it blocked Na + currents. We then sought to determine whether block by internal TEA was completely abolished on removal of K + . This could not be examined directly by applying TEA to inside-out patches since Kv2.1 runs down quickly in this configuration. Therefore, to determine whether TEA i blocked Na + currents, we did the experiments illustrated in Fig. 5 , D and E. The tip of the recording pipet was filled with internal solution lacking TEA. The pipet was then backfilled with internal solution that contained 20 mM TEA. If recordings could be made before TEA entered the cell interior, the K + /Na + conductance ratio should equal that observed in Fig. 5 A, where no TEA was added to the pipet solution. As the TEA entered the cell interior, current magnitude would decrease as the current was blocked. If the intracellular TEA concentration reached the same as that when the entire pipet was filled with TEA-containing solution , current magnitude would change until the K + /Na + conductance ratio equaled that of Fig. 5 B. In practice, we were unable to add enough TEA-free solution to the pipet tip to obtain the initial TEA-free conductance ratio and also have sufficient TEA enter the cell to attain the final conductance ratio, all in the same cell. However, Fig. 5 , D and E, illustrates two cells in which the beginning and end points were achieved. Inward Na + (○) and K + (•) currents were observed alternately. In Fig. 5 D, the pipet tip was filled with enough TEA-free internal solution so that recordings could be made before TEA entered the cell. The first Na + current (at time t = 0) was recorded after break in, series resistance, and capacitance cancellation and series resistance compensation. After three evoked Na + currents, the external solution was switched to the K + -containing solution. The initial K + current recorded was ∼50% larger than the initial Na + current. As recordings continued, the K + current diminished to 30% of its initial magnitude, while over the same time period the Na + current magnitude remained constant. Fig. 5 E illustrates a cell recorded with a pipet that contained less TEA-free solution loaded into the tip. Over the time period studied, the K + current magnitude decreased by 58% while the Na + current magnitude remained unchanged. At the end of this time period, the K + /Na + conductance ratio equaled that obtained in the experiments of Fig. 5 B. Similar results were obtained in four cells. In the absence of internal TEA, K + currents did not diminish; over an 8-min recording period, K + current magnitude was 101 ± 2% of control ( n = 3). Taken together, these data demonstrate that TEA i did not affect Na + currents, and that the change in K + /Na + conductance ratio resulted from an 85% block of K + currents by 20 mM TEA i . We did the same type of experiment as described in Fig. 5 on the double lysine mutant, to examine whether the lysine mutations to the outer vestibule prevented the cation-dependent conformational alteration responsible for loss of block by TEA i . In the absence of TEA i , K + current magnitude (with 30 mM external K + ) was 2.5× the Na + current magnitude . In the presence of 20 mM TEA i , K + current magnitude was just 0.25× that of the Na + current magnitude . Thus, as in Kv2.1, internal TEA blocked K + currents much more potently than it blocked Na + currents. We then tested whether K + currents but not Na + currents diminished in the double mutant as TEA entered the cell interior from the pipet. Fig. 6 D illustrates data from one cell in which Na + current magnitude (○) remained constant while K + current magnitude decreased by 50% (•). Similar data were obtained from five cells, three of which had initial K + current magnitudes greater than initial Na + current magnitudes. The change in conductance ratio , combined with the lack of effect on Na + current magnitude, indicates that, at a concentration that did not block Na + currents, TEA i blocked K + currents by ∼90%. Thus, mutation of the two outer vestibule lysines did not reverse the loss of TEA i block produced by removal of K + . Furthermore, the similar block of K + currents in Kv2.1 and the double lysine mutant by internal TEA indicates that the mutations to the outer vestibule had little or no effect on the internal TEA binding site. The preceding observations raised two questions of fundamental interest for understanding the nature of the conformational changes that occurred upon removal of K + . First, did K + prevent the conformational changes associated with shifts in internal and external TEA potency by interacting with a single site or with multiple sites in the pore? Second, did K + influence these conformational changes by interacting with the selectivity filter cation binding site(s), which are involved in cation-dependent changes in slow inactivation rate ? We determined the [K + ] dependence of (a) TEA o block in Kv2.1 K382V, which assayed primarily the conformational change specifically associated with Lys 356; (b) TEA o block in Kv2.1 K356G, K382V, which assayed the conformational change in the outer vestibule not associated with interference by Lys 356 or Lys 382; and (c) TEA i block in wild-type Kv2.1, which, measured the conformational change internal to the selectivity filter. We also compared these [K + ] to those required to block Na + current through the channel, which, by definition, occurs at one or more sites associated with ionic selectivity. If the [K + ] dependence of these four functional measurements were the same, the interpretation would be that K + influenced these events by interacting with the same or indistinguishable sites, and that these sites were associated with the selectivity filter. Block of Na + current by K + assays the interaction of K + with the highest affinity K + binding site(s) in the channel. Even if K + exits by passing through the channel, measurable block of Na + current by K + puts an upper limit on the minimum [K + ] required to interact with the pore. Fig. 7 A illustrates inward currents through Kv2.1 K382V, recorded at 0 mV, in the presence of 165 mM external Na + and three external [K + ]. Na + currents were significantly inhibited by 0.1 mM K + , and current magnitude decreased as [K + ] was elevated to 3 mM . These data indicate that K + interacts with the selectivity filter cation binding site(s) in Kv2.1 K382V at [K + ] as low as 0.1 mM, and that relative occupancy of the selectivity filter binding site(s) by K + increased as [K + ] was increased to 3 mM. In Kv2.1 K382V, the most sensitive measure of the change in TEA o potency was obtained with 3 mM TEA, which blocked Na + currents by <5% and blocked K + currents by ∼80% . Therefore, to determine the [K + ] dependence of the change in TEA o potency, we examined the K + -dependent increase in block by 3 mM TEA o . From a control solution that contained 165 mM Na + , the external solution was switched to one containing 165 mM Na + plus K + at one of six concentrations between 0.01 and 3 mM. Fig. 7 C illustrates current block by 3 mM TEA o in the presence of three different [K + ]. The control currents, which were normalized to each other for visual purposes, were recorded in solutions that contained the indicated [K + ] plus 0 TEA o . As [K + ] was increased, block by 3 mM TEA o increased, which reflects an increase in TEA o potency. Fig. 7 D illustrates the block by 3 mM TEA o over the entire tested range of [K + ]. Block by 3 mM TEA was significantly increased at [K + ] as low as 0.1 mM , and continued to increase as [K + ] was raised to 3 mM. To increase the sensitivity of this assay at 0.03 mM [K + ], we examined the block by 30 mM TEA, which inhibited Na + currents in Kv2.1 K382V by 13.0 ± 1.7% . Even with this [TEA] o , which was well into the rising phase of the concentration–response curve, 0.03 mM K + did not influence TEA o potency (TEA o blocked currents in the presence of 0.03 mM K + by 14.3 ± 3.0%, n = 4). These results indicate that the minimum [K + ] required to influence TEA o potency was 0.1 mM, which coincided with the minimum [K + ] required to measurably interact with the selectivity filter cation binding site(s) . In the double mutant, 1 mM TEA blocked currents by 9% in the absence of K + and 71% in the presence of 140 mM K + . Therefore, we used 1 mM TEA to examine the [K + ] dependence of the conformational change in the double mutant. The lowest [K + ] that significantly enhanced block by 1 mM TEA in the double mutant was 30 μM . The minimum [K + ] required to significantly block Na + current was also 30 μM. . Thus, as in Kv2.1 K382V, a significant increase in TEA o potency in the double mutant was produced by the same low [K + ] required to block Na + currents. TEA i did not block Kv2.1 in the absence of K + , but blocked the channel by ∼85% in the presence of 30 mM K + . Our goal in these experiments was to determine the lowest [K + ] at which TEA i blocked the channel. To determine this, we examined block of inward Na + currents by external K + in the presence and absence of TEA i . In the absence of TEA i , K + will not only block the channel but also potentially pass through the channel and exit to the inside. In the presence of TEA i , if TEA binds to the channel, it will prevent or slow K + flux through the channel and thus increase the effective [K + ] entering the channel. Consequently, whether due to block by TEA i , K + , or both, currents will be inhibited more at a given [K + ] in the presence of TEA i than in the absence of TEA i , if TEA interacts with the channel. Fig. 9 illustrates the block of Na + current by external K + in the absence (○) and presence (•) of 20 mM internal TEA, measured in wild-type Kv2.1. Channel block by K + was enhanced by TEA i at a [K + ] of 0.1 mM, which indicates that the minimum [K + ] at which TEA i interacted with the channel was 0.1 mM. This concentration was also the minimum required to block Na + currents through Kv2.1 . Although it did not reach significance at 0.03 mM K + , it appeared that block by TEA i may have been facilitated at [K + ] somewhat lower than 0.1 mM . This would be anticipated if an effect of TEA i is to increase the effective [K + ] in the channel by inhibiting K + exit to the cytoplasmic side of the channel. In summary, the conformational change associated with Lys 356, the conformational change measured in the double mutant, and the conformational change that influenced block by internal TEA, were all influenced by similar low [K + ]. Furthermore, in all cases, the minimum [K + ] required to influence the conformational change in a particular channel was identical to that required for block of Na + current through that channel. These results suggest that K + acted at the same site to influence each of these three conformational changes. The correspondence of these low [K + ] to those associated with block of Na + current are consistent with the site of K + action being at selectivity filter cation binding site(s). The data above are consistent with the hypothesis that the conformational change that altered TEA sensitivity was due to changes in K + occupancy of the pore. In these experiments, however, the conformational changes occurred at very low [K + ], in channels that were conducting predominantly Na + . The following experiments were designed to determine whether the observed conformational changes occurred in K + -conducting channels under conditions that manipulated K + occupancy of the pore. These experiments were conducted in the complete absence of Na + (NMG + substitute). As in previous experiments, we examined TEA o potency as a measure of pore conformation. First, we examined the block of inward K + currents by TEA o in the presence and absence of 20 mM internal TEA . In these experiments, TEA o would be expected to block all channels, with or without internal TEA, with the highest possible potency (IC 50 ∼ 4.5 mM for Kv2.1). The reasoning behind this prediction is as follows. In the absence of internal TEA, the entire pore of conducting channels is occupied by K + . In the presence of internal TEA, channels will be in one of two conditions: blocked or not blocked (A, 1) by internal TEA. In either condition, the open channel from the external vestibule to the TEA i binding site will be occupied by K + . However, in blocked channels, the channel pore on the cytoplasmic side of the TEA i binding site will often be unoccupied. If occupancy of the pore external to the TEA i binding site prevents the conformational alteration that lowers TEA potency, few or no channels should change conformation and external TEA will block with the highest possible potency. Fig. 10 B illustrates the effect of 10 mM external TEA (TEA o ) on inward K + currents through wild-type Kv2.1, in the absence (0 TEA i ) and presence (20 TEA i ) of internal TEA. Fig. 10 C illustrates the complete [TEA] o -response curves for these experiments. Channels carrying inward K + current were blocked identically regardless of whether internal TEA was included or not. The difference in inactivation kinetics observed between the control currents in the presence and absence of internal TEA confirms that internal TEA was present in the experiment illustrated in the right panel (the effect of internal TEA on inactivation kinetics will not be addressed in this manuscript). Thus, in either the absence or presence of internal TEA, no conducting channels underwent the conformational change that leads to decreased TEA potency. Similar results were obtained with [K + ] as low as 30 mM , which indicates that at 30 mM, the pore is sufficiently occupied by K + to prevent the conformational change associated with reduction in TEA o potency. These experiments also confirm that the site at which K + prevented the change in conformation was external to the TEA i binding site. Next, we examined the block of outward K + currents in Kv2.1 by TEA o in the presence and absence of internal TEA . In this experiment, there are expected to be multiple K + -conducting states, at least one of which is blocked by TEA o with high potency and at least one that is in a conformation with lower TEA o potency. The reasoning for this is illustrated in the diagram in Fig. 11 A. . In the absence of block by internal TEA , the K + -conducting channel is in the “high potency” conformation. When TEA i binds to the channel, it stops the flow of K + into the pore . In a percentage of channels, K + leaves the pore of the TEA i -blocked channel through the unblocked outer vestibule . In our experiments below, we maximized this transition by using an internal [TEA] that produced ∼90% block. As occupancy of the pore by K + decreases, the conformational change that results in the lowering of TEA o potency occurs . Since binding by internal TEA is also decreased or eliminated as K + leaves the pore, internal TEA comes off of the channel and K + flows through the channel that is in the low potency state . The measured TEA o potency would reflect the equilibrium between conducting channels in low and high potency states. The purpose of Fig. 11 A is to demonstrate that under these experimental conditions there will be at least two K + -conducting states, one with high TEA o potency (1) and one with lower potency (5). States 2–4 are all nonconducting. Clearly, this does not represent a complete state diagram. Transition into other possible states, which are not shown, would serve to change the percentage of channels that enter into the illustrated states but would not change the fundamental principle that the channel can exist in multiple conducting states, one or more of which have a high potency interaction with TEA o and some of which are in lower potency TEA states. Fig. 11 B illustrates two sets of outward K + currents, recorded in the absence (top) and presence (bottom) of internal TEA. Currents were carried by 100 mM K + . In the absence of internal TEA, 10 mM TEA o blocked currents by 65% . In the presence of 20 mM internal TEA, 10 mM TEA o blocked outward K + currents by just 20% . [TEA] o dependence between 1 and 100 mM under these conditions is illustrated in Fig. 11 C. The calculated IC 50 for block of outward currents by TEA o shifted ∼10-fold, from 5.3 ± 1.8 mM ( n = 3) in the absence of internal TEA to 65.1 ± 3.4 mM ( n = 3) in the presence of internal TEA. The hypothesis presented in Fig. 11 A postulated that the shift in TEA o potency was due to a change in the occupancy of the pore by K + and not by a direct antagonistic interaction between internal and external TEA . Our hypothesis predicts that under conditions where application of internal TEA produced a greater reduction in K + occupancy of the pore, TEA o would be shifted more. Under conditions where K + occupancy of the pore remained higher in the presence of internal TEA, TEA o potency would be shifted less. To test this, we examined the TEA i -induced shift in TEA o potency at different internal and external [K + ]. If the TEA i -induced shift in TEA o potency depends on K + occupancy, a larger shift would be expected when currents are recorded with lower internal [K + ]. Two effects could account for this. First, channels would empty faster once internal TEA blocked the channel . This could be caused by two processes. It is possible that with lower average occupancy, fewer ions would be in the channel and, consequently, fewer would have to leave during the on time of internal TEA. In addition, and perhaps more likely in these macroscopic current experiments, the smaller K + flux associated with lower [K + ] would produce less K + accumulation at the external mouth of the channel. Consequently, due to the greater concentration gradient at the outer mouth of the pore, K + would exit faster from the blocked channel. Second, at lower internal [K + ], channels would fill more slowly when TEA came off of its internal binding site . Conversely, recording outward currents in the presence of higher internal [K + ] would be expected to reduce the TEA i -induced shift in TEA o potency, because for reasons analogous to those above, average channel occupancy would be higher. Fig. 12 (•) illustrates that indeed, the TEA o potency shift produced by internal TEA was highly dependent on internal [K + ]. With low (30 mM) internal [K + ], internal TEA shifted TEA o potency >30-fold, from ∼5 mM to >170 mM ( n = 3). With high (140 mM) internal [K + ], internal TEA shifted TEA o potency only approximately twofold, to 11.5 ± 0.8 mM ( n = 3). As described in Fig. 11 , with the intermediate internal [K + ] of 100 mM, internal TEA produced an intermediate (∼10-fold) shift in TEA o potency. The results above were consistent with the hypothesis that the TEA i -induced shift in TEA o potency was related to changes in occupancy of the pore by K + . However, they did not rule out the possibility that this effect was due to an antagonistic interaction between internal and external TEA, or between K + and TEA in the pore. For example, lowering internal [K + ] could allow more TEA i to reach its internal binding site and thus result in greater antagonism between internal and external TEA. To test these possibilities, we examined the effect of external K + on the TEA i -induced shift in TEA o potency. If the TEA o potency shift were due to a reduced occupancy of the pore by K + , elevation of external K + would be predicted to increase K + occupancy of the pore and thus reverse the shift. In contrast, the hypothesis that the larger TEA i -induced shift in TEA o potency at lower internal [K + ] resulted from a decreased competition between internal K + and internal TEA would not predict a reversal in the TEA o potency shift upon elevation of external [K + ]. At both 30 and 100 mM internal K + , elevation of external [K + ] to 5 mM largely reversed the TEA i -induced shift in TEA o potency . With 100 mM internal K + and 20 mM TEA i , elevation of external [K + ] to 5 mM resulted in a 45 ± 1% block by 10 mM TEA o ( n = 3). This corresponds to a calculated IC 50 of 13 mM, as compared with 65 mM obtained under these same conditions except in the absence of external K + . With 30 mM internal K + and 20 mM TEA i , elevation of external [K + ] to 5 mM resulted in a 33 ± 1% block by 10 mM TEA o ( n = 3). This corresponds to a calculated IC 50 of 24 mM as compared with >170 mM in the absence of external K + . These results support the hypothesis that the potency shift was due to a change in K + occupancy. Our data indicate that occupancy of the selectivity filter by K + influences the conformation of the pore both internal and external to the selectivity filter. Since the rate of slow inactivation in “C-type” inactivating channels is influenced by occupancy of the selectivity filter by K + , we examined whether the conformational differences that affected TEA potency were associated with differences in slow inactivation rate in Kv2.1. Fig. 13 A illustrates five superimposed, normalized K + currents from wild-type Kv2.1, evoked by 8-s depolarizations to +40 mV. The five traces represent currents recorded under conditions designed to result in different K + occupancy of the pore. One current was carried by Na + in the absence of K + . Three currents were recorded in the presence of internal [K + ] ranging from 3 to 100 mM in the absence of external K + . The fifth current was recorded with 140 mM internal and 50 mM external K + . As internal [K + ] was elevated from 0 to 100 mM in the absence of external K + , there was a marked increase in inactivation rate . This increased rate of inactivation was associated with a corresponding increase in block by TEA o . To further increase the occupancy of the channel by K + , we recorded currents in the presence of 140 mM internal K + and elevated external [K + ]. Elevation of external K + to 10 mM resulted in an additional increase in inactivation rate and TEA block . Upon further elevation of external K + to 50 mM, both TEA o block and inactivation rate were unchanged . The correlation of changes in TEA block and inactivation rate, combined with the saturation of TEA block and inactivation rate at similar [K + ], support the hypothesis that the conformational differences associated with changes in TEA block influenced the rate of channel inactivation. Our results demonstrate that occupancy of the pore by K + alters the conformation of the pore at locations both internal and external to the selectivity filter. Our data suggest the following: (a) that this conformational change extends to the external edge of the outer vestibule, and includes a change in position of Kv2.1 Lys 356 (equivalent to Shaker F425) relative to the conduction pathway, (b) that the selectivity filter is the site at which occupancy by K + influences the described conformational changes, (c) that this conformational change can occur in K + -conducting channels as K + exits the pore, and (d) that the conformational changes produced by changes in K + occupancy of the selectivity filter can influence the rate of slow inactivation. In contrast to “classical” C-type inactivating channels, however, occupancy of the selectivity filter by K + speeds inactivation in Kv2.1. In addition, our data indicate that in Kv2.1, internal TEA alters the potency of external TEA not by direct repulsion between the two TEA molecules in the pore but as a result of changes in occupancy of the pore by K + ; as K + in the pore is reduced, TEA potency decreases. The results that mutation of Lys 356 largely restored TEA block in the absence of K + could be explained by one of two possibilities. The first possibility, which we favor, is that the conformational change that occurs upon removal of K + altered the position of Lys 356 relative to the conduction pathway. Movement of the positively charged lysine towards the center of the conduction pathway would interfere with the ability of TEA to enter the vestibule and/or bind to its binding site deeper in the pore. This effect might be especially profound if the lysines on each channel subunit moved relative to the central axis of the pore . Consequently, neutralization and/or reduction in the size of residue 356 would remove or reduce the interference between this residue and TEA binding, even if the mutation did not affect the K + -dependent conformational change. This possibility is directly supported by two observations. First, neutralization of this residue resulted in a half- log increase in TEA potency for block of K + currents, which, consistent with earlier studies , suggests that this lysine is in the conduction pathway. Second, fluorescent tagging experiments demonstrated directly that Shaker residue 425, which is equivalent to Kv2.1 Lys 356, and the adjacent residue 424 move relative to their environments in association with channel gating . As our data indicate for Kv2.1 residue 356, movement of Shaker residue 424 was K + dependent. It must be noted that we have not directly measured a conformational change precisely at the location of Lys 356, and that it is possible that mutation of residue 356 altered the ability of another nearby residue to change its position relative to the pore. The alternative possibility was that the cation-dependent conformational alteration that affected TEA potency was local to regions associated with the external TEA binding site (i.e., near residue Y380), and mutation of Lys 356 influenced the nature of the cation- dependent conformational change local to this region. Although we cannot unequivocally rule this out, several observations argue against this possibility. First, mutation of Lys 382, which is located two residues from the TEA o binding site, had similar effects on TEA block of K + and Na + currents. This suggests that movement of this residue was not primarily responsible for loss of TEA potency in the absence of K + . Second, the rate of slow inactivation, which involves a conformational change of the selectivity filter and presumably residues in the region of Y380 , was unaffected by the mutation. Third, the internal TEA binding site in the presence of K + , and the loss of internal TEA binding in the absence of K + , was unaffected by the outer vestibule mutations. Fourth, the mutant channel remained highly K + selective in the presence of K + , conducted Na + upon removal of K + , and had the same voltage dependence and time course of activation as wild-type Kv2.1. The proximity of the internal and external TEA binding sites to the selectivity filter , the integrity of both the selectivity filter and the internal TEA binding site in the presence of K + , and the similar inactivation rates, suggest that neither the structure of nor the ability of this part of the channel to change conformation was dramatically affected by the mutation. Our data strongly suggest that the selectivity filter was the site at which K + influenced the channel conformation. This conclusion is based on two sets of results: (a) the similar [K + ] dependence for block of Na + currents and change in TEA sensitivity and (b) studies that addressed the physical location of K + action . We observed the K + -dependent conformational change with three different measurements. The [K + ] dependence of each indicator of conformational change was identical to that associated with block of Na + current through the channel. Since block of Na + current is defined as occurring at one or more cation binding sites associated with channel selectivity, these data indicate that the conformational change was dependent on occupancy of a selectivity filter binding site by K + . This interpretation is further supported by the observation that in the different channel constructs used, K + appeared to have slightly different potencies for block of Na + current, and the K + dependence of the conformational change shifted similarly. The data in Figs. 10 – 12 , taken together, suggest that K + influenced the outer vestibule conformation by acting at a site between the internal and external TEA binding sites. When inward K + currents were studied, TEA o potency did not change with or without internal TEA at [TEA] i that blocked currents by at least 85% . In these experiments, occupancy of the channel external to the TEA i binding site was not interrupted by internal TEA, but, presumably, occupancy of the vestibule internal to the TEA i binding site was reduced in channels blocked by internal TEA. When outward K + currents were studied, a decrease in K + occupancy of the pore region external to the TEA i binding site resulted in the reduction of TEA o potency . In these experiments, the pore region internal to the TEA i binding site was always exposed to high [K + ], and this constant exposure did not prevent the loss of TEA o potency. Together, these data strongly suggest that K + influenced TEA o potency in the pore region external to the TEA i binding site. When outward K + currents were studied in the absence of internal TEA, external TEA always blocked channels with the highest potency . In these experiments, occupancy of pore regions internal to the TEA o binding site would presumably be unaffected by external TEA, but the pore region external to the TEA o binding site would be largely unoccupied in channels blocked by external TEA. These data suggest that emptying K + from the region external to the TEA o binding site did not result in the conformational change that led to a reduction of TEA o potency. Consequently, these data suggest that TEA o potency was influenced by an interaction of K + with a site internal to the TEA o binding site. When combined, these results indicate that the site at which K + influenced TEA potency was between the external and internal TEA binding sites. Although we have no direct evidence that allows us to determine whether the inner and outer vestibule conformational rearrangements occurred as a unit, it is interesting to consider the location of the apparent conformational changes in light of the structural data of Doyle et al. . By analogy with the Streptomyces lividans channel, Kv2.1 Lys 356 is in the “turret” of the outer vestibule, two residues away from the “pore helix.” The residue associated with internal TEA binding, Kv2.1 Thr 372, is located within two residues of the other end of the pore helix. Following the P loop from Thr 372 towards the central axis of the pore leads to the selectivity filter, three residues from Thr 372 . Inactivation data from both Shaker and Kv2.1 suggest that the selectivity filter structure can change conformation, and that binding of K + to the selectivity filter alters the rate of this conformational change . Thus, the structural data is consistent with the possibility that the loop that extends from the selectivity filter to Thr 372 to Lys 356 moves as a unit depending on occupancy of the selectivity filter by K + , and that this rearrangement influences the slow inactivation process. It was demonstrated previously that in Shaker and Kv1.1, internal and external TEA antagonized block by each other . This effect was attributed to mutual repulsion between TEA molecules when both internal and external sites were occupied. In these channels, increasing internal [K + ] also resulted in a decreased TEA o potency, which suggested that K + antagonized the effects of external TEA due to repulsion within the pore . Our data also demonstrate that, in Kv2.1, the TEA o potency decreased in the presence of internal TEA. However, our data are not consistent with this effect being due to mutual repulsion between TEA molecules across the selectivity filter. Our data indicate that this effect was due to the TEA i -induced reduction in K + occupancy, and the consequent effect of lowering K + occupancy on external TEA potency. The evidence for this conclusion is as follows. In the presence of internal TEA, TEA o potency for block of outward K + currents decreased as internal [K + ] was reduced. One could argue, from these results, that at lower internal [K + ], more channels became bound by internal TEA and thus repulsion between internal and external TEA increased. However, the reduction in TEA o potency by internal TEA was reversed by addition of 5 mM external K + . Addition of external K + would not be expected to affect the competition between internal K + and internal TEA for entry into the channel from the inside. It is also unlikely that addition of 5 mM external K + would increase TEA o potency due to repulsion between external K + and internal TEA, because at the concentration of internal TEA used, inward currents carried by 30 mM K + were blocked by ∼85%. Finally, in contrast to an effect that would be expected as a result of repulsion between K + and TEA, TEA potency always increased under conditions where K + occupancy of the pore was increased, whether from the inside or outside of the pore. We have interpreted our findings in terms of cation- dependent changes in the permeation pathway. An alternative possibility, however, is that cation-dependent differences in TEA potency resulted from cation- or TEA-dependent changes in channel gating. Observed TEA potency differences cannot be attributed simply to differences in open probability, since conductance– voltage curves in the presence of K + , Na + , and TEA are essentially identical in Kv2.1 , and essentially identical between Kv2.1 and the double lysine mutant (data not shown). A more complex interpretation might suggest that mean open time was dramatically different in different channels or under different ionic conditions. For example, if TEA only bound to the open state of the channel, and in low [K + ], mean open time became brief relative to TEA on rate, TEA potency would be reduced. Several experimental findings argue against this possibility. (a) External TEA apparently binds to the closed state of the channel . (b) TEA is a very fast K + channel blocker . In the frog skeletal muscle delayed rectifier, which is blocked by external TEA with the same potency as Kv2.1, the mean block and unblock times for TEA at the K d were estimated to be 3.7 and 2.1 μs, respectively . The mean open time of Kv2.1 in the presence of physiological [K + ] is ∼13 ms . Consequently, the mutation- induced increase in TEA potency in the presence of K + cannot be attributed to an increase in mean open time. (c) TEA blocked Na + currents in two of the mutant channels with nearly the same potency as it blocked K + currents in wild-type Kv2.1, which suggests that the mean open time in the presence of Na + was sufficient for full TEA access. Despite relatively potent TEA block of currents in the absence of K + , elevation of K + produced a further increase in TEA potency. (d) Finally, a significant effect of mutations on mean open time would be expected to change internal TEA potency since TEA binds inside the activation gate . However, our results demonstrate that mutations that markedly altered external TEA potency did not influence internal TEA potency in the presence of either K + or Na + . The K + -dependent conformational alteration that resulted in a change in TEA o potency was correlated with the effect of K + on inactivation rate. As [K + ] was increased, TEA o potency and inactivation rate increased . Importantly, the effects of K + on inactivation rate saturated at the same [K + ] as the effect on TEA o potency. These results suggest that the different channel conformations, which are associated with different TEA potency, can affect the rate of inactivation. Whether the conformational differences internal or external to the selectivity filter were associated with the change in inactivation rate is unknown. However, studies in Shaker support the possibility that conformational changes at or near Lys 356 are involved in this effect . Shaker residues 424 and 425 in the outer vestibule, which both move in conjunction with channel activity, appear to be associated with different aspects of channel function . Movement of residue 424 appears to be primarily associated with the inactivation process , whereas movement of residue 425 appears to be associated with the activation process . Kv2.1 Lys 356 is in the position equivalent to Shaker 425. Although the K + -dependent conformational differences associated with Lys 356 were associated with both inactivation rate and TEA o potency, TEA o potency does not appear to decrease during inactivation . These results suggest that, as proposed for Shaker 425, the conformational differences that involved Lys 356 do not result directly from the inactivation process but somehow influence the inactivation process. Recently, Klemic et al. suggested that both Kv2.1 and Shaker undergo an inactivation process that differs from C-type inactivation. During this process, called U-type inactivation, channels are proposed to enter the inactivated state from the closed state, whereas, in C-type inactivation, channels are proposed to inactivate from the open state. Entry into the U-type inactivated state is influenced by both voltage and [K + ], and is proposed to involve a mechanism unrelated to constriction near the TEA binding site and selectivity filter . It will be interesting to determine how the change in conformation associated with Lys 356 influences inactivation, whether the K + -dependent conformational changes we observed are related to inactivation by the U-type mechanism, and whether conformational changes that involve Shaker 425 affect inactivation similarly. Our results also relate to the potential influence of intracellular channel blockers on both channel gating and the potency of extracellular channel blockers. Baukrowitz and Yellen suggested that the rate of C-type inactivation in Shaker may be influenced by intracellular channel blockers due to the effects of intracellular channel block on occupancy of the pore by K + . A similar effect is observed in Kv2.1, except that, for as yet unknown reasons, the rate of slow inactivation in Kv2.1 is increased with increasing K + occupancy . Furthermore, however, our data suggest that, in Kv2.1, intracellular channel blockers may influence the potency of pharmacological agents that act in the outer vestibule via changes in K + occupancy of the pore. Whether similar effects occur in other K + channels remains to be explored. | Study | biomedical | en | 0.999998 |
10352034 | The inositol 1,4,5-trisphosphate (InsP 3 ) 1 –gated Ca channel plays a critical role in the regulation of intracellular Ca concentrations after hormonal activation of the phosphoinositide cascade in a wide variety of cell types . The mechanisms underlying regulation of the InsP 3 -gated channel are poorly understood; however, the involvement of both cytosolic Ca and InsP 3 in the regulation of this channel is well established . That is, InsP 3 is absolutely required for the opening of the channel, though channel activity can be modified by cytosolic Ca. Elevating the cytosolic Ca concentration from 0.01 to 0.3 μM in the presence of 2 μM InsP 3 , for example, resulted in a dramatic increase in channel activity . Further elevation of cytosolic Ca, however, decreased channel activity, with near complete inhibition of the channel in the presence of 5 μM Ca . Three basic models have been proposed to explain this bell-shaped Ca dependence of the channel differing in terms of the effects of InsP 3 on the bell-shaped Ca dependence. In one model , the peak of the bell-shaped Ca dependence is unaffected by InsP 3 concentration. The other two models predict either a shift to the left or the right in the peak of the bell-shaped Ca dependence of channel activity as InsP 3 concentrations increase. Recent studies indicate that the InsP 3 concentration influences the Ca dependence of InsP 3 -induced Ca release, with an increased sensitivity to Ca-dependent inhibition occurring at low InsP 3 concentrations . Thus, the peak of the bell-shaped Ca dependence of InsP 3 -gated channel activity shifted to the right as InsP 3 concentration was increased from 0.2 to 2 μM . Further elevation of InsP 3 concentration to 180 μM overcame the Ca-dependent inhibition of channel activity , which may reflect the presence of a second low affinity InsP 3 binding site on the channel complex. Recently, we modeled this complex steady state behavior of the InsP 3 -gated channel by extending a basic 8-state model proposed previously to 16-states, assuming that there are two InsP 3 (with dissociation constants of 0.3 and 10 μM) and two Ca binding sites per InsP 3 receptor in the tetrameric channel complex . In the present report, we explored in further detail the Ca dependence of InsP 3 binding, as well as the influence of Ca and InsP 3 on the mean open times of the InsP 3 -gated channel, and incorporated these data into a novel mathematical model of the channel. The proposed model differs conceptually from previous models in that it focuses on the tetrameric channel complex as a unit, but more importantly it provides a better fit of the steady state channel and binding data, and simulates the kinetics of channel activation. Microsomes were isolated from canine cerebellum, as described previously . To minimize proteolysis, all solutions used for the isolation/storage of the cerebellar microsomes included a protease inhibitor cocktail (5 μg/ml leupeptin, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 0.1 mM PMSF). In brief, cerebellum was homogenized in 6 vol of buffer A (20 mM HEPES, 0.1 mM EGTA, 5 mM NaN 3 , pH 7.2, 4°C) using a Brinkman polytron. The suspension was centrifuged at 4,000 g for 20 min, and then the supernatant was centrifuged at 100,000 g for 30 min. The pellet from the latter centrifugation was resuspended in 3 vol of buffer B (20 mM HEPES, 0.6 mM KCl, 20 mM Na 4 P 2 O 5 , 5 mM NaN 3 ; pH 7.2, 4°C), and then centrifuged at 4,000 g for 20 min. The resulting supernatant was centrifuged at 100,000 g for 30 min. The final pellet was resuspended in buffer C (10 mM MOPS, 10% sucrose, pH 7.0) to a protein concentration of ∼6 mg/ml , quick frozen in liquid nitrogen, and stored at −80°C. InsP 3 binding was determined by a centrifugation technique , using buffer identical to that used for the InsP 3 -gated channel activity measurements . Binding media contained 250 mM HEPES, 110 mM Tris, 1 mM ATP, 0.5 mM EGTA, pH 7.35, 0.6–1,200 nM 3 H-InsP 3 , and CaCl 2 (to yield the specified free Ca concentrations). InsP 3 binding was initiated by addition of cerebellar membranes to the above buffer. After an 8-min incubation, InsP 3 binding was terminated by centrifugation (40,000 g for 10 min). The supernatant was removed by aspiration. The pellets were dissolved in Soluene 350, and then assayed for radioactivity by liquid scintillation counting. Nonspecific binding was determined in the presence of 40–160 μM nonradioactive InsP 3 . Scatchard analysis of InsP 3 binding was performed using an iterative curve-fitting routine for two binding sites (SigmaPlot; Jandel Scientific). All binding studies were replicated three to five times, with different microsomal preparations. Calculations of total Ca needed to obtain a specified free Ca concentration were based on previously published association constants , adjusted to pH 7.35 (at the specified temperature; 0° or 22°C). The apparent association constants (M −1 ) for Ca-EGTA and Ca-ATP used for these calculations were 1.055 × 10 7 and 7.595 × 10 3 , respectively, at 0°C, and 1.244 × 10 7 and 6.312 × 10 3 , respectively, at 22°C. InsP 3 -gated channel activity was measured as described previously , using 53 mM Ba in the trans chamber as current carrier. The planar lipid bilayer contained phosphatidylethanolamine and phosphatidylserine (3:1 wt:wt; Avanti Polar Lipids), dissolved in decane (20 mg/ml). Cytosolic bilayer solutions contained 500 μM Na-ATP, 500 μM EGTA, 110 mM Tris, and 250 mM HEPES, pH 7.35. Luminal solutions contained 53 mM Ba(OH) 2 , 250 mM HEPES, pH 7.35. Calibrated CaCl 2 was added to the cytoplasmic solution to obtain the desired free Ca concentration . The free Ca concentration was checked by spectrofluorometric measurements using BTC (Molecular Probes, Inc.). InsP 3 -gated channel activity was initiated by addition of InsP 3 (at the specified concentration). The number of channels in each experiment was estimated from the maximum number of channels observed simultaneously in the bilayer . The InsP 3 dependence of the open probability, measured at a fixed Ca concentration, was used to correct for variations in the maximum open probability among individual channels. Transmembrane voltage was maintained at 0 mV and the single channel current was amplified (Warner Instruments) and stored on VHS tape ( Instrutech Corp .). Data were filtered at 1 kHz and digitized at 5 kHz for computer analysis using pClamp 6.0 ( Axon Instruments ). Modeling InsP 3 -gated channel activity was done by considering the tetrameric channel complex as one functional unit having four medium affinity InsP 3 binding sites ( K d = 0.3 μM at 22°C), four low affinity InsP 3 binding sites ( K d = 10 μM at 22°C), and four Ca binding sites. The mathematical model assumes mass action kinetics with first order on ( k + ) and off ( k − ) rates for the binding of any ligand ( L ) to a specific site on the receptor monomer subunit ( R ), which was described by ordinary differential equations of the form: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{d[RL]}{dt}=k_{+}[R][L]-k_{-}[RL].\end{equation*}\end{document} At equilibrium, the distribution of the receptor states is a function of the ligand dissociation constant ( K d = k − / k + ) and free concentration of ligand. The four InsP 3 receptor subunits comprising the channel complex were considered identical and positionally equivalent, and a state transition reaction scheme was constructed for the 125 possible states of the channel tetramer complex ( T ) (see results ). The various K d values fully define the steady state distribution of states and allowed us to calculate the relative abundance t i, c, j = [ T i, c, j ]/[ T total ] (of tetramers with i and j InsP 3 molecules bound to the medium and low affinity site, respectively, and c Ca molecules bound) by solving a linear system of first degree equations that result from the equilibrium condition for formation/degradation of each state. The no–flux equilibrium condition, as well as several assumptions regarding binding interactions, were used to constrain the various dissociation constants that were not directly inferred from binding experiments (see results ). Channel states characterized by the binding of one to two Ca and two to four InsP 3 molecules were assumed to be “active;” i.e., exhibit a high open probability. InsP 3 binding to the low affinity site was assumed to create an active state, independent of InsP 3 and/or Ca binding to other sites within the channel complex. Channel activity was described as a transition between two substates, open and closed (Scheme I). First order reaction rates were assumed for both transitions, and the resulting open probability for any given state of the channel is: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P^{\;\;open}_{\;\;i,\;c,\;j}=\frac{{\alpha}_{\;\;i,\;c,\;j}}{{\alpha}_{\;\;i,\;c,\;\;j}+{\beta}_{\;\;i,\;c,\;j}}.\end{equation*}\end{document} ( scheme i ) Iterative curve-fitting of the InsP 3 -gated Ca channel open probability data given the Ca dependence of InsP 3 binding using a standard quasi-Newtonian algorithm with forward differencing and quadratic extrapolation was then performed to determine the values of unknown parameters. The open time histograms were used to further constrain the model and facilitate kinetic simulations. Diffusion-limited on rates of binding were assumed in all steps for both Ca and InsP 3 binding and each off rate was determined by multiplication with the respective dissociation constant. Channel opening and closing rates were determined from the open probabilities of the respective active states given the constraints imposed on closing rates in order to match the experimental distribution histogram of open states. Simulations of the single channel recordings were performed by stochastic modeling of state transition events of the channel complex, using a Markovian model of probability matrix decomposition to predict state transitions over a given time period. The behavior of the channel is calculated from the matrix differential equation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{dP(t)}{dt}=P(t)Q,\;\end{equation*}\end{document} where the elements p m,n ( t ) of the matrix P ( t ) are the probabilities of a channel to get from state m to n over the time period t and the elements of the matrix are the rates of transition from state m to n. The solution of this equation is: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P(t)=e^{Qt}=I+Qt+\frac{(Qt)^{2}}{2!}+\frac{(Qt)^{2}}{3!}+{\ldots},\end{equation*}\end{document} where I is a unit matrix. Relaxation from a random state (picked according to steady state distribution probability) was simulated for the desired time length to obtain channel state data sets at the chosen time step. Predicted traces were generated for the simultaneous presence of up to 10 channels in the membrane from individual current functions: i ( t ) = − A · χ( t ) + S ( t ), where A is the current amplitude through the open channel, χ( t ) is either 0 or 1, depending on whether the channel is in one of the closed or one of the open states, respectively, and S ( t ) is a standard noise spectrum function chosen to match the experimentally observed noise in any given trace. There have been conflicting reports on the influence of Ca on InsP 3 binding. Initial reports indicated that micromolar Ca concentrations could completely inhibit InsP 3 binding, with half-maximal inhibition occurring at 0.3 μM Ca . On the other hand, Ca-dependent inhibition of cerebellar membranes can also occur as an artifact resulting from activation of an endogenous phospholipase C . We have re-investigated the influence of Ca on InsP 3 binding, with the goal of relating Ca-dependent modulation of InsP 3 binding to the bell-shaped Ca dependence of channel activity. As shown in Fig. 1 A, micromolar Ca decreased InsP 3 binding when using media identical to that used for channel activity measurements. Ca concentrations as high as 100 μM resulted in only partial inhibition of InsP 3 binding , in contrast to the near complete inhibition previously reported . The inhibitory effects of Ca on InsP 3 binding were reversed by addition of EGTA , ruling out contributions of Ca-dependent activation of an endogenous phospholipase C . Moreover, the presence of 100 μM free barium, an inhibitor of phospholipase C , did not alter this Ca-dependent inhibition of InsP 3 binding (data not shown). Scatchard analyses of InsP 3 binding indicated that this decreased InsP 3 binding was attributable to a Ca-dependent increase in the K d for InsP 3 binding. The Scatchard plots were nonlinear, suggesting the presence of multiple InsP 3 binding sites. Data (which were obtained over an InsP 3 concentration range of 1–600 nM) could be fit assuming two InsP 3 binding sites (with K d values of 1 and 50 nM in the absence of Ca). The concentration of InsP 3 used was too low to observe the low affinity InsP 3 binding site discussed below . The 50-nM binding site was abundant, representing 99% of the total sites in the absence of Ca. As the Ca concentration was increased from 0.0004 to 10 μM, the apparent K d for this site increased 3.5-fold . Half maximal inhibition occurred at 0.3 μM Ca with a Hill coefficient of 1.5. The high affinity InsP 3 binding site, by contrast, was in very low abundance, representing <1% of the total sites at all Ca concentrations examined, and its K d (1 nM) appeared to be Ca independent . To more closely resemble the conditions used for channel activity determinations, InsP 3 binding assays were repeated at room temperature again using media identical to that employed in the single channel experiments. Increasing the temperature to 22°C resulted in a 67% reduction in InsP 3 binding; an inhibition that was reversed by reducing the temperature to 0°C . Scatchard analyses of InsP 3 binding at 0° and 22°C show that the increase in temperature results in a decrease in InsP 3 binding affinity, with no apparent effect on the maximal number of InsP 3 binding sites. The calculated Q 10 for this temperature-dependent change in InsP 3 binding affinity was 2.3, consistent with that calculated for InsP 3 binding to rat basophilic leukemia cells . The Ca dependence of InsP 3 binding was also reexamined at 22°C , and yielded results similar to those observed at 0°C . As shown in Fig. 3 A, micromolar Ca concentrations reduced InsP 3 binding (measured in the presence of 170 nM InsP 3 ) by 55% (▪), and this inhibition was completely reversed by Ca chelation with EGTA (□, dotted line). Increasing the Ca concentration to 100 μM did not increase the extent of the inhibition (data not shown), consistent with our previous observations in aortic smooth muscle microsomes . The Ca-dependent inhibition at 22°C showed a high level of cooperativity (Hill coefficient, 2.8; half-maximal inhibition at 0.45 μM Ca) , and was attributable to a Ca- dependent increase in the K d for InsP 3 binding . The K d for InsP 3 binding at 22°C increased from 0.30 to 1.78 μM in the presence of a high Ca concentration (•; 50 μM free Ca) . There was no evidence for an effect of Ca on the maximum number of InsP 3 binding sites at either 0° or 22°C. The Scatchard analyses in Figs. 2 and 3 show the presence of only one InsP 3 binding site ( K d = 50 nM at 0°C and 312 nM at 22°C) due to the range of InsP 3 concentrations used (5 nM–1 μM), where neither the high ( K d = 1 nM) nor the low ( K d = 10 μM) affinity site can be resolved. These sites are observed only if one extends the InsP 3 concentration range below 1 nM and above 5 μM . Three InsP 3 binding sites were observed in cerebellar microsomes and purified InsP 3 receptor preparations, purified by either heparin-agarose chromatography or immunoprecipitation . In all assays, the 1-nM InsP 3 binding site was a minor component, whereas the medium and low affinity InsP 3 binding sites were abundant. At 0°C, the calculated dissociation constants for InsP 3 binding to these two predominant InsP 3 binding sites were 50 nM (medium affinity site) and 10 μM (low affinity site) . At 22°C, the dissociation constant of the 50-nM site increased to ∼300 nM, whereas the dissociation constant for the 10-μM InsP 3 binding site could not be accurately ascertained. It appeared to remain in the 10-μM range, suggesting that the low affinity InsP 3 binding site was largely temperature insensitive. We previously reported that the Hill coefficient for InsP 3 -induced Ca release was 1.3, whereas the single channel activity determinations suggested a Hill coefficient 1.0 . The previous experiments underestimated the Hill coefficient because the lowest concentration of InsP 3 used activated the channel to 10% of the maximum, a level of activity too high for accurate measurements of cooperativity. Reinvestigation of the channel activity at lower InsP 3 concentrations indicate that the Hill coefficient for channel activity measurements is at least 1.7 . This value for the Hill coefficient may reflect the requirement for InsP 3 to bind to at least two of the four InsP 3 receptors to activate the channel. Open-time histograms of InsP 3 -gated channels at two different cytoplasmic Ca concentrations were fit assuming an exponential with two time constants . Increasing the number of time constants to three did not statistically improve the fit. In the presence of 0.01 μM Ca and 2 μM InsP 3 , the appropriate time constants were 1.8 and 9.3 ms. As the Ca concentration was increased to 0.1 μM , the time constants decreased to 0.6 and 4.7 ms. Thus, the channel open times are Ca dependent. Similar analyses were done at a number of Ca and InsP 3 concentrations, with the appropriate time constants incorporated into the mathematical model (described below). Recent studies indicate that the cerebellar InsP 3 -gated Ca channel exhibits a complex regulation by Ca and InsP 3 . Data points from this earlier report are shown in Fig. 6 ; lines through the points represent the fit using the model described in the present report . For comparison, the fit obtained using the 16-state model is also shown , but fails to accurately predict channel activity at low InsP 3 concentrations. The current model provides an excellent fit of the channel data over a broad range of InsP 3 and Ca concentrations. Simple mass-action kinetics was assumed for ligand binding to the sites on the receptor monomer subunits (see methods ). Since the functional unit of the channel is the tetrameric complex (T), this analysis was extended by introducing apparent dissociation constants for sequential binding to the four monomer subunits. A total of 2 4 = 16 possible different states of the channel can be generated by binding of up to four ligand molecules to the same site on the four different monomer subunits of the tetramer. We considered the subunits identical and positionally equivalent, which reduces the 16 states to only 5 different states according to Scheme II. The resulting apparent dissociation constants for sequential binding are: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}_{d_{1}}=\frac{1}{4}K_{d_{1}}\;K^{\prime}_{d_{2}}=\frac{2}{3}K_{d_{2}}\;K^{\prime}_{d_{3}}=\frac{3}{2}K_{d_{3}}\;K^{\prime}_{d_{4}}=4K_{d_{4}}.\end{equation*}\end{document} This general framework was applied to InsP 3 and Ca binding to the channel complex, assuming that each monomer in the tetrameric channel complex contains the equivalent of one Ca regulatory site (C), one medium affinity InsP 3 site (I), and one low affinity InsP 3 site (J). This results in five different states of the tetrameric channel complex (zero, one, two, three, or four sites occupied) for each of the three classes of sites (C, I, and J). A total of 5 3 = 125 different states per channel complex are predicted ( T i,c,j , where i, c, and j represent the number (zero to four) of ligand molecules bound to the I, C, and J sites, respectively). Fig. 7 shows the state transition scheme for the 25 states in the absence of InsP 3 binding to the low affinity site ( T i,c,0 ). The apparent dissociation constants of binding of InsP 3 to the I site were labeled \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}^{I}_{d_{i,\hspace{.167em}c}}\end{equation*}\end{document} (for a tetramer that has i other I sites and c of the C sites occupied) and of Ca binding to the C site were labeled \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}^{C}_{d_{i,\hspace{.167em}c}}\end{equation*}\end{document} (for a tetramer that has c other C sites and i of the I sites occupied). This reaction scheme was extended upwards by drawing four additional planes to include binding to the low affinity InsP 3 binding site (J). The “gating” or “regulatory” action of ligand binding to the various sites on the channel is in effect determined by the differences in the opening/closing transition rates of the different states (see methods ; i.e., a state with α ≈ 0 is an “inactive” state). Essentially, “active” states were hypothesized to be channels having either two or more medium affinity InsP 3 sites and one or two Ca sites occupied . Additionally, active states were channels having two or more low affinity InsP 3 sites (J) and any number of Ca sites occupied . The activation of the channel through J was thus assumed to overcome Ca-dependent inhibition of the channel, consistent with experimental observations . At any given Ca and InsP 3 concentration, the only parameters needed to calculate the steady state relative distribution of the different states and the overall channel open probability were the various apparent dissociation constants \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K\;\;^{\prime}^{C}_{D_{c,\;\;j}},\;K\;\;^{\prime}^{I}_{D_{i,\;c}},\;and\;K\;\;^{\prime}^{\;J}_{D_{j}},\end{equation*}\end{document} and the open probabilities of the various states \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P^{open}_{i,\hspace{.167em}c,\hspace{.167em}j}\end{equation*}\end{document} . Some of these parameters could be inferred from measured dissociation constants based on several assumptions and constraints. In accordance with the thermodynamic principle of detailed equilibrium , reaction rates between any combination of states that form a complete cycle were constrained to yield no net flux at equilibrium. Thus, the apparent dissociation constants \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(K^{\prime}_{d})\end{equation*}\end{document} of binding to the C and I site were constrained as follows: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}^{C}_{d_{c,\;i}}{\cdot}K^{\prime}^{I}_{d_{i,\;c+1}}=K^{\prime}^{I}_{d_{i,\;c}}{\cdot}K^{\prime}^{C}_{d_{c,\;i+1}}\;{\forall}i,\;c\;{\in}\;\;\{0,\;1,\;2,\;3\}.\end{equation*}\end{document} With regard to interactions when binding to the same site on different monomer subunits, we assumed that Ca binding is cooperative \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(K^{C}_{d_{c,\hspace{.167em}i}}>K^{C}_{d_{c+1,\hspace{.167em}i}}),\end{equation*}\end{document} whereas InsP 3 binding is independent \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{C}_{d_{i,\hspace{.167em}c}}=K^{C}_{d_{i+1,\hspace{.167em}c}}\end{equation*}\end{document} ; for simplicity, we assumed that the cooperative effect is the same for each step during sequential binding of Ca; i.e., the ratio \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{C}_{d_{c,\hspace{.167em}i}}>K^{C}_{d_{c+1,\hspace{.167em}i}}\end{equation*}\end{document} is the same for c = 0...3 (the value of this ratio is a parameter we named cooperativity factor, κ Ca ). The dissociation constants for InsP 3 were derived directly from the binding data . For the I site, we used \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}^{I}_{d_{i,\hspace{.167em}0}}\end{equation*}\end{document} = 0.3 μM (no Ca bound) and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}^{I}_{d_{i,\hspace{.167em}c}}\end{equation*}\end{document} = 1.05 μM (for c > 0, reflecting the noncompetitive inhibition of InsP 3 binding by Ca). Binding of InsP 3 to the low affinity site appears independent of binding to any other sites with a dissociation constant \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{J}_{d}\end{equation*}\end{document} = 10 μM. Given the above constraints and InsP 3 binding parameters, all the dissociation constants for Ca binding could be derived from only two parameters, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{C}_{d_{0,\hspace{.167em}0}}\end{equation*}\end{document} and κ Ca . The values of these as well as of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P^{open}_{i,\hspace{.167em}c,\hspace{.167em}j}\end{equation*}\end{document} were obtained through standard curve-fitting algorithms for the predicted channel open probability data (Table I ). The predicted K d of Ca binding to the first site is 0.7 μM, which approximates the reported K d for Ca binding to a cytoplasmic region of the type 1 InsP 3 receptor . Table I summarizes all the parameters used to construct the model. A sample recording of InsP 3 -gated channel activity obtained in the presence of 0.2 μM InsP 3 and 0.1 μM Ca is shown for comparison with channel activity predicted by the model (B). Using constants derived from the InsP 3 binding analyses, channel open time histograms, and the steady state open probability data, the mathematical model described above was used to generate a time course of channel activity in the presence of 0.2 μM InsP 3 and 0.1 μM Ca. The predicted channel activity data closely resemble the actual channel recording (A); moreover, quantitative comparisons show that the dwell time histogram of a simulated trace could be fitted with a double exponential curve using identical time constants as those used to fit the experimental data obtained at the same Ca and InsP 3 concentrations . The parameter list used for the steady state analysis (and values) is not sufficient to calculate the values required to fill the transition rate matrix (see methods ). This requires explicit numbers for the on and off rates ( k + and k − values) and opening and closing rates (α and β values). The on rates for binding were based assuming diffusion limitation for both Ca and InsP 3 (10 and 250 μM −1 s −1 , respectively; this includes the effect of buffering for Ca) binding. The channel closing rates were based on the single channel open time distribution histograms. A minimum of two different closing rates is demonstrated by the biexponential fits of the dwell times. To accurately predict the apparent time constants and relative ratios, as well as their change with increasing Ca concentration , we used four different closing rates ranging from 150 to 1,000 s −1 (Table I ) whereby the faster rates were assigned to the active states with two Ca bound and the slower rates to the active states with one Ca bound. The corresponding binding off rates and channel opening rates were then calculated using the parameter values from the steady state simulations: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{I}_{-_{i,\;c}}=K^{I}_{d_{i,\;c}}{\cdot}k^{I}_{+_{i,\;c}};\;k^{C}_{-_{i,\;c}}=K^{C}_{d_{i,\;c}}{\cdot}k^{C}_{+_{i,\;c}};\;\;\;{\alpha}_{i,\;c}={\beta}_{i,\;c}{\cdot}\frac{P^{\;\;open}_{\;\;i,\;c}}{1-P^{\;\;open}_{\;\;i,\;c}}.\end{equation*}\end{document} To maintain the necessary matrix algebra at a manageable size, we limited ourselves to simulations of channel activity under conditions with [InsP 3 ] ≤ 2 μM, so that the effect of the J site on channel activity is negligible and only the 25 states depicted in Fig. 7 (and the corresponding substates of the active states) have to be included in the calculations. To produce simulated traces of channel current recording, we generated predicted relaxation data. An initial state was chosen for time 0, either arbitrarily or the most probable state for the given Ca and InsP 3 concentration (as indicated by the steady state distribution values). We calculated the probability matrix P ( t ) for a time value equal to the chosen sampling interval in single channel records (τ = 40 μs), and generated data sets of predicted channel states at the given sampling rate for a desired length of simulation. Fig. 8 B shows a representative segment of a simulated trace with 2.1 pA open channel current amplitude and normally distributed baseline noise of 1 kHz median frequency. The current mathematical model is thus capable of modeling both steady state open probability data, ligand binding data, and channel kinetics. Recent studies in our laboratory indicate that Ca and InsP 3 interact to increase the dynamic range of the InsP 3 -gated Ca channel . At a resting intracellular Ca concentration of 0.05–0.1 μM, agonist-induced production of InsP 3 would be expected to activate the InsP 3 -gated Ca channel, with a subsequent rise of intracellular Ca (up to 0.3 μM) promoting further activation of the channel through a positive feedback mechanism . Negative feedback of Ca on the InsP 3 -gated channel, however, will limit the rise of intracellular Ca, with near complete inhibition of the channel at 5 μM Ca, even in the presence of 2 μM InsP 3 . Increasing the InsP 3 concentration results in a rightward shift in the bell-shaped curve . Thus, the channel is more sensitive to the inhibitory effects of Ca when the InsP 3 concentration is low. This may be important physiologically, as it would further limit the rise in intracellular Ca in the presence of low InsP 3 concentrations. That is, it would promote a more gradual rise in intracellular Ca concentration over the InsP 3 concentration range 0.02–2 μM. Conversely, very high InsP 3 concentrations can largely overcome this Ca-dependent inhibition of the channel , preventing closure of the channel in local/confined areas such as those near the site of InsP 3 production. An important question concerns the mechanism(s) responsible for this complex regulation of the InsP 3 -gated channel. Our working hypothesis has been that the previously reported Ca-dependent decrease in InsP 3 binding is involved. Although micromolar Ca has been reported to completely inhibit InsP 3 binding to cerebellar membranes , our results reveal only a partial (<50%) inhibition of InsP 3 binding. As 95–99% of the InsP 3 receptors in the cerebellum have been shown to be of the type 1 isoform , it is unlikely that this partial inhibition is due to heterogeneity of InsP 3 receptors. Instead, the Ca-dependent decrease in InsP 3 binding appears to reflect a change in the affinity for InsP 3 . This was first suggested in a report showing that the K d for InsP 3 binding to rat cerebellar membranes increased from 0.04 to 0.12 μM . Such a shift in InsP 3 affinity has also been observed in aortic smooth muscle membranes . Using binding conditions identical to those used for the channel activity determinations, the present study also shows a Ca-dependent shift in InsP 3 affinity, though the calculated K d s are substantially higher than those previously reported. Thus, the binding affinities for InsP 3 reported in the present study are comparable to the calculated InsP 3 affinity for channel activation. The reason for the relatively high K d values in the present study involves in large part the high temperature dependence of the binding constant ( Q 10 = 2.3 for the cerebellar membranes). That is, InsP 3 binding studies are typically done on ice (0°C), whereas channel activity determinations are done at room temperature (23°C). In the present study, however, efforts were made to closely match both the InsP 3 binding and channel analyses. A similar Q 10 (2.0) has been reported for the K d of InsP 3 binding to rat basophilic leukemia cells , which was useful for modeling Ca release using InsP 3 binding data. The eight-state model originally proposed for the regulation of the InsP 3 -gated Ca channel represents one possible explanation for the bell-shaped Ca dependence of channel activity at low InsP 3 concentrations. Yet this eight-state model is unable to explain the relief of Ca-dependent inhibition of InsP 3 -gated channel activity at high InsP 3 concentrations. We hypothesized that a low affinity InsP 3 binding site may be involved, and provided evidence for its existence using ligand binding assays. To fit the data at high InsP 3 concentrations, the eight-state model was therefore extended to include the 10 μM InsP 3 binding site, yielding a 16-state model . In an effort to both explain the steady state channel data and extract kinetic information on InsP 3 -gated Ca release, we used a novel approach that involves consideration of the channel as a unit. Previous models have focused on an individual InsP 3 receptor, predicting various states of the monomer, and then calculating probabilities of each combination of states in a tetrameric complex. The latter approach was used when extending the previously reported eight-state model of the InsP 3 receptor to 16 states , and we obtained reasonable fits of the channel data. A stochastic model has recently been proposed , though it assumed only one InsP 3 binding site per channel complex, which is likely an oversimplification. The present model provides a more realistic representation of the channel with four medium-affinity InsP 3 binding sites per channel complex , and yields superior fits of the data over a broad range of InsP 3 and Ca concentrations compared with our previous 16-state model . Moreover, rate constants (rather than dissociation constants) and mean open times were incorporated into the current model, which facilitates kinetic analyses of channel activity. By focusing on the channel complex (rather than individual InsP 3 -receptor subunits), the number of possible states of the channel could be greatly reduced, thereby allowing for the kinetic simulations of channel activity. The mathematical model in the present study requires just four Ca binding sites per tetrameric channel complex. This is consistent with our initial observation that in the presence of 2 μM InsP 3 the bell-shaped Ca dependence of channel activity could be fit assuming that two molecules of Ca were needed to open the channel, whereas two molecules of Ca appeared to close the channel. A similar stoichiometry of four divalent cation sites per channel complex was obtained when Mn was substituted for Ca . Examination of Ca binding to the cloned/expressed receptor fragments raises the possibility of up to eight Ca binding sites per InsP 3 receptor monomer . Seven of these Ca binding sites appear to reside on the cytosolic region of the InsP 3 receptor , though the Ca affinity of only one of these sites ( K d 0.8 μM Ca, Hill coefficient 1.0) has been determined . The predicted K d for Ca binding in the present study (0.7 μM, Table I ) is consistent with the latter report. It has been hypothesized, based on the cation selectivity of the stimulation and inhibition of InsP 3 -induced Ca release , that Ca binding to at least three distinct cytosolic sites regulates InsP 3 -gated channel activity . For simplicity of the mathematical model, it is assumed that there are four distinct Ca binding sites on the channel, with Ca binding to one site altering the Ca affinity of the other sites in the channel complex. Inclusion of multiple Ca binding sites per InsP 3 receptor monomer in the model is possible, and would be expected to increase the freedom in the model and provide even better fits of the channel and binding data, but at the cost of a considerably larger matrix calculation. As the identities of regulatory Ca binding sites become available, the mathematical model will be refined to more accurately represent the molecular details of channel structure. Although the proposed model provides an accurate simulation of steady state InsP 3 -gated channel activity and InsP 3 binding data, it is important to ascertain the physiological relevance of an InsP 3 binding site with a dissociation constant of ∼10 μM, particularly if the medium affinity InsP 3 binding site has an affinity 30× lower (0.3 μM) at 22°C. It should be appreciated, however, that the medium affinity InsP 3 binding site exhibited a strong temperature dependence ( Q 10 = 2.3), so that at 37°C, the dissociation constant of the medium affinity InsP 3 binding site is predicted to approach 1 μM. The temperature dependence of the low affinity InsP 3 binding site was difficult to measure, but it appeared to be relatively temperature independent. There are reports of ligand binding sites with low (or reversed) temperature dependence, where binding affinity is mainly entropy driven; it is not possible to accurately predict a priori if a given binding site will have a high or low temperature dependence of ligand binding . The low level of specificity of the low affinity site for 1,4,5-InsP 3 versus 1,3,4,5-InsP 4 is in part consistent with the apparent low temperature dependence of this site. Thus, the difference in dissociation constants between the medium and low affinity InsP 3 binding sites may be as low as 10-fold at the physiological temperature of 37°C (i.e., 1 vs. 10 μM). At first glance, dissociation constants of 1–10 μM seem to be unexpectedly high, although review of the literature indicates that at physiological temperatures, rather high InsP 3 concentrations may be needed to release Ca . In the case of cerebellar Purkinje cells, flash photolysis studies indicated the need for at least 9 μM intracellular InsP 3 to induce Ca release, with Ca release increasing progressively as InsP 3 concentration was elevated up to 80 μM . Moreover, it has been reported that sustained elevation of intracellular Ca can be observed using high concentrations (100 μM) of either InsP 3 or the poorly hydrolyzed InsP 3 analogue InsPS 3 . Furthermore, relatively high InsP 3 concentrations have been noted in some cell types under basal (0.1–3 μM) and agonist-induced (1–20 μM) conditions . Thus, InsP 3 dissociation constants of 1–10 μM at 37°C may be of physiological significance. In summary, the present study provides a simple mathematical model of the cerebellar InsP 3 -gated channel, which provides an accurate simulation of InsP 3 -gated channel activity over a broad range of InsP 3 and Ca concentrations. The model incorporates the well known “medium affinity” InsP 3 binding site, and takes into account its temperature and Ca dependences. The model also includes a low affinity InsP 3 binding site that exhibits little Ca or temperature dependence. Individual rate constants were incorporated into the model to allow kinetic simulations. It is expected that this novel model will provide a foundation for subsequent studies aimed at elucidating the molecular mechanisms underlying the complex behavior of the InsP 3 -gated channel. | Study | biomedical | en | 0.999998 |
10352035 | The production of the intracellular signaling factor inositol 1,4,5-trisphosphate (IP 3 ) 1 and the subsequent release of Ca 2+ stored in intracellular organelles is a fundamental cellular signaling function . The form of the resultant change in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is highly variable. Specific, well-characterized response types include a single, large increase in [Ca 2+ ] i , which may virtually deplete the stores, and smaller, maintained oscillations of [Ca 2+ ] i . Often both patterns may be observed in a single cell type, depending on the agonist concentration. The release of Ca 2+ by IP 3 occurs through the activation of a specific receptor for IP 3 , which is located on the endoplasmic reticulum (ER) surface, and which is also a functional calcium channel . In addition to its IP 3 binding site (at the NH 2 -terminal end) and its pore-forming region (the COOH-terminal end, containing six membrane-spanning regions), the IP 3 receptor (IP 3 R) also contains a large regulatory domain between the NH 2 - and COOH-terminal regions. A number of cytosolic factors have been suggested to modulate IP 3 R activity, including, but not restricted to, PKA, PKC, Ca 2+ -calmodulin kinase II (CaMK-II), adenine, and possibly guanine nucleotides. These additional factors may permit the wide range of response types that are seen for IP 3 -induced Ca 2+ release. In addition to factors such as kinases modulating IP 3 R activity, Ca 2+ itself plays a fundamental role. A number of studies have shown that the steady state open probability of the type-I IP 3 R displays a bell-shaped dependence on [Ca 2+ ] i , with a peak at approximately [Ca 2+ ] i = 300 nM , while for type-III IP 3 receptors the open probability curve is a monotonically increasing function of [Ca 2+ ] i . However, the steady state behavior of the receptor is much less important than the dynamic response of the receptor to a sudden increase of either [Ca 2+ ] i or [IP 3 ]. When exposed to a sudden change in [Ca 2+ ] i or [IP 3 ], the receptor responds by a transient increase in open probability, followed by adaptation or recovery, to a lower level . Direct injection of IP 3 , or the use of nonhydrolyzable IP 3 analogues, has shown that in mouse pancreatic acinar cells oscillations of [Ca 2+ ] i can occur when the IP 3 concentration is constant . Thus, dynamic regulation of the receptor by Ca 2+ may represent the mechanism by which oscillations of [Ca 2+ ] i arise, with Ca 2+ initially promoting its own release, and then secondarily inhibiting further release. In the discussion , we discuss the possible effects on the model of changing IP 3 concentrations. A number of mathematical models have been developed to simulate IP 3 -induced Ca 2+ release , although, to date, all models have been based on the properties of the type-I receptor. In the model of De Young and Keizer , each subunit of the tetrameric IP 3 R contains one binding site for IP 3 and two binding sites for Ca 2+ , an activating site and an inactivating state. It was assumed that Ca 2+ flux occurred only when the IP 3 binding site and the activating Ca 2+ binding site were occupied, and the Ca 2+ inactivating site was unoccupied. This formulation resulted in an eight-state model, only one of which was a conducting state. With rate constants for interconversion between the eight states chosen, where possible, to correspond to experimental values, this model displayed oscillatory behavior over a range of [IP 3 ] . A fundamental feature of this model, necessary to allow oscillations to occur, is that IP 3 and Ca 2+ binding to the activation site are fast processes, whereas Ca 2+ binding to the inhibitory site is a slow process. Other models of IP 3 R kinetics are based on the same premise of fast activation and slow inhibition by Ca 2+ , and Tang et al. showed that these are essentially equivalent formulations. A model of [Ca 2+ ] i dynamics in pituitary gonadotroph cells is perhaps the most highly developed of these in terms of the specific cellular mechanisms controlling [Ca 2+ ] i and equivalence of the model behavior to experimental single cell data. Experimental data on the relative rates of activation and inactivation of the IP 3 R by Ca 2+ is consistent with inactivation being a slower process . However, careful analysis of data with higher kinetic resolution suggests that the difference between the rates of activation and inactivation is not as great as is typically used in mathematical models. Furthermore, despite the fact that inactivation is apparently too slow in the models, the models are unable to reproduce long-period, baseline oscillations (up to 2 min or longer in period) observed in cells such as hepatocytes and pancreatic acinar cells . Recent models have addressed the issue of long-period oscillations , but their relevance to any particular cell type is, as yet, unclear. From the point of view of modeling Ca 2+ oscillations and waves, pancreatic acinar cells present particular difficulties. Firstly, in response to different agonists, pancreatic cells exhibit markedly different Ca 2+ responses . Application of acetylcholine (ACh) to pancreatic acinar cells results in the generation of [Ca 2+ ] i oscillations that are roughly sinusoidal with a frequency of ∼4–6/min. The [Ca 2+ ] i oscillations are superimposed on a raised baseline such that [Ca 2+ ] i does not fall to basal levels before the generation of the next peak . In contrast, the [Ca 2+ ] i response to cholecystokinin (CCK) is very different, consisting of baseline spikes of much longer period. Despite these differences, there is much evidence that both types of oscillations result from agonist- dependent activation of phospholipase C, and the resulting increase in intracellular [IP 3 ] (see discussion ). How might these two very different patterns of Ca 2+ release occur, with apparently the same basic intracellular processes? In particular, how do the long baseline spikes observed with CCK occur, given that [IP 3 ] is thought to be maintained above basal levels throughout the period of stimulation, whereas [Ca 2+ ] i , which initially provides the negative feedback signal to end Ca 2+ flux from the stores, has returned to its basal values long before the next spike occurs? In studying these questions, it is important to keep in mind that pancreatic acinar cells contain a preponderance of type-III IP 3 receptors , and thus any model to explain the observed oscillations should be based on data from the correct receptor subtype wherever possible. Fortunately, data on the steady state properties of type-III IP 3 receptors have recently become available, although their adaptational and time-dependent properties are still unknown in detail. Our goal is to understand the mechanisms underlying these different oscillatory patterns and provide a single explanation for these seemingly diverse phenomena. Of course, such an explanation can be simplistic at best and cannot take into account all the complexities of the real cell. Nevertheless, it will provide a framework to help us understand how a set of relatively simple cellular interactions can result in diverse and complex behavior. We begin by showing (experimentally) that in mouse and rat pancreatic acinar cells physiological concentrations of CCK cause rapid phosphorylation of the type-III IP 3 R, but physiological concentrations of ACh do not. Assuming that the phosphorylated receptor does not pass Ca 2+ current (see discussion ), it follows that the rate at which the IP 3 R recovers from inactivation depends crucially on the agonist. Secondly, we show that the rate of Ca 2+ removal from the cytoplasm is an order to magnitude slower after application of CCK than after application of ACh. These data are then incorporated into a model of the type-III IP 3 R, and thus into a whole-cell model of the Ca 2+ response. The model provides a unified explanation of seemingly disparate results; it agrees with the rates of activation and inactivation of the IP 3 R by Ca 2+ , as measured by Finch et al. and Dufour et al. , but can nevertheless produce oscillations of very long period and generate oscillations of the two observed types. Furthermore, the steady state open probability of the model type-III IP 3 receptor is an increasing function of Ca 2+ , as found experimentally by Hagar et al. , thus showing that such monotonic steady state curves are consistent with oscillatory behavior, a fact that is not always appreciated. Predictions of the model are then tested, and confirmed, experimentally. Fura-2/AM was purchased from Molecular Probes, Inc., collagenase (CLSPA grade) from Worthington Biochemicals, bovine serum albumin (fraction V) from ICN Immunobiologicals, and minimal essential amino acid supplement from GIBCO BRL . [ 32 P]orthophosphate (9,000 Ci/mmol) was obtained from Dupont NEN . Monoclonal antisera directed against the type-III inositol 1,4,5-trisphosphate receptor was obtained from Transduction Laboratories. All other materials were obtained from Sigma Chemical Co. Acini were prepared by methods previously described . In brief, pancreata were excised from freely fed adult male Sprague-Dawley rats (200–250 g) or mice. Acini were prepared by enzymatic digestion with purified collagenase, followed by mechanical shearing. Acini were then filtered through 150 μm Nitex mesh, purified by sedimentation through 4% BSA in HEPES ringer, and then suspended in a physiological salt solution containing 10 mg/ml bovine serum albumin, 0.1 mg/ml soybean trypsin inhibitor, and (mM): 137 NaCl, 4.7 KCl, 0.56 MgCl 2 , 1.28 CaCl 2 , 1.0 Na 2 HPO 4 , 10 HEPES, 2 l -glutamine, 5.5 d -glucose, essential amino acids. The pH was adjusted to 7.4 and equilibrated with 100% O 2 . Isolated acini were incubated with 2.5 μM fura-2/AM at ambient temperature for 30 min, and then washed and resuspended in fresh physiological salt solution without BSA. For measurement of [Ca 2+ ] i , fura-2–loaded acini were transferred to a chamber, mounted on the stage of an Axiovert 35 microscope ( Carl Zeiss, Inc. ), and continuously superfused at 1 ml/min with PSS without BSA. Solution changes were rapidly accomplished by means of a valve attached to an eight-chambered superfusion reservoir, which was maintained at 37°C. Determination of [Ca 2+ ] i was performed using digital imaging microscopy with an ATTOFLUOR ratiovision system (ATTO Inc.) as previously described . Briefly, excitation at 340/380 nm was alternately achieved by a computer controlled filter and shutter system and the resultant emission at 505 nm was recorded at the rates indicated in the figures by an intensified CCD camera, and subsequently digitized. Mean gray values obtained by excitation at 340 and 380 nm, in user-defined areas of interest, were used to compute 340/380 ratios. Calibration of fluorescent ratio signals was accomplished as previously described according to the equation of Grynkiewicz et al. by comparing the fluorescence of known standard Ca 2+ buffers containing fura-2. Acini were labeled with 0.3 mCi/ml 32 PO 4 for 2 h in HEPES ringer devoid of added phosphate. Aliquots of labeled acinar cells were then treated as indicated. At the end of the incubations, the acini were rapidly centrifuged in a microcentrifuge and the pellets were resuspended in ice-cold lysis buffer containing 100 mM NaF, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM benzamidine, 1% Triton X-100, 10 μg/ml leupeptin, and 10 μg/ml pepstatin, at pH 7.4). The homogenates were then sonicated. After 30 min on ice, the samples were precleared by addition of 100 μl of protein A-agarose beads and rotation for 2 h at 4°C. The protein A beads were removed by centrifugation and the protein content of the samples was assayed. Type-III IP 3 R was immunoprecipitated from the samples of equal protein by incubation with 1 μg antibody/mg of protein (Transduction Laboratories) for 3 h. After incubation, the beads were washed five times with the lysis buffer by centrifugation and resuspension. After the final wash, the beads were boiled in Laemmli buffer and immunoprecipitated proteins separated by electrophoresis on 5% SDS-PAGE gels. The gel was dried and exposed to a Phosphoimager intensifing screen (Bio-Rad Laboratories) for visualization and analyzed by molecular analyst software for quantification (Bio-Rad Laboratories). Aliquots of acini (in duplicate), metabolically labeled with 32 P, were stimulated with secretagogues for 2 min and the extent of phosphorylation of the receptor was assessed by immunoprecipitating the receptor, separation on SDS-PAGE, and then autoradiography. Acini were initially treated with varying concentrations of CCK. In unstimulated acini, phosphorylation of one major band of ∼300 kD, corresponding to the type-III IP 3 R, could be detected. An increase in the degree of phosphorylation could be demonstrated at 10 pM CCK (170 ± 15% above basal) and reached a maximum at 100 nM CCK (252 ± 23% of control). Phosphorylation of the receptor was not significantly greater at 1 or 10 nM CCK ( n = 4 rat and 2 mouse preparations gave qualitatively similar results). It should be noted that the onset and maximum phosphorylation of the receptor achieved coincides with concentrations of CCK that can be demonstrated to induce calcium oscillations . Fig. 1 shows a typical experiment. The extent of receptor phosphorylation was also investigated upon stimulation by the muscarinic agonist, carbachol. In this series of experiments, an increased phosphorylation of the type-III IP 3 R was also consistently observed 2 min after stimulation with agonist. Phosphorylation could be detected at 1 μM CCh (160 ± 22%) and reached a peak at 10 μM (203 ± 28% of control). In contrast to stimulation by CCK, no significant phosphorylation of the receptor was observed at concentrations of CCh below 1 μM, concentrations that can be demonstrated to induce an oscillatory calcium signal . A typical experiment is shown in Fig. 2 . It is known that, in pancreatic acinar cells, CCK activates both the adenylate cyclase pathway as well as the phospholipase C pathway, while ACh activates only the latter pathway . Thus, a plausible working hypothesis is that the CCK-induced receptor phosphorylation is caused by the activation of PKA, a pathway that is not stimulated by ACh, or at least not to the same extent. To investigate the mediator generated upon agonist stimulation that results in phosphorylation of the type-III IP 3 R, duplicate aliquots of acini were incubated for 5 min with agents known to activate or be a mediator in discrete second-messenger pathways: acini were incubated in either cyclopiazonic acid (CPA), which leads to an elevation of [Ca 2+ ] i by inhibition of the Ca 2+ -ATPase present on intracellular calcium stores, TPA, an activator of protein kinase C, or CPT-cAMP, a cell-permeable cAMP analog. In three experiments ( n = 2 rat and 1 mouse preparation) no enhanced phosphorylation of the type-III IP 3 R was ever observed from acini incubated with either TPA or CPA, indicating that a calcium-dependent kinase or protein kinase C is apparently not responsible for phosphorylation of the receptor. A marked increase in phosphorylation was, however, always observed when the acini were incubated with CPT-cAMP. The extent of phosphorylation, >427 ± 52% above basal, was greater than observed with phospholipase C–coupled agonists. These data indicate that a cAMP-dependent pathway is capable of phosphorylating the receptor. A typical experiment is shown in Fig. 3 . Previous mathematical models of the IP 3 R have tended to treat IP 3 as a permissive factor; its binding is regarded as obligatory for channel activation, but beyond that it has a passive role, with [Ca 2+ ] i providing the dynamics underlying oscillations. However, two lines of experimental evidence suggest that IP 3 binding may play a more important, dynamic role. Firstly, binding studies have shown the affinity of IP 3 for its receptor is dependent on the [Ca 2+ ] i . For type-I receptors, increasing [Ca 2+ ] i causes either a decrease in IP 3 -binding affinity or a biphasic effect , whereas, for the type-III receptor, increasing [Ca 2+ ] i enhances IP 3 binding . Secondly, there is evidence that IP 3 itself may inactivate the IP 3 R. For example, experiments with high kinetic resolution performed under Ca 2+ -clamp conditions have shown that during continuous perfusion with a particular medium-Ca 2+ concentration, the introduction of IP 3 causes a transient release of 45 Ca 2+ from microsomes derived from rat brain synaptosomes or rat hepatocytes . This suggests either that IP 3 can inactivate the receptor or that at least some of the Ca 2+ -induced inactivation occurs only during permeation of the channel. The Mn 2+ quench experiments of Hajnóczky et al. demonstrate that IP 3 can inactivate the receptors without permeation of Ca 2+ . Furthermore, they found that Ca 2+ enhanced IP 3 -induced channel inactivation. We assume that the complete IP 3 R is composed of four, functionally identical, independent subunits. The reaction scheme governing transitions of each subunit is shown in Fig. 4 . S denotes the fraction of subunits in the shut state (S), in which the receptor channel is closed and IP 3 is not bound. Binding of IP 3 causes the receptor to be converted to the open state, O, and we let O denote the fraction of receptors in state O. Although several subconductance states of IP 3 R have been observed, the channels most often open to just one of these . We therefore assume that IP 3 must be bound to all four subunits for the receptor to be in the conducting state. Thus, the fraction of conducting receptors is O 4 . To explain the apparent IP 3 -induced inactivation (described above), we propose that the O state is relatively unstable, and the subunits will progress through to the more stable I 1 (inactivated) state, in which IP 3 is still bound but the channels do not conduct. Thus, binding of IP 3 to a single binding site accounts for both channel activation and at least one aspect of inactivation. We assume that the reverse transition (I 1 → O) has a very slow rate, and so exclude it from our reaction scheme. It is reasonable to consider that a direct transition from S to I 1 could occur. However, we suggest that the S → O transition is greatly favored, and will therefore strongly dominate the S → I 1 transition, and so we make the simplifying assumption of setting the rate of the latter transition to zero. In our model, we do not specifically include any binding sites for Ca 2+ . Although it appears there may be several of these on the IP 3 R , these have not been well characterized. Because of this, we instead incorporate the effects of Ca 2+ by making the rate of the S to O transition a function of [Ca 2+ ] i . By doing this, we are able to correlate model behavior with experimentally observed effects of [Ca 2+ ] i , without having to speculate on the number or nature of Ca 2+ binding sites on the receptors. This approach has the added advantage that the reaction scheme is kept simpler. However, a more complex model that assumes that Ca 2+ governs the interconversion of the receptor between two different states, one with a high IP 3 affinity, the other with a low IP 3 affinity , behaves in exactly the same manner (Sneyd, LeBeau, and Yule, manuscript submitted for publication). In fact, it can be shown that, if it is assumed that Ca 2+ binding is fast, the model presented here can be derived from this more complex model. We set the transition S → O to be an increasing function of [Ca 2+ ] i . This is based on two separate experimental observations. First, raising [Ca 2+ ] increased the rate of 45 Ca 2+ flux in superfusion experiments . Second, in permeabilized hepatocytes, raising the medium [Ca 2+ ] reduced the EC 50 for IP 3 -induced 45 Ca 2+ release from internal stores . Both of these experimental observations are consistent with Ca 2+ enhancing the affinity of IP 3 for the receptors. More than 80% of hepatocyte IP 3 R are type II, with the remainder being type I , suggesting that IP 3 binding to type-II receptors is enhanced by increasing [Ca 2+ ], similar to type-III receptors . The pancreas (most of which is exocrine acinar cells) contains mostly type-III receptors, with a significant number of type-II receptors, but few type-I receptors . Thus, we have assumed that Ca 2+ enhances IP 3 binding to the receptors in the model. The transition I 1 → S represents the normal pathway by which the IP 3 R recovers from inactivation, with IP 3 dissociating from its binding site. The receptor can then rebind IP 3 and repeat the cycle. We refer to this pathway (involving S, O, and I 1 only) as the “intrinsic” receptor pathway, on the basis that this reflects the direct effects of IP 3 and Ca 2+ on receptor activity. However, we also include a transition from I 1 to I 2 , where I 2 represents a second inactivated state of the receptor in which IP 3 is no longer bound (i.e., IP 3 dissociates during this transition). This pathway is agonist specific and involves phosphorylation of the IP 3 R. As shown above, physiological concentrations of CCK cause rapid phosphorylation of the IP 3 R, (possibly via the activation of PKA), while physiological concentrations of ACh do not. Hence, to model the application of CCK, k 4 is set to be nonzero, while the application of ACh is modeled by setting k 4 = 0. Phosphatase actions eventually dephosphorylate the receptor, converting it to the S state, whereupon it may rebind IP 3 . A crucial element of this scheme is that while in the I 2 state the receptor is unaffected by either the IP 3 or Ca 2+ concentrations. We refer to the pathway involving all four states of the channel as the “full” pathway, with the inclusion of the generation of I 2 representing a “mediated” effect, as distinct from the intrinsic effects described above. The distinction between intrinsic and mediated effects is largely for descriptive convenience. By the law of mass action differential, equations for the various receptor states can be determined. The equation for the fraction of receptor subunits in the open state ( O ) is 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{d{\mathit{O}}}{dt}=k_{1}p{\mathit{S}}-k_{-1}{\mathit{O}}-k_{2}{\mathit{O}},\end{equation*}\end{document} where p is [IP 3 ]. The variable S can be eliminated using the conservation law S = 1 − ( O + I 1 + I 2 ). Here, I 1 and I 2 are the fraction of receptors in states I 1 and I 2 , respectively. As previously stated, we assume that all four of the subunits must be in this state before the receptor will pass any Ca 2+ current. Thus the conducting state is O 4 . The equation for I 1 is given by 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{dI_{1}}{dt}=k_{2}{\mathit{O}}-(k_{3}+k_{4})I_{1},\end{equation*}\end{document} and for I 2 by 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{dI_{2}}{dt}=k_{4}I_{1}-k_{5}I_{2}.\end{equation*}\end{document} As described above, the rate k 1 is a function of [Ca 2+ ] i , which is denoted by c : 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{1}=\frac{{\alpha}_{1}c^{3}}{{\beta}^{3}_{1}+c^{3}},\end{equation*}\end{document} whereas k −1 , k 2 , k 3 , and k 5 are constants. The constant α 1 is the maximum rate of the S to O transition, and β 1 is the Ca 2+ concentration at which the rate of the S to O transition is half its maximum rate. The function k 1 is an increasing function of c , representing our assumption that [Ca 2+ ] i enhances the affinity of IP 3 binding. Little is known regarding the mechanism(s) by which Ca 2+ exerts its effects on the IP 3 R, and therefore, although Ca 2+ may act by directly binding to the receptor, our reaction scheme does not explicitly indicate such a mechanism. However, as discussed above, the present model can be derived from a more general model in which Ca 2+ regulates the binding affinity of IP 3 . Since we have not suggested explicit mechanisms representing the transitions governed by k 1 , k 2 , and k 3 , we use the simplest possible functions. Thus, we chose k 2 and k 3 as constants, and let k 1 be a sigmoidal function of c , with a Hill coefficient of 3. If k 1 is assumed to have a smaller Hill coefficient, it is more difficult to get oscillations in the model. It thus appears that cooperativity in the action of Ca 2+ on IP 3 binding may be an important physiological mechanism. These choices for the rate constants are easily modified to give bell-shaped steady state open probability curves if one wishes to model other types of IP 3 receptors. For instance, with a bell-shaped open probability curve, oscillations occur in the model even when k 1 has a Hill coefficient of 1 (computations not shown). The rate constant k 4 is given by 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{4}=\frac{{\alpha}_{4}p}{{\beta}_{4}+p},\end{equation*}\end{document} and models the action of a kinase (possibly PKA) on the IP 3 R, and is assumed to be dependent on the type of agonist used to initiate Ca 2+ oscillations. The constant α 4 denotes the maximum rate of the reaction attained at high p , while β 4 denotes the value of p at which the rate is half maximal. When activated, k 4 shunts the receptors through the I 2 pathway. Since it is reasonable to assume that greater levels of agonist stimulation will result in greater kinase activity, we assume that k 4 is a saturating function of the IP 3 concentration. As can be seen from Table I , β 4 is chosen to be relatively small, so that the rate of phosphorylation saturates quickly, and, in most physiological regimes, is approximately constant. To determine reasonable values for the transition rates, we simulated experiments in which a superfusion system was used to determine the rapid kinetics of IP 3 R activation and inactivation. This technique has the feature that extravesicular [IP 3 ] and [Ca 2+ ] can be rapidly changed under otherwise clamped conditions . Unfortunately, such data are not available from type-III receptors, and so it is unknown whether their time- dependent behavior is similar to that of type-I or -II receptors. In the absence of evidence to the contrary, we shall assume it is, although the model does point out some likely important differences, as we shall see. The experimental observations we are attempting to reproduce are shown in Fig. 5 , A and B. Fig. 5 A depicts 45 Ca 2+ efflux for various extravesicular [Ca 2+ ], with a constant [IP 3 ] of 10 μM. Initially, increasing the bathing [Ca 2+ ] increased both the initial rate and peak response of 45 Ca 2+ efflux, but these factors decreased at high [Ca 2+ ]. The cumulative release was also found to increase initially, but then decrease at higher [Ca 2+ ] . Fig. 5 B shows the results of similar experiments in which extravesicular [IP 3 ] was changed, while the [Ca 2+ ] was fixed at 400 nM. Here, again, increasing [IP 3 ] caused an increase in both the initial rate and cumulative 45 Ca 2+ release, although in this case neither factor was observed to decline at high [IP 3 ] . Simulations of these experiments are shown in Fig. 6 . Since only direct effects of IP 3 and Ca 2+ would be expected to affect IP 3 R activity, only the intrinsic pathway was used for these simulations (i.e., generation of I 2 was excluded by setting k 4 = 0). The simulated experiments used the same concentrations of agonists, although the full range was not used. The qualitative behavior of the simulations was in good agreement with the experimental data, with one significant difference. The rate of inactivation of the receptor is an increasing function of [Ca 2+ ] i in the data but not in the model. In other words, the data from type-II receptors indicates that k 2 is an increasing function of c , but this was not included in the model. The principal reason for this is that we found, in order to obtain a monotonic steady state open probability curve (see below), it was simplest to set k 2 to be a constant, rather than an increasing function of c . Although the dynamic properties of type-III IP 3 receptors have not yet been measured, our model predicts that a monotonic steady state curve will likely be associated with receptors whose rate of inactivation is independent of [Ca 2+ ] i . The simulated dose responses of the model also agree qualitatively with the results of Marchant and Taylor . For type-I IP 3 receptors, the steady state open probability has been found to have a bell-shaped dependence on [Ca 2+ ] , and this can easily be reproduced by our model (computations not shown). However, recent results have shown that the type-III IP 3 R has a steady state open probability curve that is a monotonic increasing function of Ca 2+ . Since it appears that Ca 2+ oscillations in pancreatic acinar cells are governed by Ca 2+ release through type-III IP 3 R, we chose parameter values so as to obtain a monotonically increasing steady state open probability curve as a function of Ca 2+ concentration . Fig. 7 shows that as [IP 3 ] is increased, the point of inflexion (i.e., where the slope is greatest) shifts to the left. Since the measurement of the steady state open probability curve has been measured at only a single IP 3 concentration, this is a prediction from our model that has not yet been tested. In the Appendix , we show that, at least in most current models, including those of Tang et al. , De Young and Keizer , Atri et al. , Bezprozvanny , and Li and Rinzel , the position of the steady state open probability curve as a function of IP 3 concentration is crucially dependent on the assumed effects of Ca 2+ on IP 3 binding. When an increased Ca 2+ concentration enhances IP 3 binding to the receptor, the models predict that the steady state curve will move to the left as the background [IP 3 ] increases. Conversely, when an increased Ca 2+ concentration decreases IP 3 binding to the receptor, the steady state curve is predicted to move to the right as [IP 3 ] increases. The rightward shift of the peak of the bell-shaped steady state curve observed by Kaftan et al. came from studies of the type-I IP 3 R. Binding of IP 3 to the type-I receptor is decreased by raising [Ca 2+ ] , whereas IP 3 binding to type-III receptors was enhanced by raising [Ca 2+ ] . Thus, from analysis of our model, the rightward shift of the steady state curve observed by Kaftan et al. is the expected effect for type-I receptors, but the opposite should occur in cases where Ca 2+ enhances IP 3 binding, which appears to be the appropriate situation for pancreatic acinar cells. We emphasise another crucial point that is often overlooked. The exact nature of the steady state open probability curve has little effect on the dynamic properties of the receptor, and thus has little influence on whether or not the receptor is able to mediate Ca 2+ oscillations. This is because it is the initial fast activation of the IP 3 R, followed by a slower inactivation, that is the crucial mechanism underlying Ca 2+ oscillations. As we shall see, a model with a monotonic steady state curve is quite capable of generating oscillations, and in fact these oscillations are almost the same as those of a similar model in which the steady state open probability curve is bell shaped (computations not shown). We now incorporate the receptor model into a description of acinar cell [Ca 2+ ] i responses, endeavoring to keep the model as simple as is reasonably possible so as to retain the focus on the kinetics of the IP 3 R. Hence, we do not include any possible effects of ryanodine receptors (see discussion ). The equation for [Ca 2+ ] i ( c ) is given by 6 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{dc}{dt}=J_{rel}-\;J_{pump}+\;J_{influx},\;\end{equation*}\end{document} where J rel represents the IP 3 -induced release of Ca 2+ from the ER and is given by 7 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}J_{rel}=k_{flux}{\mathit{O}}^{4},\end{equation*}\end{document} in which k flux is the maximum rate of Ca 2+ release from the ER. J pump represents the combined activity of all Ca 2+ pumps that remove Ca 2+ from the cytosol and is given by 8 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}J_{pump}=\frac{V_{p}c^{2}}{K^{2}_{p}+c^{2}},\end{equation*}\end{document} where V p represents the maximum rate of pumping and K p is the [Ca 2+ ] i for half-maximal pumping. The Ca 2+ ATPase pump is assumed to operate in a cooperative manner, with a Hill coefficient of 2 . J influx is an adjustable parameter that represents a constant rate of Ca 2+ influx into the cytosol. This term includes both influx from outside the cell and any leak of Ca 2+ from intracellular stores such as the ER and mitochondria. The standard parameter values are given in Table I . The values in brackets correspond to those values used to model CCK-induced oscillations, as described in more detail below. To keep the model as simple as possible at this stage, we do not consider effects such as spatial variation in the [Ca 2+ ] i signals, separation of either Ca 2+ influx versus leak from internal stores, or the various forms of Ca 2+ pumping both into internal stores or out of the cell. We also do not consider the aspect of store filling state and the dependence on this of [Ca 2+ ] i signals, nor do we include capacitative Ca 2+ entry. Thus, we are concerned only with the initial phases of the [Ca 2+ ] i signals (i.e., the generation of the responses), when the filling state of the Ca 2+ stores will not yet have been compromised, as opposed to the sustained phases of the responses. We leave such aspects for further investigation. In addition to our need to keep the model as simple as possible at this stage, there is another very good reason to omit consideration of spatial effects at this stage. It is well known that, to understand the behavior of a reaction–diffusion system (such as this would be in the presence of Ca 2+ diffusion), it is necessary first to understand the behavior of the model in the absence of diffusion; i.e., the reaction kinetics must be understood in detail before it is possible to understand how diffusion will affect the system. Although diffusion and spatial heterogeneities will modulate the amplitude and period of the oscillations, they are unlikely, by themselves, to explain the gross features of the different oscillation types. Only a study of the reaction mechanisms will do that. Finally, we assume that Ca 2+ buffering is fast and linear, so that all the fluxes in the model are effective fluxes of free Ca 2+ . In the absence of any specific information on the properties of calcium buffers in pancreatic acinar cells, this is the simplest assumption. The parameter values used in the model simulations are presented in Table I . The values for the rates of activation and inactivation of the IP 3 receptor by Ca 2+ (α 1 , β 1 , α 2 , β 2 , γ 2 , α 3 , β 3 , k −1 , and k 2 ) were determined by ensuring the model agrees with the results of Dufour et al. . This was not done by a detailed fitting procedure, only approximate agreement was required. The parameters governing the rate of phosphorylation of the IP 3 receptor by PKA (α 4 and β 4 ), and the recovery from the I 2 state ( k 5 ), were not determined from experimental data; the values in Table I were chosen so as to give reasonable agreement with the observed CCK-induced oscillations. For ACh-induced oscillations, α 4 was set to zero to mimic the much decreased rate of receptor phosphorylation. To measure the approximate rate of Ca 2+ ATPase activity, exponential functions were fit to the downward slopes of the oscillations induced by either ACh or CCK (data not shown). Although this assumes that the decline in Ca 2+ concentration during the decreasing phase of the oscillation is due to Ca 2+ pumping, and although it assumes the pumping has linear kinetics, these assumptions will have little, if any, qualitative effect on the result, and are accurate enough for our purposes; we are not doing a detailed fit of the model to data, merely using the data to determine approximate values for the parameters. ACh-induced oscillations have a declining phase that approximates an exponential curve, with a decay rate that varies from ∼0.2 to ∼0.4 s −1 . On the other hand, the decay rate for the decreasing phase of CCK- induced oscillations ranges from ∼0.03 to ∼0.06 s −1 . Based on these experimentally measured time constants for the removal of Ca 2+ from the cytoplasm, we chose two different values for V p , the maximum pumping rate, corresponding to the two different agonists. For ACh, we chose V p = 2.6 μM s −1 , while for CCK we chose V p = 0.2 μM s −1 (Table I ). Note that, although V p does not correspond directly to the time constants measured by fitting an exponential to the experimental data, these maximum pumping rates will give rates of decline of the correct orders of magnitude for the two different agonists. Based on the data of Lytton et al. , the pump was assumed to operate in a cooperative fashion, with a half-maximal rate occurring at 0.54 μM. The parameter k flux was determined by requiring a physiologically reasonable resting Ca 2+ concentration. There remains only one parameter, the rate of Ca 2+ influx, to discuss. We hypothesise that this parameter is agonist dependent. In addition to the very different kinetics of [Ca 2+ ] i transients evoked by the two agonists, the transients also differ greatly in their sensitivity to removal of external Ca 2+ . ACh-induced transients are very sensitive, such that if the medium bathing cells is switched from normal (1.2 mM [Ca 2+ ]) to nominally Ca 2+ -free during repetitive [Ca 2+ ] i oscillations, the oscillations are abolished within ∼30 s . This behavior has also been observed by Muallem et al. , and for bombesin-stimulated oscillations in rat pancreatic acinar cells by Xu et al. . The same treatment has only a slow effect on CCK- induced oscillations . Here, the oscillations may continue for up to 20 min, with the peak of the transients slowly reducing. The slow decay, and eventual loss, of CCK-induced oscillations probably reflects Ca 2+ store depletion, which is normally prevented, when Ca 2+ is present in the medium, by capacitative Ca 2+ entry , which is not included in the model at this stage. It seems unlikely that the same explanation can account for the effects of removal of external Ca 2+ on ACh- induced oscillations because of the contrasting rapidity of the loss of oscillations. Instead, the experimental observations suggest that ACh may activate a Ca 2+ entry pathway that is necessary for the generation of oscillations. This is consistent with the raised baseline Ca 2+ concentrations observed during ACh-induced oscillations. Although it is clear that the mechanisms of Ca 2+ entry are much more complicated than our model assumes , we ignore these complications for now and just treat Ca 2+ influx as a simple leak. It is unclear what qualitative effect this will have on the model conclusions. Based on the above argument, we assume that ACh increases the rate of Ca 2+ influx from outside the cell, while CCK does so to a much lesser extent, and thus, to simulate the two different types of oscillations, we use the two different values shown in Table I . It is important to note that only three parameters are dependent on the agonist. All other parameters are invariant for all the simulations. Thus, we hypothesise that the differences between oscillations induced by ACh, and those induced by CCK, are due to the following two things. (a) CCK activates PKA, and causes greater phosphorylation of the IP 3 receptor, thus shunting it through the closed state I 2 . ACh-induced phosphorylation is much less significant, and thus the state I 2 is bypassed. (b) ACh increases the rate of Ca 2+ turnover much more than CCK does. Thus, the rate of Ca 2+ influx from outside the cell, and the rate of Ca 2+ removal from the cytoplasm, are greater in the presence of ACh than in the presence of CCK. We investigated the effects of increasing [IP 3 ] on [Ca 2+ ] i responses in the model, with the pathway regulated by k 4 switched off. The rates of Ca 2+ influx and pumping were set to 0.4 and 2.6 μM s −1 , respectively (see Table I ). Raising [IP 3 ] to 0.66 μM caused the generation of [Ca 2+ ] i oscillations with a period of ∼15 s . During the oscillatory behavior, the nadir of the oscillations occurred at ∼300 nM, well above the resting [Ca 2+ ] i . For the simulation in Fig. 8 , [IP 3 ] was not raised immediately to its final level, but was instead raised gradually, with a time contant of 10 s. Thus, our simulations include the effects of a continually varying IP 3 concentration, as would be expected to occur upon agonist stimulation. Although slow variations in [IP 3 ] modulate the exact shape of the oscillations, their overall behavior can still be predicted by considering the behavior in response to a constant [IP 3 ]. Similarly, if [IP 3 ] is allowed to decrease gradually, the oscillations eventually disappear (computations not shown). The crucial point to notice is that, although [IP 3 ] is changing slightly throughout the course of an oscillation (particularly during the first few peaks), such changes are not a crucial part of the oscillatory mechanism. Oscillations can occur in the presence of a fixed [IP 3 ] in the oscillatory range. The behavior of the system over a full range of [IP 3 ] can be seen in Fig. 9 . In this figure, the effect of increasing [IP 3 ] progressively on the behavior of the system is shown. In particular, this analysis examines the effect of [IP 3 ] on [Ca 2+ ] i responses, showing where they are stable and where they are oscillatory. Starting at low [IP 3 ] values, the system is stable, as depicted by the solid line. As [IP 3 ] is progressively increased, [Ca 2+ ] i gradually rises, but it still stable (i.e., it remains at a single value over time). However, at [IP 3 ] = 0.62, a critical point is reached where the steady state solution becomes unstable and a stable periodic solution (i.e., oscillations) emerges. The maximum and minimum amplitudes of the periodic solution are depicted by the solid lines. This “oscillatory regime” reflects values of [IP 3 ] at which steady oscillations of [Ca 2+ ] i are observed. When [IP 3 ] exceeds 0.67, the periodic solution collapses back to a stable, steady state value that is raised considerably compared with that at [IP 3 ] before the periodic region. From Fig. 9 , two other important features are apparent. The first is that, at [IP 3 ] just before the oscillatory regime, [Ca 2+ ] i is raised relative to the resting level and, for [IP 3 ] values that give oscillations, the minimum value of [Ca 2+ ] i during the oscillations also remains elevated compared with the resting [Ca 2+ ] i . This feature therefore corresponds to the raised baseline [Ca 2+ ] i during oscillations that are observed in both the experimental and simulated oscillations . The second feature from Fig. 9 is that the range of [IP 3 ] over which oscillations are generated is very narrow (≈50 nM). This feature is consistent with the experimental observation that only a narrow range of ACh concentrations is able to elicit oscillations of [Ca 2+ ] i . It seems reasonable to assume that the narrow range of ACh concentrations generates a relatively narrow range of [IP 3 ], although assays for IP 3 are not yet sensitive enough to test this prediction. The experimental results indicate that CCK (but not ACh) activates PKA, which phosphorylates the inactivated (I 1 state) receptors, converting them to the I 2 state. The receptors remain in the I 2 state, where they are insensitive to [Ca 2+ ] i , until the action of phosphatases converts them back to the S state. The action of PKA was modeled by increasing k 4 to 0.05 s −1 . Furthermore, we reduced the rate of Ca 2+ turnover by decreasing V p , the maximum pump rate, to 0.2 μM s −1 , while the rate of Ca 2+ influx was reduced to 0.025 μM s −1 . Simulated CCK-induced [Ca 2+ ] i oscillations are shown in Fig. 10 . At 0.6 μM [IP 3 ], after a large initial spike, repetitive [Ca 2+ ] i transients occur with a period of ∼150 s. The [Ca 2+ ] i spike kinetics are consistent with the experimental data and the spikes originate from close to the basal [Ca 2+ ] i , in contrast to the ACh- induced oscillations. Again, for this simulation, [IP 3 ] was raised gradually with a time constant of 100 s to mimic more realistically the effects of agonist application. The bifurcation diagram for the full (CCK-activated) model is shown in Fig. 11 . The threshold [IP 3 ] for generating oscillations occurs at ∼0.56 μM, at which large oscillations occur. Before this, raising [IP 3 ] causes little increase in [Ca 2+ ] i . As [IP 3 ] is increased through the oscillatory regime, the nadir of the periodic solution (i.e., the minimum [Ca 2+ ] i value during repetitive oscillations) remains close to the resting value, corresponding to the baseline nature of the oscillations. The minimum [Ca 2+ ] i only starts to increase as the periodic solution collapses to the stable steady state solution at ∼0.77 μM [IP 3 ]. In contrast, the maximum [Ca 2+ ] i during oscillations is large at the threshold [IP 3 ] and falls markedly as [IP 3 ] is further increased. It should be noted though, that the maximum amplitude of the initial [Ca 2+ ] i spike will depend on the rate of IP 3 accumulation and will typically overshoot the maximum value of the periodic solution. The bifurcation diagram shows that simulated CCK-induced oscillations occur for a larger range of [IP 3 ] than is the case of ACh . This is consistent with experimental observations that ACh- induced oscillations occur for only a very narrow concentration range, whereas a wider range of CCK concentration is effective at generating oscillations (Yule, D.I., unpublished data). Finally, Fig. 11 shows that, for [IP 3 ] above ∼0.77 μM, a stable steady state solution corresponding to a maintained plateau [Ca 2+ ] i occurs. We used the model to investigate the relationship between the rate of Ca 2+ influx and generation of ACh-induced [Ca 2+ ] i oscillations. By reducing J influx , we can simulate the effect of reducing extracellular [Ca 2+ ]. It turns out that, as J influx is decreased, the range of IP 3 concentrations for which oscillations are observed shifts to the right; i.e., to higher [IP 3 ]. Thus, a decrease in calcium influx would be expected to abolish oscillations, while an increase in [IP 3 ] would be expected to restore them. This behavior is demonstrated in Fig. 12 A. The values for J influx and [IP 3 ] are indicated at the top of the figure. At the normal J influx value of 0.4 μM s −1 , oscillations are induced when the concentration of IP 3 is raised to 0.66 μM. Reducing J influx to 0.35 μM s −1 abolishes the oscillations, but these are recovered when [IP 3 ] is increased to 0.75 μM. Experiments were conducted to test these model predictions . Acinar cells were exposed to 70 nM ACh, which evoked repetitive oscillations in the presence of 1.2 mM external Ca 2+ . At t = 280 s, the external Ca 2+ concentration was reduced to 100 mM, which abolished oscillations within one to two oscillation periods. An increase in the ACh concentration (to 100 nM) at 350 s, still in the presence of low external Ca 2+ , restored the oscillations, this time with an increased frequency. The increased frequency is also sometimes seen in the model (computations not shown). Comparison of Fig. 12 , A and B, shows that the model predictions are confirmed by the experimental data. In the presence of CCK, removal of external Ca 2+ has no immediate effect on the Ca 2+ oscillations , and this effect is also reproduced by the model (computations not shown). Using the model, we studied the effects of phosphorylating the IP 3 receptors during ACh-induced oscillations (computations not shown). Oscillations are initiated by 0.66 μM IP 3 , with the other parameters the same as for ACh-induced oscillations (Table I ); in particular, α 4 = 0 to simulate the absence of phosphorylation. Then, at 100 s, α 4 was increased to 0.05 s −1 , to simulate the cAMP-induced phosphorylation of the receptor by PKA. Oscillations are quickly abolished. This prediction is confirmed by existing experimental data and our own experiments, in which oscillations induced by 100 ACh are quickly abolished by the addition of 0.1 mM CPT-cAMP (Yule, D.I., unpublished data). Although phosphorylation of the receptor can lead to long-period oscillations in the absence of fast membrane transport of Ca 2+ (recall that CCK does not increase the membrane transport of Ca 2+ as much as ACh does), it is unable to do so in the presence of large fluxes of Ca 2+ across the membrane. Essentially, the Ca 2+ ATPase pumps remove Ca 2+ so quickly from the cytoplasm that the Ca 2+ flux through the IP 3 R (which, it must be recalled, is now sitting in a closed phosphorylated state much of the time) cannot keep up the oscillatory cycle. It is only when the IP 3 R is not shunted into the phosphorylated state that the receptor cycle is fast enough to maintain the oscillations. Based on old and new experimental data, we have constructed a new model of the type-III IP 3 R and incorporated it into a whole-cell model for intracellular Ca 2+ oscillations in pancreatic acinar cells. This model agrees with recent data on the rates of activation and inactivation of the IP 3 R by Ca 2+ , and can reproduce both the short-period, raised baseline oscillations induced by ACh in pancreatic acinar cells, as well as the long-period baseline spiking induced by CCK. The steady state open probability of the model IP 3 R is a monotonically increasing function of [Ca 2+ ] i , as shown experimentally by Hagar et al. . The ability of the model to reproduce this wide variety of experimental data is based principally on two things. (a) Physiological concentrations of CCK cause rapid phosphorylation of the IP 3 R while physiological concentrations of ACh do not. Phosphorylation of the IP 3 R does not appear to be Ca 2+ dependent, and can also result from the addition of CPT-cAMP. Hence, we hypothesize that CCK, via production of cAMP and activation of PKA, shunts the IP 3 R through a phosphorylation pathway that prevents rapid recovery from receptor inactivation. Inactivation of the IP 3 R by Ca 2+ is rapid, as indicated by the data of Dufour et al. , and then receptor phosphorylation holds the receptor in an inactivated state. The resulting long-period oscillations are then governed by the time taken to recover from the phosphorylated state. In the absence of phosphorylation (i.e., after stimulation by ACh), the oscillation period is governed principally by the rate of recovery from Ca 2+ -induced inactivation of the receptor. (b) In the presence of ACh, the flux of Ca 2+ in both directions across the plasma membrane is greater than in the presence of CCK. Measurement of the rates of Ca 2+ removal during the downstroke of the oscillations indicates that the rate of Ca 2+ ATPase activity is much greater in the presence of ACh than in the presence of CCK. Furthermore, it appears that ACh increases the rate of Ca 2+ entry from outside the cell much more than does CCK. Indirect evidence for this has appeared before; Yule et al. showed that removal of external Ca 2+ abolishes ACh-induced oscillations, but has little effect on CCK-induced oscillations. By making this assumption in the model, we were able to predict the effect of both reducing external Ca 2+ and increasing agonist concentration. The fact that the model predictions were confirmed by the experimental data lends support to this hypothesis, although this evidence is still indirect. It has been known for some years that PKA is able to phosphorylate the IP 3 R; more recently, Wojcikiewicz and Luo have shown that type-I, -II, and -III receptors are differentially susceptible to phosphorylation in intact cell lines (AR4-2J rat pancreatoma and RINm5F rat insulinoma cells). Furthermore, it is also known that CCK activates both the adenylate cyclase and phospholipase C pathways in pancreatic acinar cells, while ACh does not appear to activate the adenylate cyclase pathway . However, what is far less clear is the exact effect of IP 3 R phosphorylation. In some cell types, it appears that phosphorylation of the IP 3 R by PKA inhibits Ca 2+ release , while in hepatocytes the opposite effect occurs . In platelets, PKA phosphorylation of the IP 3 R causes a 30% inhibition of IP 3 -induced Ca 2+ release , while more recent data shows that PKA inhibits IP 3 -induced Ca 2+ release in megakaryocytes . In a renal epithelial cell line, kinase activators and phosphatase inhibitors decrease the response to carbachol, while kinase inhibitors increase the response to carbachol , results that are consistent with the assumptions of our model. Our experiments on the phosphorylation of the IP 3 receptor were performed in both rats and mice, and similar results were obtained. Thus it is reasonable to assume that the model of the IP 3 receptor is applicable to both rats and mice. However, there are quantitative differences between the Ca 2+ responses of rat and mice pancreatic acinar cells to agonist stimulation. In the study of Tsunoda et al. in rat pancreatic acinar cells in intact acini, Ca 2+ responses to application of CCh took the form of low period baseline spiking, similar to the responses to application of CCK. Since most of the studies showing clear differences between the responses to CCK and ACh have been performed in single isolated mouse pancreatic acinar cells , it is not clear whether our conclusions are also applicable to rat pancreatic acinar cells in an acinus. For a start, it is known that gap junctional communication modulates the observed Ca 2+ oscillations in rat pancreatic acini , but whether this is sufficient to account quantitatively (or even qualitatively) for the differences in the responses of coupled and isolated cells is not known. Furthermore, it is likely that variations in crucial parameter values, such as pumping and influx rates, will lead to quantitatively different model behavior, and such differences may be one way to explain the differing responses of rat and mice pancreatic acinar cells. However, we have not done exhaustive parameter studies and so cannot say for certain. Note, however, that baseline spiking in response to application of ACh is also seen in mouse pancreatic acinar cells, as long as the concentration of ACh is low enough. Hence, our model is consistent with the data from rat pancreatic acinar cells in this low concentration limit. It is thus plausible that the basic mechanisms are similar in both rat and mice pancreatic acinar cells, differing only in the details of certain parameter values. In our model, we have assumed that phosphorylation of the IP 3 R shunts it into a closed state; it is important to note that this is assuming that phosphorylation neither inhibits nor potentiates Ca 2+ release. Hence, the assumptions underlying our model are consistent with both effects of PKA. This can easily be seen by the following argument. If k 3 were small, then the intrinsic recovery of the IP 3 R from the inactivated state would be slow; if the rates of phosphorylation and dephosphorylation ( k 4 and k 5 ) were large enough, it is possible that shunting the receptor through the phosphorylated I 2 state could increase the steady state open probability. Conversely, if k 3 were large, and thus the intrinsic recovery from inactivation was fast, then shunting the receptor through the phosphorylated state could decrease the steady state open probability. In fact, we can derive an explicit relationship between k 3 , k 4 , and k 5 that will determine the exact effects of phosphorylation on the steady state open probability. The steady state proportion of open receptors, O , can be easily calculated as 9 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{O}}=\frac{{\phi}p}{\frac{k_{-1}+k_{2}}{k_{1}}{\phi}+p},\end{equation*}\end{document} where 10 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\phi}=\frac{1}{1+\frac{k_{2}}{k_{3}+k_{4}} \left( 1+\frac{k_{4}}{k_{5}} \right) }.\end{equation*}\end{document} Thus, the function φ controls the sensitivity of the IP 3 R to IP 3 ; as φ increases, the sensitivity of the IP 3 R decreases, and vice versa. A short calculation now shows that φ is an increasing function of k 4 when 2 k 3 > k 5 − k 4 , and is a decreasing function of k 4 when 2 k 3 < k 5 − k 4 . It follows that, depending on the values chosen for k 3 , k 4 , and k 5 , the model can reproduce either an increase or a decrease of sensitivity of the IP 3 R upon phosphorylation. Oscillations in the model occur for constant [IP 3 ], and are the result of cycles of activation and inactivation of the IP 3 R. At present, there is no direct evidence for our assumption that oscillations in [IP 3 ] are not necessary to obtain [Ca 2+ ] i oscillations. From experiments using nonmetabolizable analogs of IP 3 , there is indirect evidence that the observed [Ca 2+ ] i oscillations are not governed by underlying oscillations in [IP 3 ] . However, a complete resolution of this question awaits an experimental determination of the kinetic behavior of [IP 3 ] during the course of a [Ca 2+ ] i oscillation. During application of an agonist, [IP 3 ] will certainly rise and fall as IP 3 is produced and degraded. We do not build these features into our model directly, but just assume that IP 3 is produced and degraded with given rates. Thus, in the simulations, [IP 3 ] increases and decreases gradually. This has little effect on the oscillatory behavior of the model, which can be well understood by considering the behavior at fixed [IP 3 ]. Although it appears that ryanodine receptors do exist in pancreatic acinar cells, we have not included in the model any possible effects of ryanodine receptors or IP 3 -independent Ca 2+ -induced Ca 2+ release. Kasai et al. have shown that the response to ACh is eliminated by heparin, although there is still Ca 2+ -induced Ca 2+ release from the granular area. Thus, although it is likely that IP 3 -independent factors are important for modulating the shape of the [Ca 2+ ] i oscillations, it appears that they cannot, by themselves, support oscillations. Nathanson et al. have also shown that Ca 2+ -induced Ca 2+ release has an effect on the speed of ACh and CCK-stimulated intracellular [Ca 2+ ] i waves. However, neither caffeine nor ryanodine eliminates the waves, although they do decrease the wave speed. Furthermore, the intracellular waves are initiated at the apical zone of the cell, which is where the type-III IP 3 receptors are mostly found . We interpret these results to mean that the oscillatory period and other fundamental oscillation properties are determined primarily by the properties of the IP 3 receptors in the apical zone, with other forms of Ca 2+ -induced Ca 2+ release playing only a modulatory role. Hence, for simplicity, in this model we consider only IP 3 receptors. One major difference between our model and previous ones is in the assumption of how Ca 2+ affects the IP 3 receptor. Rather than assuming actual Ca 2+ binding sites on the receptor (which are not well characterized), we assume that the actions of Ca 2+ arise from its effect on IP 3 binding. Thus, although [IP 3 ] is constant during an oscillation, IP 3 nevertheless plays an active dynamic role, as oscillations in [Ca 2+ ] i are driven by cycles of IP 3 binding and unbinding from the receptor. In the case of CCK-induced oscillations, the receptor also undergoes periodic cycles of phosphorylation and dephosphorylation; the oscillation period is then set by the rate of receptor dephosphorylation, rather than by the rate of receptor recovery from inactivation. Calcium-induced calcium release also plays an important role. As [Ca 2+ ] i increases, the rate of IP 3 binding to the receptor is increased (S to O transition), leading to an effective calcium activation of the receptor. Another major difference from previous models is our use of the phosphorylation pathway, through which some agonists will shunt the receptor. It has been recognized for some time that a third mechanism, in addition to Ca 2+ activation and inactivation of the IP 3 R, is required to explain the long interspike intervals that occur in some cells . Indeed, phosphorylation and dephosphorylation of the IP 3 R has previously been proposed as a possible mechanism for setting the interspike interval . We have shown that such a hypothesis is quantitatively consistent with the observed oscillations, we have collected experimental data that show agonist-dependent rates of phosphorylation of the IP 3 receptor and have shown that a single mechanism can be used as a framework to understand widely differing experimental results in a single cell type. Ours is not the first model to generate long interspike intervals. Laurent and Claret constructed a model of Monod-Wyman-Changeux type and showed that, with appropriate choices of the parameters, the model could reproduce long-period oscillations, while Dupont and Swillens postulated the existence of an intermediate Ca 2+ domain around the mouth of the receptor to achieve a similar result. Our model differs from these in that we present a single mechanism that can generate a wide variety of oscillatory patterns, and then show experimentally that the necessary elements of the model exist in a particular cell type. The simplicity of our model can, in some circumstances, be a disadvantage. Our assumption of independent subunits is unlikely to be correct , and a more detailed allosteric model of the effects of Ca 2+ on IP 3 binding would presumably increase the model's accuracy. However, we believe that the additional complications introduced by such procedures are unlikely to be a worthwhile expenditure of effort at this stage. More detailed models of the receptor states and phosphorylations can wait until the properties of the pancreatic acinar IP 3 receptors are known in more detail, and until spatial effects are better understood. Zhu et al. have presented a qualitative model that, in many respects, is similar to ours. They conclude that histamine-stimulated Ca 2+ oscillations in HeLa cells result from cycles of phosphorylation and dephosphorylation of the IP 3 R by CaMK-II. In their model, just as in ours, phosphorylation of the IP 3 R inhibits Ca 2+ release by closing the channel. They showed that inhibition of the phosphatase by calyculin A or okadaic acid (which corresponds to a reduction in k 5 in our model) increases the oscillation period, and this result is reproduced by our model (computations not shown). This emphasizes the importance of receptor dephosphorylation for setting the period of CCK-induced oscillations. In addition, our model is easily adapted to simulate phosphorylation of CaMK-II by letting k 4 = c 4 /(0.5 4 + c 4 ). This assumes that phosphorylation by CaMK-II is modulated by Ca 2+ in a cooperative fashion, and thus includes the possibility of feedback from the Ca 2+ signal to receptor phosphorylation. In this case, the model again exhibits long-period baseline spiking (computations not shown). One principal feature of our model that is supported only indirectly is the assumption that ACh increases membrane transport of Ca 2+ , while CCK increases it to a lesser extent. In other words, when the cell is at steady state (i.e., in the absence of agonist stimulation) there is a continual, but small, flux of Ca 2+ through the cytoplasm, as Ca 2+ leaks into the cell and is quickly removed. Upon ACh stimulation, overall membrane transport of Ca 2+ is greatly increased, in both directions, and this, in combination with activation of the IP 3 R, results in raised-baseline, sinusoidal, oscillations that are dependent on external Ca 2+ . CCK, on the other hand, increases membrane transport of Ca 2+ only slightly, and thus CCK-induced oscillations depend on external Ca 2+ much less. It has been known for some years that the rate of Ca 2+ turnover is low at steady state, and that agonists such as CCh and CCK increase the rate of Ca 2+ pumping out of the cytoplasm, as well as increasing the rate of Ca 2+ influx . However, what is not clear is whether ACh increases the rate of Ca 2+ turnover more than does CCK. Our model makes the assumption that it does. Although we used our model to make predictions about the effects of removing external Ca 2+ and subsequently increasing [ACh], and these predictions were confirmed experimentally, and although the model provides a consistent framework that can possibly explain the different effects of low extracellular Ca 2+ on ACh- or CCK-induced oscillations, this is still only indirect evidence for this assumption. One important test of the model will be to compare transmembrane and trans–ER Ca 2+ fluxes directly, in the presence of CCK or ACh, or neither. Lawrie et al. have shown that application of ACh in the absence of external Ca 2+ will initially cause oscillations, but these die away within a few minutes. This particular result is not reproduced by our model because of the simplified way in which it treats Ca 2+ pumping. When Ca 2+ is removed from the cytoplasm by pumps, it can be removed either to the outside or to the ER. It is easy to see that the rate of removal to the ER has no effect on the long term steady state [Ca 2+ ] i of the cell, which can only be affected by Ca 2+ transport across the plasma membrane. When ACh is applied to a cell in low [Ca 2+ ] i , the kinetics of the IP 3 R can still lead to cycles of Ca 2+ release and uptake from the ER, but each time Ca 2+ is released into the cell cytoplasm, a fraction of it is lost to the outside. Eventually, the cell runs down and oscillations stop, as observed. Thus, over a longer time scale, depletion of the ER plays a role in terminating Ca 2+ oscillations. The only way to model this is to treat Ca 2+ transport across the ER membrane separately from Ca 2+ transport across the plasma membrane, a feature that is omitted from our model for the sake of simplicity. However, we have constructed an extended version of the model, in which we take into account depletion of the ER (LeBeau, Yule, and Sneyd, unpublished data). The extended model behaves in a similar manner to the model presented here, but can account for a wider array of experimental results, including long-time oscillatory behavior and the application of ACh in the absence of external Ca 2+ . Details of this model and comparison with experimental results will be presented in a later paper. Another important feature of the model is the assumption that an increase in [Ca 2+ ] i causes an increase in the binding affinity of IP 3 to its receptor. The experimental data on the properties of IP 3 binding as a function of Ca 2+ are not completely consistent. Yoneshima et al. claim that Ca 2+ increases the binding affinity of IP 3 to type-III receptors, but decreases the binding affinity to type-I receptors. However, Cardy et al. , although agreeing that the major effect of Ca 2+ on IP 3 binding is stimulatory for type-III and inhibitory for type-I receptors, claim that these effects are mediated by changes in the maximal binding (although, for type-III receptors, more complex effects occur at higher Ca 2+ concentrations). Nevertheless, they conclude that Ca 2+ regulates the interconversion of the IP 3 R between two different states, one state with a high IP 3 affinity, and the other with a low affinity. This interpretation is entirely consistent with our results, as our model can in fact be rigorously derived by considering just such a receptor mechanism (Sneyd, LeBeau, and Yule, manuscript submitted for publication). As we show in the Appendix , a decrease in IP 3 binding affinity with increasing Ca 2+ implies that the peak of the steady state open probability curve shifts to the right as [IP 3 ] increases. Conversely, an increase in the IP 3 binding affinity with increasing Ca 2+ implies that the steady state open probability curve will shift to the left with increasing [IP 3 ]. Thus, since type-I receptors have a decreasing IP 3 binding affinity with increasing Ca 2+ , the model predicts that the steady state open probability curve will shift to the right with increasing [IP 3 ], exactly as seen by Kaftan et al. . On the other hand, since type-III receptors have an increasing IP 3 binding affinity with increasing Ca 2+ , we predict that the steady state open probability curve will shift to the left with increasing [IP 3 ]. These measurements have yet to be performed. No models to date (at least that we know of) have investigated the effects of changes in the maximal binding. In this context, it is important to note that the open probability curve at infinite [IP 3 ] does not give the maximal binding curve. In our model (and all other receptor binding models that we have investigated), the maximal binding fraction is always 1 and, as in the presence of large amounts of IP 3 , all receptors will bind IP 3 . As well as the temporal responses to ACh and CCK being quite different, the spatial characteristics of the response are also agonist dependent . ACh- induced oscillations are initiated at the secretory pole of the cell, and spread from there across the cell, with the response at the basal pole being consistently of smaller amplitude and slightly delayed. During the course of this intracellular wave, significant Ca 2+ gradients are maintained in the cell cytoplasm. CCK, on the other hand, causes an increase in [Ca 2+ ] i that is simultaneous across the entire cell, with no change in amplitude from secretory to basal poles. Furthermore, these intracellular Ca 2+ waves can be transmitted intercellularly when individual cells in acini are coupled to their neighbors via gap junctions . A prerequisite for the study of such intra- and intercellular waves is a detailed understanding of the kinetics underlying the oscillatory response in each individual cell. Thus, although the current study does not address these questions, our model provides a useful framework that can be used to study the problem of wave propagation in this cell type. | Study | biomedical | en | 0.999997 |
10352036 | Cyclic nucleotide-gated (CNG) 1 channels form a unique family of ion channels that are activated by the binding of cGMP or cAMP . These channels are thought to be formed by the association of four subunits , each containing a COOH-terminal binding site for ligand . In retinal photoreceptors and olfactory receptor neurons, two types of subunits (α and β) coassemble to form heteromultimeric channels . Activation of these channels is allosteric in nature, thus the binding of several cyclic nucleotide molecules to the cytoplasmic binding domains induces conformational changes that cause the channel pore to open . CNG ion channels are excellent proteins in which to study allosteric activation because the binding sites for cGMP are readily accessible in excised inside-out patches, conformational changes induced by ligand binding can be observed in a single protein molecule in real time, and there is no detectable desensitization in the continued presence of ligand. Although structure–function studies are beginning to shed light on which parts of the protein are involved in activation , the sequence of events leading to channel opening remains largely unclear. A complete kinetic model is required to piece together the structural changes that occur. Various allosteric models have been proposed that can fit dose–response data. However, as demonstrated for other allosteric proteins, equilibrium or steady state data are not sufficient to support one model to the exclusion of others. Even when kinetic transitions are studied at the single channel level, the constant binding and unbinding of cyclic nucleotides makes it difficult to correlate any particular event to a specific number of ligands bound. As a result, the intermediate states of activation, in particular, are poorly understood. Hence, previously proposed mechanisms tend to be oversimplified due to the limitations of the assays. We have shown previously that these problems can be circumvented by locking single channels into each possible liganded state with the use of a photoaffinity analogue of cGMP, 8- p -azidophenacylthio-cGMP . Two criteria were used to establish the number of ligands covalently attached to each channel. First, dose–response relations for free cGMP were measured before and after covalent attachment of ligand. Four discrete shifts from the control relation were observed corresponding to the attachment of one to four ligands. These shifted relations reflected graded changes in both the Hill coefficient and the effective concentration of cGMP ( K 1/2 ). Second, the liganding assignments were supported by the obvious changes in opening behavior. It was then possible to accumulate minutes of data at each level of liganding, resulting in sufficient representation of all conformational states. We found that the channel locked in a certain liganded state could assume multiple conductance states. In other words, although ligands were fixed in their binding sites, the channel was not frozen into a single conformation, or conducting state. This was the key observation that allowed us to rule out the simple concerted allosteric model Monod-Wyman-Changeux and the sequential model Koshland-Nemethy-Filmer . Recently, a complementary approach for determining the contribution of individual binding events to activation of CNG channels was described by Liu et al. . Multiple binding site mutations were made that apparently destroy binding to the retinal channel subunit. Heteromeric channels were expressed in Xenopus oocytes by coinjecting RNA for this binding site–deficient subunit with RNA coding for an “intact” retinal subunit, in which the pore sequence was replaced by that from the higher conducting catfish olfactory channel. Single channel patches were then isolated and the unaltered binding sites in each channel were saturated with cGMP. Since the higher conducting pore region accompanied each unmutated binding site, different levels of conductance reported different numbers of active binding sites present in each channel. The findings in this study agree with our previous results that there is significant opening in partially liganded channels. However, there are discrepancies in the degree of opening for some of the liganded states. These differences are discussed below. The major purpose of this paper is to present the first kinetic analysis of a CNG channel in every liganded state. Surprisingly, at each level of liganding, as many as five closed states were revealed, and each conducting state exhibited transient and sustained conformations. This information provides further evidence against the simple, limiting mechanisms mentioned above, and allows us to propose a more complete model that describes the opening process of the retinal rod cGMP-gated channel. Xenopus laevis oocytes were injected with cRNA encoding the α subunit of the bovine retinal rod cGMP-gated channel. After 3–5 d of incubation at 15°C, single channels were isolated in excised inside-out membrane patches. Electrodes were coated with Sylgard and resistances varied from 15 to 20 MΩ. All patches were studied in symmetrical control solution containing (mM): 130 NaCl, 2 HEPES, 0.02 EDTA, 1 EGTA, pH 7.6 with NaOH. For recording, channels were held at ±50 mV for at least 10 s, and switched to 0 mV for at least 15 s in between. Multiple segments at +50 mV were recorded under each condition so that at least 30 s of channel activity was used for all analyses. For dose– response assays, cGMP was added to the control solution. Patches that contained single channels were identified by the lack of multiple openings stacked on top of each other at high concentrations of cGMP. Although at low concentrations step-wise openings between subconducting levels were observed, single transitions from closed to fully open and vice versa occurred much more frequently than would be expected if the larger openings arose from multiple low conductance channels. The procedure at saturating cGMP normally took about 5 min, which has been shown to be a sufficient period of time to avoid spontaneous shifts in K 1/2 . Furthermore, during dose–response assays, most concentrations were checked at least twice. After each channel was subjected to a dose–response assay, a nearly saturating concentration (20 μM) of APT-cGMP was perfused onto the patch, and UV light (360 nm) was shone for timed periods (10–180 s) (Scheme I). APT-cGMP binds to the channel's binding pockets and, upon UV photolysis, covalently attaches to the channel. In this way, cGMP becomes “locked” into the binding sites . After UV exposure, patches were washed with control solution for at least 20 min to remove completely all unattached nucleotides from the patch . After this treatment, behavior of partially liganded channels was recorded in control solution in the absence of free cGMP. Finally, a second dose–response assay was performed that proved to be shifted from the first if covalent attachment of ligand had occurred. It should be noted that UV exposure alone did not produce any shifts in the dose–response relation: in three patches, the relations were identical before and after typical exposure times. The number of ligands attached to individual channels was determined by the change in the slope of the dose–response relation (Hill coefficient) and by the change in behavior of the partially liganded channel in the absence of free ligand . Sometimes the channel entered into long closed states on the order of several hundreds of milliseconds, some as long as tens of seconds . Since these sojourns were infrequent, we did not acquire enough events to analyze them adequately. However, they could be easily distinguished from “normal” channel activity with the use of stability plots , and were excluded from the analyses. Single channel data were filtered at 50 kHz with a four-pole Bessel filter in the patch-clamp amplifier (Axopatch 200A; Axon Instruments ), subsequently filtered at 5 kHz with an eight-pole Bessel filter, digitized at 88 kHz (Neuro-corder DR-484 PCM unit; Neuro Data Instruments), and stored on VHS tape. For most analyses, data were played back, filtered at 1 kHz (eight-pole Bessel filter), and sampled at 5 kHz. In some cases, the data were filtered at 5 kHz and sampled at 25 kHz. One record (from a triply liganded channel) was later digitally filtered at 500 Hz for compiling events. Each opening or closing event was idealized by simultaneously measuring the amplitude and dwell time (Pclamp6; Axon Instruments ). Events were comprised of consecutive sample points that occurred within a single conductance class. The amplitudes of the sample points were averaged and the durations were summed to give the mean amplitude and dwell time of an idealized event. When a sample point fell into a different conductance class, a new event began. For identifying the conductance class of each event, three mean current amplitudes were typically set with horizontal lines at 0 (baseline), 0.3–0.4 (O1), 0.7–0.9 (O2), and 1.2-1.4 (O3) pA. These ranges reflect patch-to-patch variations; there were no systematic differences in current amplitudes between locked channels and channels activated by free ligand. Thresholds fell half way between the horizontal lines that defined the conductance classes. At a bandwidth of 1 kHz, events <0.6 ms were marked as “short” events with uncertain amplitudes (Pclamp6). The baseline was adjusted when it varied by more than ±0.015 pA for more than a few milliseconds. Once the event was accepted, its average amplitude and dwell time were added to the events list, and the conductance class was recorded. Noise and artifacts were excluded by eye during this process. After compiling the events list, dwell times were converted into probabilities (event time/total record time) for plotting against amplitudes in the amplitude histograms. The entire events list was binned for the amplitude histogram, including the short events. I / I max was calculated as the mean current divided by the maximum current measured at saturating cGMP, on the same patch. Conductance states were plotted separately for dwell-time fitting. Distributions were plotted as the square root of the normalized observations against the log 10 of the dwell times . All distributions were fit with the maximum likelihood method. The “goodness of fit” for multiple components was determined by fitting a distribution with different numbers of components. The extent to which the addition of a component improved the fit was evaluated by the log-likelihood ratio test (Pclamp6). The rise-time of the filter ( T r ) was 0.34 ms at 1 kHz, calculated as described . Dwell times <2 T r (0.6 ms) were not included in the exponential fits. The amplitude of the baseline noise at 1 kHz was typically ±0.1–0.15 pA about the mean, with a standard deviation (root mean square) of 0.02–0.03 pA (the false event detection rate was 1.6 × 10 −19 s −1 ). No corrections were made for missed events. However, the number of components in a fit are usually not affected by missed event errors, although the time constants may be somewhat overestimated . The distributions provide only a lower bound for the number of states. In addition, errors arising from missed events are not nearly as severe when multiple thresholds are employed . Adjacent events were analyzed to determine the connections between states. The resolution of fast events was limited by the cutoff frequency of the filter. Thus, fast events classified as O1 or O2 might actually be O3 or closed events that were cut off during the rise time of the filter. This was handled at several different steps in the analysis. For adjacent state analysis, transitional events (on a rising or falling phase) less than T r were combined with the following event. Peaks that were too brief to ascertain an amplitude were not removed initially, because they marked an interruption in an opening or closing event. Next, adjacent events were grouped by current amplitude (e.g., closed-O1 pairs would comprise one group). The first events in the pair were then sorted into kinetic classes based on the time constants and areas of the exponential fits. Subsequently, the second events were sorted into kinetic classes; again, the approximate number of events was dictated by the areas of the exponential fits. To rule out uncertain openings or closings, all open events shorter than 2 T r were thrown out. The resolution of closings could be more precise (> T r ) because any event with an amplitude lower than 2 SD below the O1 state must be a closed event . Thus, only reliable events were used to determine the number of observed adjacent events for each kinetic class. When one connection occurred more often than any other, it was taken as a direct connection (see Appendix ). These results were supported by component dependency calculations . In brief, component dependency is the percentage of observations that one state, i (which for simplicity is defined as one exponential component), was followed by another state, j , compared with the independent probability that those two states would occur adjacent to each other. This is calculated as follows: for any two states, i and j , \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}component\;dependency=[Obs(ij)-Ex(ij)]/Ex(ij),\end{equation*}\end{document} where Obs( ij ) is the observed number of adjacent events for states i and j , and Ex( ij ) is the expected number of adjacent events that would be observed if the two events occurred independently of one another. Ex( ij ) is the product of the probabilities that the two events could occur individually: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}Ex(ij)=P_{i}^{*}P_{j}^{*}\;total\;number\;of\;events,\end{equation*}\end{document} where P i and P j are the individual probabilities: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{i}=(total\;number\;of\;events\;in\;statei)/(total\;number\;of\;events\;in\;all\;states),\end{equation*}\end{document} and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{j}=(total\;number\;of\;events\;in\;state\;{\mathit{j}})/(total\;number\;of\;events\;in\;all\;states).\end{equation*}\end{document} For simulations of the connected state models, the rate constants between each pair of connected states were put into a matrix format. As an initial screen for candidate models, steady state occupancies and mean currents were computed as described in order to predict the I / I max value (mean current/maximum current). For further testing, current traces were simulated by starting at the longest closed state. The dwell time of each event was a randomly generated number within the exponential distribution for that kinetic state. The following event was chosen based on the probability of going to each connected state. The amplitude of an open event was randomly chosen from a Gaussian distribution with a mean and standard deviation based on experimental amplitude histograms (constructed from idealized events). This accounted for an observed variation in mean amplitudes (±10%) that was not included in the model. The amplitude of each event was stored along with the dwell time. The list of events stopped when the sum of the dwell times reached the maximum time of the record designated by the operator. Next, the simulated data were sampled with the same protocol employed for the experimental data. Events were sampled at 25 kHz, and random Gaussian noise was added to the sampled data (±0.25 pA, about the mean amplitude of the baseline, the noise typically observed at 5 kHz). These data were put through a Gaussian filter program with a 5-kHz cutoff frequency. To simulate the playback step, data were resampled at 5 kHz and filtered at 1 kHz. The resulting simulated data could be plotted as single channel traces (amplitude versus time). Events lists were compiled as described above, and subsequently analyzed just as those obtained from experimental data. Once the simulation of a connected state model produced parameters that came close to the experimental parameters, the rate constants in the connected state diagram were adjusted by small degrees, where necessary, to reproduce the parameters more closely. In all cases, the rates between states were adjusted to comply with the principle of microscopic reversibility. For this, the lifetimes of individual states were constrained, but the proportions of events that went to adjacent states were adjusted. It should be noted that a single exponential component may harbor several hidden states, so what we have called a single state could be a group of states with an average lifetime represented by a single time constant. Fig. 1 shows the opening behavior of single CNG channels locked in each liganded state. When one ligand was bound, the channel opened with very low probability and mostly to a low conducting level. With two ligands, the channel opened to multiple conducting levels, and entered into a bursting behavior characterized by frequent openings separated by brief closures. Three ligands caused significant opening, and each conducting level exhibited transient and sustained life times (see also below). In the fully liganded channel, the probability of opening approached unity. Moreover, the most prominent opening was to the highest conducting level. Overall, the behavior of the channel locked into any particular liganded state was intricate, indicating an intrinsic flexibility of the protein. The flexible nature of the protein was particularly clear in triply liganded channels, where the probability of opening was large enough to allow examination of the myriad states and behaviors. Fig. 2 A shows a longer record (∼5 s out of 100 s total) of a triply liganded channel on an expanded time scale. The small horizontal lines at the right of each trace indicate the mean current levels for the closed state (C), two subconducting states (O 1 and O 2 ), and fully open state (O 3 ). A fascinating and prominent feature was the tendency to open to subconducting states. In fact, the triply liganded channel preferred opening to these states over the fully open state. Many of the openings to subconducting states were quite stable, as shown in the stretch between the two asterisks. These states did not arise from overfiltering of fast events as shown in the same stretch filtered at 5 kHz instead of 1 kHz . Note in particular the long sojourns into the O 2 state. In the catfish olfactory CNG channel, rapid subconductance states have been shown to arise from protons binding in the pore . However, the entire record in Fig. 2 argues against subconductance states being the result of proton block in the rod channel. For example, if proton block were responsible for some of the briefer openings to subconductance states as in row 2 of Fig. 2 A, then it would be difficult to explain the absence of proton block in row 8 and elsewhere during long openings. We cannot completely rule out the possibility that different conformational states could have different susceptibilities to proton block; however, this scenario would simply support our contention that locked channels are flexible and can assume a variety of conformational states. It should be recognized, however, that proton block of this channel has never been demonstrated at this pH (7.6) and membrane potential (+50 mV) , and that subconducting behavior was indistinguishable at pH 8.6 and +50 mV . These latter conditions have been shown to eliminate proton block in the catfish olfactory CNG channel . There are several features apparent in locked channels that are usually assumed to arise from the binding and unbinding of free ligands. For comparison, the properties of single channels activated by free cGMP are illustrated in Fig. 3 . At low ligand concentrations, the channel exhibited bursting behavior, openings to the same three current levels , and both transient and long-lived openings; at saturating ligand concentrations , long, stable openings (*) occurred. Bursts are normally thought of as periods of repeated openings arising from highly liganded states, while the intervals between bursts are attributed to latency of binding. However, Figs. 1 and 2 illustrate that bursting also occurred in locked channels, in the absence of binding and unbinding of free ligands. Subconductance states are thought on occasion to be an obligatory consequence of a multiply liganded channel losing one or more ligands . However, in each liganded state, the locked channel freely moved between three conductance states without the loss or gain of ligands. Finally, both transient and sustained events were observed in locked channels. With three ligands attached , the appearance of stable openings in the middle of a burst of rapid transitions is striking. Such different lifetimes are usually thought to reflect transient and stable binding events. However, the channel can exhibit these behaviors independent of ligand binding and unbinding. There are two reasons to believe that these locked channel behaviors do not arise from tethered cGMP moieties momentarily “falling out” of the binding site. First, the length of the linker chain in APT-cGMP is very short (<10 Å) so that if the cGMP moiety unbinds, its effective concentration is expected to be on the order of hundreds of millimolar at that single cGMP binding site. Thus, unbinding events would be undetectable (<0.1 μs) within our time resolution. Second, if the effective concentration (which would dictate the tendency to rebind after unbinding) were much less than expected, we should be able to measure an increase in opening probability when free ligand is added to locked channels that are fully liganded. However, addition of 2–1,000 μM cGMP never increased the open probability of fully liganded channels. It is clear that locked channels exhibit many of the same properties as channels activated by free ligand. This indicates that intricate behaviors are intrinsic to the channel protein. We now consider subconductance states in more quantitative detail. All partially liganded channels showed a preference for opening to subconductance states over the fully open state. This is demonstrated in the amplitude histograms in Fig. 4 A, which represent extended periods of channel behavior at each level of liganding. Channel opening with one ligand attached was similar to spontaneous channel opening, even though singly liganded channels required fewer ligands to activate fully . These openings, however, were very infrequent and did not allow for a detailed analysis. In doubly, triply, and even fully liganded channels, it is clear that the channel opened to multiple conductance states. A sum of four Gaussian functions, including one for the closed state, was required to fit the histograms with two, three, and four ligands attached . All-points amplitude histograms, although naturally broader than histograms of idealized events (see materials and methods ), fully support the existence and prominence of subconductance states. Fig. 4 C shows a comparison of these two types of histograms for the same triply liganded channel record. Interestingly, different channels that had equal numbers of ligands attached (not shown) sometimes showed a preference for one subconductance state over the other (O1 or O2). However, partially liganded channels always had a higher probability of opening to subconductance states than opening to the fully conducting state. The overall degree of opening ( I / I max ) in partially liganded channels also varied . When four ligands were attached , the channel favored opening to the fully conducting state (O3) at the expense of the subconducting states. Thus, opening to any given conducting state is dependent on how many ligands are bound. This is summarized in Fig. 5 A. In locked channels, both subconductance states peaked with three ligands attached. An important control for these experiments is the response of untethered channels to free ligand . The individual dose–response relations revealed that the probabilities of observing both subconducting states peaked at the same concentration of cGMP. This is predicted from the results in Fig. 5 A. It should be noted that the probability of observing a subconductance state of the native rod channel also peaks at a subsaturating concentration of cGMP . Overall, the findings in Figs. 1 – 5 strongly support the notion that subconducting states are fundamental intermediate steps in the process of gating. As a further test of whether data acquired from locked channels reflect the channel's natural response to cGMP, we determined whether the open probabilities predict the single channel dose–response relation. It is important to use a single channel dose–response relation for this analysis because the typically low Hill coefficient (∼2.0) observed in macropatches does not reflect the consistently higher Hill coefficient (∼3.0) observed in single channel patches . A dose–response relation can be generated from locked channel data with the use of a minimum model that simulates the opening of locked channels as well as the binding of cGMP. We assumed initially that cGMP binds independently to the four identical subunits when the channel is in the closed state, and each liganded closed state opens to the degree predicted from the locked channel data. To simplify this analysis, the multiple open states we observed were contracted into a single open conformation. This is reasonable, because for a dose– response relation it is only necessary to know the overall equilibrium between open and closed states. The degree of opening in locked channels can be expressed as the ratio of the mean current to the maximal current at saturation ( I / I max ). These values were as follows: singly liganded, 9.6 × 10 −6 ; doubly liganded, 0.0097; triply liganded, 0.33; and fully liganded, 1.00. For this application, these values were converted to open probabilities and equilibrium constants as described in Fig. 6 A, legend. Fig. 6 B shows dose–response relations from 16 single channel patches. I / I max is the fractional current activated by cGMP. Because K 1/2 varied significantly between individual single channel patches, the cGMP concentrations were expressed relative to each channel's K 1/2 . This simply aligned the dose–response relations along the x axis. The overall dose–response relation was fit with a Hill coefficient of 2.9, and the Hill coefficients for each single channel were also very high, ranging from 2.5 to 3.9. The solid curve shows a simulation from the model, which could be described by a Hill coefficient of ∼2.8. As mentioned above, the opening of partially liganded channels exhibited some variability from patch to patch. For this simulation, the highest values were used because we felt these were the most reliable . However, the lowest values (given in the legend) produced simulations that could be described by a Hill coefficient of ∼3.1, which falls within the observed range of single channel Hill coefficients. The only free parameter in the model is the ligand dissociation constant, and the value of the Hill coefficient was virtually insensitive to changes in K d . The reproduction of the dose–response curve is consistent with the idea that locked channels open normally, and is further evidence that the assignment of the numbers of tethered ligands in those channels is correct. Liu et al. reported the following open probabilities for the different liganded states of mutated channels (see introduction ), which they regard as behaving like wild-type channels: singly liganded, 0.017; doubly liganded, 0.16; triply liganded, 0.32; and fully liganded, 0.95. Before this, the same group reported spontaneous open probabilities in unliganded channels of 1.25 × 10 −4 , which contrasts with an I / I max value of 6.8 × 10 −6 in our experiments . The numbers from the former group suggest more opening of unliganded, singly, and doubly liganded channels. When we applied the same minimum model to test their open probabilities, the dashed curve in Fig. 6 B was obtained, which exhibits pronounced curvature and clearly cannot explain the wild-type single channel dose–response relations. Again, the simulation was insensitive to the value assumed for K d . We also tested the effects of adding different degrees of binding cooperativity to closed states in the mechanism in Fig. 6 A, using K d values that decreased progressively with each bound ligand. (It should be noted that favorable opening already confers a significant degree of binding cooperativity to the open states.) Using the opening numbers of Liu et al. , the upper portions of the experimental relation (which are most sensitive to binding cooperativity) could be fit, but these simulations still deviated markedly from the foot of the relation (low I / I max values). In contrast, adding an equivalent amount of binding cooperativity using our open probabilities yielded a good fit to the upper part of the curve and a slightly steeper fit to the foot of the relation that was still well within the spread of the data. The foot of the relation primarily reflects the number of ligands that have to bind for substantial activation, and significant deviations indicate discrepancies in the opening of lower liganded states. Finally, we tested whether a coupled dimer model proposed by Liu et al. could fit the single channel relations. In this model, four subunits associate as two functional dimers. Each dimer undergoes a concerted transition, and the channel opens when both dimers are activated in this way. Simulated data from this model produced the dotted curve shown in Fig. 6 B. Although this is slightly closer to the wild-type dose– response behavior, it is still too shallow, and also exhibits pronounced curvature at low values of I / I max . This model incorporates a significant degree of closed-state binding cooperativity like that described above. It comes closer to the foot of the experimental dose–response relations in Fig. 6 B largely because it predicts lower open probabilities in unliganded and singly liganded channels than those observed in the experiments of Liu et al. . Later, we discuss possible reasons why their approach may have yielded somewhat distorted numbers for channel opening at different levels of liganding. In summary, locked channels appear to reflect accurately the activity of normally liganded channels; thus, we are confident in the reliability of this approach for dissecting the allosteric mechanism of channel opening. Eigen pointed out that the KNF sequential model (diagonal box) and the MWC concerted model (two vertical boxes) are both limiting cases of a more general allosteric model shown in Fig. 7 . In this model, there are four subunits in a protein, each capable of undergoing a single conformational change (from □ to ○). The bound ligand is represented by G. In the KNF sequential model, ligand binding is required for conformational changes to occur; thus, for a tetrameric channel, different open states would arise with different numbers of ligands bound. Early studies of single retinal CNG channels seemed to support the sequential model, because subconducting states were observed at low cGMP concentrations and exhibited a dose dependence that suggested that they reflect intermediate steps in activation . However, the sequential model is inadequate to describe the multiple open states observed at every level of liganding in locked channels . The MWC concerted model is a simple and appealing scheme that has been used to describe steady state, macroscopic current data for CNG channels. In this model, a channel protein assumes only two conformations, closed and open. The interconversion involves a synchronous change in all four subunits. Ligand binding increases the open probability by stabilizing the open conformation. Spontaneous channel openings in the absence of ligand have been reported for CNG channels . This finding is consistent with a concerted mechanism of opening; however, spontaneous openings are also predicted by the general allosteric model (see below). The very existence of subconductance states described above indicates that a two-state model is insufficient. Furthermore, at the level of resolution afforded by locked channels, it is clear that subconductance states were the most prominent open states in four of the five liganded conditions . On occasion, the simple concerted model has been expanded to include two or three conformational transitions. An important prediction of any strictly concerted mechanism is that the channel opening equilibrium constant should increase by a constant factor with each ligand that binds. However, such models cannot describe our data because the overall equilibrium constants for each open (conductance) state did not change by a constant factor for each ligand that bound (Table I ). The equilibrium constants ( K o ) were calculated as the ratio P o / P c , where P o is the probability of observing a particular open state, and P c is the probability of observing the closed state. It is striking that for the two subconductance states the equilibrium constants not only did not change by a constant factor with each ligand (shown by the ratios of K o between liganded states), but these factors actually varied by more than two orders of magnitude. The data for the fully conducting state are less complete since we rarely observed (and in some patches did not observe) this state with zero ligands or one ligand attached. However, kinetic data presented below argue strongly against concerted models even when the channel's opening behavior is simplified to consider only the fully conducting state. In contrast, the general allosteric model in Fig. 7 captures some of the complex behavior that we have observed in single channels. For example, multiple channel conformations are allowed with a fixed number of ligands bound. In each row, the channel undergoes the same conformational changes regardless of the number of ligands bound. Thus, spontaneous opening of the unliganded channel is easily accommodated. The effect of ligand binding is to enhance the probability that those conformational changes occur. The actual scheme is more complex than the diagram shown, because a channel with a ligand bound to a subunit in a square conformation is different than a channel with a ligand bound to a subunit in a circle conformation. The total number of distinct states depends on assumptions about symmetry . For the assumption of fourfold rotational symmetry, 55 distinct channel states are expected. This is a reasonable assumption for a channel with a single pore comprised of four identical subunits. With this assumption, adjacent subunits perform differently than diagonally opposed subunits. That is, if conformational changes occur in adjacent subunits, the channel behaves differently than if they occur in opposing subunits. Similar considerations apply to ligand binding to adjacent or opposed subunits. However, when ligands are locked into binding sites, only one binding configuration can be considered. This affects only the doubly liganded states (here, adjacent binding is assumed) and reduces the original 55 states to 46 total states (or 45 total states if two ligands bind to diagonally opposed subunits). Each state shown in the diagram represents the number of subunits that have undergone a conformational change (□ to ○) and the number of ligands bound (G). The number of distinct states is indicated in parentheses above each representative state. For example, consider the case with three ligands bound, and three of the subunits have undergone conformational changes to a circle (row 4, column 4). There are three nonequivalent configurations possible. First, all three subunits with ligand bound could be in the circle conformation, as shown. Alternatively, the unoccupied subunit could be a circle, and one of the three bound subunits a square. For the latter case, there are two possibilities. One is when the two bound circles are adjacent to each other, and the other is when the two bound circles are opposed to each other. At each step of liganding, conformational changes that occur in one, two, three, or all four subunits are predicted to give rise to identifiable closed and open states. In evaluating the general allosteric model, locked channels allow us to examine channel opening behavior in each row, because the number of ligands attached is constant. We begin by examining channel behavior in the absence of ligand. The closed times for unliganded channels were fit with only one time constant (τ c ∼ 15 s, not shown). Interestingly, spontaneous openings exhibited multiple conductance states. Although the most favorable was the lowest conductance state, the other two open states were occasionally observed, indicating that all three conductance states are an intrinsic property of the protein. The observation of multiple states is consistent with the general allosteric model; in fact, the model predicts more conformational states (6) than we observed (4). This may be due to the low probability of opening ( P o ∼ 10 −5 ). Channel behavior with one ligand bound was not significantly different than the behavior observed in unliganded channels. Again, the low number of events may not allow for a meaningful test of the model. For doubly, triply, and fully liganded channels, all states were sufficiently populated. Dwell-time histograms for closed states and each conductance state (O1, O2, O3) were constructed and plotted in Fig. 8 as the square root of the number of observations versus the log 10 of the dwell intervals . This method allows a wide range of dwell times to be displayed. An additional advantage is that each exponential peaks at the value of its time constant. Interestingly, in the doubly liganded channel, the closed time distribution was fit with five exponentials. This indicates that the channel can assume at least five distinct closed state conformations. Triply and fully liganded channel closed time distributions required only three exponentials for a reasonable fit. Although we have already identified three open states based on different conductances, fits to individual open dwell-time distributions revealed multiple kinetic states. In some panels in Fig. 8 (e.g., C, F, and G), the need for two exponentials is not readily apparent. However, a single exponential would not accommodate all the long and short events; thus, some excess events would have to be omitted. First, we cannot justify omitting excess long-lived events because they contribute significantly to the overall open probability. Second, when a single exponential is constrained to accommodate the longest dwell times, excess brief events must be omitted. This results in simulated data with markedly fewer fast transitions, a prominent feature of single channel behavior . For the fully liganded channel, a third long-lived open state was observed that was not apparent in partially liganded channels . As an important control, the dwell times for O3 were fit with the same three exponentials in channels activated by saturating free cGMP, though the proportion of the longest-lived state (O3 LL ) was slightly lower than that observed in locked channels . Table II lists the time constants and their fractional contributions for all liganded states that were fully analyzed. Since the fully liganded channel record was filtered at 1 kHz, we were concerned that very short events in between O3 open times could have been missed, thus giving rise to artifactually long open events. To check whether the longest component was real, the same record filtered at 5 kHz was analyzed. The open channel noise was high (root mean square = 0.28 pA), and it was difficult to distinguish noise spikes from rapid events (false event detection rate ∼450 s −1 ). Thus, we chose to compile indiscriminately all events that crossed a threshold set midway between the bottom of the open channel noise and the O2 level. Afterwards, a resolution of 80 μs was imposed (the filter rise time is 70 μs at 5 kHz, as opposed to 340 μs at 1 kHz). The maximum likelihood fits (not shown) to all O3 events detected in this analysis still required three exponentials with the following time constants and proportions: 1.6 ms and 0.113; 7.8 ms and 0.867; and 23 ms and 0.02 (7,128 total events). The proportion of the longest component was lower; however, this was expected with such a high false detection rate and a threshold set very close to the noise level. The important result was that three components were still measured. Thus, the filtering at 1 kHz did not produce a third component that was not real. It should be noted that when records like this are corrected for missed events, the number of components usually does not change, although the time constants and proportions may be altered . Overall, the kinetic analysis indicates that there were 11 distinguishable states in doubly liganded channels, 9 in triply liganded channels, and 10 in fully liganded channels. The general allosteric model described above predicts 10 states in doubly liganded channels, 12 in triply liganded channels, but only 6 in fully liganded channels. (It should be noted that an alternative assumption of twofold symmetry still predicts only seven states in the fully liganded channel.) This model comes close to accounting for the number of states observed in our single channel data. However, the large number of states in the fully liganded channel points out that we need to expand on the general allosteric model by allowing for more than one conformational change per subunit. To evaluate fully the general allosteric model or any expanded version, it is necessary to determine how the various channel states are connected to each other and what the transition rates are. The first question is whether each conducting level can arise directly from a closed state, or whether closed states always open to a particular conducting level. A simple inspection of the raw records from doubly, triply, and fully liganded channels indicates that all conducting states can directly follow a closed state. The second question is whether the different conducting states are connected to each other. Again, simple inspection indicates that the conductance states are connected to each other. Finally, given that there are multiple closed states and open states, we would like to know which individual states are directly connected to each other. We considered using maximum likelihood methods, but the large number of states and the presence of stable subconductance states made these methods impractical. Thus, we examined the connections between individual closed and open states by means of an adjacent state analysis . An assessment of bursting behavior was used to corroborate the adjacent state analysis. We then used simulations to test the models, which also provide a realistic correction for missed events. The Appendix describes how the adjacent state analysis, burst analysis, and model simulations led to the development of a connected state model. We found that the single connected state model shown in Fig. 9 A, with rate constants given in Table III , could explain the kinetic data for doubly, triply, and fully liganded channels. The solid lines indicate connections that were used in all three liganded conditions, while the dashed lines indicate connections that were necessary to simulate data in only one or two of the liganded conditions. An additional closed state was observed in doubly liganded channels (C LL ) and an additional open state was observed in fully liganded channels (O3 LL ). Furthermore, different connections between long-lived open states were used to simulate the behavior in doubly liganded versus triply and fully liganded channels. In triply and fully liganded channels, long-lived openings to the O3 states were interrupted by rapid transitions to and from the subconductance and closed states . This pattern was simulated most easily by connecting state O3 L and the two short-lived conductance states (O2 S and O1 S ), instead of connecting the long-lived open states. Comparisons between real and simulated single channel traces for the three liganded conditions are shown in Fig. 10 . Amplitude histograms are compared in Fig. 4 , A and B, dwell-time constants are compared in Table II , and the adjacent state analyses (see Appendix ) are compared below in Fig. 11 and Table IV . Clearly, the connected state diagram in Fig. 9 A reproduces most of the single channel characteristics. We do not propose that this connected state diagram is unique. For instance, equivalent states could have been missed by equating the number of exponentials with the number of distinct states. However, adding more states does not change the main conclusion that locked channels can assume a large number of stable kinetic states. Including a larger number of states would probably obviate the need for diagonal connections in Fig. 9 A. Also, it may have allowed us to capture some of the more complex single channel behavior. For example, the tendency for short- and long-lived events to occur in clusters is not fully reproduced. Also, the bursting properties were similar, but not as robust as observed in the data (see Table V ). At this resolution, however, there is not enough information to include more states. The fact that the data at each liganded level is reproduced by similar diagrams lends credence to the notion that the channel assumes a similar set of conformational states. Thus, this feature of the general allosteric model shown in Fig. 7 is supported by the data, where each row could be represented with the connected state diagram. However, a single conformational change as depicted in the general allosteric model is insufficient to account for all the states observed in the connected state model. A plausible extension of the general allosteric model is discussed below. Although there are details of our model that remain uncertain, there are several salient features that emerge from this analysis. First, subconductance states are ligand dependent and are particularly prominent in triply liganded channels. Thus, any successful model will have to include subconductance states as critical intermediate steps in gating. Furthermore, models will have to include multiple kinetic states at all conducting levels. Another noteworthy feature is the tendency for bursting behavior to transpire without binding and unbinding of ligand. This bursting occurs at intermediate levels of liganding, when the channel is observed to leave absorbing closed states, and then shuttle between activated closed and open states. This suggests that in locked channels two or more ligands give rise to bursts by overcoming an energy barrier between the absorbing closed states and the activated states. Conversely, in fully liganded channels, open states are so favorable that returning to absorbing closed states is rarely observed. This behavior is described by the connected state model and a mechanism for bursting very much like this will have to be incorporated into any successful model. Another feature that is essential to any model is that in the fully liganded channel there is a direct, favorable path from an activated closed state to a stable, long-lived fully open state. That is, opening to the O3 state rarely occurs in a staircase fashion (through subconductance openings). Such a sequential mechanism appears to be operating in glutamate receptors . However, in those channels, even when one conductance state predominates (proposed to arise from a particular liganded state), there are brief transitions to other states. These transitions might reflect a flexibility in those channels similar to that observed in the rod CNG channel. To determine the effects of ligand binding on conformational changes in allosteric proteins, investigators have generally been limited to adding different concentrations of free ligand. Even in single channel recording, in which there is an unprecedented resolution of conformational states, it is difficult with this limitation to assign any observed behavior to a particular liganded state. At any instant, it is virtually impossible to know how many ligands are bound. Taking advantage of a method we developed for covalently tethering ligands to single CNG channels , we have presented here a complete kinetic analysis of single rod CNG channels locked in each liganded state. Remarkably, we have found that channels with a fixed number of ligands in place exhibit interconversions among 9 or 10 different states: 4 different conductance levels (including closed) and more than 1 kinetically distinguishable state at each of those conductance levels. Many of these states were stable on the millisecond time scale. The large number and complex behavior of states cannot be explained by simple phenomena such as proton block, which has been shown to cause two rapid subconductance states in catfish olfactory CNG channels . The number of conducting states we observed at each liganded level is clearly inconsistent with either simple concerted (MWC) or sequential (KNF) allosteric models. An attempt to explain the behavior with a somewhat more complicated concerted model in which each conducting state arises from a separate concerted transition from the closed state also fails: when each conducting level is treated as a single state, the apparent channel opening equilibrium constant does not change by a constant factor with each ligand that binds (Table I ). Most importantly, the observation of two to five kinetically distinguishable states at each conducting level violates both the letter and intent of strictly concerted models, which were proposed for their simplicity. However, with some limitations to our resolution of the precise rate constants for the different kinetic states, and lacking detailed structural information, we cannot rule out that there are some concerted transitions in the channel's activation pathway . The fact that approximately the same number of states were observed in doubly, triply, and fully liganded channels suggests that the channel undergoes the same series of conformational changes. On this idea, different numbers of bound ligands would favor certain states over others. The success of the connected state model in simulating the intricate behavior in each liganded state lends strong support to this overall hypothesis. The number of distinct states that were observed at each level of liganding is most easily explained by assuming that there are activating conformational changes in each individual subunit. The general allosteric model shown in Fig. 7 postulates only a single conformational change per subunit, and yet it comes close to providing enough states. The only condition in which it obviously falls short is the fully liganded channel. The model postulates 6 states with an assumption of fourfold and 7 states for twofold rotational symmetry, while 10 states were observed. If this model is part of activation, the fact that it provides enough states at lower levels of liganding could indicate either that there are additional conformations in fully liganded channels, or that these additional states exist in lower liganded channels but were not resolved. The latter could be due to limited kinetic resolution, to functionally equivalent states, or to some states not being sufficiently populated. In comparing the general allosteric model with the connected state model (or, for that matter, with the behavior of any channel), it is not clear which states would be closed and which would be open. Given that there are four observed conductance states and five stages of activation in the model, the following assumptions seemed quite reasonable : channels with zero and one activated subunits are closed; channels with two adjacent subunits activated give rise to an open state (O1), and channels with diagonally opposed subunits activated are still closed; channels with three and four activated subunits give rise to different open states (O2 and O3, respectively). This can account for all of the closed states and is enough (or more than enough) to account for the short-lived open states. However, multiple O3 states, which probably require all four subunits to be activated, are not easily explained. Furthermore, in the fully liganded channel, there are four states missing. We assume therefore that the four long-lived open states require additional conformational changes. The simplest way to expand the general allosteric model is by introducing a second conformational change per subunit. Such a scheme has been proposed for the activation of Shaker K + channels . In this scenario, the fully liganded state would be permitted 21 distinct conformations. While possible, this seems a bit of an overcompensation since the fully liganded state lacks only four open states. An alternative way to expand the model is by introducing a conformational change that requires a change in the association of two adjacent subunits that have undergone a square-to-circle conformational change . In this speculative model, we can limit the number of total states by requiring that this association occurs only on adjacent subunits that have ligands bound. This predicts long-lived open states will occur only after at least two ligands are bound to the channel, in accordance with our observations. The longest-lived open state (O3 LL ) would arise from the association of two pairs of subunits in unison, which is possible only in the fully liganded channel. Thus, the size of the conductance state (O1, O2, O3) could be determined by the number of subunits in the active conformation (circle conformations), and the lifetime (O S , O L , or O LL ) could be determined by whether or not adjacent subunits were interacting. Altogether, this adds three conformations each to the doubly and triply liganded states and four conformations to the fully liganded state. Disparate regions of the channel protein have been reported to move during gating (see introduction for references). This is not surprising given that binding of ligand in the COOH-terminal tail induces opening of the pore some distance away. These findings suggest that there is an intrinsic flexibility in each subunit, and lend support to our proposal that subunits undergo conformational changes in the absence or presence of ligand. The connected state model allows us to examine whether these apparent subunit-based changes occur independently of each other or cooperatively. As closed states proceed from inactive to activated states (that lead to open states), it is clear that there is a progressive increase in rates, apparent in all liganded states (Table III ). This contrasts with the expected decrease in rates (4α, 3α, 2α, α) that would be predicted by subunits behaving independently. This suggests that as each subunit undergoes a conformational change, it increases the probability that another subunit will do the same. Thus, these subunit-based conformational changes appear to be occurring cooperatively. Similarly, the rates from closed to open states do not appear to follow the pattern for independence. This makes it more difficult to predict the underlying structures for each state. The speculative structural scheme in Fig. 9 B implies that some of the conformational changes can occur in more than one subunit simultaneously. These can be thought of as concerted transitions that occur alongside single subunit-based changes. Two types of concerted transitions are depicted in Fig. 9 B: diagonal lines indicating square-to-circle changes that occur simultaneously, and crossed lines indicating associations between adjacent subunits. Sometimes the concerted transitions would dominate (e.g., the transition between C S and O3 S in the fully liganded channel), and other times they would be relatively minor (e.g., the transition between C S and O1 S in the fully liganded channel). Regarding the proposed changes in association between pairs of subunits, there is evidence that interactions between adjacent subunits can stabilize the open state . These authors showed that Ni 2+ potentiation, which stabilizes the open state, requires two histidine residues in adjacent subunits. In another study , activation properties were different between homotetrameric channels that had two mutated subunits adjacent to each other compared with channels with the same two mutated subunits diagonally opposed to each other. Recently, Liu et al. proposed a mechanism that employs adjacent subunit interactions (coupled dimer model) as the only means of opening the channel. This model, however, was not based on any kinetic analysis. Here, we have shown that there are many more kinetic states than can be explained by their two-step mechanism; however, we incorporate adjacent subunit interactions as a plausible means to extend the total number of conformational states of the general allosteric model. It is important to realize that, although the structural scheme depicted in Fig. 9 B can explain the number of observed states with only two types of conformational changes, there is no direct evidence for these particular structures. Liu et al. have recently taken issue with our assignment of the number of ligands attached to channel binding sites, based on discrete shifts (in K 1/2 ) in the cGMP dose–response relations . They have suggested that spontaneous shifts in our dose–response relations led to mistaken liganding assignments; however, such spontaneous shifts have been ruled out in our experiments (see materials and methods ). An advantage of our method is that we are able to assess behavior in the same channel before and after tethering ligands. A large number of dose– response relations for single channels superimposed in the control condition , making the subsequent shift caused by the attachment of one ligand unmistakable . Most importantly, the P o values obtained from locked channel data can reconstruct the dose–response relation obtained from unmodified single channels . In contrast, Fig. 6 also demonstrates that it is difficult to reconstruct the single channel dose–response relation from wild-type channels using the data of Liu et al. . This fit to unmutated, unmodified single channel data is crucial since both approaches introduce modifications to the normal ligand-bound channel. We suggest that the data of Liu et al. are not consistent with the opening of wild-type channels either because extensive mutagenesis has altered channel gating or because their assignment of the number of active binding sites is incorrect. It remains a possibility that slightly different experimental conditions (e.g., high KCl on both sides of the membrane in their experiments) could have contributed to the observed differences. Nonetheless, the effect on gating that might be caused by substituting a foreign pore region into retinal channels (RO133 subunits containing a catfish olfactory CNG channel pore) has been studied only in homomultimeric channels and only at the macroscopic current level . This is insufficient to determine whether single channel behaviors change. In this vein, subtle single amino acid mutations in the pore have been shown to alter gating . In addition, the mixing of replaced pore regions and multiple binding site mutations may have unforeseen effects on gating. For example, in the study by Liu et al. , a subunit with a double binding site mutation and a retinal pore expressed, while a subunit with the same double binding site mutation and an olfactory pore did not express. Regarding the assignment of the number of active binding sites, Liu et al. may have missed singly liganded channels because they were limited to searching for robust channel activity with a well-defined conductance level. We have found that a singly liganded channel rarely opens, and when it did open it was usually to brief subconducting levels. Thus, discrimination between channels with a single active binding site and those with no active binding sites would be challenging. Their use of Ni 2+ to improve the resolution of channel conductance may not be reliable because some channel constructs could not be potentiated, and it has not been shown which open states Ni 2+ will stabilize. The variability of Ni 2+ potentiation among channel constructs further suggests that the extensive mutagenesis affects gating. The low levels of channel activity (1–5%) that comprise the dark current of rod outer segments suggest that under physiological conditions subconductance states are likely to play a role in phototransduction. Fig. 4 D shows an amplitude histogram averaged over five different channels in which free cGMP produced activation between 1 and 5% (3% average). Subconductance states were occupied roughly half the time [ P o (O1 + O2) = 0.014 and P o (O3) = 0.013], and contributed about one third of the total current. Recently, Hackos and Korenbrot reported the intriguing result that the Ca 2+ /Na + permeability ratio changes as a function of cGMP concentration in retinal rod outer segments, suggesting that subconductance states may play an important role in regulating internal Ca 2+ concentrations. Using a powerful approach that allows us to tether one ligand at a time to single CNG channels, we have presented a thorough kinetic analysis of channel gating at every level of liganding. The richness of channel behavior we observed indicates a complex mechanism of gating that has not been recognized before. Simple concerted and sequential mechanisms, as well as the simple coupled dimer model proposed recently, are easily ruled out. Instead, we propose that the same 10-state mechanism, including two subconducting levels and a fully conducting level, can explain gating in each liganded state. In structural terms, such a model can be accounted for by invoking more than one conformational change in each subunit. | Study | biomedical | en | 0.999997 |
10352037 | Voltage-gated K + (K V ) 1 channels help establish the resting membrane potential and modulate the frequency and duration of the action potentials in excitable cells. Molecular biology techniques have identified several mammalian genes encoding the pore-forming α subunits of K V channels that can give rise to delayed rectifier or A-type currents upon expression in heterologous systems . The functional and structural diversity of the K V channels' α subunits is further increased by their capacity to form functional heterotetrameric structures and to associate with modulatory β subunits . For example, association of β subunits with some members of the Shaker subfamily results in αβ heteromultimers with inactivation kinetics more rapid than those of the corresponding α homomultimers , and, even further, some of these β subunits can convert a delayed rectifier into a rapidly inactivating channel . In some tissues, K + currents exhibit specific properties, such as regulation by oxygen levels . It has been hypothesized that O 2 sensitivity of K + currents could be intrinsic to the channels themselves or, alternatively, that a membrane-bound O 2 sensor or a regulatory subunit of the K + channels confers the observed sensitivity . In the present work, we have used an heterologous expression system to study the association of the auxiliary subunit Kvβ1.2 (formerly Kvβ3) with some cloned K V channels and its possible contribution to the hypoxic sensitivity of the heteromultimers. The K V channels used ( Shaker B and Kv4.2) express rapidly inactivating currents comparable to the oxygen-sensitive K + currents described in some preparations . We found subfamily-specific functional interactions between Kvβ1.2 and the different K V channels studied, so that Kvβ1.2 coexpression is able to regulate the amplitude of the endogenous HEK293 K V currents, the rate of inactivation of the Shaker currents, and the redox and oxygen sensitivity of the Kv4.2 currents. The hypoxic response of the Kv4.2+Kvβ1.2 heteromultimers was unaffected by application of reduced glutathione (GSH) in the pipette solution or in the bath, but was prevented by treatment with DTT (1,4 dithiothreitol) and restored with DTDP (2,2′-dithiodipyridine), suggesting that reduction of some, but not all, of the residues susceptible to redox modulation can disrupt the mechanism underlying the low pO 2 regulation of these channels. Hypoxic inhibition was reverted by carbon monoxide (suggesting the presence of an hemoproteic O 2 sensor in HEK cells) and remains in excised membrane patches, indicating that the mechanism of low pO 2 inhibition is restricted to the plasma membrane. HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum ( GIBCO BRL ), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l -glutamine. Cells were grown as a monolayer and plated on squared coverslips (24 × 24 mm) placed in the bottom of 35-mm Petri dishes at a density of 2–4 × 10 5 cells/dish the day before transfection. Transient transfections were performed using the calcium-phosphate method with 1 μg of plasmid DNA encoding the drosophila Shaker B (H4) K + channel α subunit (into pRcRSV; Invitrogen Corp.), or the Kv4.2 K + channel α subunit (into E42c) alone or in combination with 2 μg of plasmid DNA encoding the Kvβ1.2 subunit into pREP4. In a group of experiments, the cells were only transfected with 2 μg of Kvβ1.2 subunit. In all cases, 0.2 μg of green fluorescent protein (GFP) in a CMV-promoter expression plasmid (GFPPRK5), was included to permit transfection efficiency estimates (10–40%) and to identify cells for voltage-clamp analysis . Voltage-clamp recordings revealed typical inactivating currents in 100% of the cells expressing GFP. A group of control cells was obtained by analyzing the currents present in cells transfected with GFP alone or in untransfected cells. All plasmids used in this study were generously provided by Drs. E. Marban and G.F. Tomaselli (John Hopkins University, Baltimore, MD). K + currents were studied using either the whole-cell or the outside-out configuration of the patch-clamp technique. The holding potential was −60 or −80 mV, respectively. Isolated HEK cells were studied 1–3 d after transfection. The coverslips with the attached cells were transferred to a small recording chamber (0.2 ml) placed in the stage of an inverted microscope and perfused by gravity with (mM): 141 NaCl, 4.7 KCl, 1.2 MgCl 2 , 1.8 CaCl 2 , 10 glucose, 10 HEPES, pH 7.4 with NaOH. The bath solution was connected to ground via a 3 M KCl agar bridge and a Ag-AgCl electrode. Patch pipettes were double pulled (PP-83; Narishige Co.) and heat polished (MF-83; Narishige Co.) to resistances ranging from 1.5–3 MΩ for whole-cell experiments to 10–15 MΩ for cell-free recordings when filled with a internal solution containing (mM): 125 KCl, 4 MgCl 2 , 10 HEPES, 10 EGTA, 5 MgATP, pH 7.2 with KOH. Hypoxia was achieved by bubbling the reservoir that fed the perfusion chamber with 100% N 2 . The final pO 2 level in the perfusion chamber was below 10 mmHg. The time course of the fall in the pO 2 was complete within 1 min of solution exchange. In selected experiments, the control solutions were also bubbled with air to exclude potential artifactual effects due to the bubbling of the solutions. Whole-cell currents were recorded using an Axopatch 200 patch-clamp amplifier, sampled at 10 and filtered at 2 kHz (−3 dB, four-pole Bessel filter). The series resistance (ranging from 4 to 10 MΩ) was routinely compensated by 60–80%. Data were leak subtracted on line by a P/4 protocol. K + currents from macropatches in the outside-out configuration were registered several minutes after excision and were taken as the difference between the current recorded in a 50-ms depolarizing pulse to +40 mV from a holding potential of −80 mV and the average current obtained applying four pulses to +40 mV after inactivating the K + channels with 200-ms prepulses to the same potential. To facilitate the subtraction of capacitative transients, the potential was held at −80 mV during 1 ms between prepulse and pulse. Currents were sampled at 5 and filtered at 1 kHz. Records were digitized with a Digidata-1200 A/D converter ( Axon Instruments ), and stored on disk using PCLAMP version 6.02 software. All the experiments were done at room temperature (20–22°C). Analysis of the data was performed with the CLAMPFIT subroutines of the PCLAMP software and ORIGIN 4.0 software (Microcal Software, Inc.). Pooled data are expressed as mean ± SEM. Statistical comparisons between groups of data were carried out with the two-tailed Student's t test for paired or unpaired data, and values of P < 0.05 were considered statistically significant. The analysis of the differences between two groups of data when comparing more than one variable was done with a fully factorial analysis of variance [(M)ANOVA] using commercial software (SYSTAT; Systat Inc.). DTT, DTDP, and GSH were obtained from Sigma Chemical Co. DTT and GSH were prepared fresh and dissolved in the bath or in the pipette solution, and DTDP was first dissolved in ethanol to a concentration of 500 mM, and then diluted in bath solution to a final concentration of 100 μM. Untransfected or mock-transfected (GFP alone) HEK293 cells show K V currents of variable size, ranging from 100 to 600 pA at +60 mV. As shown in Fig. 1 A, over the length of a 100-ms pulse, this endogenous current exhibited almost no inactivation. Outward current was observed at potentials above −20 mV, and peak current typically showed a plateau at potentials more positive than +40 mV. When HEK293 cells were transfected with Kvβ1.2, there was a significant increase in the current amplitude. The peak current amplitude at +100 mV increased from 0.32 ± 0.07 nA in the mock-transfected cells ( n = 7) to 1.38 ± 0.32 in the Kvβ1.2-transfected cells ( n = 8). The averaged current–voltage relationships were statistically different [ P = 0.006 with (M)ANOVA], suggesting that Kvβ1 subunit could be exerting a chaperon-like effect on the HEK293-endogenous K + currents. Transfection of HEK293 with Shaker or Kv4.2 α subunit cDNA gave rise to large, rapid inactivating currents in all the cells studied . Kvβ1.2 coexpression did not modify significantly the current amplitude, as shown in the averaged peak current–voltage relationships. It has been demonstrated that Kvβ1.2 is able to modulate the rate of inactivation of some of the members of the Shaker family when coinjected in Xenopus oocytes . However, it is not known whether this subunit is able to functionally associate with the K + channels of the Shal subfamily (Kv4) and modulate their electrophysiological properties. In our expression system, Kvβ1.2 also modulates the kinetics of the recombinant Shaker channels. The traces in Fig. 2 A show that β subunit coexpression accelerates the rate of inactivation and decreased the amplitude of the currents at the end of a 100-ms pulse. The inactivation time course for both Shaker and Shaker +β K + channels was best fitted to a biexponential function with time constants that exhibited little voltage dependence. The presence of β subunit produced an acceleration of inactivation due to a significant decrease in the fast time constant at all the voltages [ P = 0.02 with (M)ANOVA] and a less-pronounced decrease in the slow time constant that was not significant in our analysis ( P = 0.054). However, this slow time constant is very likely distorted by the contribution of endogenous currents. Fig. 2 B shows a similar analysis of the kinetics of the Kv4.2 and Kv4.2+β recombinant channels. The time course of inactivation was also fitted to a biexponential function, but in this case it was not changed by the presence of β subunit. The same lack of effect on the activation and inactivation kinetics was observed for the endogenous channels upon association with β subunit (data not shown). The absence of effect of β subunit coexpression on the kinetics of the Kv4.2 channels has two possible explanations: either there is a lack of association between the two subunits, or, alternatively, β subunit associates with Kv4.2 α subunits to modulate other properties of the channel. Among these other properties, we have studied their sensitivity to sulfhydryl group reagents. Chemical redox modulation has been demonstrated for different native K + channels as well as for several cloned K + channel α subunits . Furthermore, a redox mechanism has been shown to modulate the effects of β subunit on the rate of inactivation of Kv1.4 K + channels . In our work, the application of the membrane-permeable reducing agent DTT or the oxidizing agent DTDP to HEK cells expressing Shaker or Shaker +β had effects similar to those reported in the aforementioned works using other redox agents. Fig. 3 A shows one of four cells in which application of 100 μM DTDP markedly decreased the rate of inactivation of the Shaker +β currents in an irreversible way, whereas treatment with 2 mM DTT had the opposite effect. As an indicator of the change in the rate of inactivation, the average modification in the amplitude of the currents at the end of a 100-ms depolarizing pulse was calculated for three more cells expressing Shaker +β channels . DTT treatment reduced the amplitude of the current at the end of the pulse by 20.85 ± 7.1%, whereas DTDP increased this amplitude by 65.5 ± 12.1% ( n = 4). The same modifications in the rate of inactivation were observed in cells transfected with Shaker alone (data not shown). On the contrary, treatment with DTT or DTDP failed to modify the amplitude or the kinetics of the currents expressed in cells transfected with Kv4.2 alone , in agreement with previous results showing the resistance of channels of the Kv4 subfamily to treatment with other redox agents . When the same experiment was carried out in Kv4.2+β-transfected cells , we could observe that application of 2 mM DTT produced an irreversible reduction of the current amplitude without any significant change in the kinetics of the currents. Treatment with DTDP (100 μM) did not modify in a significant way the amplitude of the currents, but was able to revert the inhibition induced by DTT. Similar effects were observed in five more cells expressing Kv4.2+β channels . These results demonstrate that Kvβ1.2 is capable of associating with Kv4.2 α subunits and, upon coexpression, confers sensitivity to redox modulation to the Kv4.2+β heteromultimers. Regulation of ion channels by low pO 2 was first demonstrated in chemoreceptor cells of the rabbit carotid body, where an inactivating K + current was shown to be reversibly inhibited by hypoxia . It is not known whether the oxygen-sensitive K + channels have an O 2 sensing domain or, alternatively, the oxygen-sensitive cells have other sensor structures that can affect channel function. To address this question, we have studied the effect of lowering pO 2 on the cloned K + currents, as well as the possible contribution of Kvβ1.2 to the O 2 modulation of the channels. Fig. 4 A shows typical records obtained in depolarizing pulses to +40 mV from a holding potential of −60 mV for each channel (endogenous, Shaker , and Kv4.2) alone or in combination with Kvβ1.2. In each case, N 2 -equilibrated solutions were applied for 2–5 min. Hypoxia did not modify in a significant way the amplitude or the time course of the endogenous currents in the mock-transfected ( n = 5) or β-transfected ( n = 6) cells. The same lack of effect was observed in all cells expressing Shaker channels alone ( n = 8). In cells transfected with Shaker +β, hypoxia produced a 10% reduction in the current amplitude only in one of eight cells studied (12.5%) and, in Kv4.2-transfected cells, there was a 9% reduction in the current amplitude in 2 of 13 cells (15%). However, when the effect of hypoxia was studied in Kv4.2+β-cotransfected cells, 38 of 41 cells studied (93%) showed a reduction in the peak current amplitude that ranged from 10 to 40% and averaged 15.48 ± 0.93% at +40 mV (mean ± SEM, n = 38). Even though in some of the cells low pO 2 application seemed to slow down the time course of inactivation, this effect was not found to be statistically significant. The time course of hypoxic inhibition of the Kv4.2+β cotransfected cells is shown in Fig. 4 B, where the peak current amplitude at three different voltages is represented against time. The effect of hypoxia was fully achieved within 1 min after the exchange of the solution and was readily reversible upon washout with the control solution. The hypoxia-induced inhibition of Kv4.2+β K + currents was voltage independent , excluding a possible spurious effect due to a shift in the current–voltage relationship. The association with Kvβ1.2 invests Kv4.2 channels with two new properties, sensitivity to redox modulation and responsiveness to low pO 2 stimulation, making attractive the hypothesis that the redox status of the Kv4.2+β channels could be involved in the effect of hypoxia. This hypothesis was explored by studying the effect of hypoxic solutions on these channels after application of reducing or oxidizing agents. Fig. 5 A shows that application of 2 mM DTT produces an irreversible reduction of the current amplitude after which the response to hypoxia is lost. In these cells the effect of hypoxia was modified from a 13 ± 1.2% inhibition in control conditions to a 1.6 ± 0.6% inhibition after DTT treatment. The same protocol was used to study the effect of the oxidizing agent DTDP (100 μM) on the hypoxic inhibition of the channels . In this case, we can see that DTDP did not modify the response to hypoxia of the Kv4.2+β currents (hypoxic inhibition averaged 16.1 ± 2.7% before and 14 ± 1.7% after DTDP treatment, n = 6), although it was able to recover the hypoxic response of cells previously exposed to DTT (data not shown). These results indicate that the residues of the Kv4.2+β heteromultimers sensitive to DTT and DTDP treatment are involved in the response of the channel to acute hypoxia. Although the previous data suggest that the redox state could be one of the mechanisms involved in the hypoxic modulation of Kv4.2+β channels, we have explored whether physiological redox modulators such as GSH have effects comparable to DTT on the currents and on their response to hypoxia. It is noteworthy that these two reducing agents modify in a similar way the time course of inactivation of cloned Shaker K + channels . Additionally, due to its lower membrane permeability, GSH could be helpful in indicating whether the effects are mainly due to modification of an intracellular or an extracellular site. We performed a series of experiments to explore the effect of pipette application of 5 mM GSH on the amplitude and the kinetics of Kv4.2+β currents and on their response to low pO 2 exposure. Parallel experiments in the same cultures with our normal pipette solution were used as controls. We found that inclusion of 5 mM GSH in the pipette solution did not change the amplitude of the currents (the peak current at +40 mV averaged 2.57 ± 0.46 nA in control versus 2.37 ± 0.49 nA in GSH-treated cells, n = 9) nor the inactivation time course (the two time constants were 11.6 ± 2.9 and 120 ± 6 ms in control cells versus 15 ± 4 and 135 ± 10.2 ms in the presence of GSH, n = 9). When the cells were bathed in a N 2 -equilibrated solution, all cells in the two groups showed a reduction of the peak current amplitude, averaging 16.25 ± 1.6% in control and 16.86 ± 1.83% in GSH-treated cells. The effects of extracellular application of 5 mM GSH are shown in Fig. 6 , where the peak current amplitude in depolarizing pulses to +40 mV is plotted against time. In this cell, application of a N 2 -equilibrated solution produced the same reduction of the current amplitude before and after bath application of GSH (23 and 22%, respectively). Treatment with 5 mM GSH reversibly decreased the amplitude and rate of inactivation of Kv4.2+β currents, but did not modify the magnitude of the effect of hypoxia. Similar results were observed in four more cells, in which the reduction produced by 5 mM GSH was somehow variable, averaging 33 ± 7%. The fact that DTT effects could not be reproduced by treatment with GSH suggests that these two agents are modifying different thiol groups. To further elucidate the underlying molecular mechanisms of hypoxic inhibition of Kv4.2+β channels, we have performed some experiments in excised membrane patches, devoid of potential intracellular mediators. Fig. 7 shows the effect of hypoxia on the peak current amplitude recorded with the protocol described in materials and methods in an outside-out patch obtained from a Kv4.2+β-transfected cell. The current traces corresponding to the numbers in the plot are also shown in Fig. 7 . As in the whole-cell experiments, perfusion with N 2 -equilibrated solution produced a reduction in the amplitude of the current that reverted upon washout with normoxic solution. The same inhibition was observed in five more patches, with an average effect of 22.0 ± 2.5%. The average peak currents in these six patches was 106.3 ± 31.0 pA. These data indicate that acute hypoxia is acting through a membrane-delimited pathway to produce the inhibition of Kv4.2+β currents, suggesting that either the Kv4.2+β channel proteins are intrinsically oxygen sensitive or, alternatively, there is a closely associated but distinct oxygen-sensing element that is endogenously expressed in the membrane of the host cell. The available evidence argues in favor of this latter possibility in several oxygen-sensitive tissues , as well as in heterologous expression systems such as COS cells and HEK293 cells . To explore this possibility, we have studied the effect of carbon monoxide on the hypoxic response of Kv4.2+β. CO is a very inert gas that in biological systems only reacts with hemoproteins. Fig. 8 shows the effect of CO on the inhibition of Kv4.2+β currents by low pO 2 . Fig. 8 shows the peak current amplitudes at two different voltages obtained in a cell while perfusing with control solution (pO 2 = 150 mmHg), with a hypoxic solution equilibrated with 100% N 2 (pO 2 < 10 mmHg), with a hypoxic solution equilibrated with a gas mixture containing 20% CO in N 2 (pO 2 < 10 mmHg, estimated pCO = 150 mmHg), and after returning to the control solution. We found that CO is reverting in a significant extent the inhibition observed with hypoxia with a time course even faster that the onset of hypoxic application, indicating that CO is able to successfully replace O 2 at the O 2 -sensing molecule, albeit with smaller affinity. The same reversion was observed in 10 more cells studied, in which CO prevented or reversed by 69.5 ± 3.2% the low pO 2 -induced inhibition of Kv4.2+β currents, so that the average hypoxic inhibition decreased from 16.2 ± 1.4% to 5.04 ± 0.9% . The expression of recombinant channels in heterologous systems has proved a useful tool to characterize ionic channels in isolation. Transient transfection of HEK293 cells provides an efficient expression of channel proteins in a mammalian cell background, and offers an additional advantage for the present work, due to the absence of endogenous Kvβ subunits in HEK293 cells . The presence of endogenous α subunits did not represent a problem due to the clear differences, both in amplitude and kinetics, between these endogenous currents and the currents through transfected K V channels . The significant increase in the endogenous current amplitude obtained in Kvβ1.2-transfected cells suggest that this subunit interacts with the endogenous α subunits. A chaperon-like effect has been reported for several β subunits acting on specific K V channel α subunits . Studies on β subunit–mediated effects on K + channels have been primarily focused on the modifications induced in the inactivation kinetics of the heteromultimers. These studies indicate that several β subunits (Kvβ1, Kvβ3, and the Drosophila homologue Hk ) are able to increase the rate of inactivation of specific members of the Shaker subfamily that express A-type currents or convert delayed-rectifier currents into A-type currents (see introduction ). This interaction between Kvβ1 and members of the Shaker subfamily has been confirmed with immunohistochemical studies that show the association and colocalization of these α-β complexes . It has also been reported the existence of selective interaction between both Kvβ1 and Kvβ2 and the mammalian Shal homologue Kv4.2 , but functional analysis has failed to reveal a change in the inactivation properties of the members of the Shal subfamily when coexpressed with Kvβ1 subunit . In agreement with these reports, we found that coexpression with Kvβ1.2 produces a significant change in the rate of inactivation of the Shaker channels due to a decrease in the fast time constant. Besides, the fact that the acceleration of the channel inactivation by Kvβ1.2 does not reduce the peak current amplitude suggests that Kvβ1.2 is also increasing the surface expression of Shaker channels. Also in agreement with previous data, we found no changes in the inactivation rate of the Kv4.2 currents upon Kvβ1.2 coexpression. However, the association is functionally assessed by the acquisition by the Kv4.2+β currents of new property, namely the sensitivity to sulfhydryl group reagents . Another proof of this functional association of Kv4.2 with Kvβ1.2 is the capability of Kv4.2+β currents to respond to low pO 2 . This response was only consistently observed in our expression system with this particular α+β subunit combination, and consisted in a reversible reduction of the current amplitude upon exposure to hypoxic solutions . One important aspect to consider regarding this effect is whether we are dealing with a metabolic or an allosteric-type mechanism. Given the speed of the effect of hypoxia, and the presence of 5 mM ATP in the intracellular solution, a direct action of hypoxia seems more likely than a response to altered cellular metabolism. Actually, hypoxic inhibition of native A-type K + channels has been slow to occur in excised membrane patches , and the data presented in Fig. 7 , showing the same effect of hypoxia in excised patches in the absence of potential intracellular mediators, strongly suggest that the response to hypoxia is a membrane-delimited mechanism. In addition, the persistence of the low pO 2 inhibition in a cell-free preparation confirms that Kv4.2 is able to coassemble with Kvβ1.2. The modifications in the hypoxic response after application of freely membrane-permeable oxidizing and reducing agents suggest that hypoxic sensitivity can be modulated by the redox status of the channel proteins and that the same cysteine residues modified by DTT and DTDP are involved in the low pO 2 regulation of the Kv4.2+β channels. However, the absence of effect of GSH when applied intracellularly argues against a role for redox modulation under physiological conditions, and also excludes the possibility that the effect of low pO 2 on the Kv4.2+β channels could be attributable to the redox status of the cytoplasmic β subunits. On the other hand, extracellular GSH application does not interfere with the hypoxic response of the channel, supporting the idea that hypoxia and reducing agents can inhibit Kv4.2+β currents through different mechanisms. The fact that Shaker and Shaker +β channels are also modified by these agents, but insensitive to hypoxia, stresses out the fact that the effect of low pO 2 as a physiological stimulus is not simply achieved by the reduction of a sulfhydryl group. Redox modulation of Shaker or Shaker +β currents was able to change their rate of inactivation, but none of these maneuvers rendered the channels sensitive to hypoxia (data not shown). Furthermore, in contrast with DTT effect, application of hypoxic solutions did not modify the rate of inactivation of the channels . These observations indicate that O 2 sensing must have some specific structural requirements that seem to be achieved in our expression system by the combination of Kv4.2 α subunits with Kvβ1.2 subunits. With respect to the molecular nature of the O 2 -sensing mechanism, there are two possibilities: first, the Kv4.2+β channels themselves are the O 2 -sensing devices and, second, there is some other O 2 -sensing molecule endogenously present in HEK293 cells capable of interacting with Kv4.2 α subunits only when a β subunit is also present. Data on the literature showing that other structurally distinct channels are also O 2 sensitive in this cell line support the second possibility, and data on the present study locate this O 2 sensor in the plasma membrane. Since the only known targets of CO in biological systems are reduced hemoproteins with accessible iron sites, our observation that CO is able to interact with this putative O 2 sensor, replacing O 2 and preventing the inhibition of K + currents , strongly suggests that the intrinsic O 2 sensor of HEK293 cells is a hemoprotein. The physiological relevance of the findings reported here is difficult to evaluate because neither the molecular nature of the O 2 -sensitive K + channels nor the distribution and coexpression of Kv4.2 with Kvβ1 in native tissues are known. However, our results showing evidence that β subunits provide hypoxic sensitivity to specific K V channel α subunits put forth the interesting possibility of the existence of tissue-specific modulatory subunit(s) that confer hypoxic sensitivity to the expressing tissues. This idea is supported by a recent report by Patel et al. in which a new K channel subunit, Kv9.3, that does not form a channel itself, is able to coassemble with Kv1.2 and increase the probability of the heteromultimeric channels to be modulated by hypoxia. Finally, our findings provide new clues in the search for the molecular mechanisms of O 2 sensing in hypoxia-sensitive tissues, raising a completely new set of questions requiring further investigation. | Study | biomedical | en | 0.999998 |
10352038 | It has been known since the work of Knop and Sachs over 130 yr ago that plants cannot grow in the absence of potassium . It is their most abundant inorganic constituent, contributing importantly to the osmotic potential and electrolytic character of cytoplasm. The plasma membrane is typically more permeable to K + than to other ions, so the difference in its concentration across the membrane has a large influence on the membrane potential and, hence, cell physiology. Another reason for its essentiality is that some enzymes require K + as a cofactor. The mechanism by which cells concentrate K + from dilute extracellular sources such as soil has received considerable attention because plant growth depends directly on it. Early kinetic studies by Epstein et al. gave evidence of two distinct uptake mechanisms: a high affinity system operating over micromolar concentration ranges and a low affinity system that predominates when [K + ] ext is in the millimolar range. Recent measurements of K + electrochemical potential gradients were incorporated into this classical model to create the widely held view that active transport is necessary when [K + ] ext is less than ∼300 μM, but that a passive mechanism suffices at higher values of [K + ] ext . This important thermodynamic information was readily integrated with ground-breaking molecular advances occurring at about the same time. Genes encoding passive K + channels and active K + cotransporters were cloned by complementation of yeast mutants, functionally characterized after heterologous or ectopic expression, and demonstrated to be expressed in roots . These advances collectively gave rise to the dominant view that transporters such as HKT1 and the KUP family are responsible for “high affinity” K + uptake and that inward-rectifying K + channels such as AKT1 mediated uptake when K + was more concentrated than ∼300 μM. This paradigm was shown to require modification when an Arabidopsis mutant lacking detectable AKT1 channel activity ( akt1 ) was found to be defective in K + uptake and growth on solutions as dilute as 10 μM K + , a concentration previously thought to be well outside the realm of possibilities for channels . However, measurements of membrane potentials more negative than −230 mV in Arabidopsis roots demonstrated that uptake of K + from 10 μM solutions by channels was indeed energetically feasible, at least in cells near the root apex . Now it seems reasonable to view inward-rectifying K + channels as passive uptake mechanisms capable of conducting growth-supporting K + fluxes in the high-affinity concentration range, provided that the K + electrochemical potential gradient is inward. The existence of a mutant lacking inward-rectifying K + channels in the root provides an opportunity to dissect genetically the channel-mediated contribution to K + uptake from that of other transporters, and to determine the significance of each under various ionic conditions a plant may encounter. A condition meriting close attention in this respect is the presence of NH 4 + , as Hirsch et al. found it must be present to observe the akt1 phenotype (poor growth relative to wild type on [K + ] ext < 0.1 mM). In the absence of NH 4 + , mutant and wild type grow similarly. This would be expected if NH 4 + inhibited a K + transport mechanism that operates in parallel with AKT1 and is necessary for growth when AKT1 activity is lacking. There is much support in the literature for this possibility. Inhibitory effects of NH 4 + on K + uptake have been noted and, in a study of maize roots, Vale et al. found that K + uptake was comprised of NH 4 + -sensitive and NH 4 + -insensitive components. Smith and Epstein presented evidence that NH 4 + inhibited K + uptake by competing for a binding site on the transporter in maize leaves. However, the converse (K + inhibition of NH 4 + uptake) does not seem to occur, a result that at least one authority considered “quite surprising” . The present work takes advantage of the akt1 mutation to produce an explanation of this relationship between K + , NH 4 + , and growth. A related and somewhat controversial topic is the role of Na + in K + uptake . The renewal of interest in Na + –K + relationships is due to the finding that the HKT1 transporter of barley functions as a Na + -coupled K + symporter , and to genetic advances in understanding the relationship between the ability of a plant to resist Na + stress and K + nutritional status . The akt1 mutant was used here in studies that shed light on how the uptake mechanisms responsible for growth-sustaining K + fluxes are importantly influenced by NH 4 + and Na + . Measurements of membrane potential (V m ) in apical root cells were made with an intracellular microelectrode as described in Hirsch et al. in order to assess the permeability of the membrane to K + . Eq. 1 is a simplified description of the ionic basis of V m in plant cells: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}V_{m}=\frac{G_{K}{\cdot}E_{K}+G_{X}{\cdot}E_{X}-I_{pump}}{G_{tot}}.\end{equation*}\end{document} G K and E K are the conductance and equilibrium potential for K + , G X and E X represent the conductance and equilibrium potential for all other ions lumped together, and I pump is the current created by an electrogenic pump (the H + -ATPase in the case of plants). G tot is the total conductance of the membrane. Shifts in extracellular KCl concentration ([KCl] ext ) were imposed on the root while V m was recorded continuously. The change in V m resulting from shifts in [KCl] ext is described by: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\Delta}V_{m}= \left( \frac{{\mathit{G}}_{K}{\cdot}{\Delta}E_{K}}{{\mathit{G}}_{total}} \right) + \left( \frac{{\mathit{G}}_{X}{\cdot}{\Delta}E_{X}}{{\mathit{G}}_{total}} \right) - \left( \frac{{\Delta}I_{pump}}{{\mathit{G}}_{total}} \right) .\end{equation*}\end{document} Assuming that an imposed shift in [KCl] ext affects only the K + and Cl − components, Eq. 2 simplifies to: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\Delta}V_{m}= \left( \frac{{\mathit{G}}_{K}{\cdot}{\Delta}E_{K}}{{\mathit{G}}_{total}} \right) + \left( \frac{{\mathit{G}}_{Cl}{\cdot}{\Delta}E_{Cl}}{{\mathit{G}}_{total}} \right) .\end{equation*}\end{document} Increasing [KCl] ext caused positive shifts in V m , demonstrating that the membrane was more permeable to K + than the counterion Cl − , as is typical of plant cells. In the extreme case of a negligible Cl − conductance, Eq. 3 reduces to: 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{{\Delta}V_{m}}{{\Delta}E_{K}}=\frac{{\mathit{G}}_{K}}{{\mathit{G}}_{total}}.\end{equation*}\end{document} For the purposes of determining the effects of the akt1 mutation and various ionic treatments, the assumptions implicit in Eq. 4 were adopted. Thus, the magnitude of the ΔV m resulting from shifts in [KCl] ext is interpreted here as a measure of the relative K + permeability of the membrane. The solutions used to bathe the roots were exactly the same solutions used for the growth experiments (below), except agarose was omitted. For experiments that tested the effects of NH 4 + , Na + , and H + , the mounted seedlings were bathed in the test solution for ∼2 h before impalement. Rb + fluxes were also performed exactly as described by Hirsch et al. . Percent inhibition by NH 4 + was calculated so that the results of independent trials involving different specific activities could be averaged. 24 surface-sterilized seeds of either akt1 or the Wassilewskija wild type were sown with equal spacing across square Petri plates containing media (described below) solidified with 0.8% agarose. They were maintained in darkness at 4°C for 48 h before being placed in a growth chamber set to deliver 16 h days and 8 h nights at 21°C. Germination was assayed after 72 h when [K + ] ext was 10 or 100 μM , but after only 48 h when [K + ] ext was 1,000 μM because of the faster embryo growth in this condition. A seed was considered to have germinated if emergence of the radicle from the seed coat could be detected with the aid of a 40× dissecting scope. After 4 d of growth, the fresh weight of the group of seedlings was determined to the nearest 0.1 mg, and at 8 d the harvesting/weighing procedure was repeated with a separate plate of seedlings. The difference in mass between the two time points was divided by the number of intervening days to obtain an average growth rate for the group of seedlings between days 4 and 8. Experiments spanning 12 d of growth produced similar results. All data shown are the averages of at least three independent trials. The following base solution was used for studying the effects of NH 4 + : 2.5 mM NaNO 3 , 2.5 mM Ca(NO 3 ) 2 , 2 mM MgSO 4 , 0.1 mM NaFeEDTA, 80 μM Ca(H 2 PO 4 ) 2 , 25 μM CaCl 2 , 25 μM H 3 BO 3 , 2 μM ZnSO 4 , 2 μM MnSO 4 , 0.5 μM CuSO 4 , 0.5 μM Na 2 MoO 4 , 0.01 μM CoCl 2 , 0.5% sucrose, and 2.5 mM Mes. NH 4 + was added as NH 4 H 2 PO 4 to achieve the desired amount and 1 mM Ca(H 2 PO 4 ) 2 was added to the 0 NH 4 + solution to balance the phosphate concentrations. K + was added as KCl. The pH of the mixture was adjusted to 5.7 with NaOH and autoclaved for 10 min. Longer autoclaving frequently produced a crystalline precipitate that probably contained NH 4 + because it formed copiously in solutions containing >1 mM NH 4 + , and not at all in its absence. Also, the normal inhibitory effect of NH 4 + on growth was not observed when solutions containing the precipitate were used in experiments. This is a very important technical detail. The following base solution was used for studying the effects of Na + : 2.5 mM Ca(NO 3 ) 2 , 2 mM MgSO 4 , 0.1 mM EDTA, 0.1 mM FeCl 2 , 80 μM Ca(H 2 PO 4 ) 2 , 25 μM CaCl 2 , 25 μM H 3 BO 3 , 2 μM ZnSO 4 , 2 μM MnSO 4 , 0.5 μM CuSO 4 , 0.5 μM Na 2 MoO 4 , 0.01 μM CoCl 2 , 0.5% sucrose, and 2.5 mM Mes or 2.5 mM HEPES when the intended pH was basic. K + was added as KCl and Na + was added as NaCl. The pH was adjusted to 5.7 for growth experiments or otherwise to the indicated value with BTP. Note that the nominally 0 Na + treatment has 1 μM Na + from the Na 2 MoO 4 and any contaminating Na + in the water or chemicals. Also noteworthy, growth was inhibited when BTP {1,3-bis [tris(hydroxymethyl)methylamino]propane} was used instead of NaOH to adjust the pH of media containing specific Na + concentrations. Experiments demonstrated that this amount of BTP, all else equal, inhibited growth rate by 50%. This is another important technical detail. The first goal was to compare the effect of NH 4 + on the K + permeability of the plasma membrane in wild-type and akt1 root cells. Fig. 1 A shows a typical recording of V m made by impaling a root cell ∼150 μm from the apex of the cap with a microelectrode. After the voltage stabilized at −236 mV, the continuously flowing bathing solution containing 10 μM K + was switched to one containing 100 μM K + , a treatment referred to as Δ[K + ] 10–100 , and then subsequently to 1,000 μM K + . The change in steady state V m that occurred in response to these shifts (ΔV m ) is related to the K + permeability of the plasma membrane as discussed in materials and methods . The permeability detected by this method in akt1 mutant roots may be attributed to non-AKT1 activities because of the evidence that the mutant allele is functionally a null, despite the transferred-DNA being inserted in what might appear to be a dispensable cytoplasmic tail . The contribution of AKT1 channel activity to the K + permeability of wild-type roots may be inferred by subtracting the ΔV m measured in akt1 roots from the wild-type ΔV m . Following this reasoning, Fig. 1 B shows that the wild-type K + permeability, in the absence of NH 4 + , was ∼63% due to AKT1 channel activity and 37% due to non-AKT1 activities when the shift was Δ[K + ] 10–100 . When assayed at the higher concentration (Δ[K + ] 100–1,000 shift), the AKT1 component was a similar 55% of the now larger wild-type K + permeability . Such accounting of the membrane's K + permeability permitted an examination of which components were affected by NH 4 + . Approximately 50% of the wild-type ΔV m resulting from a Δ[K + ] 10–100 shift was inhibited by 2 mM NH 4 + . The NH 4 + -sensitive component of the wild-type response was very similar in magnitude to the minus-NH 4 + akt1 response, which was completely blocked by 2 mM NH 4 + . Thus, the ΔV m in wild-type roots, a parameter related to K + permeability, behaves as the quantitative sum of a NH 4 + -insensitive AKT1 component and a NH 4 + -sensitive non-AKT1 component. This simple quantitative relationship did not persist when [K + ] ext was increased from 100 to 1,000 μM . Instead, it appeared that 2 mM NH 4 + inhibited only 50% of the non-AKT1 component, as opposed to 100% at the lower [K + ] ext . The actual steady state value of V m for each genotype in each condition is shown in Table 1. The finding that the degree of inhibition by 2 mM NH 4 + depended on [K + ] ext prompted a more detailed investigation of the K + and NH 4 + concentration interdependence of the phenomenon. Fig. 2 demonstrates that 2 mM NH 4 + inhibited ∼50% of the ΔV m caused by Δ[K + ] 100–1,000 in akt1 roots, consistent with the data in Fig. 1 C. Only 0.5 mM NH 4 + was needed to inhibit 50% of the smaller response to Δ[K + ] 10–100 and 2 mM was completely inhibitory, consistent with Fig. 1 B. The large ΔV m response to shifting [K + ] ext from 1 to 10 mM was much less sensitive to this range of [NH 4 + ] ext . Taken together, the results in Figs. 1 and 2 indicate that AKT1 channel activity accounts for 50–60% of the K + permeability of the root plasma membrane, with the remainder resulting from one or more NH 4 + -sensitive transporters. Furthermore, the data in Fig. 2 may be taken as evidence that the non-AKT1 transporter has a K + -binding site to which NH 4 + may competitively bind when it is in large excess, preventing K + transport. The 50% block of the response to Δ[K + ] 100–1,000 by 2 mM NH 4 + may be taken as evidence that the K + -binding site of the non-AKT1 mechanism has a 50% probability of being occupied by NH 4 + under those particular conditions. This occupancy by NH 4 + increased to 100% when [K + ] ext was 10-fold lower, and it decreased to near negligible levels when [K + ] ext was in the millimolar range. Experiments were performed to determine if the decrease in K + permeability caused by NH 4 + characterized in Fig. 2 actually resulted in decreased K + ( 86 Rb + ) uptake at the whole-root level. Fig. 3 demonstrates that ∼90% of Rb + uptake into akt1 roots from 10 μM solutions was inhibited by treatment with 4 mM NH 4 + . This inhibition by NH 4 + was [Rb + ] ext dependent, being only 20% at 1,000 μM Rb + . Thus, fluxes at the organ level and electrical changes at the membrane both indicate that NH 4 + competitively inhibits one or more important K + -transport mechanisms detectable in the absence of AKT1 channel activity. The data presented thus far indicate that the wild-type root employs at least two K + -uptake mechanisms operating in parallel, each contributing significantly to the total flux even from 10 μM external solutions. One of these “high affinity” transporters is the passive AKT1 channel and the other is an NH 4 + -sensitive transporter of unknown molecular identity. Most important to the field of plant mineral nutrition is whether both of these K + -transport activities mediate fluxes of sufficient magnitude to be relevant to growth. If so, akt1 plants should grow more slowly than wild-type when K + is limiting, and their growth should be NH 4 + sensitive in a manner similar to the membrane permeability and fluxes presented in Figs. 1 – 3 . This idea was tested by measuring the growth of mutant and wild-type plants at various concentrations of K + and NH 4 + and at two stages of plant development— germination and seedling establishment. Germination is a consequence of, among other processes, the rapid expansion of cells already present in the mature embryo. Fig. 4 shows that at 10 μM K + , germination of akt1 seeds was strongly inhibited by increasing [NH 4 + ] ext compared with wild type. In the presence of 1 mM NH 4 + , no akt1 seeds had germinated 72 h after stratification, compared with 80% of the wild type. Half of the maximal akt1 germination was inhibited by 0.76 mM NH 4 + . The lower germination rate of akt1 seeds provided with 10 μM K + in the absence of NH 4 + (47 vs. 100% of wild-type seeds) is evidence that post-imbibition embryo growth depends upon AKT1-mediated K + uptake when [K + ] ext is low. Note that these germination percentages were determined at one point in time. Not shown is that nearly all seeds eventually germinated except those in the most inhibitory conditions (low K + with high NH 4 + ). When 100 μM K + was present and NH 4 + absent, 100% of both akt1 and wild-type seeds germinated within 72 h. This indicates that AKT1 activity is not required in this situation and that non-AKT1 activities were sufficient to meet the demands imposed by embryo growth. Increasing the [NH 4 + ] of this higher K + medium significantly inhibited akt1 germination while only modestly affecting the wild type. Germination of akt1 seeds was 50% inhibited by 3.8 mM NH 4 + , and nearly complete inhibition of akt1 germination was achieved by 10 mM NH 4 + . Increasing [K + ] ext from 100 to 1,000 μM further protected germination rates from inhibition by NH 4 + . Thus, as with the membrane permeability assays in Fig. 2 and the fluxes in Fig. 3 , increasing [K + ] ext lessened the inhibitory effect of NH 4 + on this earliest stage of akt1 plant growth. Growth rates of seedlings were also determined under the same conditions. Fig. 5 A demonstrates that in the absence of NH 4 + , akt1 seedlings grew more slowly than wild type on 10 μM K + , as was the case with the embryo growth responsible for germination . This is evidence that the K + flux conducted by AKT1 channels contributed significantly to growth even when [K + ] ext was 10 μM. Submillimolar NH 4 + added to the 10 μM K + medium inhibited the growth rate of akt1 seedlings, which was too low to measure reliably at concentrations >700 μM. The faster wild-type growth was not inhibited by NH 4 + in this concentration range. Embryo growth, assayed as germination rate, behaved similarly with respect to inhibition by NH 4 + . When [K + ] ext was increased to 100 μM , wild-type and akt1 seedlings grew several times faster than at 10 μM K + , and similar to each other in the absence of NH 4 + (as was also the case for embryos). Increasing [NH 4 + ] ext from 0 to 2 mM strongly inhibited the growth rate of akt1 seedlings without affecting the wild-type rate. This inhibition of akt1 growth rate by NH 4 + displayed a concentration dependence very similar to the NH 4 + inhibition of membrane K + -permeability assayed by Δ[K + ] 100–1,000 . This result, along with those in Figs. 2 and 3 , supports the idea that NH 4 + inhibits growth of akt1 seedlings by inhibiting K + permeability and fluxes mediated by one or more non-AKT1 transporters. Increasing [K + ] ext to 1,000 μM markedly reduced the amount of inhibition caused by NH 4 + . Thus, protection against NH 4 + inhibition by increasing K + was observed for seedling growth as it was with K + permeability, Rb + fluxes, and embryo growth. This is consistent with the notion that the K + transport activity supporting growth in the absence of AKT1 channel activity employs at least one substrate (K + ) binding site for which NH 4 + can compete under physiologically relevant conditions. The lack of inward-rectifying channel activity in akt1 roots was exploited in experiments designed to reveal information about what energizes the parallel, NH 4 + -sensitive, non-AKT1 activity. The approach was to measure V m in cells of akt1 roots in the absence of NH 4 + and administer shifts in [K + ] ext . Specifically, the hypothesis to be tested was whether the non-AKT1 K + -transport activity behaved as a coupled transporter, such as a H + -K + cotransporter or a Na + -K + cotransporter . Fig. 6 demonstrates that the presence of 2 mM Na + more than doubled the ΔV m induced by Δ[K + ] 10–100 when the pH of the medium was buffered at 5.7. Decreasing the proton concentration to pH 7.7 significantly reduced the magnitude of the ΔV m (K + permeability) of akt1 roots, but Na + stimulation was still observed. Reducing the proton concentration further (pH 8.7) essentially eliminated the response to Δ[K + ] 10–100 in the absence of Na + , though a measurable ΔV m could be observed in the presence of Na + . At higher K + concentrations (Δ[K + ] 100–1,000 ), a significant pH dependence of K + permeability was not detected. The Na + effect was relatively weaker than observed in the lower K + conditions, and not significant at the P = 0.05 level. These results are consistent with the non-AKT1 K + transport occurring by a symport mechanism that is energized by the electrochemical potential gradient of Na + and H + . Perhaps separate Na + -K + and H + -K + symporters function in parallel to actively transport K + . If so, the substrate-binding sites of both must have an affinity for NH 4 + . Alternatively, a single K + symporter may be capable of using electrochemical potential gradients of either Na + or H + as an energy source. It is also possible that the non-AKT1 transporter has an obligate requirement for both Na + and H + to actively transport K + , as our nominally 0 Na + conditions contain trace amounts (see materials and methods ). The results in Fig. 6 formed the basis of another test of the hypothesis that the K + permeability detected electrophysiologically in the absence of AKT1 channels represents the uptake pathway upon which growth of akt1 plants depends. Na + should stimulate growth of akt1 plants if the K + required for growth is taken up by this Na + -stimulated, NH 4 + -sensitive, non-AKT1 activity. Furthermore, the growth rate of wild-type plants should be less Na + dependent, given that a significant portion (50–60%) of wild-type K + permeability was attributed to AKT1 channels . Fig. 7 A demonstrates that both of these predicted results were observed when seedlings were grown on 10 μM K + . The growth rate of akt1 plants increased by 119% as [Na + ] ext was increased to 1,000 μM. Wild-type seedlings also benefited from increasing [Na + ] ext , though not to the same relative extent. At stressful levels of Na + (50– 100 mM), the growth rates of wild-type and akt1 plants were relatively equally inhibited (data not shown), indicating that the akt1 phenotype is distinct from that of the salt overly-sensitive mutants . The growth rate of akt1 plants was not stimulated by Na + when [K + ] ext was 100 μM. This is consistent with the relatively weaker stimulatory effect of Na + on K + permeability when assayed at this higher [K + ] ext and the evidence that, when >100 μM, [K + ] is not limiting growth rate . Our interpretation of the results presented here is that AKT1 channels mediate K + uptake across the plasma membrane of root cells in parallel with one or more genetically distinct K + transporters that are inhibited by NH 4 + . The concentration of NH 4 + that forces growth to depend on AKT1-channel activity depends on the K + status of the soil solution, and is in agreement with the roughly millimolar levels found to follow fertilizer application . It seems reasonable to suppose that other soil conditions encountered by plants may impair AKT1 function, shifting the bulk of K + -uptake activity to the non-AKT1 mechanism. The conclusion that AKT1 and non-AKT1 mechanisms mediate K + uptake in substantially overlapping concentration ranges seems inescapable, though different than the conclusions of Maathuis and Sanders , which were based on studies performed before a null mutant was available to exploit. Both AKT1 and non-AKT1 mechanisms clearly contribute in the absence of NH 4 + when [K + ] ext is 10 or 100 μM . This is somewhat surprising, given that the enhancement of K + permeability by Na + and H + at low [K + ] ext suggests that the non-AKT1 mechanism is an active symporter with K + transport coupled to the electrochemical potential gradient of one or both of those ions. It would be surprising, though not a violation of any thermodynamic law, if a cotransport mechanism contributed significantly to fluxes that could be conducted by passive channels. It is possible that the non-AKT1 mechanism is also passive and the enhancement of ΔV m by Na + and H + is due to a faster transport cycle, higher open probability, or the recruitment of more transporters into action. Interestingly consistent with this notion is the demonstration that Na + positively modulates the kinetics of AKT1 without permeating the channel . Rigorous voltage-clamp studies and Na + flux measurements are needed to distinguish whether Na + affects the kinetics or thermodynamics of the non-AKT1 transport mechanism(s). Such a study may reveal that Na + affects both because the two possibilities are not mutually exclusive. Regardless of how the non-AKT1 transport activity is energized, its inhibition by NH 4 + and stimulation by Na + were mirrored in most conditions by the effects of these ions on the growth of akt1 and, to a much lesser extent, wild-type plants. These close positive and negative correlations constitute evidence that the K + permeability detected electrically in akt1 roots is due to an activity that supports growth when the AKT1 mechanism is inoperative. The results also indicate that the relative contributions to plant growth of genetically distinct K + transport systems depend on ionic variables of the sort and magnitude encountered in soils. This finding may be relevant to the agronomic practice of managing plant nutrients. There is every reason to believe that continuing the combined electrophysiological and reverse-genetic approach will lead to a more complete and useful molecular-level accounting of the K + -transport activities supporting growth. The reverse-genetic approach to studying the non-AKT1 contributor requires knowing beforehand what gene or genes to eliminate. Therefore, it is now very important to consider what genes may be responsible for the non-AKT1 transport activity characterized physiologically by the present work. The recent impressive isolation and characterization of plant genes encoding proteins that perform K + transport has produced two strong candidates. The stimulation by Na + brings the HKT1 transporter originally found in wheat to the forefront as a candidate for the non-AKT1 activity. HKT1 is believed to function as a K + -Na + symporter . The earlier report of H + gradients serving as an energy source for HKT1-mediated K + transport also can be accommodated by the pH dependence of the non-AKT1 activity . Unfortunately, the present literature on HKT1 does not contain tests of NH 4 + as an inhibitor. Ideally, an Arabidopsis mutant with a disruption in an HKT1 homologue will be isolated and provide for a combined genetic and physiological test of the idea that AKT1 and HKT1 together conduct the K + fluxes needed for growth. The increase in K + permeability due to the presence of Na + was greater when assayed by Δ[K + ] 10–100 shifts than Δ[K + ] 100-1,000 shifts . The same trend was observed in akt1 seedling growth rate: Na + more than doubled the growth rate at 10 μM K + , but was without effect when [K + ] ext was 100 μM . Perhaps the non-AKT1 mechanism is more Na + coupled when the electrochemical potential gradient for K + is great, but less so when the energetics permit a passive mode of operation. Previous work has attributed a passive conductance to HKT1 that is separate from its Na + -K + symport activity , indicating that cotransporters can display such complexity of mechanism. Also, the growth rate of seedlings was limited by something other than K + at concentrations above 100 μM , so Na + may have stimulated K + uptake from 100-μM solutions, but limitations in some other factor prevented growth rate from responding. Another possible contributor to the non-AKT1 transport activity is one or more of the KUP family of K + transporters recently identified in Arabidopsis and barley. These transporters can complement K + -uptake deficiencies in mutants of Escherichia coli and yeast and can confer enhanced K + uptake into cultured Arabidopsis cells when overexpressed . A member of this family from barley is inhibited by NH 4 + , similar to the non-AKT1 activity studied here in planta . Arabidopsis KUP-mediated K + transport is also inhibited by NH 4 + (E. Kim and J.I. Schroeder, personal communication), though it is not stimulated by Na + . Thus, the Na + data currently favor HKT1, while the NH 4 + data favor KUP as the molecule(s) responsible for the non-AKT1 component of the root K + -uptake apparatus. It is also possible that the non-AKT1 activity is due to a combination of KUP and HKT1 activities insofar as both are inhibited by NH 4 + . The last point to make is that the competition between NH 4 + and K + for a binding site on the non-AKT1 transporter explains the previously observed inhibition of K + transport by NH 4 + in corn roots . The fact that plants have a specific NH 4 + transporter that is not blocked by K + explains why the converse (block of NH 4 + uptake by K + ) is typically not observed. Thus, the result that surprised Marschner receives a molecular-level explanation as a result of the present work. | Study | biomedical | en | 0.999997 |
10359571 | Male BALB/cByJ mice at 8–14 wk of age were obtained from The Jackson Laboratory . Mice were housed at least 1 wk before experimentation. Mice were cared for and handled at all times in accordance with National Institutes of Health and institutional guidelines. B-1 and B-2 lymphocytes were prepared by negative selection from peritoneal washout cells and from spleen cell suspensions, as described previously ( 35 ). The resulting B cells were cultured at 37°C with 5% CO 2 in RPMI 1640 medium (BioWhittaker) supplemented with 5% heat-inactivated fetal bovine serum ( Sigma Chemical Co. ), 10 mM Hepes (pH 7.2), 50 μM 2-ME, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. B-1 cells were 90–96% sIgM + , CD5/ Mac-1 + by flow cytometric analysis. B cells were lysed by incubation for 30 min (4°C) in ice-cold NP-40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 1 mM PMSF, 25 μg/ ml leupeptin/aprotinin, 1 mM Na 3 VO 4 , and 10 mM β-glycerophosphate) ( 22 ). Insoluble material was removed by centrifugation at 15,000 g for 15 min (4°C). Cell lysates were then incubated for 3 h with 1.5 μg nonimmune IgG or 1.5 μg anti-Cdk4 Ab, or 1.5 μg anti-Cdk6 Ab, followed by the addition of 50 μl of a 1:1 slurry of protein G–agarose. After 90 min, the immune complexes were collected, washed several times in NP-40 buffer, and separated by electrophoresis through a 10% polyacrylamide SDS gel. The resulting proteins were then transferred to Immobilon-P membrane ( Millipore ) and immunoblotted with an anti-cyclin D2 mAb (1:500 dilution in TBST) as described below. For detection of cyclin D2, cyclin D3, and retinoblastoma, B lymphocytes were solubilized in 100 μl of solubilization buffer (50 mM Hepes, pH 7.4, 15 mM EGTA, 137 mM NaCl, 15 mM MgCl 2 , 0.1% Triton X-100, 10 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 1 mM PMSF, and 1 μg/ml aprotinin/leupeptin) and NP-40 buffer supplemented with 20 mM NaF, respectively ( 36 ). Insoluble material was removed by centrifugation at 15,000 g (15 min), and 10–20 μg of total protein was separated through a 12% polyacrylamide SDS gel and transferred to Immobilon-P membrane. The Immobilon-P membrane was blocked in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk (4 h), washed several times, and then incubated 18 h with specific primary Abs. The membrane was washed extensively with TBST, incubated with anti–rabbit or mouse IgG-conjugated horseradish peroxidase Ab at 1:3,000 in TBST (90 min), and developed by enhanced chemiluminescence. B cells were sonicated at 4.0°C in Rb buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol [DTT], 0.1% Tween-20, 10% glycerol, 0.1 mM PMSF, 1 μg/ml leupeptin/aprotinin, 10 mM β-glycerophosphate, 1 mM NaF, and 0.1 mM Na 3 VO 4 ) ( 28 ). Insoluble material was removed by centrifugation, and the supernatant was incubated with 1.5 μg nonimmune rabbit IgG or 1.5 μg anti-cyclin D2 Ab. After 3 h, 50 μl of a 1:1 slurry of protein G–agarose was added and incubated for an additional 60 min. The immune complexes were then washed six times with Rb buffer and three times in a buffer of 50 mM Hepes, pH 7.4, and 1 mM DTT. The immune complexes were resuspended in 30 μl of kinase buffer (50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 5 mM MnCl 2 , 1 mM DTT, 2.5 mM EGTA, 10 mM β-glycerophosphate, 0.1 mM Na 3 VO 4 , and 10 μCi [γ- 32 P]ATP at 6,000 Ci/ mmol) in the presence of 1 μg of a truncated Rb protein substrate (p56 Rb ). The reactions were terminated after 15 min at 30°C by the addition of 2× SDS sample buffer, and the kinase mixture was separated through a 10% polyacrylamide SDS gel. Phosphorylated Rb was detected by autoradiography of the dried gel. Total RNA was isolated from primary B cells (Ultraspec RNA reagent; Biotecx Laboratories, Inc.), size fractionated by denaturing agarose gel electrophoresis, and transferred to GeneScreen Plus membranes (NEN Life Science Products, Inc.). Membranes were hybridized with radiolabeled cDNA probes specific for cyclin D2 and glyceraldehyde-6-phosphate dehydrogenase (GAPDH), generated by PCR using previously reported primer sequences ( 37 , 38 ), and developed by autoradiography. F(ab′) 2 fragments of goat anti–mouse IgM were obtained from Jackson ImmunoResearch Laboratories and used at 15 μg/ml. PMA was obtained from Sigma Chemical Co. and used at 300 ng/ml. Percoll was obtained from Amersham Pharmacia Biotech . Anti–rabbit and anti–mouse IgG-conjugated horseradish peroxidase Abs, anti-cyclin D2 Ab (sc-452), and anti-Cdk4 Ab (sc-260) were obtained from Santa Cruz Biotechnology . Anti-Cdk6 Ab was obtained from PharMingen . The production of mouse anti-cyclin D2 Ab (DCS-3 and DCS-5) and mouse anti-cyclin D3 Ab (DCS-22), used for immunoblotting, has been described ( 39 , 40 ). Antiactin Ab was obtained from Sigma Chemical Co. Rb phosphoserine 780 –specific Ab was obtained from MBL International Corp. ( 41 ). The truncated Rb substrate protein (p56 Rb ) was obtained from QED Advanced Research Technologies. Enhanced chemiluminescence reagents were obtained from Kirkegaard & Perry. Protein G–agarose was obtained from Life Technologies. To investigate intrinsic differences between B-1 and B-2 proliferative responses to phorbol ester, we evaluated the expression of D-type cyclin regulators, which function to couple mitogenic pathways to cell cycle regulatory Cdks in a number of divergent cell types ( 18 ). Because we previously identified cyclin D2 as the major D-type cyclin expressed in anti-Ig and LPS mitogenically activated mature B-2 lymphocytes, we initially focused on this G1 cyclin ( 36 ). B cells were treated with the phorbol ester PMA or anti-Ig for various periods of time, after which solubilized proteins were size fractionated by SDS-PAGE and immunoblotted with an mAb that specifically recognizes cyclin D2 ( 39 ). Stimulation of B-2 cells with anti-Ig produced substantial upregulation of cyclin D2 expression, which peaked at 24 h, as shown in Fig. 1 and as reported previously ( 36 ). In contrast, PMA treatment of B-2 cells, which fails to induce S phase entry, failed to produce any detectable increase in cyclin D2 . The results with B-1 cells were completely inverted. Stimulation of B-1 cells with anti-Ig, which fails to induce S phase entry, failed to produce a substantial increase in cyclin D2 (data not shown). However, PMA treatment of B-1 cells produced marked induction of cyclin D2 expression . The PMA-induced increase in cyclin D2 occurred quite early, reaching a peak within 2–4 h of treatment, much sooner than the onset of cyclin D2 expression in anti-Ig–stimulated B-2 cells, and significantly earlier than inducible cyclin D2 expression observed in other cells of hematopoietic origin ( 20 , 23 , 42 ). By 14 h, the level of cyclin D2 in PMA-stimulated B-1 cells had significantly declined although it was still readily detected as B-1 cells entered S phase. PMA stimulation also produced an increase in cyclin D3 expression, but this occurred much later (at 14–24 h) and took place in both B-1 and B-2 cells . Much of the delayed increase in cyclin D3 would appear to be too late to control B-1 G1-S transition, inasmuch as entry into S phase occurs at 18 h and peak S phase is found at 24–30 h of PMA stimulation ( 14 , 15 ). Cyclin D1 expression was not stimulated by PMA in B-1 or B-2 cells (data not shown). These findings indicate that cyclin D2 induction accurately reflects the divergent mitogenic responses of B-1 and B-2 cells, and strongly suggest that early cyclin D2 expression is a key feature of the B-1 cell S phase response to phorbol ester stimulation. To determine whether the early induction of cyclin D2 depends on new protein synthesis and/or new gene expression, B-1 cells were treated with PMA in the presence or absence of cycloheximide and actinomycin D for 4 h. As shown in Fig. 2 A, both cycloheximide and actinomycin D completely blocked cyclin D2 expression induced by PMA. These results suggest that cyclin D2 expression in PMA-treated B-1 cells is regulated at the level of transcription. This conclusion is supported by Northern blot analysis showing marked induction of cyclin D2 mRNA expression after B-1 cell stimulation with PMA for 1 (data not shown) and for 2 h . To evaluate whether the early transcriptional induction of cyclin D2 is accompanied by the formation of cyclin D2–Cdk4 or cyclin D2–Cdk6 holoenzyme complexes, B-1 and B-2 cells were treated with PMA or anti-Ig for various periods of time, after which extracted protein was immunoprecipitated with rabbit anti–mouse Cdk4 or rabbit anti– mouse Cdk6, resolved by SDS-PAGE, and immunoblotted for cyclin D2. As shown in Fig. 3 , the assembly of cyclin D2–Cdk4 complexes essentially paralleled the inducible expression of cyclin D2 : cyclin D2–Cdk4 complexes were readily detected in PMA-stimulated B-1 cells within 4 h; however, cyclin D2–Cdk4 complexes were not detected after anti-Ig stimulation of B-1 cells. Conversely, cyclin D2–Cdk4 complexes were readily observed in anti-Ig–stimulated B-2 cells, but were not found after B-2 stimulation with PMA. The formation of cyclin D2–Cdk6 complexes was similarly addressed: assembly of these complexes recapitulated that of cyclin D2–Cdk4 complexes although the former were much less abundant than the latter. Indeed, total Cdk4 levels exceeded Cdk6 levels in both B-1 and B-2 cells (data not shown). Notably, the observed associations between cyclin D2 and Cdk4, and between cyclin D2 and Cdk6, are specific inasmuch as no immunoreactive protein corresponding to the expected cyclin D2 molecular weight of 34 kD was detected after immunoprecipitation with (control) nonimmune serum alone (data not shown). These results indicate that cyclin D2 expression in B-1 cells is accompanied by the formation of Cdk-containing complexes. To evaluate the physiological significance of PMA-stimulated cyclin D2 expression and cyclin D2–Cdk4/Cdk6 assembly in B-1 cells, the capacity of immunoprecipitated cyclin D2 to phosphorylate exogenous Rb in vitro was assessed. As shown in Fig. 4 A, treatment of B-1 cells with PMA produced kinase-active, D2-containing complexes within 4 h, whereas treatment of B-2 cells with PMA failed to produce immunoprecipitable kinase activity. Further, B-1 cell stimulation with PMA resulted in phosphorylation of endogenous Rb within 4 h, as evidenced by the appearance of more slowly migrating species (pRb) detected by immunoblotting with anti-Rb antisera (data not shown). Moreover, endogenous Rb phosphorylation was due, at least in part, to cyclin D–Cdk4 complexes, because the cyclin D–Cdk4 phosphoacceptor Ser 780 site was phosphorylated within 4 h and the extent of this phosphorylation increased steadily over time, as measured by an anti-pRb Ser 780 Ab . Of note, Rb Ser 780 phosphorylation was not detected in B-1 cells treated with anti-Ig alone. Collectively, these results indicate that cyclin D2 is induced early by PMA in B-1 cells, and that induced cyclin D2 is capable of association with Cdks to constitute enzymatically active complexes; these results strongly suggest that cyclin D2– Cdk4 and to a lesser extent cyclin D2–Cdk6 complexes are involved in regulating PMA-mediated cell cycle progression in B-1 lymphocytes. We examined D-type cyclin expression in B-1 cells to elucidate the mechanism underlying the rapid onset of S phase produced by stimulation with phorbol ester alone. In keeping with previous results, cyclin D2 was found to be the major D-type cyclin induced by proliferative signals in B-1 and B-2 cells. PMA induced cyclin D2 early (at 2–4 h) in B-1 cells but not at measurable levels in B-2 cells, whereas in direct contrast, anti-Ig induced cyclin D2 late (at 24 h) in B-2 cells with little, if any, expression detectable in B-1 cells (data not shown). The phorbol ester–induced expression of cyclin D2 in B-1 cells is controlled at the level of transcription inasmuch as the PMA-stimulated increase in cyclin D2 protein (a) was blocked by actinomycin D, and (b) was accompanied by a rapid increase in cyclin D2 mRNA. Cyclin D3 was also induced after PMA stimulation, but this occurred much later and in both B-1 and B-2 cells. Thus, cyclin D2 expression appears to be an accurate, consistent, and early reflection of the competency of particular stimuli to induce cell cycle progression to S phase in discrete primary B cell populations. Moreover, the ease and rapidity with which a key cell cycle control protein is induced in B-1 cells may be causally related to the self- renewing characteristics of this B cell subset as well as its propensity for clonal and malignant transformation ( 1 – 6 ). The unexpected induction of cyclin D2 by PMA alone, uniquely in B-1 cells, provides a molecular basis for the observation that PMA-stimulated B-1 cells progress to S phase entry, and this is supported by the demonstration that PMA-stimulated cyclin D2 associates with Cdk4 and results in the early appearance of Rb-phosphorylating activity. The induction of kinase-active cyclin D2–containing complexes in PMA-responsive B-1 cells provides an important demonstration that only mitogenic signals induce holoenzyme formation, in this case exemplified by B cell subsets that respond differently to the same stimuli. This greatly strengthens the role of cyclin D2–Cdk4 complex formation in B cell cycle progression, previously documented by treating B-2 cells with various stimuli that produce mitogenesis, such as anti-Ig, LPS, and PMA plus ionomycin ( 36 , 43 – 45 ). It has been reported elsewhere that cyclin D2 is expressed early after murine splenic B cell (B-2 cell) stimulation ( 46 ). We do not find this to be so; instead, we find that the timing of cyclin D2 expression anticipates the timing of the S phase peak by ∼24 h in both B-1 and B-2 cells ( 14 , 15 ). The origin of the disparity in these sets of results remains uncertain, although it should be noted that in the study by Howard and colleagues, large, rather than small, B-2 cells were examined, which may reflect prior activation ( 46 ). However, the results we obtained are not simply a function of large size, inasmuch as there was little induction of cyclin D2 in B-1 cells stimulated by anti-Ig in our study (data not shown). Our earlier observation that B-1 cells progress in cell cycle to S phase in response to phorbol ester treatment, whereas B-2 cells require treatment with a calcium ionophore in addition to phorbol ester, gave rise to the idea that B-1 cells endogenously express some signaling component or growth-promoting molecule that requires calcium ionophore for expression in B-2 cells. This notion is supported by our finding that B-2 cells stimulated with anti-Ig for 2 d become responsive to phorbol ester alone ( 47 ), further suggesting that a discrete alteration, inducible by sIg signaling in mature B-2 cells, is responsible for phorbol ester responsiveness. The present results suggest that this alteration, perhaps in the form of an sIg-triggered signaling component or growth-promoting molecule that is constitutively expressed in B-1 cells, relaxes (or fulfills one of) the requirements for cyclin D2 expression. Our recent finding that B-1 cells constitutively express nuclear, activated STAT3 that is triggered by PMA plus calcium ionophore (as well as by anti-Ig) in B-2 cells ( 10 ) suggests that one or more STAT proteins may play a role in regulating cyclin D2 expression. | Other | biomedical | en | 0.999998 |
10359572 | E14 ES cells derived from strain 129/Ola were used throughout the experiment. ES bcl-x +/+ , bcl-x +/− , and bcl-x −/− cell lines were produced as described previously ( 21 ). Genomic DNA containing the bcl-x locus was isolated from a library of mouse strain 129/Sv DNA. A 1.8-kb XhoI-BamHI fragment containing most of the bcl-x coding region was replaced with either a PGK- neo polyadenylated (poly A) cassette or a PGK- hyg poly A cassette. Both targeting vectors contain 6.0-kb 5′ and 1.0-kb 3′ regions of homology with the drug-resistance markers and a PGK- tk poly A cassette. Transfection and selection were performed as described ( 31 ). DNA prepared from ES cells was digested with EcoRV, transferred to a nylon membrane, and then hybridized with the 0.4-kb KpnI-PstI probe that flanked the 3′ homology region. The expected sizes of wild-type bcl-x , mutant bcl-x with the neo targeting vector, and mutant bcl-x with the hyg targeting vector were 9.8, 7.0, and 5.5 kb, and were detected in wild-type, bcl-x +/− , and bcl-x −/− ES clones, respectively. ES bcl-x +/+ , bcl-x +/− , and bcl-x −/− cells were injected into the 3.5-d post-coitum blastocysts from C57BL/6 mice to generate chimeric mice. GPI (glucose phosphoisomerase) isozymes were used to analyze the contribution of ES cells in various organs of chimeric mice. The GPI isozyme of B6 mice and of 129/ Ola mice from which E14 ES cells were established are GPI-1A and GPI-1B, respectively. Separation and detection of GPI isoenzymes were performed essentially as described ( 32 ). Chimeric mice were perfuged with PBS to eliminate blood cell contamination, and dissected tissues were kept at –70°C. Frozen tissue samples were thawed and gently homogenized in water, and cells were then lysed by three rounds of freezing and thawing. After centrifugation of the homogenates, the supernatants were diluted with water and then electrophoresed on Titan III Zip Zone cellulose acetate plates (Helena Laboratories) in Tris-glycine buffer (25 mM Tris, 200 mM glycine, pH 8.5) for 4 h at 150 V at 4°C. The stainings were performed by overlaying the mixture consisting of 2 ml of 0.2 M Tris-HCl (pH 8.0), 0.1 ml each of 0.25 M magnesium acetate, 10 mg/ml NADP, and 100 mg/ml fructose 6-phosphate, and 0.2 ml of MTT, 0.05 ml of 2.5 mg/ml phenazine methosulfate, 5 μl glucose 6-phosphate dehydrogenase (140 U/ml; Sigma Chemical Co. ), and 5 ml of 2% agarose. The GPI isozyme bands appeared after a few minutes in the dark. Density of the bands was analyzed by densitometer. The hemoglobin type of B6 mice and 129/Ola mice are single ( Hbb s /Hbb s ) and diffuse ( Hbb d /Hbb d ), respectively, and were used to evaluate the contribution of ES cells in circulating erythrocytes of chimeric mice. These two types of hemoglobin can be distinguished by electrophoresis. Cellulose acetate electrophoresis of cystamine-modified hemoglobins was performed essentially as described ( 33 ). Whole blood in PBS containing 50 mM EDTA was layered onto 2 vol of Histopaque-1077 ( Sigma Chemical Co. ) and centrifuged at 3,500 g for 20 min at room temperature. The pellet, enriched for RBCs, was collected. 10 μl purified RBCs was added to 300 μl cystamine lysis buffer (12.5 mg/ml cystamine dihydrochloride, 1 mM dithiothreitol, 0.55% ammonium hydroxide) and agitated to lyse the RBCs. The samples were applied to Titan III cellulose acetate plates and run in TBE buffer (0.18 M Tris, 0.10 M boric acid, 0.002 M EDTA) for 40 min at 300 V. The plates were placed in staining solution (1% Ponceau S, 5% TCA) for 10 min and rinsed in three changes of 5% acetic acid for 10 min each. The percentage contributions of ES cells in adult chimera were examined using the allotype of GPI from various nonhematopoietic organs, such as the liver and kidney. The hemoglobin type analysis data were obtained from the chimera in which the contribution of ES cells to nonhematopoietic organs was >50%. ES bcl-x +/+ , bcl-x +/− , and bcl-x −/− cells were cultured on embryonic fibroblasts as feeder cells in the presence of a saturated dose of leukemia inhibitory factor using the standard procedure ( 31 ). The culture of OP9 stromal cells and the differentiation induction method were carried out as described ( 27 ). OP9 stromal cells were maintained in α-MEM (Life Technologies, Inc.) supplemented with 20% FCS (Summit) and standard antibiotics ( 27 , 34 ). 10 5 ES cells were transferred onto confluent OP9 stromal cells in 10-cm culture dishes (Nunc). After day 3 of the induction, human recombinant EPO (provided by Kirin Brewery Co. Ltd.) was added at a final concentration of 2 U/ml during the differentiation induction. The induced cells were trypsinized at day 5, and 10 6 cells were transferred onto fresh OP9 cells on a 10-cm plate. Nonadherent cells were harvested on day 6, 7, or 8 to obtain EryP. On day 10, all of the cells on individual 10-cm plates were harvested by vigorous pipetting and transferred to individual 10-cm plates with a fresh OP9 cell layer, and then both adherent and nonadherent cells were harvested to obtain EryD after day 11. To determine the differentiation capacity of bcl-x +/+ and bcl-x −/− ES cells, on day 8 of the differentiation induction 30 hematopoietic clusters were picked and transferred to semisolid culture medium containing IL-3 (50 U/ml) and EPO (2 U/ml), which promote erythroid and myeloid cell growth. As previously reported, the day 8 hematopoietic clusters have a clonal origin and can differentiate into erythroid and various myeloid lineages under these conditions ( 27 ). 5 d after transfer into this myeloid permissive semisolid media, individual colonies were picked, cytospin specimens were stained with May-Grunwald Giemsa, and the emerged blood cells were typed. More than 75% of the differentiation-induced cells between days 6 and 8 were EryP, and the same proportion of cells between days 11 and 13 were EryD. In some experiments, the purification of EryP and EryD was carried out with metrizamide step gradient centrifugation. The cells were washed once with Tyrode's buffer containing 0.1% gelatin. 1–5 × 10 6 cells in 1 ml of the Tyrode's buffer were layered on a step gradient of 2.0 ml of 30% wt/vol metrizamide (Nacalai Tesque) and 2.0 ml of 15% wt/vol metrizamide. The cells were centrifuged at room temperature for 20 min at 400 g at the interface between the 15% metrizamide and the 30% metrizamide. The cells remaining at this interface were collected and washed three times with α-MEM with 20% FCS. After the purification, >98% of the cells were dianisidine-positive erythroid cells, with a viability of 95–98%. Hemoglobin-containing cells were confirmed with dianisidine staining as reported previously ( 35 ). To examine EPO responsiveness , 3.0 × 10 5 /ml dianisidine-positive differentiation-induced cells were cultured in 6-well plates containing 20% FCS supplemented with α-MEM in the absence or presence of 2 U/ml EPO without the OP9 cell layer. The viability of the cells was examined using the trypan blue dye exclusion method and calculated by counting >200 cells. May-Grunwald Giemsa staining of cytospin specimens was also carried out to examine the morphological changes of apoptotic EryP. The number of hemoglobin-containing cells and the percentage of viable cells are reported as mean ± SD. The t test was used for statistical analysis, using StatView software. After culture for 18 h in the presence or absence of 2 U/ml EPO, 10 6 cells were harvested by centrifugation at 200 g for 10 min. Low molecular weight DNA was extracted following the method of Sellins and Cohen ( 36 ). One quarter of the extracted DNA was electrophoresed in a 2.0% agarose gel and stained with ethidium bromide. ES cells of bcl-x +/+ , bcl-x +/− , and bcl-x −/− genotypes were injected into the blastocysts of C57BL/6 mice to assess their ability to differentiate into various organs in vivo. There were no differences in the growth of parental bcl-x +/+ , bcl-x +/− , and bcl-x −/− ES cells (data not shown). Chimeric mice of >80% chimerism by coat color were analyzed for the contribution of the injected ES cells in various organs based on the activity of GPI-1 isozymes. E14 ES cell–derived cells express the GPI-1A isozyme, which is easily distinguishable from the GPI-1B isozyme of the C57BL/6-derived cells ( 37 ). As for heart, kidney, and muscle, there were no differences in the contribution of parental bcl-x +/+ , bcl-x +/− , and bcl-x −/− ES cells (Table I ). On the other hand, the contribution of bcl-x −/− ES cells to lymphoid organs such as spleen and thymus was significantly lower than that of bcl-x +/+ or bcl-x +/− ES cells. This result is compatible with a previous report on the shortened life span of bcl-x −/− immature lymphocytes ( 21 ). Two bcl-x −/− ES cell lines (clones 18 and 3a) were analyzed for the function of the bcl-x gene in hematopoiesis. Host blastocysts from the strain C57BL/6 are homozygous for the Hbb s β-globin haplotype . In contrast, 129/Ola mice, from which the ES cell line of this study was established, are homozygous for the Hbb d haplotype . The proportion of major and minor hemoglobin shows the contribution of the injected ES cells to mature circulating EryD in the chimeric mice. When bcl-x +/+ or bcl-x +/− ES cells were used for chimera production, the contribution of the ES cells to the circulating EryD was proportional to the contribution of ES cells to the other organs. However, when bcl-x −/− ES cells were used, no contribution of the ES cells to circulating EryD was detected, despite their significant contribution to the other nonlymphohematopoietic organs . These data clearly show that bcl-x has an essential role for the in vivo production of EryD. In addition, the results from the chimeric mice demonstrate that the contribution of bcl-x to EryD production is cell autonomous, since the hematopoietic microenvironment in the chimeric animal could not complement the defective EryD production from bcl-x −/− ES cells. The process of definitive erythroid lineage cell production can be divided into two stages. The earlier stage is commitment and involves differentiation from multipotential progenitor cells to committed erythroid lineage cells. The later stage is proliferation and maturation of the committed EryD progenitors. Two possibilities might account for the failure of EryD production by bcl-x −/− ES cells. One is the commitment failure of the multipotential hematopoietic progenitor cells into erythroid lineage cells, and the other is the proliferation and maturation failure of the committed erythroid cells. To analyze these possibilities, in vitro differentiation induction from bcl-x −/− and bcl-x +/+ ES cells was carried out using OP9 stromal cells. The differentiation capacity of day 8 in vitro differentiation–induced hematopoietic progenitor cells was examined. The day 8 hematopoietic clusters were of clonal origin, and most of them could differentiate into multiple hematopoietic lineages, including the definitive erythroid lineage ( 27 ). There were no differences in the number of day 8 hematopoietic clusters induced from bcl-x +/+ or bcl-x −/− ES cells (data not shown). As shown in Table II , there were also no significant differences in the types of colonies that developed from the day 8 hematopoietic clusters in methylcellulose semisolid media containing IL-3 and EPO as growth factors. These data show that bcl-x is not necessary for the differentiation of the definitive erythroid lineage from multipotential hematopoietic progenitors. Since Bcl-X is essential for the production of fully mature EryD, bcl-x seemed to play important roles during the maturation of EryD after commitment to the erythroid lineage. To further analyze the function of bcl-x in the production of erythroid lineage cells, in vitro differentiation induction into erythroid lineage cells from bcl-x −/− and bcl-x +/+ ES cells was carried out using OP9 stromal cells in the presence of EPO. The number of hemoglobin-containing dianisidine-positive cells was counted between days 6 and 8 and between days 12 and 14. As previously reported, EryP and EryD appear in the former and latter periods, respectively ( 29 ). On day 6, the number of bcl-x −/− EryP was the same as bcl-x +/+ EryP. However, on day 8, the difference between the number of bcl-x −/− EryP and bcl-x +/+ EryP became pronounced. As shown in Table III , on day 8 the number of bcl-x −/− EryP was only ∼10% that of bcl-x +/+ EryP. Moreover, the number of bcl-x −/− EryP on day 8 was ∼10% that of the day 7 bcl-x −/− EryP, suggesting that cell death occurred between these days. Similar results were obtained with EryD. There was no difference in the number of bcl-x −/− EryD and bcl-x +/+ EryD on day 12. But, the difference became significant with maturation, and the number of EryD originating from bcl-x −/− ES cells was about one quarter that from bcl-x +/+ ES cells on day 14. The percentage of viable cells was next examined, because apoptotic cell death of bcl-x −/− erythroid cells was suspected (Table IV ). Here again, there were no significant differences between the day 7 EryP and the day 12 EryD, but the differences became significant thereafter. The percentages of viable cells mainly reflect viable erythroid cells, because the vast majority of the cells during differentiation induction belong to the erythroid lineage. More than 80% of the cells harvested between days 7 and 8, and >90% of the cells harvested between days 12 and 14 were EryP and EryD, respectively, when the bcl-x +/+ ES cells were induced for differentiation. The percentage of viable bcl-x −/− EryP seems relatively high for the very low number of bcl-x −/− EryP (Table IV ). This apparent discrepancy was probably due to the removal of dead bcl-x −/− EryP by adherent macrophages. Electron microscopic features at about day 8 of the differentiation induction showed macrophages with prominently phagocytosed dead EryP (data not shown). The morphological features and DNA fragmentation of the induced cells were analyzed to confirm that the decreased number and viability of bcl-x −/− cells were due to apoptosis. Immature EryP and EryD were purified by metrizamide density gradient separation on days 6 and 12 of the differentiation induction, respectively. At these times, no differences in number and viability were detectable between the bcl-x +/+ and the bcl-x −/− erythroid cells as shown above. Using this purification method, >98% of the purified cells were erythroid lineage cells and their viability was 95–98%. These purified EryP and EryD were cultured on OP9 cells for 2 d in the presence of EPO, and the cells were harvested. Their morphological and molecular features were then examined. The bcl-x +/+ EryP were viable and had mature morphology on day 8. In contrast, the bcl-x −/− EryP had fragmented nuclei with clumped chromatin, suggestive of apoptosis. On day 14, the vast majority of the cells were enucleated mature EryD when the bcl-x +/+ ES cells were induced for differentiation, whereas enucleated EryD were rarely observed when the bcl-x −/− ES cells were induced. The hemoglobinized bcl-x −/− EryD were mainly nucleated erythroblasts. Thus, it was difficult to find viable, fully mature EryP and EryD on days 8 and 14 of the differentiation induction of bcl-x −/− ES cells, respectively, although immature EryP and EryD were equally viable on days 6 and 12, respectively. Low molecular weight DNA was extracted from the cells, and agarose gel electrophoresis was carried out . The nucleosomal DNA ladder, which is characteristic of apoptotic cells, was observed to be significantly more abundant in the bcl-x −/− erythroid lineage cells than in the bcl-x +/+ erythroid lineage cells. These data clearly demonstrate that the bcl-x −/− erythroid lineage cells underwent apoptosis during the end stage of maturation. EPO is required by immature erythroid lineage cells to prevent apoptosis. To analyze the roles of EPO and Bcl-X during the maturation of erythroid cells, an EPO deprivation experiment was performed. On days 6.5 and 11.5 of the differentiation induction, immature EryP and EryD were purified. These erythroid cells were not considered to be late-stage erythroid progenitors, but rather immature erythroid cells, because hemoglobinization had already begun but the cells still showed an immature morphology. After purification on days 6.5 and 11.5, the bcl-x +/+ and bcl-x −/− erythroid lineage cells were cultured without OP9 stromal cells in the presence or absence of EPO. 1 and 1.5 d after the culture, the viability of the cells was examined . Deprivation of EPO at this stage affected the viability of EryP much more severely than EryD; however, the results of the examination of EryP and EryD were essentially the same. In the presence of EPO, there were significant differences in the viability of bcl-x +/+ and bcl-x −/− erythroid cells . Even in the absence of EPO, the differences in the viability were significant ( P < 0.005 by t test). Furthermore, EPO deprivation decreased the viability of both EryP and EryD even in the context of bcl-x null . Taken together, EPO deprivation and the bcl-x null mutation affected cell death additively. bcl-x , a member of the bcl-2 family of apoptosis regulatory genes, can be alternatively spliced to produce two protein isoforms, Bcl-X L and Bcl-X S ( 6 , 17 , 18 ). Bcl-X L exhibits remarkable structural homology with Bcl-2 and inhibits apoptotic cell death. Evidence from studies of cell lines and transgenic mice suggests that the bcl-2 gene family plays a role in the survival of erythroid lineage ( 22 , 23 , 25 ). The expression pattern of bcl-x obtained from primary human erythroid cells and mouse erythroblasts infected with the anemia-inducing strain of Friend virus (FVA) suggests that bcl-x among bcl-2 gene family members is the principal antiapoptotic regulator during late erythroid differentiation ( 24 ). Bcl-X is strongly increased during the terminal differentiation stages of human and mouse erythroblasts in the presence of EPO, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression leads to an apparent level ∼50 times greater than the level in proerythroblasts before EPO stimulation. In contrast, neither mouse nor human erythroblasts express Bcl-2 transcript or protein. The levels of other Bcl-2 family members, Bax and Bad proteins, remain relatively constant throughout differentiation, but diminish at the end of terminal differentiation near the time of enucleation. These data on the expression pattern of the bcl-2 gene family products imply that bcl-x is the critical member of the bcl- 2 family during erythroid differentiation. Furthermore, the increased apoptotic cell death of hematopoietic cells in bcl-x −/− fetal liver and the absence of defects in the fetal liver of bcl-2 −/− mice support the hypothesis that Bcl-X, not Bcl-2, is the important factor in erythropoiesis ( 21 , 38 – 40 ). However, there is no direct evidence for the role of bcl-x in erythropoiesis, despite this circumstantial evidence. To examine the critical physiological roles of the bcl-x gene on hematopoiesis, chimeric mice production and OP9 in vitro differentiation induction were carried out using bcl-x −/− ES cells. There was no contribution by bcl-x −/− ES cells to the circulating EryD in the chimeric mice, demonstrating that bcl-x is indispensable for the full maturation of EryD. Defects in erythropoiesis were analyzed in detail using in vitro differentiation induction from ES cells by coculturing the cells on the macrophage colony-stimulating factor–deficient OP9 stromal cell line (the OP9 system ). Two waves of erythroid cell production were observed when ES cells were cocultured with OP9 stromal cells. The development of hematopoietic cells in this OP9 system is very similar to that observed in developing mouse embryos ( 27 , 29 , 41 ). The first wave of erythropoiesis, appearing between days 6 and 8 of the induction, and the second wave, appearing after day 10 of the induction, correspond to primitive and definitive erythropoiesis, respectively, by morphological and biochemical criteria ( 29 ). Our data clearly show that apoptotic cell death of bcl-x −/− erythroid lineage cells was observed only at the end of maturation in both primitive and definitive erythropoiesis. bcl-x –deficient mice die at about embryonic day 13 ( 21 ). Extensive apoptotic cell death is evident in hematopoietic cells in fetal liver. There is a threefold increase in TUNEL (for terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling)–labeled apoptotic nuclei in histologically identifiable hematopoietic cells in embryonic day 12.5 bcl-x −/− liver compared with wild-type tissue. These data suggest that erythropoiesis in the fetal liver of bcl-x −/− mice is impaired because the vast majority of fetal liver hematopoietic cells at this gestational stage are erythroid lineage cells. The data on defective EryD production by the OP9 system are consistent with these in vivo data. During in vitro differentiation, although significant numbers of bcl-x −/− erythroblasts survived, almost no enucleated erythrocytes could be detected. This result shows that the pivotal function of bcl-x is expressed at the late stage of erythroid maturation. The critical role of bcl-x seems to be brought about by the remarkable increase of Bcl-X protein at the end of erythroid maturation. In vitro differentiation induction shows that apoptotic cell death of EryP also occurred at the late stage of maturation, which is consistent with primitive erythropoiesis in the bcl-x −/− mice. The effect of bcl-x on primitive erythropoiesis was not examined extensively because of the difficulty counting EryP numbers correctly in tiny mouse embryos. However, the following two lines of evidence strongly suggest that EryP production in bcl-x −/− mice was impaired to some extent. First, bcl-x −/− mice were paler than the control mice at day 12.5 of gestation (21; our unpublished data). At this gestational stage, >95% of the erythrocytes are still EryP, although the relative percentage of EryP begins to decrease ( 42 ). Second, bcl-x −/− mice died at day 13 of gestation, which is earlier than the mutant mice lacking only definitive hematopoiesis by gene targeting of c-myb ( 43 ). The c-myb targeted mice were severely anemic by day 15; however, the mutant mice appeared normal at day 13 of gestation. On the other hand, EPO signal–deficient mice, which have a partial defect in primitive erythropoiesis and a complete defect in definitive erythropoiesis, die at day 13 of gestation, as early as bcl-x −/− mice ( 12 , 13 ). Meanwhile, it is reasonable to consider that a similar time course of cell death of bcl-x −/− EryP and bcl-x −/− EryD would reflect a similar underlying molecular mechanism of cell death caused by the null mutation of bcl-x . The cause of cell death might be explained by the relationship between massive heme synthesis at the end of maturation of erythroid lineage cells and the antioxidant function of Bcl-X L ( 24 ). Of the various methods of in vitro hematopoietic differentiation from ES cells, the OP9 system has several remarkable advantages, among which are their potential to differentiate into fully mature blood cells and the feasibility of analyzing the cells quantitatively ( 26 , 27 ). To analyze the defective erythropoiesis from bcl-x −/− ES cells, quantitative analysis of the fully mature erythroid cells was necessary. However, such analysis is almost impossible by the conventional in vitro differentiation induction method with embryoid body formation. The other substantial advantage of the OP9 system is that hematopoietic microenvironment and hematopoietic cells can be analyzed separately by this method. It is well known that hematopoiesis is maintained by the hematopoietic microenvironment, such as stromal cells. By the conventional embryoid body formation method, both hematopoietic microenvironment and hematopoietic cells are induced from ES cells and are unseparable. But with the OP9 system, hematopoietic cells are induced from ES cells while the hematopoietic microenvironment is provided by OP9 stromal cells. It is concluded from the defective EryD production in the chimeric mice that this defect is cell autonomous. In addition, the defective erythropoiesis of the bcl-x −/− genotype with the OP9 system strongly supports this conclusion. The production of definitive erythroid lineage cells is controlled by EPO ( 11 ). EPO induces the proliferation and prevents the apoptotic cell death of EryD. The antiapoptotic effect of EPO on EryD was observed from late erythroid progenitors (CFU-E) until the onset of hemoglobinization ( 2 , 14 – 16 ). In other words, EPO-deprived apoptotic cell death is hardly at all observed at the end of maturation when maximal hemoglobin synthesis occurs. On the other hand, massive apoptotic cell death of bcl-x −/− EryD was observed after day 13 of differentiation induction. It is reasonable to consider that the accumulation of Bcl-X (probably Bcl-X L ) resulting from EPO stimulation prevents the apoptotic cell death of terminally differentiated erythroid cells. However, the accumulation of Bcl-X cannot be the only way to explain the antiapoptotic effect of EPO, because EPO prevents apoptotic cell death to some extent even in the absence of Bcl-X . Taken together, it is likely that EPO has dual roles to prevent apoptotic cell death at different differentiation stages. | Study | biomedical | en | 0.999998 |
10359573 | Male Wistar rats (weighing 240–300 g) were initially screened by determining whether they could run on a motor-driven treadmill at 20–25 m/min, 0% grade, for 20 min (exercise began and ended with a 5-min “warm-up” and “cool-down” at 15 m/min and 0% grade; the duration of exercise included the warm-up and cool-down periods). At least 2 wk later, only rats that completed the exercise screening test were used for all experiments described in this study. For exercise experiments, rats received one session of exercise, which consisted of running on a treadmill at 27–30 m/min, 0% grade, for 25–30 min, including the warm-up and cool-down periods as described for the screening test. Rats in the sham-treated control group were placed on the treadmill apparatus without exercise for 30 min and allowed to recover at room temperature for a defined interval, as indicated in the figure legends. Some rats were not placed on the treadmill apparatus (untreated controls). After the exercise session, rats were anesthetized with sodium pentobarbital, and the chest of each rat was opened via a right parasternal sternomectomy. The heart was exposed, and 7-0–type silk thread was then passed around the left coronary artery (LCA) 3–4 mm distal to the origin of LCA. After a 10-min period of stabilization, the LCA was ligated. The snare was released after 20 min of coronary occlusion. Reperfusion was indicated by a change in the color of the ventricular surface. Arrhythmias were monitored by electrocardiogram. Ventricular fibrillation (VF) was defined according to the criteria of the Lambeth Conventions ( 21 ). If VF occurred during ischemia and did not resolve spontaneously within 3 s, manual cardioversion was attempted by gentle palpation of the nonischemic region of the heart. We excluded from infarct size analysis rats in which VF persisted for more than 6 s or in which cardioversion had to be performed >3 times. 48 h after reperfusion, the rats were killed by an overdose of sodium pentobarbital (100 mg/kg). The heart was excised and the size of the infarct was evaluated by a method of double staining using Evans blue dye and triphenyltetrazolium chloride (TTC) as previously described ( 22 , 23 ). The size of the myocardial infarct, i.e., the area without TTC staining, was expressed as a percentage of the ischemic area at risk, i.e., the area free of Evans blue dye. The rate–pressure product showed no significant difference among the groups before ischemia, at the end of the ischemic period, or 30 min after reperfusion (data not shown). We measured the activity and content of SODs, intrinsic radical scavengers that are supposed to protect the heart against ischemia/reperfusion injury. At the time after exercise indicated in the figures, rats in the exercise and control groups were killed by administering an overdose of sodium pentobarbital. The blood was washed out of the left and right coronary arteries with PBS from the ascending aorta in a retrograde manner. The left ventricular myocardium was rapidly frozen in liquid nitrogen and stored at −80°C. SOD activity of the myocardial samples was determined by the nitroblue tetrazolium method ( 24 ) with a modification for sample preparation ( 23 ). Mn-SOD content was measured by means of an ELISA, as we previously reported ( 12 ). The measurements of Mn-SOD activity and content were performed at least in triplicate. The activity and content of Mn-SOD was corrected for the protein concentration in the supernatant determined according to the Lowry method ( 24a ). To examine whether TNF-α and IL-1β are involved in the activation of Mn-SOD after exercise, the levels of TNF-α and IL-1β in myocardium were measured. Myocardium was homogenized in 20 mM PBS containing 1 mM EDTA and centrifuged at 900 g for 15 min. The levels of TNF-α and IL-1β in the supernatant were measured by means of ELISA kits (TNF-α, Genzyme Co. ; IL-1β, Immuno-Biological Labs. Co.). The measurements of cytokines were performed in triplicate. The levels of TNF-α and IL-1β were corrected for the protein concentration in the supernatant determined by the Lowry method ( 24a ). To manipulate the level of expression of Mn-SOD, 22-mer phosphorothioate derivative of the antisense oligodeoxyribonucleotide ([ASODN]; CACGCCGCCCGACACAACATTG), sense oligodeoxyribonucleotide ([SODN]; CAATGTTGTGTCGGGCGGCGTG), or scrambled ODN (TCTCAGTGAGCCCTCATTCTGT) was injected intraperitoneally just after exercise at a dose of 10 mg/kg. To optimize the experimental conditions for the in vivo delivery of systemically injected ODNs, we evaluated the time course of their accumulation in the heart. In experiments with 5′ FITC–labeled ASODN to Mn-SOD, we found that significant labeling of these tissues occurred at the following times after the intraperitoneal injection: at 2–4 h in endothelial cells, at 4 h in vascular smooth muscle, and at 8 h in cardiac myocytes (unpublished observation). We could not determine the subcellular distribution of ASODN in the cardiac myocytes due to technical limitations. To neutralize the increases of TNF-α and IL-1β after exercise, anti-murine TNF-α antibody (0.5 ml) and/or anti-murine IL-1β antibody (0.5 mg), obtained from Genzyme , were infused intraperitoneally 30 min before exercise. Both antibodies cross-react with rat cytokines. Recombinant murine TNF-α and recombinant murine IL-1β were obtained from Genzyme . The recombinant cytokines were diluted in pathogen-free saline on the day of injection. Rats received an intravenous injection of cytokine via the right femoral vein for 2 min at a dose of 1.5 μg/kg body weight to examine the involvement of TNF-α in exercise-induced cardioprotection. N -2-mercaptopropionyl glycine (MPG) is a low molecular weight, synthetic analogue of glutathione and a diffusible and membrane-permeable antioxidant. This agent especially scavenges hydrogen peroxide and hydroxyl radicals. To determine the involvement of reactive oxygen species during exercise in the acquisition of tolerance against ischemia/reperfusion injury, MPG (100 mg/kg) was infused intraperitoneally 10 min before the 20-min exercise session or 30 min before the injection of TNF-α. As the half-time for elimination of MPG is ∼7 min in vivo ( 25 ), MPG should not be effective during myocardial ischemia/reperfusion period. Treatment with MPG also did not alter the area at risk (data not shown), indicating that its effect on infarct size may not be mediated via changes in collateral flow. Figure data are expressed as mean ± SEM. Intergroup comparisons were assessed for significance by using one-way analysis of variance (ANOVA) with Bonferroni's post hoc test for multiple comparisons. A level of P < 0.05 was accepted as statistically significant. A total of 28 rats developed serious VF during occlusion (10 in sham-treated control group, 4 in exercise group, 3 in AODN-treated group, 2 in exercise group treated with AODN, 2 in exercise group pretreated with anti-murine TNF-α and anti-murine IL-1β antibodies, 4 in TNF-α–treated group, 2 in exercise group pretreated with MPG, and 1 in sham-treated control group pretreated with MPG) and were excluded from the evaluation of myocardial infarct size. 29 rats died prematurely (probably because of arrhythmia or heart failure) during the 48-h reperfusion period (7 in sham-treated control group, 4 in exercise group, 3 in AODN-treated group, 1 in SODN-treated group, 1 in exercise group treated with AODN, 1 in exercise group treated with scrambled ODN, 1 in exercise group pretreated with anti-murine TNF-α antibody, 2 in exercise group pretreated with anti-murine IL-1β antibody, 2 in exercise group pretreated with anti-murine TNF-α and anti-murine IL-1β antibodies, 5 in TNF-α–treated group, 1 in exercise group pretreated with MPG, and 1 in sham-treated control group pretreated with MPG). We examined the size of myocardial infarct after occlusion (20 min)/reperfusion (48 h) in the LCA in rats, which received one session of exercise. There were no significant differences in the size of the myocardial infarct for recovery intervals of 0.5–72 h among the sham-treated control groups (data not shown). As shown in Fig. 1 (top), the size of the myocardial infarct after reperfusion in the sham-treated control rats did not differ significantly from the size of the infarct observed in rats at 3, 24, and 72 h after exercise. However, rats subjected to ischemia at 0.5, 36, 48, and 60 h after the exercise session exhibited a marked decrease relative to the control rats in the size of the myocardial infarct after reperfusion. The infarct-limiting effect of exercise was slightly less at 36 and 60 h than at 0.5 and 48 h after exercise. The area at risk did not differ significantly among the groups (data not shown). Reactive oxygen species produced during ischemia/reperfusion in the heart have been believed to induce myocardial cell damage (26– 28). An intrinsic radical scavenger, Mn-SOD protects hearts against oxygen free radicals. To examine the involvement of Mn-SOD in the cardioprotective effect of exercise, we measured Mn-SOD activity after treadmill exercise. There were no significant differences in the activity and content of Mn-SOD for recovery intervals of 0.5–72 h among the sham-treated control groups (data not shown). Mn-SOD activity was unchanged at 3, 24, and 72 h after exercise but was significantly increased at 0.5 and 48 h after exercise, as compared with the sham-treated control . The biphasic time course of the change in Mn-SOD activity in the myocardium of rats exposed to exercise resembled the time course for the protection against ischemia/ reperfusion injury. The myocardial content of Mn-SOD did not change relative to the sham-treated control group within 24 h after exercise. Thereafter, however, the Mn-SOD level increased significantly, reaching 137% of the control value 48 h after exercise . The Mn-SOD level returned to control values within 72 h after exercise. The activity of the cytosolic isoform of SOD (Cu, Zn-SOD) was unaffected by exercise (data not shown). We examined the relationship between the acquisition of tolerance to ischemia/reperfusion and the induction of Mn-SOD in the myocardium 48 h after exercise. We manipulated the level of expression of Mn-SOD using ASODN. The administration of ASODN completely inhibited the increases in Mn-SOD activity and content 48 h after exercise . However, SODN did not attenuate the increases in Mn-SOD activity and content induced by exercise. As shown in Fig. 2 (top), the expected decrease in infarct size induced by exercise was also abolished in rats treated with ASODN to Mn-SOD, in which the induction of Mn-SOD was specifically inhibited. SODN, which did not attenuate the induction of Mn-SOD in the myocardium after exercise, did not abolish the protective effect of exercise. Administration of the scrambled oligonucleotide had no effect on infarct size or on Mn-SOD activity as seen with SODN. Administration of ASODN decreased the activity and content of Mn-SOD and increased the infarct size after reperfusion in sham-treated control rats. To examine the contribution of the cytokines to exercise-induced cardioprotection, we investigated the time course of the induction of TNF-α and IL-1β in the myocardium after exercise using the ELISA method. There were no significant differences in TNF-α and IL-1β contents for recovery intervals of 0–30 min among the sham-treated control groups (data not shown). As illustrated in Fig. 3 (top), an increase in TNF-α content was evident immediately after exercise. The level of TNF-α peaked 10 min after exercise and then decreased rapidly toward the baseline within 20 min. Immediately after exercise, the myocardial level of IL-1β also increased significantly, as compared with the sham-treated control group, and declined to the control level within 10 min . To determine whether the production of these cytokines after exercise may be involved in the acquisition of tolerance to ischemia/reperfusion, we administered the antibody to these cytokines intraperitoneally 30 min before exercise. The intraperitoneal administration of anti-murine TNF-α and anti-murine IL-1β antibody 30 min before exercise inhibited the observed increase in levels of TNF-α and IL-1β, respectively, after exercise . As shown in Fig. 4 (top), the administration of an antibody to TNF-α did not influence the size of infarct at 0.5 or 48 h after exercise. The administration of an antibody to IL-1β also did not alter the size of the myocardial infarct at 0.5 or 48 h after exercise. However, the simultaneous administration of the antibodies to TNF-α and IL-1β abolished the protection against ischemic damage at 0.5 and 48 h after exercise. Antibody to TNF-α or IL-1β had no effect on the increase in Mn-SOD activity induced by exercise . The simultaneous administration of the antibodies to these cytokines eliminated the activation of Mn-SOD at 0.5 and 48 h after exercise. We next investigated whether TNF-α could mimic biphasic cardioprotection. In the untreated control groups, the size of myocardial infarct and Mn-SOD activity remained constant after vehicle (saline) injection in the time course of the experiment (data not shown). As shown in Fig. 5 (top), the infusion of murine rTNF-α reduced the size of the infarct in a biphasic pattern. The first phase of protection occurred 0.5 h after the administration of TNF-α. When such tolerance had disappeared, the second phase of protection was observed. Peak protection during the second phase was achieved 48 h after administration of a bolus injection of TNF-α. The time course was identical to that for the cardioprotection induced by exercise . IL-1β also mimicked this biphasic cardioprotective effect (data not shown). The activity of Mn-SOD increased significantly in a biphasic manner 48 as well as 0.5 h after the bolus injection of TNF-α . No change in Mn-SOD activity was observed 3, 24, and 72 h after TNF-α administration. IL-1β also activated Mn-SOD in a biphasic manner (data not shown). The time course of cardioprotection coincided with that for the increase of Mn-SOD activity after the administration of TNF-α. To determine whether the generation of reactive oxygen species during exercise may be involved in the acquisition of tolerance to ischemia/reperfusion, we administered an antioxidant, MPG, 10 min before exercise. Although this agent did not alter the size of the myocardial infarct in the sham-treated control rats, MPG completely abolished the protection against ischemia/reperfusion injury at 0.5 and 48 h after exercise . MPG did not alter Mn-SOD activity in sham-treated control rats, whereas it completely abolished the early (0.5-h) and late (48-h) peaks in Mn-SOD activity that followed either exercise or the injection of TNF-α . MPG completely abolished the exercise-induced increase in TNF-α and IL-1β in myocardial tissue . A substantial body of epidemiologic evidence demonstrates that exercise reduces cardiovascular mortality. Although exercise can improve other known risk factors for CAD, such as elevated plasma lipid level, obesity, and glucose intolerance, exercise appears to exert an independent cardioprotective effect ( 1 , 2 ). The present results provide the first demonstration that the physiological stimulus of exercise has a directly beneficial effect on myocardial ischemia/reperfusion injury in a biphasic manner in an animal model. Exercise appears to induce structural and/or functional adaptation, leading to an increase in coronary vascular transport capacity ( 3 ). Bloor et al. reported that exercise training could contribute to the development of the collateral circulation and the tissue salvage in the myocardial ischemia seen in pigs ( 29 ). However, we observed no difference in the area at risk among the experimental groups, indicating that the beneficial effects of exercise are unlikely to be related to the development of collateral circulation. The mechanism that underlies the cardioprotection observed at both the early phase and the late phase after exercise appeared to be related to an increase in Mn-SOD activity. A mechanism remains to be elucidated for the activation of Mn-SOD at the early phase (0.5 h) after exercise, where there was no difference in Mn-SOD at the protein level between the exercise and sham-treated control groups. These results indicated that a precursor or inactive form of Mn-SOD that possesses antigenicity but lacks enzyme activity may be modulated by exercise. The increase of Mn-SOD activity disappeared by 3 h after exercise, suggesting that a rapid inactivation should follow the activation of Mn-SOD. It is possible that a reversible phenomenon, such as a phosphorylation–dephosphorylation mechanism, might be involved in this step. Inhibition of Mn-SOD induction at the late phase by the administration of ASODN to Mn-SOD abolished the protection against ischemia/reperfusion injury induced by exercise. Therefore, at the late phase, the induction of Mn-SOD leads to an increase in its enzyme activity, resulting in the acquisition of cardioprotection against ischemia/reperfusion injury. Previous reports ( 20 ) and this study showed that TNF and IL-1 are produced after exercise. Our results clearly showed that TNF-α and IL-1β are involved in exercise-induced cardioprotection via activation of Mn-SOD. Because the effects of TNF-α and IL-1β show some redundancy ( 30 ), either of these cytokines is sufficient for both biphasic Mn-SOD activation and cardioprotection, and the blockade of both cytokines is necessary for the inhibition of Mn-SOD activation and cardioprotection induced by exercise. Although the mechanism for the activation of Mn-SOD appears to be different between early and late phases after exercise, these cytokines can activate Mn-SOD at both phases. It has been known that exercise produces reactive oxygen species in the heart ( 18 , 19 ). Our results indicated that (i) reactive oxygen species induce TNF-α and IL-1β, (ii) the activation of Mn-SOD by these cytokines is mediated by the reactive oxygen species, and (iii) the cardioprotective effect of TNF-α administration is mediated through the production of reactive oxygen species. Based on these observations, we could elucidate the mechanism for the exercise-induced cardioprotection as follows. Reactive oxygen species, produced during exercise, increase the levels of TNF-α and IL-1β in myocardium. Then, the cytokines activate Mn-SOD during the early phase and induce Mn-SOD during the late phase of cardioprotection, possibly via the production of reactive oxygen species. Reactive oxygen species are present upstream as well as downstream of the cytokines in the exercise-activated signaling pathway. It is unclear whether the loop of these cytokines and reactive oxygen species is necessary for the activation of Mn-SOD. The production of reactive oxygen species by the cytokines may take part in a positive feedback mechanism in this signal transduction system. The role of the reactive oxygen species might be dose dependent. A high dose of reactive oxygen species causes damage to cardiac myocytes, whereas a low dose of such species acts as a signal transduction messenger in cells. The transcriptional factors activator protein 1 and nuclear factor κB are subjected to redox regulation ( 31 – 36 ). The activation of nuclear factor κB induces such cytokines as TNF-α and IL-1β ( 37 – 39 ). TNF-α and IL-1β also stimulate the production of reactive oxygen species in cells (40– 44). The induction of Mn-SOD is regulated under a redox state ( 45 ) and is mediated by cytokines ( 46 ). Exercise may activate these transcriptional factors via the production of reactive oxygen species and lead to cytokine production. However, the relationship between the activation of these transcriptional factors and Mn-SOD as observed in the early phase of cardioprotection remains to be established. The time course of the cardioprotection induced by exercise resembles that produced by a brief period of cardiac ischemia (ischemic preconditioning) ( 4 – 7 ). This tolerance seems to be related to the production of reactive oxygen species ( 8 – 11 ). The induction of Mn-SOD by a brief period of ischemia is responsible for the preconditioning phenomenon ( 12 – 14 ). TNF and IL-1 are both reported to be involved in cardioprotection against ischemia/reperfusion injury ( 15 – 17 ) as well as the radioresistance induced by sublethal ionizing radiation or LPS ( 47 ). The sequence and nature of the signal transduction steps in the preconditioning phenomenon and radioresistance remain to be elucidated. The acquisition of cardioprotection following sublethal stress such as brief ischemia, exercise, ionizing radiation, and LPS may involve a common mechanism that functions through an induction and activation of Mn-SOD via the production of reactive oxygen species and cytokines. | Study | biomedical | en | 0.999999 |
10359574 | For construction of the lexA DNA binding domain fusion, the vector plex202 (provided by R. Brent, Harvard Medical School, Boston, MA) was used. The coding sequences for the cytoplasmic domain of TNFR-I (aa 205–426) or for the truncated form (aa 206–345) were cloned in frame into plex202. A Jurkat cDNA library fused to a synthetic activation domain ( 16 ) was screened. The yeast strain EG48/ JK103 ( 17 ) was instrumental. For analysis of the interaction of the cytoplasmic tail of (cyt)TNFR-I with full-length Grb2 and the SH3 or SH2 domain– mutated forms of Grb2, the matchmarker two-hybrid system was instrumental ( Clontech ). The coding sequence for the cytoplasmic domain of TNFR-I (aa 205–426) or the PLAP deletion mutant was fused to the DNA binding domain of Gal4 by subcloning pGBT9. The coding sequences for Grb2 ( 18 ), ΔNSH3Grb2, ΔSH2Grb2, or ΔCSH3Grb2 was fused to the activation domain of Gal4 by subcloning into pGAD424. Here, the yeast strain HF7c was used. Lysis of the cells was performed on ice in 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.05% Triton X-100, 1 mM PMSF, 0.2 U/ml aprotinin, 100 μg/ml leupeptin, and 1 mM orthovanadate. The lysate was clarified by centrifugation, and the supernatant was preabsorbed by 30-min incubation with protein A/G–agarose ( Santa Cruz Biotechnology ). Purified supernatants were incubated with antisera specific for c-TNFR-I ( Santa Cruz Biotechnology ), for full length TNFR-I, Grb2, or son of sevenless (SOS)1 (poly- and monoclonal sera from Santa Cruz Biotechnology and signal transduction laboratories), respectively. The complexes were precipitated by addition of protein A/G– agarose. The precipitates were washed twice with 0.5 M LiCl and 0.1 M Tris, pH 7.4, and once with 0.01 M Tris, pH 7.4. For Western blot analysis, proteins were separated by SDS-PAGE ( 19 ) and transferred onto cellulose nitrate membranes (0.45 μm; Schleicher & Schuell). Membranes were washed for 30 min with PBST and preblocked for 15 min in PBST containing 10% nonfat dry milk powder (blocking solution). Incubation with the first antibody diluted in blocking solution was performed for 2 h at room temperature. After extensive washing with PBST, the bound antibody was detected by peroxidase-conjugated secondary antibodies ( Amersham Corp. ). After 45 min of washing, the blots were developed using the ECL Western blotting detection system ( Amersham Corp. ). The sequence coding for full length human TNFR-I was amplified from pHup60 (provided by P. Goodwin, Immunex Corp. ) by PCR using a forward primer, which introduced the coding sequence for an NH 2 - terminal histidine tag and subcloned into pCDNA.3. The sequence coding for the cytoplasmic domain of TNFR-I (aa 205– 426) was subcloned into the bacterial expression plasmid pQe8 (QIAGEN Inc.). For construction of the PLAP deletion mutant, fragment I, nucleotide 256–1040, and fragment II, nucleotide 1050–1618, were amplified by PCR. In the case of fragment I, the backward primer introduced a HindIII site; in the case of fragment II, the forward primer harbored a HindIII site. The HindIII-restricted fragments were ligated and subcloned into the pCDNA.3 expression plasmid, generating pTNFR-IΔPLAP. The coding sequences for Grb2 and the dominant negative mutants of the SH3 and SH2 domains (provided by J. Duyster, University of Ulm) were subcloned into pCDNA.3. 0.8 × 10 6 70Z/3 and 293 cells were transfected by lipofection using DOTAP ( Boehringer Mannheim ) or the calcium phosphate procedure. For transfections, 5 μg of the expression constructs was used, with the exception of 3xAP-1(activator protein 1)-chloramphenicol acetyltransferase (CAT), where an amount of 1 μg was sufficient. Transfection efficiency was determined by transfection of the cells with green fluorescent protein. The transfection efficiency was 70–80%. Transfection efficiency was normalized for by transfection with a plasmid coding for the luciferase gene (pLuc). The CAT-ELISA was performed according to the instructions of the manufacturer ( Boehringer Mannheim ). Protein purification of the hexa-His–tag fusion proteins was performed as described previously ( 20 ). 15 min after stimulation with TNF-α (100 U/ml; in the case of 70Z cells, 200 U/ml) or EGF (5 ng/ml), cells were lysed in 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.2 mM EDTA, 1 mM EGTA, 10 mM sodium-β-glycerol-phosphate, 50 mM sodium fluoride, 0.5% Triton X-100, 1 mM sodium-orthovanadate, 0.25 M sucrose, 0.5 mM PMSF ( Sigma Chemical Co. ), 0.15 U/ml aprotinin, and 2 μg/ml leupeptin. Insoluble material was removed by centrifugation at 20,000 g at 4°C for 15 min. The protein concentration of the supernatant was determined by a Bradford assay. 500 μg total protein was incubated for 15 min at 4°C with protein A/G–agarose. The agarose beads were removed by centrifugation, and the supernatant was incubated for 2 h with c-Raf-1–specific antiserum. To precipitate the antigen–antibody complex, protein A/G–agarose was added again and incubated for 1 h at 4°C. The complexes were sedimented by centrifugation, and the pellet was washed twice in a buffer containing 100 mM Tris/HCl, pH 7.4, and 500 mM LiCl followed by a wash in 10 mM Tris/HCl, pH 7.4. The pellet was resuspended in kinase buffer as described previously ( 21 ). To elucidate the mechanism of TNF-α–dependent activation of c-Raf-1 kinase, a yeast two-hybrid screen using the lexA system was performed to identify adapter molecules interacting with the membrane proximal region of TNFR-I. Using a human Jurkat T cell cDNA library ( 16 ), a clone encoding the COOH-terminal part of the adapter protein Grb2 (aa 113–217) was identified. The coding sequence consists of a truncated middle SH2 domain and a complete COOH-terminal SH3 domain (Table I , top). The complete adapter protein Grb2 consists of a middle SH2 domain known to bind to phosphotyrosine residues and an NH 2 - and COOH-terminal SH3 domain. The TNFR-I/Grb2 interaction was confirmed using the Gal4 system by cotransfection of the yeast strain HF7c with pGAD424Grb2 and pGBT9cytTNFR-I (Table I , bottom). Only double-transfected cells grew under the selection conditions. To further confirm the interaction of Grb2 with the cytoplasmic domain of TNFR-I in another experimental system, coprecipitation experiments using highly purified, bacteria-derived Grb2 and cytTNFR-I were carried out. These experiments demonstrated that (i) Grb2 is indeed a binding partner of TNFR-I and (ii) no additional factors are necessary for the binding of Grb2 to TNFR-I . The in vivo interaction of endogenous Grb2 with endogenous TNFR-I was studied by immunoprecipitation of cellular lysates derived from CCL13 cells. Grb2 was coprecipitated by a TNFR-I–specific antibody and, vice versa, TNFR-I was coprecipitated by a Grb2-specific antiserum . These results demonstrate that Grb2 binds to TNFR-I in vivo. Moreover, binding of Grb2 to TNFR-I seems to be constitutive, as the presence of TNF-α did not influence the amount of coprecipitated TNFR-I by a Grb2-specific antiserum. In the next set of experiments, the structural basis of the observed Grb2/TNFR-I interaction was analyzed. SH3 domains are known to bind to PXXP motifs ( 22 – 24 ). The membrane-proximal domain of TNFR-I harbors a PLAP motif within aa 237–240 as a potential binding site for the COOH-terminal SH3 domain of Grb2. Indeed, increasing concentrations of peptides covering this sequence (functional peptide TTKPLAP) were able to compete the interaction of Grb2 with the cytoplasmic domain of TNFR-I, as demonstrated by coprecipitation experiments in vitro . The significance of this PLAP motif for the Grb2/ TNFR-I interaction was confirmed using the yeast two-hybrid system. A deletion mutant of cytTNFR-I lacking the PLAP motif (pGBT9cytTNFR-IΔPLAP) failed to interact with full length wild-type Grb2 (pGAD424Grb2) (Table I , bottom). Furthermore, in vivo Grb2 could not be coprecipitated by Ni-NTA (nitrilotriacetic acid)–agarose from the lysate of transfected 293 cells overproducing hexa-His– tagged TNFR-I lacking the PLAP motif . Ni-NTA–agarose specifically precipitated the hexa-His–tagged mutated receptor but not the wild-type receptor. Essential structural requirements of the Grb2 adapter protein for interaction with TNFR-I were analyzed using the yeast two-hybrid system. Functional inactivating mutations of the NH 2 -terminal SH3 domain (pGADGrb2ΔNSH3) or of the middle SH2 domain (pGADGrb2ΔSH2) did not affect the interaction of Grb2 with cytTNFR-I. The inactivating mutation of the COOH-terminal SH3 domain (pGADGrb2ΔCSH3), however, abolished the interaction with cytTNFR-I under these conditions (Table I , bottom). These results confirm the dependence of the Grb2/TNFR-I interaction on the COOH-terminal SH3 domain of Grb2. The significance of TNFR-I/Grb2 interaction for TNF-α–dependent activation of c-Raf-1 kinase was investigated by different experimental approaches. Cell-permeable peptides were applied to disrupt the TNFR-I/Grb2 complex. These peptides consist of two domains, a signal sequence derived from the third helix of the antennapedia homeodomain, which mediates membrane translocation ( 25 ), and the functional domain covering the PLAP sequence of TNFR-I. Presence of these peptides abolished TNF-α–dependent activation of c-Raf-1 kinase . This effect is specific, as the EGF-dependent activation of c-Raf-1 kinase was not affected by the presence of these peptides. Moreover, a mutated peptide (KLAP) did not affect the TNF-α–dependent activation of c-Raf-1 kinase. In addition, transfection of 70Z/3 cells, which lack functional TNFR-I, with an expression plasmid encoding wild-type TNFR-I restores the capability of TNF-α–dependent activation of c-Raf-1 kinase. However, 70Z/3 cells transfected with an expression plasmid encoding the PLAP deletion mutant of TNFR-I failed to respond to TNF-α stimulation with an activation of c-Raf-1 kinase . Finally, transfection of 293 cells with expression plasmids encoding inactivating point mutations in the NH 2 - or COOH-terminal SH3 domain of Grb2 in both cases caused a loss of TNF-α–dependent induction of c-Raf-1 activity. The mutation of the middle SH2 domain, however, did not affect the TNF-α–dependent activation of c-Raf-1 kinase . In the case of tyrosine kinase receptor–dependent activation of c-Raf-1 kinase, the SH3 domains of Grb2 are known to interact with the SOS proteins, which interfere with Ras ( 22 , 26 , 27 ). Based on the observation that integrity of the NH 2 -terminal SH3 domain of Grb2 is essential to trigger TNF-α–dependent activation of c-Raf-1 kinase, it was investigated whether Grb2, while bound to TNFR-I with its COOH-terminal SH3 domain, still interacts with SOS1/2 proteins with its NH 2 -terminal SH3 domain. Hexa-His–tagged cytoplasmic domain of TNFR-I was added to cellular lysates derived from 293 cells. The mixture was precipitated using either anti-Grb2– or anti-SOS1/2–specific antibodies, or Ni-NTA–agarose, which specifically precipitates 6H-cytTNFR-I. Western blot analysis of the precipitates demonstrated that cytTNFR-I indeed coprecipitates with both Grb2 and SOS . Transfection of 293 cells with an expression plasmid coding for a transdominant negative Ras mutant (RasN17) caused a loss of TNF-α–dependent activation of c-Raf-1 kinase. These results demonstrated that the TNF-α–dependent activation of c-Raf-1 kinase requires the integrity of the TNFR-I-Grb2-SOS-Ras pathway. The physiological significance of Grb2-mediated activation of c-Raf-1 kinase upon stimulation with TNF-α was investigated by reporter gene assays. Cotransfection of an AP-1–driven reporter gene plasmid (p3xAP-1–CAT) ( 28 ) with the expression plasmids encoding the three Grb2 mutants harboring the inactivating point mutations in the NH 2 - or COOH-terminal SH3 domain or those in the middle SH2 domain, with a transdominant negative Ras mutant (pRasN17), or with a transdominant negative mutant of c-Raf-1 kinase (pHCR13.1) ( 29 ) caused, with exception of pΔSH2Grb2, a loss of TNF-α–dependent induction of the reporter gene . This shows (i) that the integrity of the TNFR-I/Grb2 interaction is a prerequisite for the TNF-α–dependent activation of AP-1, (ii) that, in addition to the COOH-terminal SH3 domain, the NH 2 -terminal SH3 domain of Grb2 is essential for the activation of AP-1 by TNFR-I, and (iii) that Ras is involved in TNF-α–dependent signal transduction. The PLAP-dependent Grb2/TNFR-I interaction is essential but not sufficient for TNF-α–dependent activation of c-Raf-1 kinase ( 15 ). Transfected 70Z/3 cells producing a deletion mutant of TNFR-I (TNFR-IΔ308–340), which lacks the ability to activate nSMase, fail to respond with activation of c-Raf-1 kinase upon stimulation of TNFR-I ( 8 , 12 ). This mutant lacks the FAN binding domain ( 13 , 30 ). FAN was recently discovered to mediate the TNF-α–dependent activation of nSMase ( 12 , 13 ) . In addition, disruption of the TNFR-I/FAN interaction by cell-permeable peptides ( 25 ) covering the binding domain blocked the TNF-α–dependent activation of c-Raf-1 kinase . These data confirm the relevance of TNF-α–dependent activation of nSMase for induction of c-Raf-1 kinase activity. Based on our results, we propose that the TNF-α–dependent activation of c-Raf-1 kinase is mediated through at least two cooperative domains that are both indispensable. The cytoplasmic tail of TNFR-I interacts via a PLAP motif with the COOH-terminal SH3 domain of the adapter protein Grb2. The NH 2 -terminal SH3 domain of Grb2 interferes with SOS, therefore linking TNFR-I to c-Raf-1 kinase involving Ras ( 26 ). In contrast to the situation, in the case of tyrosine kinase receptors, in which Grb2 interacts with its middle SH2 domain with phosphotyrosine, this Grb2/SOS/Ras-dependent pathway is not sufficient for activation of c-Raf-1 kinase. A second domain is required. This domain was recently shown to bind FAN ( 12 , 13 ). The TNFR-I/FAN interaction is essential for TNF-α–dependent activation of nSMase ( 8 , 12 , 13 , 31 , 32 ). The hydrolysis of sphingomyelin catalyzed by nSMase generates ceramide, which can finally activate ceramide-activated protein (CAP) kinase. CAP kinase recently has been shown to bind to TNF-α–dependent activated c-Raf-1 kinase ( 15 ). Both pathways, the Grb2-SOS-RAS pathway and the activated CAP kinase pathway, might cooperatively mediate TNF-α–dependent activation of c-Raf-1 kinase . Although TNFR-I does not belong to the tyrosine kinase receptor family, Grb2 has been identified as a binding partner. In contrast to the interaction with phosphotyrosine, which is mediated through its middle SH2 domain, Grb2 interacts here with TNFR-I via its COOH-terminal SH3 domain. Although there is an obvious difference on the molecular level of the receptor/adapter interaction, in both cases, binding of Grb2 to the receptor is essential for triggering activation of c-Raf-1 kinase. Whereas for tyrosine kinase receptors the Grb2-SOS-Ras pathway seems to be sufficient for activation of c-Raf-1 kinase, in the case of TNFR-I, a second pathway via CAP kinase is involved. One possible explanation is that the interaction of SOS with the NH 2 -terminal SH3 domain of Grb2 is not strong enough to recruit c-Raf-1 kinase to the membrane and, in addition, the membrane-associated CAP kinase is required. Moreover, the binding of Grb2 to the PLAP motif in the cytoplasmic domain of TNFR-I is constitutive. Therefore, TNF-α–dependent activation of nSMase provides a trigger for the TNF-α–dependent activation of c-Raf-1 kinase. In conclusion, we demonstrate that the tyrosine kinase adapter protein Grb2 is a novel binding partner of TNFR-I and is essential for TNF-α–dependent activation of c-Raf-1 kinase. | Study | biomedical | en | 0.999995 |
10359575 | COS-1 cells were maintained in DMEM (BioWhittaker) supplemented with 10% FCS, 1 mM L-glutamine, and 50 μg/ml gentamycin (Life Technologies, UK). The murine IIA1.6 B cell line that coexpresses FcαRI and the FcR γ chain has been described previously ( 21 ). cDNAs encoding the complete FcαRI coding region and a mutant cDNA encoding a soluble form of FcαRI were gifts from Dr. C. Maliszewski ( Immunex Corp. , Seattle, WA) ( 19 , 31 ). cDNA for bFcγ2R has been described previously ( 7 ). Chimeric cDNAs were constructed by overlap extension PCR ( 21 ). Although the genomic structure of bFcγ2R is unknown, the high homology to FcαRI allowed us to infer intron–exon boundaries because amino acid residues that link the FcαRI exons are identical to those at comparable positions in bFcγ2R . Primers were thus designed to allow the fusion of exons at these residues. To construct the bEC1 (1–50) -FcαRI mutant, primers were designed to allow the fusion of the first 50 amino acids of bFcγ2R to FcαRI at isoleucine 50; both FcαRI and bFcγ2R have isoleucine residues at this position, which lies almost exactly in the middle of the EC1 domains. The integrity of all chimeric cDNAs was confirmed by sequence analysis. Chimeric FcR cDNAs were cloned into the pCDNA3 mammalian expression vector (Invitrogen, The Netherlands) before transfection. The pCMV-GFP plasmid, which directed the expression of green fluorescent protein (GFP), was constructed by inserting the CMV promoter region from pCDNA3 into the multiple cloning site of the pEGFP-1 vector ( Clontech ). COS-1 cells were transiently transfected with 2 μg chimeric FcR cDNA constructs by means of Fugene 6 transfection reagent ( Boehringer Mannheim , Germany) according to the manufacturer's instructions. In some experiments, 1 μg pCMV-GFP was cotransfected together with the FcR constructs. Cells were incubated at 37°C in a humidified CO 2 atmosphere for 48 h before harvesting. Uncoated magnetic M-450 Dynabeads ( Dynal , Norway) were coated, according to the manufacturer's instructions, with either human serum IgA (hIgA) or bovine IgG2 (bIgG2), which were purified as previously described ( 7 , 32 ). Due to low transfection efficiency of some DNA constructs (see Results), transfected COS-1 cells were first enriched for those becoming positive for gene expression by cotransfection of the FcR and pCMV-GFP constructs. Experiments showed that most fluorescent (GFP + ) cells had also taken up both plasmids, thus expressing the chimeric FcR together with GFP (see Results). Therefore, binding assays were performed as follows: 5 × 10 4 GFP + COS-1 cells (which had also been cotransfected with an FcR construct) were purified in a FACSVantage ® cell sorter ( Becton Dickinson ) and mixed with Ig-coated Dynabeads in a final volume of 50 μl per well in V-bottomed microtiter plates. After a 15-min incubation at room temperature, the plate was spun at 50 g for 1 min and incubated for an additional 45 min at room temperature. Cells and beads were resuspended and examined for the presence of rosettes, using a combination of light and fluorescent microscopy, in a Nikon Eclipse E800 microscope. Rosettes were defined as GFP + cells binding four or more Ig-coated beads and at least 200 GFP + COS-1 cells were counted for each determination. For blocking studies, cells were incubated with either mAb My43 (50 μl culture supernatant) or CC-G24 (50 μl ascites fluid diluted 1:4) for 30 min at room temperature before the addition of Ig-coated beads. A cDNA that encodes a soluble form of FcαRI was expressed in Chinese hamster ovary cells by means of the pEE14 expression system (Lonza, UK), and the protein was isolated from the culture supernatant by affinity chromatography with Sepharose-bound human IgA (van Zandbergen, G., and C. van Kooten, unpublished data). The previously described FcαRI mAbs My43 (murine IgM), A3, A59, A62, and A77 (all murine IgG1) were used in this study ( 33 , 34 ). My43 and A62 were gifts from Dr. Li Shen (Dartmouth Medical School, Lebanon, NH) and Dr. Max Cooper (University of Alabama, Birmingham, AL), respectively. A77 was supplied by Medarex Europe (The Netherlands), and A3 and A59 were purchased from Immunotech (France) and Research Diagnostics Inc. respectively. The bFcγ2R mAb CC-G24 (murine IgM) was generated by immunizing mice with bFcγ2R protein purified from cattle leukocytes, and the specificity was confirmed by staining COS-7 cells transfected with cDNA encoding the bFcγ2R or bovine FcγRII (Howard, C.J., unpublished data). To obtain new FcαRI mAbs, female BALB/C mice were immunized with purified soluble FcαRI. Splenocytes isolated from immunized animals were fused with myeloma cells (SP20) in the presence of 50% polyethylene glycol. The cell suspension was diluted in IMDM supplemented with 10% FCS, hypoxanthine (100 μmol), aminopterin (0.4 μM), thymidine (16 μM), 500 pg/ml IL-6, 100 U/liter penicillin, and 100 μg/ml streptomycin. Cells producing antibodies to FcαRI were subcloned by limiting dilution. Five clones producing mAb to FcαRI were expanded and the specificity was determined by FACS ® analysis (see below). To define the capacity of these new mAbs to inhibit binding of IgA to FcαRI, blocking studies were performed as follows: FcαRI mAbs or control mAbs of the same isotype were diluted in FACS buffer and incubated together with FcαRI-transfected IIA1.6 cells for 15 min at 4°C. Purified human serum IgA, which had previously been heat-aggregated for 1 h at 63°C (aIgA), was then added for 1 h at 4°C. Cells were washed and bound aIgA was detected by incubation with a goat anti– human IgA F(ab) 2 -PE polyclonal antibody conjugate (Southern Biotechnology Associates, Inc.) in FACS ® analysis. Isotype control antibodies for murine IgG1 were purchased from Becton Dickinson , and those for murine IgM were provided by Dr. Robert Burns (Scottish Agricultural Science Agency, Edinburgh, UK). Cells (5 × 10 5 ) were washed twice with FACS buffer (PBS/0.5% BSA/0.02% azide) and incubated with either FcαRI mAb (murine IgM or IgG1) culture supernatant or the appropriate isotype control supernatant for 1 h at 4°C. Cells were then washed twice with FACS buffer and incubated for 1 h at 4°C with either goat anti–mouse (GAM) IgM-FITC conjugate (1:150 final dilution), or a GAM IgG1-PE conjugate (1:150 final dilution) (both from Southern Biotechnology Associates, Inc.). In experiments where GFP + cells were analyzed for chimeric FcR expression, a GAM IgG1 Tricolor secondary reagent (1:200 final dilution) (Caltag Labs.) was used. After washing twice with FACS buffer, cells were fixed in PBS-buffered 1% (wt/vol) paraformaldehyde at 4°C and analyzed on a FACScan ® . Data acquisition was conducted with Lysis II software ( Becton Dickinson ), and data analysis was performed using WinMDI software (available from The Scripps Research Institute, La Jolla, CA). To map the ligand-binding domains of FcαRI and bFcγ2R, we generated five chimeric receptors as follows: hEC-bFcγ2R, consisting of the two EC domains of FcαRI fused to the transmembrane/cytoplasmic (TM/C) domain of bFcγ2R; bEC-FcαRI, the two bovine EC domains fused to the TM/C domain of FcαRI; hEC1-bFcγ2R, the EC1 domain of FcαRI fused to the EC2 TM/C region of bFcγ2R; bEC1-FcαRI, the EC1 domain of bFcγ2R joined to the EC2 TM/C region of FcαRI; and bEC1 (1–50) -FcαRI, the first 50 amino acids of bFcγ2R fused at isoleucine 50 to FcαRI . Together with wild-type FcαRI and bFcγ2R cDNAs, individual chimeric FcR constructs were transfected to COS-1 cells, and their cell surface expression was assessed by FACS ® analysis and by a specific binding assay using Ig-coated beads (see Materials and Methods). Initial experiments revealed the transfection efficiency of individual constructs to be quite variable. The most efficient construct directed expression of FcαRI on the surface of ∼30% of COS-1 cells, whereas the least efficient (bEC1-FcαRI) was expressed by only 3% of the transfectants . Therefore, we developed a method to selectively enrich for transiently transfected cells by cotransfecting a plasmid that directed expression of GFP (visualized by green fluorescence) together with the chimeric FcR constructs. Most GFP + COS-1 cells cotransfected with FcαRI were recognized by mAb A62 , and formed rosettes with hIgA-coated beads . By this procedure we obtained an approximately twofold enrichment of chimeric FcR-expressing cells, which in the case of FcαRI resulted in >60% of cells being reactive with FcαRI mAb and further able to form rosettes with hIgA coated beads . It should be noted that GFP − COS-1 almost never formed rosettes, most likely because transfectants expressing FcαRI alone accounted for only ∼1% of the total cells . We, furthermore, demonstrated the specificity of our binding assay by using blocking mAbs specific for FcαRI or bFcγ2R to inhibit rosette formation . The finding that the inhibition obtained with My43 was only partial (∼50%) was most likely explained by the use of culture supernatant other than a higher concentration of purified antibody which was not available. FcαRI does not bind bIgG2, and bFcγ2R does not bind hIgA (reference 7 and this paper); accordingly, we neither observed rosettes when hIgA-coated beads were mixed with bFcγ2R transfectants, nor when bIgG2-coated beads were mixed with FcαRI transfectants . Binding studies with COS-1 cells enriched for FcR expression as described above, showed not unexpectedly that wild-type FcαRI and bFcγR2 transfectants produced the highest levels of rosette formation with hIgA- and bIgG2-coated beads, respectively . Transfectants expressing chimeras coding for the entire EC portions of the receptors (hEC-bFcγ2R and bEC-FcαRI) bound their respective Ig-coated beads efficiently, although at a slightly lower level than their wild-type counterparts . The fact that the hEC1-bFcγ2R chimera retained IgA-binding capacity demonstrated that the binding site of FcαRI lies within the membrane-distal EC1 domain. Furthermore, because this chimera did not form rosettes with bIgG2-coated beads, our finding further suggested that the binding site for bIgG2 in bFcγ2R was not located within the EC2 domain of this receptor. Conversely, the bEC1-FcαRI chimera did form rosettes with bIgG2-coated beads, but not with beads coated with hIgA. Altogether these results showed that, in common with FcαRI, the ligand-binding site of bFcγ2R appeared to lie within the EC1 domain. It should be noted, however, that the level of binding obtained with the two EC1 chimeras (hEC1-bFcγ2R and bEC1-FcαRI) was reduced when compared with the wild-type receptors, and the EC chimeras (hEC-bFcγ2R and bEC-FcαRI) . Futhermore, to better localize the Ig-binding sites of these two receptors, a further chimera was constructed in which the first 50 amino acids of the EC1 domain were from bFcγ2R, while the remaining EC1 (49 amino acids) and the rest of the receptor were from FcαRI [bEC1 (1–50) -FcαRI]. Although this chimera was expressed at the cell surface and could be recognized by the majority of FcαRI mAb, it bound neither hIgA- nor bIgG2-coated beads. To confirm that the IgA-binding site of FcαRI lies within the EC1 domain, we mapped the specific epitopes for a number of blocking and nonblocking mAbs. Of the previously described FcαRI mAbs, only My43 (murine IgM) was able to block the binding of hIgA to FcαRI ( 33 ). Four others (A3, A59, A62, and A77, all murine IgG1) did not inhibit binding ( 34 ). We also included a number of new FcαRI mAbs (2E6, 2D11, 7G4, 2H8, and 7D7, all murine IgG1), raised against a soluble form of FcαRI. These mAbs were shown to be specific for FcαRI by reaction with IIA1.6 cells expressing this receptor . They were next assayed for ability to block binding of heat-aggregated hIgA to FcαRI: mAbs 2E6, 2D11, 7G4, and 2H8 produced such inhibition while 7D7 did not . Accordingly, we presumed that the blocking mAbs would prove to be EC1-specific, whereas the nonblocking ones would be EC2 specific. To test this hypothesis, we screened the reactivity of all mAbs against the panel of chimeric FcR expressed in COS-1 cells by FACS ® analysis. Indeed, all mAbs capable of blocking the binding of heat-aggregated hIgA to FcαRI , mapped to the EC1 domain . Also, all nonblocking mAbs were directed against the EC2 domain, except for mAb A3 that apparently recognized an epitope depending on parts of both domains. Unfortunately, because only one mAb against bFcγ2R was available, a similar detailed study could not be performed for this receptor. We showed that mAb CC-G24 only recognized wild-type bFcγ2R and the bEC-FcαRI chimera . Thus, like mAb A3, it is most likely directed against a conformational epitope depending on both EC1 and EC2. By means of a panel of chimeric FcRs, we identified conclusively for the first time the ligand-binding sites of FcαRI and bFcγ2R. Surprisingly, these sites were found to be located in the EC1 domains. FcαRI and bFcγ2R are highly homologous both at the protein and nucleotide level (41 and 56% identity, respectively), but show much less homology with other human and bovine FcRs ( 7 ). This suggests that FcαRI and bFcγ2R evolved from a common ancestral gene (also shared by KIR, ILT, MIR, LIR, LAIR-1, HM18, PIR, and gp49B1 genes) and not shared by other human or bovine FcRs ( 7 ). Mapping of the bFcγ2R gene to bovine chromosome 18, which corresponds to human chromosome 19, further supports this notion ( 35 ). Our finding that the Ig-binding sites of both FcαRI and bFcγ2R are located within their EC1 domains, was based on the fact that rosetting with Ig-coated beads was only seen for the corresponding FcαRI or bFcγ2R EC1 chimera. In both cases, however, a reduction in binding activity was seen compared with that obtained for the wild-type receptors and for the comparable EC chimeras . Although these differences in part may be attributed to the expression levels of individual constructs (especially for the bEC1-FcαRI chimera, see above), it is also possible that the EC2 domains and membrane-proximal regions of the receptors contribute either directly (by forming “secondary” contact sites) or indirectly (by preserving three- dimensional structure) to the affinity and stability of the ligand interactions. This would be analogous to the activity of other two-domain FcRs, namely FcγRII, FcγRIII, and FcεRI in which the ligand-binding sites are located in the membrane-proximal EC2 domains, whereas structures within the EC1 domains contribute to the binding process ( 29 , 30 ). It should also be noted that the region of hIgA interacting with FcαRI has recently been mapped to the Cα2/ Cα3 boundary ( 36 ). This is in contrast to the region of human IgG responsible for interaction with FcγRs, which is proposed to lie much closer to the hinge ( 30 ). Therefore, in terms of the evolution of Ig/FcR interactions, it should be interesting to map the region of bIgG2 that binds to bFcγ2R. Because recombinant bIgG2 is available, experiments can readily be designed to determine whether this region lies close to the hinge region as in human IgG or in a position analogous to that of hIgA ( 37 ). To substantiate our observation that hIgA binds to the EC1 domain of FcαRI, we mapped the epitopes for a panel of blocking and nonblocking FcαRI mAbs that bound equally to the EC parts of wild-type FcαRI and the hEC-Fcγ2R chimera. The nonblocking mAbs (A59, A62, A77, and 7D7) were shown to react with the membrane-proximal EC2 domain because they bound only to wild-type FcαRI and the hEC-bFcγ2R, bEC1-FcαRI, and bEC1 (1–50) -FcαRI chimeras. The only exception was the nonblocking mAb A3 that bound only to wild-type FcαRI and the hEC-bFcγ2R and bEC1 (1–50) -FcαRI chimeras, suggesting that its epitope is conformational and depends on regions of both EC1 and EC2 (similar to the bFcγ2R mAb CC-G24; see Results section). In contrast, all blocking mAbs (My43, 2E6, 2D11, 7G4, and 2H8) were shown to react with the EC1 domain of FcαRI because their binding activity was retained with the hEC1-bFcγ2R chimera. The epitopes recognized by My43 and 2D11 were further localized to the region of EC1 directly adjacent to EC2, because they were shown to bind the bEC1 (1–50) -FcαRI chimera. 2E6 and 2H8 on the other hand, bound only weakly to this chimera, whereas 7G4 did not bind at all. Similar mapping studies with blocking mAbs have previously been used to localize the IgG-binding sites of FcγRII and FcγRIII to their EC2 domains ( 25 , 27 ). A number of FcαRI mRNAs have been isolated and shown to encode splice variants of the receptor ( 38 – 41 ). One such report described cell surface expression of an FcαRI variant that lacked the complete EC2 domain, and suggested the EC1 domain to be involved in hIgA binding ( 40 ). However, in contrast to our results, mAb My43 was proposed to react with EC2, and mAb A59 with EC1. We believe that observation to be spurious either due to incorrect cell surface expression of the splice variant and/or aberrant receptor structure caused by lack of the complete EC2 domain. This possibility was supported by our attempts to express various FcαRI splice variants in COS cells with no success ( 38 ). Additionally, chimeras constructed between FcαRI and FcγRII were not expressed efficiently (Morton, H.C., and J.G.J. van de Winkel, unpublished observations), possibly reflecting a degree of structural incompatibility between these two FcRs. In fact, our unsuccessful experience with those approaches led us to construct chimeras between FcαRI and bFcγ2R as reported here, because their levels of homology (and hence presumably their overall structure) are more similar than for other FcRs. Thus swapping of highly homologous regions should have minimal affect on the overall structural integrity of the resultant chimeras. Therefore, we feel that the present approach is more physiological than previous attempts to this end. The surprising difference seen between the ligand-binding sites of FcαRI and bFcγ2R versus those of other leukocyte FcγRs and FcεRI may have interesting implications in terms of Ig interactions. As mentioned above, this disparity could simply reflect the proposed evolution of FcαRI and bFcγ2R from an ancestral gene distinct from that giving rise to other FcγRs and FcεRI. This notion is supported by the observation that residues within the membrane-distal domain of two KIR proteins determine their ability to bind to their respective ligands, the two groups of HLA-C allotypes ( 42 ). Moreover, due to their high levels of homology, the three-dimensional structure of FcαRI and bFcγ2R might more closely resemble that of the KIR proteins than that of more distantly related FcRs ( 43 ). Indeed, more detailed mutational analysis, directed by modeling studies using the recently published three dimensional structure of the p58 KIR as a template for the protein backbones of FcαRI and bFcγ2R, are currently underway in our laboratory to further localize the Ig-binding sites within these two receptors. An alternative evolutionary explanation possibly applicable at least for FcαRI might be that its ligand-binding site developed to ensure interaction with all molecular forms of IgA: monomeric IgA, dimeric IgA (including J chain), and secretory IgA (including J chain and secretory component). FcαRI is reported to bind all these ligand variants ( 44 , 45 ). Therefore, because the site of interaction with FcαRI at the Cα2/Cα3 boundary appears to be accessible to the receptor in all these forms of IgA, FcαRI could have evolved to accomplish this interaction via its EC1 domain to avoid potential problems of steric hindrance of a more membrane-proximal binding site in relation to large IgA polymers. In conclusion, we have shown that the closely related FcαRI and bFcγ2R bind their ligands via sites located in their membrane-proximal EC1 domains. The difference in the Ig-binding sites of these two receptors versus other leukocyte FcγRs and FcεRI, may reflect the proposed divergent evolutionary pathway from a distinct genetic precursor, or (at least in the case of FcαRI) a specific adaptation for efficient interaction with large molecular forms of IgA. | Study | biomedical | en | 0.999998 |
10359576 | Peptides were synthesized by Quality Controlled Biochemicals, Inc. and subjected to quality control by reverse-phase HPLC and mass spectrometry. cDNA constructs were made by reverse transcriptase PCR using RNA from NOD spleens. Fos and Jun dimerization domains were attached to the 3′ end of the I-A g7 α and β extracellular domains, respectively, through a seven-amino acid linker [Val-Asp-(Gly) 5 ] that contained a SalI restriction site. The cDNAs encoding the signal peptides and extracellular domains of I-A g7 α and β chains were amplified first and cloned separately into the EcoRI/SalI site of the pRmHa-3 vector under the control of the inducible metallothionein promoter ( 18 ). The Fos and Jun segments were excised with SalI from similar constructs made for HLA-DR2 ( 19 ) and cloned into the SalI site of these vectors (I-Aα-Fos, I-Aβ-Jun). The orientation of the Fos and Jun segments and the sequence of the constructs were confirmed by dideoxy-sequencing. The following oligonucleotides were used for the amplification of I-Aα and I-Aβ segments. Forward primer for I-Aα, 5′ AAA AAA gAA TTC ATg CCg TgC AgC AgA gCT CTg 3′; reverse primer for I-Aα, 5′ AAA AAA gTC gAC TTC TgT CAg CTC TgA CAT gg 3′; forward primer for I-Aβ, 5′ AAA AAA gAA TTC ATg gCT CTg CAg ATC CCC AgC 3′; reverse primer for I-Aβ, 5′ AAA AAA gTC gAC CTT gCT CCg ggC AgA CTC ggA 3′. The EcoRI and SalI sites in the forward and reverse primers, respectively, are underlined. Drosophila melanogaster S2 cells (4 × 10 6 ) were transfected with 20 μg of each plasmid, 0.5 μg of hygromycin selection vector pUC-hygMT, and 20 μl of liposomes (Invitrogen) in 1 ml medium without FCS for 4 h. After transfection, cells were grown in Schneider medium ( Sigma Chemical Co. ) supplemented with 10% of FCS. Selection was performed by addition of hygromycin to a final concentration of 0.1 mg/ml. Approximately 2 wk after transfection, cells were cloned by limiting dilution in 96-well plates. Clones were expanded and tested for secretion of I-A g7 by Western blot analysis after induction of expression with copper sulfate (1 mM). The clone with the highest expression level was scaled up in roller bottles using Schneider medium supplemented with 5% FCS and 0.1 mg/ml of hygromycin. The supernatant was harvested 4 d after induction of protein expression with copper sulfate at 1 mM ( Sigma Chemical Co. ). Supernatants were concentrated by ultrafiltration using a YM30 spiral membrane cartridge (Amicon). The I-A–specific 10-2.16 mAb (20; hybridoma from the American Type Culture Collection) was coupled to cyanogen bromide–activated Sepharose 4B beads ( Pharmacia Biotech ). The concentrated supernatant was passed through a Sepharose 4B precolumn and the 10-2.16 column at a flow rate of 9 ml/h. The column was then washed with 100 mM phosphate buffer, pH 8.0, and the protein was eluted with 2 bed volumes of 50 mM glycine, pH 11.5. Fractions were immediately neutralized by addition of 2 M Tris, pH 8.8, and dialyzed against PBS. Protein concentration was determined with the Coomassie Plus protein assay (Pierce). Antisera specific for the Fos and Jun dimerization domains were generated by immunizing Lewis rats with synthetic peptides (provided by Dr. P. Kim, Whitehead Institute, Cambridge, MA). Fos peptide sequence, Ac-CGGTDTLQAETDQLEDEKYALQTEIANLLKEKEKL-CONH 2 ; Jun peptide sequence, Ac-CGGIARLEEKVKTLKAQNYELASTANMLREQVAQL-CONH 2 . For Western blot analysis, proteins were separated under reducing conditions by SDS-PAGE (12%) and transferred to a polyvinylidene difluoride membrane ( Millipore Corp. ). Membranes were blocked overnight in 3% non-fat dry milk in TBST-buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.2% Tween 20). Membranes were probed with Fos antiserum (1:1,000) and/or Jun antiserum (1:500). Bound Fos and Jun antibodies were detected by incubating the membranes with horseradish peroxidase–conjugated anti–rat IgG (1:3,000). The blots were developed by ECL ( Amersham ). HPLC gel filtration analysis of I-A g7 was performed using a Bio-Gel SEC 30-XL column (300 × 7.8 mm; Bio-Rad) with phosphate buffered saline, pH 7.8, at a flow rate of 0.8 ml/min. The following proteins were used as molecular mass standards: thyroglobulin (669 kD), aldolase (158 kD), BSA (67 kD), ovalbumin (43 kD), and chymotrypsinogen A (25 kD). Purified I-A g7 (0.6 μg) was incubated with biotinylated test peptide at 37°C for 18 h in a final volume of 50 μl. Buffers were either 20 mM sodium acetate/100 mM NaCl, pH 5.0, or PBS, pH 7.4, both containing 1 mM PMSF and 1 mM EDTA. In parallel, a 96-well plate (Fluoronunc; Nunc) was coated overnight at 4°C with 50 μl of 10 μg/ml of the I-A–specific antibody 10-2.16 in sodium bicarbonate buffer, pH 9.6. Nonspecific binding sites were blocked with 10% FCS in PBS/0.05% Tween 20 (PBS-Tween) for 2 h at room temperature. Wells were washed three times with PBS-Tween and I-A g7 –peptide samples were added to the wells. After a 1-h incubation, wells were washed four times with PBS-Tween and europium-labeled streptavidin (Wallac Oy, Finland) was added (1:2,000 in PBS/0.5% BSA, 100 μl/well) for detection of I-A g7 –bound biotinylated peptide. After a 1-h incubation at room temperature, wells were washed six times with PBS-Tween and Delfia enhancement solution (Wallac Oy) was added. Fluorescence was quantitated after 30 min in a fluorescence plate reader . For competition assays, 0.6 μg of soluble I-A g7 was incubated with biotinylated transferrin peptide (1 μM) at pH 5.0 in the presence of nonbiotinylated competitor peptide (8 nM–25 μM). I-A g7 –bound biotinylated transferrin peptide was quantitated as described above. For CLIP and CLIP 98D dissociation assays, soluble I-A g7 was loaded with biotinylated CLIP or CLIP 98D (1 μM) at 37°C for 18 h at pH 7.4. An equal volume of 20 mM sodium acetate, pH 4.5, or PBS, pH 7.4, was added in the presence of nonlabeled mouse serum albumin peptide (100 μM). The final pH was either 5.0 or 7.4. At different time points (5–120 min), the reaction was stopped by freezing the samples at −20°C in the presence of BSA (200 μM). For quantification of bound biotinylated peptide, samples were mixed with an equal volume of 40 μM Tris, pH 7.4, and captured with 10-2.16 antibody–coated wells at 4°C for 1 h. All wells were developed by incubation for 1 h at 4°C with streptavidin-europium (dilution of 1:2,000). Fluorescence was quantitated as described above. For other peptide dissociation assays, soluble I-A g7 was incubated with biotinylated peptide (1 μM) at 37°C for 18 h at pH 5.0 in a final volume of 50 μl. Then the pH of the samples was either maintained at pH 5.0 or adjusted to pH 7.4 (by addition of Tris, pH 8.8). Nonlabeled mouse serum albumin peptide (100 μM) was added to prevent rebinding of biotinylated peptides. At different time points (5 min to 72 h), reactions were stopped by freezing the samples at −20°C in the presence of BSA (200 μM). Bound biotinylated peptides were then quantitated as described above. 7-wk-old female NOD mice were immunized subcutaneously with 100 μg of Herpes simplex virus (HSV)-2 VP16 peptide in CFA. Draining lymph nodes were removed 10 d after immunization and lymph node cells were seeded at 5 × 10 5 per well in 96-well U-bottomed plates in serum-free media (AIM-V; GIBCO BRL ) containing 5.5 × 10 −5 M of 2-ME. HSV-2 VP16 peptide was added to a final concentration of 5 μM. After 3 d of culture, the medium was replaced with RPMI supplemented with 10% FCS, 5 U/ml recombinant human IL-2 ( Boehringer Mannheim ), 2-ME, and nonessential amino acids ( Sigma Chemical Co. ). T cells were grown for 6–8 d in this media and cells were fed every third day. The T cell line was maintained in culture by repeated cycles of a 3-d restimulation with irradiated NOD spleen cells and 5 μM HSV-2 VP16 peptide in AIM-V, followed by a 7-d resting period in RPMI supplemented with 10% FCS and recombinant IL-2. T cell proliferation assays were set up in triplicates in 96-well U-bottomed plates with 2.5 × 10 4 T cells and 2.5 × 10 5 irradiated NOD spleen cells per well in AIM-V containing 2-ME. For blocking experiments, antibodies were added to a final concentration of 10 μg/ml. After 72 h of culture, 1 μCi of [ 3 H]thymidine (NEN) was added to each well and incorporated radioactivity was quantitated in a beta-scintillation counter after harvesting of cells onto glass fiber filters (Wallac Oy). Soluble I-A g7 was expressed in Drosophila Schneider cells by replacing the transmembrane and cytoplasmic segments of I-Aα and I-Aβ with leucine zipper dimerization domains from the transcription factors Fos and Jun, respectively. A short, flexible linker [Val-Asp-(Gly) 5 ] that encoded a SalI restriction site was placed between the extracellular domains of I-Aα and I-Aβ and the Fos/Jun segments . Leucine zipper dimerization domains were previously shown to promote the assembly of HLA-DR2 and I-A d ( 19 , 21 ). The I-Aα–Fos and I-Aβ–Jun constructs were separately cloned into the pRmHa-3 vector under the control of the copper-inducible metallothionein promoter ( 18 ). These constructs were cotransfected with a hygromycin selection plasmid into S2 cells and transfectants were cloned by limiting dilution after initial selection with hygromycin. Expression was induced by addition of copper sulfate to the growth medium and the protein was purified from concentrated supernatants by affinity chromatography using mAb 10-2.16. The yield was ∼0.7–1.6 mg/liter of culture. SDS-PAGE of the purified protein demonstrated two bands with an apparent molecular mass of 43.3 and 33 kD . The identity of these bands as I-Aα–Fos and I-Aβ–Jun, respectively, was confirmed by Western blot analysis using antisera specific for the Fos and Jun leucine zippers . The migration of the two chains was similar to that observed for α and β chains of HLA-DR2 expressed with Fos/Jun dimerization domains ( 19 ). Purified I-A g7 eluted from a HPLC gel filtration column with a molecular mass appropriate for soluble I-A g7 . Importantly, the protein did not form high molecular mass aggregates, indicating that it was indeed soluble in the absence of detergent. Soluble I-A g7 was used to examine the binding of naturally processed I-A g7 peptides as well as peptides from islet autoantigens under detergent-free conditions . This is important because detergents can have a major effect on peptide binding by MHC class II molecules. For example, detergents with unbranched intermediate length hydrocarbon chains were found to promote dissociation of CLIP from HLA-DR ( 22 ). Two different binding assays were performed. In the direct binding assay, biotinylated peptides were incubated with soluble I-A g7 and bound biotinylated peptides were quantitated with europium-labeled streptavidin after capture of complexes with an I-A–specific antibody. In the competition assay, unlabeled peptides were used at different concentrations to compete for binding of a biotinylated transferrin peptide to I-A g7 . A naturally processed peptide from mouse albumin (residues 560–574) was the best binder in both assays . This peptide competed for binding of the biotinylated transferrin peptide at very low peptide concentrations and also gave very high counts in the direct binding assay, even when the concentration of the biotinylated albumin peptide was <100 nM. Strong binding could also be demonstrated for another naturally processed peptide from transferrin (res. 55–68), as well as for a peptide from HSP60 (res. 441–460) . The murine CLIP peptide showed little or no binding at this pH (pH 5). Several peptides that were identified as T cell epitopes of islet antigens competed for binding to I-A g7 only at relatively high peptide concentrations, indicating that they were relatively poor binders. This included all three peptides from GAD 65 (res. 247– 266, 509–528, and 524–543) as well as HSP60 (169–180) and insulin (9–23). The HSP60 (169–180), insulin (9–23), and GAD65 (249–263) peptides were also synthesized with an NH 2 -terminal biotin group and tested in the direct binding assay. These peptides gave only low counts that were close to background (data not shown). It is important to note that the majority of the peptides that have been described as T cell epitopes actually represent the human rather than the murine sequences. Since there are sequence differences in the sequences of some of these peptides, it is possible that some of the murine peptides are stronger binders. The immunodominant myelin basic protein peptide (MBP, Ac1-11) that induces experimental autoimmune encephalomyelitis in mice with the H-2 u haplotype is also a low affinity binder, whereas the immunodominant T cell epitope of MBP in mice with the H-2 s haplotype (res. 87– 99) is a high affinity binder ( 24 ). The transferrin peptide bound to I-A g7 at both an acidic and a neutral pH . The binding kinetics were characteristic for an “empty” MHC class II molecule and similar to those previously observed for soluble HLA-DR2 . In contrast, CLIP bound to I-A g7 at a neutral pH; at an acidic pH little or no binding was detected . Analysis of human and murine CLIP peptides has demonstrated a binding mode in which two methionine residues of CLIP occupy the P1 and P9 pockets of the MHC class II peptide binding site. These residues correspond to positions 90 and 98 of mouse as well as residues 91 and 99 of human invariant chain, respectively ( 25 – 27 ). Since the transferrin peptide carried a negative charge at the P9 position, the corresponding residue of CLIP (methionine 98) was substituted by aspartic acid (CLIP 98D). This single amino acid substitution resulted in strong binding both at a neutral and at an acidic pH . These results demonstrated that the binding characteristics of CLIP to I-A g7 were due to the interaction of methionine 98 with the P9 pocket of I-A g7 . MHC class II molecules assemble with invariant chain at a neutral pH in the endoplasmic reticulum and are then transported through the Golgi complex to an endosomal compartment that has a low pH. In this compartment, the invariant chain is proteolytically cleaved, leaving the CLIP peptide in the binding site. The removal of CLIP is catalyzed by H-2M, which allows binding of antigenic peptides ( 28 – 32 ). Therefore, it was important to examine the stability of complexes that were assembled at a neutral pH and then shifted to an acidic pH. For that purpose, I-A g7 was incubated with biotinylated CLIP at a neutral pH at 37°C. An unlabeled competitor peptide was then added in order to prevent rebinding of biotinylated CLIP and the pH was changed for different periods of time (5–120 min) (Table II ). After this incubation period, the sample was neutralized and the amount of bound biotinylated peptide was quantitated. This experimental design in which complexes were formed at a neutral pH and neutralized again after incubation at pH 5 for different periods of time allowed the kinetics of CLIP dissociation to be analyzed. These experiments demonstrated that even a brief incubation (5 min) at pH 5.0 before neutralization resulted in complete dissociation of CLIP from I-A g7 (Table II ). Even with shorter incubation times at pH 5 (<2 min), no bound CLIP was detected (data not shown). When the complex was kept at pH 7.4, dissociation was considerably slower and bound CLIP was still detected after 120 min. The rapid, pH-dependent dissociation of CLIP was due to methionine 98 of CLIP, which occupies the P9 pocket of MHC class II molecules ( 25 ). Substitution by aspartic acid (CLIP 98D) greatly increased the stability of the complex (Table II ). These results indicated that the stability of I-A g7 –CLIP complexes was pH dependent, and that the P9 pocket of I-A g7 was critical in determining this property. A possible explanation for the pH-dependent dissociation of CLIP was that the interaction of peptide side chains with the P9 pocket was pH dependent. Therefore, a panel of CLIP analogue peptides in which methionine 98 was substituted by all naturally occurring amino acids was examined in the competition assay (Table III ). These experiments demonstrated three important properties of I-A g7 : (i) At both an acidic and a neutral pH, I-A g7 has a strong preference for a negatively charged residue at P9. (ii) The interaction of I-A g7 and CLIP is relatively weak at a neutral pH, and there is little or no interaction at an acidic pH. (iii) The interaction of some peptide side chains with the P9 pocket is pH dependent. Particularly striking is the observation that a CLIP analogue with histidine at P9 competes well at pH 7.4 (IC 50 of 4 μM), but not at pH 5.0 (IC 50 of >125 μM). Since it has been hypothesized that the interaction of peptides with I-A g7 is short lived ( 17 ), we determined the dissociation rates of several peptides, which included the naturally processed transferrin and serum albumin peptides, the CLIP 98D analogue, and peptides that have been identified as T cell epitopes in NOD mice . Purified I-A g7 was first incubated with one of the biotinylated peptides (1 μM), then the pH was adjusted to pH 7.4 or maintained at pH 5.0 and mouse serum albumin peptide was added to a final concentration of 100 μM to prevent rebinding of dissociated biotinylated peptide. The GAD 65 peptide dissociated very rapidly and a t 1/2 value could not be estimated. In contrast, complexes of I-A g7 and several other peptides were long lived, particularly at a neutral pH. The estimated t 1/2 value for the serum albumin peptide was >72 h and for the transferrin peptide ∼45 h at pH 5.0 and >72 h at a neutral pH. The dissociation of the CLIP 98D analogue was faster, with a t 1/2 value of ∼23 h at pH 5.0 and ∼70 h at pH 7.4 . These results demonstrated that I-A g7 forms long-lived complexes with naturally processed peptides and that the dissociation rate of peptides was affected by pH. Nevertheless, I-A g7 –peptide complexes were not resistant to SDS . I-A g7 (0.6 μg) was loaded at pH 5.0 with four different peptides (transferrin, serum albumin, CLIP, or CLIP 98D analogue) by overnight incubation at 37°C. Fractions of I-A g7 –peptide complexes (0.1 μg) were examined for the formation of SDS-resistant dimers by Western blot analysis using polyclonal antisera specific for the Fos and Jun segments. An SDS-resistant dimer could not be detected with any of the peptides tested. Therefore, the SDS assay does not determine whether peptides were bound to I-A g7 before addition of detergent. Soluble HLA-DR1 expressed in insect cells forms SDS-stable dimers with the influenza hemagglutinin (307–319) peptide ( 33 ). This indicates that SDS-sensitivity is not a general property of soluble MHC class II molecules expressed in insect cells. Since several of the peptides that were identified as T cell epitopes of islet autoantigens were poor binders, we examined if peptides with such binding properties could induce a T cell response. We chose the peptide from HSV-2 VP16, which has been reported to bind to HLA-DQ8 ( 34 ), since it represented a foreign antigen. This peptide showed little binding in the direct binding assay, but competed for binding of the biotinylated transferrin peptide at high peptide concentrations (data not shown). NOD mice were primed with this peptide in CFA and a T cell line specific for the HSV-2 peptide could be established from draining lymph nodes by restimulation with the HSV-2 peptide . This T cell line was I-A g7 restricted since the response could be completely blocked by mAb 10-2.16, but not by a control antibody (Y3-P) . Stimulation of this T cell line required the continuous presence of the peptide in the culture. Even a single wash of the APCs after overnight incubation with the peptide completely eliminated the T cell response . These results demonstrate that T cells from NOD mice can be primed even by a peptide that is a poor binder for I-A g7 . Soluble I-A g7 was used to define the biochemical requirements for peptide loading by this MHC class II molecule, which confers susceptibility to type I diabetes in NOD mice. Several peptides, including two naturally processed peptides from exogenous antigens, bound to I-A g7 . The interaction with the invariant chain–derived CLIP peptide was relatively weak and pH dependent. CLIP bound to I-A g7 at a neutral pH, but rapidly dissociated at an endosomal pH. This property of CLIP was due to methionine 98, which occupies the P9 pocket of the MHC class II peptide binding site ( 25 ). Substitution of this residue of CLIP greatly increased the stability of the complex, both at an acidic and neutral pH. These characteristics of I-A g7 result in the rapid generation of empty molecules at an endosomal pH. Several mutant cell lines have been described that are defective in antigen presentation due to a lack of DM expression. In these cell lines, MHC class II–CLIP complexes accumulate because DM does not catalyze the removal of CLIP ( 32 ). For several human and murine MHC class II molecules, CLIP peptides are abundant among naturally processed peptides in normal cell lines, reflecting a relatively stable interaction ( 35 , 36 ). For example, CLIP peptides have been eluted from purified I-A b and I-A d . CLIP was found to bind with a relatively high affinity to I-A d (IC 50 of 14 nM) and was one of the best binders identified for this class II molecule ( 36 ). These results are important, since I-A g7 and I-A d share the same I-A α chain ( 12 ). Thus, the binding properties of CLIP to I-A g7 appear to be due to the unique I-A g7 β chain. The requirement for DM in the presentation of antigens by I-A g7 has been analyzed using T cell hybridomas specific for chicken ovalbumin ( 37 ). For all T cell hybridomas, the antigen was presented by transfectants that expressed only I-A g7 and invariant chain, but not H-2M, indicating that the murine DM homologue is not essential for antigen presentation by I-A g7 . For two out of four hybridomas with a distinct epitope specificity, presentation was enhanced by coexpression of H2-M. These results indicate that presentation of certain epitopes by I-A g7 can be enhanced by H-2M. This may be due to “editing” of peptide content as well as stabilization of empty I-A g7 molecules by H-2M, as described for human MHC class II molecules ( 30 , 31 , 38 ). The recently solved crystal structure of I-A d demonstrates several features of I-A–peptide interactions that are important in this context ( 16 ). Detailed analysis of the I-A d peptide binding motif demonstrated that only two peptide residues in a 6-amino acid core were important for binding ( 39 ). These residues are located in the P4 and P9 pockets of the I-A d crystal structure. I-A d and I-A g7 have the same α chain sequence, but differ at 17 positions of the β chain. An important difference is the presence of an interchain salt bridge between β57 Asp and α76 Arg in I-A d , which is absent in I-A g7 (β57 Ser). In I-A g7 –peptide complexes, a salt bridge may instead be formed by P9 Asp or P9 Glu of bound peptides and α76 Arg. This would explain the strong preference for negatively charged residues at the P9 position, which was first noted among naturally processed peptides eluted from I-A g7 ( 23 ). In contrast, I-A d has a preference for alanine or serine at the P9 position. An I-A g7 peptide binding motif has been proposed, in which a preference for aliphatic residues was noted at P6 and for hydrophobic residues at P9 ( 40 ). Also, at P11 binding was enhanced by the presence of aspartic acid. It appears that these data are in general agreement with other data on I-A g7 – peptide interactions, provided that the relative positions are reassigned such that these data reflect the preferences of the P4, P7, and P9 pockets of I-A g7 . Are I-A g7 molecules poor peptide binders or are only some of the peptides that have been identified thus far poor ligands? This may be a separate issue from the lack of SDS resistance. The analysis of soluble I-A g7 clearly demonstrates that this MHC class II molecule binds peptides. Two naturally processed peptides from extracellular antigens, mouse albumin and transferrin, are strong binders, indicating that I-A g7 does not have a global defect in presentation of exogenous antigens. Peptide binding by purified I-A g7 has only been reported in two studies that used detergent-solubilized molecules. In one of these studies, little or no specific peptide binding could be demonstrated ( 17 ). In the other study, high detergent concentrations (2% NP-40) were used in the peptide binding assay ( 40 ). Binding was demonstrated for a hen egg lysozyme peptide (res. 9–27), but not for the mouse albumin peptide that had been eluted from purified I-A g7 and that represents the best binder for soluble I-A g7 ( 23 ). It is possible that these difficulties in examining peptide binding by I-A g7 are related to the isolation method, in particular to the use of certain detergents. The naturally processed transferrin and serum albumin peptides formed long-lived complexes with I-A g7 , in particular at a neutral pH. However, several peptides that have been identified as T cell epitopes of islet antigens were found to be poor binders. A similar observation has been made in the experimental autoimmune encephalomyelitis model, since the immunodominant MBP peptide (Ac1-11) in mice with the H-2 u haplotype is a very weak binder ( 24 ). Taken together, our data indicate that some peptides form long-lived complexes with I-A g7 . However, it is possible that a smaller fraction of I-A g7 molecules represent long-lived class II–peptide complexes and/or that the average affinity of I-A g7 bound peptides is lower than that of peptides bound to other I-A molecules. I-A g7 immunoprecipitated from splenocytes of NOD mice does not form SDS-resistant dimers ( 17 ). This property was also observed for soluble I-A g7 , even when the molecules were loaded with peptides. Therefore, it appears that SDS-sensitivity of the dimer is an intrinsic property of I-A g7 and does not directly reflect peptide content. SDS sensitivity of the I-A g7 dimer could be due to polymorphic residues at the dimerization interface of I-Aα and I-Aβ. Mutational analysis of I-A g7 indicated that β56 and β57 do not determine SDS-sensitivity ( 17 ). The absence of the salt bridge between β57 and α76 is therefore not responsible for the lack of SDS resistance. It is also important to keep in mind that instability of the dimer in SDS does not indicate that the dimer is also unstable under native conditions. In fact, analysis of I-A g7 purified from B cells did not indicate that the dimer was unstable in the absence of SDS ( 17 ). The pH-dependent interaction of I-A g7 results in the rapid generation of empty molecules at an endosomal pH. These results demonstrate a novel mechanism by which the presentation of antigenic peptides can be affected by a disease-associated MHC class II molecule. | Study | biomedical | en | 0.999997 |
10359577 | The modified HIV-1 transduction vector was derived from pHIV-PLAP, which has a backbone of the HIV-1 NL4-3 provirus with a frameshift in env and substitution of the human placental alkaline phosphatase (PLAP) gene in place of the nef gene ( 37 ). This vector was further manipulated to remove vif , vpr , vpu , and env genes as previously described ( 38 ). Genes of interest were introduced between the 5′ NotI and 3′ XhoI sites within the nef open reading frame, thus replacing the PLAP cDNA. An additional EcoRI site was introduced 3′ of the NotI site to facilitate subcloning of different cDNAs. The murine (m)CD28 cDNA (gift of J. Allison, University of California, Berkeley, CA) was subcloned into pBluescript (Stratagene) and the cytoplasmic mutant version was prepared by site-directed mutagenesis techniques through overlap PCR amplification using mutated oligonucleotides as previously described ( 39 ). All constructs were verified by DNA sequencing and were subcloned into the HIV vector. The mCD4 cDNA has been described previously ( 40 ). The enhanced green fluorescent protein (EGFP), purchased from Clontech , was subcloned into the HIV vector by introducing 5′ NotI and 3′ XhoI sites through PCR amplification. A bicistronic construct was prepared by ligation of the EGFP to the 3′ end of the encephalomyocarditis virus internal ribosomal entry site (IRES) sequence ( 41 ) and was similarly subcloned into the EcoRI-XhoI sites within the HIV vector. A GFP-encoding, replication-competent HIV-1 provirus was constructed by subcloning EGFP in place of the nef gene in the R5-tropic HIV-1 (BAL) backbone. The EGFP was also subcloned into the MLV-derived vector, pMX, provided by Dr. Toshio Kitamura (Tokyo University, Tokyo, Japan). PBMCs were separated from buffy coats of healthy donors (New York Blood Bank) through Ficoll-Hypaque ( Pharmacia , Sweden). Monocytes were removed by plastic adherence for 2 h at 37°C. To obtain highly purified resting CD4 + and CD8 + T cells, PBMCs depleted of monocytes were incubated with anti-CD4 or anti-CD8 conjugated with Dynabeads ( Dynal , Norway) at a 1:4 target/bead ratio. After 30 min of incubation at 4°C and continuous shaking, the bead-bound cells were recovered using a magnet ( Dynal ). The bead-bound cells were then washed at least four times to remove unbound cells and the CD4 + or CD8 + cells were detached from the beads using Detachabead ( Dynal ) according to the manufacturer's instructions. These cells were incubated with anti– HLA-DR and anti-CD69 antibodies, and then by Dynabeads conjugated with goat anti–mouse IgG, followed by magnetic removal of bead-bound preactivated cells. This purification protocol typically resulted in 99.5% purity of positively selected cells, as determined by postpurification FACS ® analysis. In some experiments, CD4 cells were negatively sorted into CD45RA + RO − and CD45RO + RA − subsets by incubating with anti-CD45RO and anti-CD45RA antibodies, respectively, followed by anti–mouse IgG Dynabead separation as described above. B and NK cells were purified by pre-incubating the cells with purified anti-CD19 ( Becton Dickinson ) or hybridoma supernatants of anti–human (h)CD16 (American Type Culture Collection [ATCC]), respectively, followed by magnetic separation using Dynabead-conjugated goat anti–mouse antibody. Bead-conjugated cells were then cultured at 37°C for 2 h and separated from released beads by a magnet ( Dynal ). The culture media used in all experiments was RPMI 1640 ( GIBCO BRL ) supplemented with 10% FCS (Hyclone), penicillin (50 U/ml; GIBCO BRL ), streptomycin (50 μg/ml), sodium pyruvate (1 mM; GIBCO BRL ), and glutamine (2 mM; GIBCO BRL ). T cell lines were prepared by activation of purified resting T cells with allogeneic PBMCs, treated with 50 μg/ml mitomycin C ( Sigma Chemical Co. ) for 30 min at 37°C and 5 μg/ml PHA ( Sigma Chemical Co. ). Alternatively, T cells were activated by cross-linking with plate-bound anti-CD3 antibody (OKT-3) and soluble anti-hCD28 antibody ( PharMingen ). Cells were split 3 d after activation, expanded, and maintained in culture media supplemented with 200 U/ml recombinant IL-2 (Chiron). Culture of T cells with cytokine combinations has been described previously ( 42 , 43 ). All cytokines were purchased from Genzyme or R&D Systems. Macrophages were differentiated in vitro after adherence to plastic. Dendritic cells (DCs) were generated as previously described ( 44 ). Activation of human B cells was performed by noncognate stimulation with activated T cells as previously described ( 42 ). The EBV-transformed B cell line was generated by infection of PBMCs with supernatants from the B95-8 cell line (ATCC) in the presence of 1 μg/ml Cyclosporin A ( Sigma Chemical Co. ). Replication-incompetent HIV particles pseudotyped with VSV-G were generated by calcium phosphate transfection of HEK-293T cells (ATCC) with 20 μg of proviral HIV vector and 12 μg of pL-VSV-G plasmid ( 36 ) per 3 × 10 6 cells seeded on 10-cm plates. The transfection media was replaced after 8 h with fresh DMEM supplemented with 10% FCS. Supernatants were collected at 48 h after transfection. The virus-containing supernatants were centrifuged for 10 min at 1,200 rpm to remove cells, then passed through 0.4-μm filters to remove fine debris. Supernatants either were used immediately for infections or were frozen in aliquots at −80°C. The viral titers were determined by infection of the human T cell line Hut78 with serially diluted virus supernatant. Typically, viral titers had a range of 3–10 × 10 6 infectious units (ifu)/ml. T cells were infected at a multiplicity of infection (MOI) of 10–20 in 24-well plates in the presence of 10 μg/ml polybrene ( Sigma Chemical Co. ). After an additional day of culture with virus supernatants, cells were washed or sedimented through Ficoll and resuspended in fresh culture media. R5-tropic replication-competent viruses were prepared similarly by transfecting 293T cells, and titers of 2–5 × 10 5 ifu/ml were obtained. VSV-G–pseudotyped MLV-based viruses were similarly prepared by transfecting 293T cells with 12 μg each of pMX.EGFP, pJK3 (expressing MLV gag and pol genes), and pL-VSV-G as well as 3 μg of pCMV-Tat plasmids. Flat-bottomed 96-well microtiter plates were coated for 2 h at 37°C with goat anti–mouse IgG (Caltag) at 20 μg/ml. Plates were washed twice with PBS and coated with anti-CD3 (OKT3, purified from ascites; ATCC) at different concentrations for 2 h at 37°C. Wells were washed an additional two times with PBS, and T cells at 10 5 cells/well were added to plates. Purified anti-mCD28 or anti-hCD28 antibodies ( PharMingen ) were added to cultures at 1 μg/ml in a 150 μl final volume. After 48–64 h, cells were pulsed for 16 h with 1 μCi of [ 3 H]thymidine, and [ 3 H]thymidine incorporation was measured with a beta counter (Wallac). To determine IL-2 production, culture supernatants were collected 36 h after initiation of stimulation and IL-2 activity was assayed using the CTLL-2 indicator cell line (obtained from ATCC). The proliferation of CTLL cells were determined by [ 3 H]thymidine incorporation for the last 4 h of a 24-h culture. Cells were stained with the relevant antibody on ice for 30 min in PBS buffer with 2% FCS and 0.1% sodium azide. Staining or GFP expression was analyzed on a FACScan ® using the CellQuest software ( Becton Dickinson ). Live cells were gated based on forward and side scatter. Cell sorting was performed at the New York University Medical Center core facility using a Coulter fluorescence-activated cell sorter. The following antibodies with PE, FITC, TC, or PercP conjugations were used for staining: anti-mCD28, anti-mCD4, anti-hCCR5 and anti-hCXCR4 ( PharMingen ), anti-hCD4 and -hCD45RO (Caltag), anti-hCD3, -hCD8, -hCD14, -hCD16, -hCD19, -hCD25, -hCD56, -hCD69, -hCD45RA, and -hHLA-DR (all from Becton Dickinson ). Bromodeoxyuridine (BrdU) incorporation was performed by culturing the cells with 10 μM BrdU ( Sigma Chemical Co. ) for the indicated time. After washing in staining buffer, the cells were permeabilized using FACS ® permeabilizing solution ( Becton Dickinson ) and stained with anti-BrdU–PE ( PharMingen ) in the presence of DNAse I ( Sigma Chemical Co. ). Analyses were performed using a FACScan ® . To achieve stable gene expression in primary human T cells using an HIV-1–derived vector (HDV) system, we used an NL4-3–based HIV-1 provirus that has deletions in the envelope glycoprotein ( env ) gene and the accessory genes vif , vpr , vpu , and nef ( 38 ). The GFP gene was subcloned in place of the nef open reading frame, and this vector was pseudotyped with the VSV-G envelope to produce a high titer, replication-incompetent virus. To determine the efficiency and stability of HIV-1–mediated gene transduction, mitogen-activated T cells were challenged with HDV-EGFP viruses at an MOI of 10–20. After 3 d in IL-2–containing medium, during which time the cells continued to expand, the majority of the T cells (∼65%) expressed GFP . The GFP + cells were then sorted by a FACS ® and subsequently expanded in IL-2 with bimonthly PHA stimulation to determine the stability of expression of the transduced gene. High levels of GFP expression were detected in T cell lines that were expanded in culture for up to 4 mo . The expression of the introduced gene was also stable in individual T cell clones that were established from HDV– transduced T cell lines (data not shown). Furthermore, it was possible to introduce (sequentially or simultaneously) three different genes (GFP, mCD28, and mCD4) into the same cells while maintaining their long-term expression (data not shown). Therefore, it is feasible to exploit the HDV system to express multiple gene products within a given primary T cell population. We next assessed the efficiency of HDV-mediated gene transduction into different subsets of T cells. To increase the sensitivity of detecting transduced cells, we constructed a modified vector, HDV-IRES.EGFP. Both spliced and unspliced transcripts encoded by this vector permit efficient translation of EGFP through the IRES, resulting in up to threefold higher expression levels (data not shown). CD4 + and CD8 + T cell subsets were purified from PBMCs, activated with PHA, and infected with HDV-IRES.EGFP virus. Analyses of GFP expression 5 d after infection consistently demonstrated a higher transduction efficiency (indicated as percentage of GFP expressing cells) of activated CD4 + T cells relative to CD8 + cells . Similar results were observed in separate experiments with other reporter genes and cells from different donors (data not shown). Transduction efficiencies of other subsets of lymphoid and myeloid lineage cells were also examined. Activated γ/δ-TCR + T cells, IL-2–activated NK cells, and in vitro– derived DCs were efficiently and stably transduced as shown by GFP expression 2–3 wk after infection with HDV-IRES.EGFP virus. However, we were unable to detect transduction of resting B cells, B cells stimulated through noncognate interaction with activated T cells, or B cells cultured with IL-4, IL-2, or IL-6 . Interestingly, EBV-transformed B cells were susceptible to infection albeit at a lower efficiency than activated T cells (∼20% of cells were transduced at comparable MOI, data not shown). Freshly isolated monocytes were also resistant to transduction using the HDV system but in vitro differentiation into macrophages rendered them susceptible (data not shown). It is well established that HIV challenge of quiescent T cells is blocked before completion of reverse transcription ( 22 ). We have shown that TCR-activated T cells are very efficiently and stably transduced with the HDV system. However, for many experimental applications it would be desirable to transduce resting T cells in the absence of TCR-induced activation, without affecting their differentiation state. To determine the parameters of resting T cell infection, CD4 + and CD8 + T cells were purified from PBMCs that were also negatively sorted for the activation markers HLA-DR and CD69. The purity of sorted cells was 99.5% for CD4 + T cells and 95–98% for CD8 + T cells, as determined by FACS ® analyses. The purified cells were cultured overnight and the resting status of these cells was confirmed by the negligible expression levels of activation molecules (HLA-DR and CD69) and the lack of cycling cells (data not shown). These cells were then infected with HDV-IRES.EGFP at an MOI of ∼20. As expected, we found very few T cells expressing GFP 5 d after infection (<0.3%), and concluded that freshly isolated resting T cells are resistant to transduction with the HDV system. Certain cytokines, such as IL-2, IL-4, IL-7, or proinflammatory cytokines, such as TNF-α or IL-6, have been shown to potentiate HIV replication in vitro ( 45 , 46 ). Some of these cytokines (IL-2, IL-4, and IL-7) also promote long-term survival of resting T cells in vitro, while maintaining the maturational state of these cells ( 43 , 47 ). We hypothesized that the signals delivered through cytokine receptors alone may cause resting naive or memory T cells to overcome the resistance to HDV-mediated transduction. To examine this possibility, CD4 + T cells were cultured in the presence of various cytokines or in media alone for 4 d before challenge with HDV-IRES-EGFP. 5 d after infection, robust GFP expression was observed in CD4 + cells that were cultured in IL-2, IL-4, IL-7, or IL-15, and a lower but significant enhancement was detected in the presence of IL-6 , but none with TNF-α, IL-12, or IFN-γ (data not shown). CD4 + human T cells can be divided into naive and memory subsets based on expression of RA and RO isoforms, respectively, of the CD45 molecule ( 48 ). These subsets are phenotypically and functionally distinct and have different activation requirements for antigen-specific stimulation ( 48 ). In contrast to memory T cells, naive cells exhibit few effector functions (e.g., cytokine production), are less susceptible to activation induced cell death, and display more robust proliferation in response to TCR-mediated signals. To assess transduction efficiencies of naive versus memory subsets of resting T cells stimulated with cytokines, we purified naive T cells by removal of CD45RO + T cells using bead selection. The purified population (100% RA + , 98% RO − ) was prestimulated with IL-2, IL-4, or IL-7 and challenged with HDV-IRES.EGFP in the presence of the same cytokines. Effective transduction of naive T cells cultured with IL-4, IL-7, and, to a lesser extent, IL-2, was observed . In contrast, memory T cells were more efficiently transduced when stimulated with IL-2 and IL-15 (see below and data not shown). Naive T cells cultured in media alone did not display any GFP expression upon infection (data not shown). Transduction of resting CD8 + cells was also achieved in the presence of the same cytokines at similar or better efficiencies than that observed with resting CD4 + cells . Interestingly, this result contrasts those observed with transduction of mitogen-activated CD8 + T cells, which were less efficiently transduced than CD4 + T cell lines . It has been shown that TNF-α and IL-6, in the presence of IL-2, IL-4, or IL-7, can fully activate resting T cells in an antigen-independent manner ( 42 , 43 ). These cytokine combinations had a small effect in increasing transduction efficiencies of CD4 + or CD8 + T cells, but greatly boosted infection of the naive subset . The activation status of resting CD4 + T cells stimulated with individual cytokines was determined by staining infected cells with antibodies specific for activation markers. In each condition, >95% of GFP + cells were negative for the activation molecules CD69 or HLA-DR (data not shown). Continuous BrdU labeling of cytokine-treated resting cells for 8 d revealed that a total of 5–7% of cells treated with IL-2 or IL-4 and ∼15% cells treated with IL-7 or IL-15 had entered the cell cycle during this period (data not shown). We then wished to determine whether transduction occurs only in the cells that have entered the cell cycle. Therefore, resting T cells were cultured continuously for 8 d with BrdU during cytokine stimulation and HIV infection. Although the presence of BrdU drastically reduced the efficiency of HIV infection (possibly due to adverse effects during reverse transcription), more than half of the cells that expressed GFP had not incorporated BrdU . This result shows that entry into the S phase of the cell cycle is not required for infection of the cytokine-stimulated resting T cells. In contrast to cytokine stimulation, almost all of the T cells activated through the TCR incorporated BrdU after 2 d of culture . Although few cytokine-stimulated T cells enter into the cell cycle, especially in the presence of IL-7 and IL-15, we asked whether MLV-derived vectors could also transduce these cells. Cytokine-stimulated T cells were challenged with a VSV-G–pseudotyped, GFP-expressing MLV-based vector, pMX.EGFP. 4 d after infection, <0.5% of the cytokine-stimulated T cells and ∼1.5% of the TCR-stimulated T cells expressed GFP, whereas nearly 100% of transformed human T cell line, Jurkat, were transduced (data not shown). These results clearly distinguish the efficiency of the HDV in the transduction of primary human T cells. Another important difference between cytokine-versus TCR-mediated stimulation of resting T cells is that, in contrast to TCR triggering, cytokine signals do not cause differentiation of naive T cells towards the memory phenotype ( 43 , 49 ). To confirm this, we cultured HDV-IRES.EGFP– infected resting T cells for 1 mo in the presence of IL-2, IL-4, IL-7, or IL-15. These cells were then stained with CD45RO antibody to discriminate memory (RO + ) and naive (RO − ) T cells. The proportion of naive to memory T cells was comparable to that in the starting population (∼1:1) in all conditions, although some bias was apparent in favor of memory or naive T cells with IL-2 and IL-4, respectively . The HDV-transduced naive T cells also retained their phenotype and IL-4 stimulation favored infection of these cells . To gain insight as to the level at which cytokines influence HIV infection of T cells, we challenged these cells with HDV-IRES.EGFP in the absence of cytokines. 3 d later cells were extensively washed to remove any remaining virus and were cultured in the presence or absence of cytokines for an additional 4 d. In the absence of cytokines, there were very few GFP + cells (<0.3%). However, addition of IL-2, IL-4, or IL-7 after infection revealed GFP expression in 1–3% of cells. We also infected resting cells in the presence of IL-2 or IL-4 plus IFN-α, which is known to block HIV infection at a preintegration step ( 50 , 51 ). Indeed, IFN-α completely blocked the transduction of resting T cells cultured with IL-2 or IL-4 (data not shown). Addition of IFN-α to cultures after 4 d of transduction in the presence of cytokines did not have any effect on GFP expression, even after 10 d of culture (data not shown). Taken together, these results suggest that HIV can enter into a few resting T cells, but is arrested at a preintegration stage of the viral life cycle. Cytokine signals are likely to overcome this preintegration block. Finally, we asked whether cytokine-activated T cells can also be infected with replication-competent HIV-1, since this may be an important mode of infection of T cells in vivo and in the establishment of latency in resting cells. We used a CCR5-tropic GFP-expressing replication-competent HIV-1 to infect cytokine-stimulated resting T cells under conditions similar to those used for the HDV transduction. Infection of cytokine-stimulated resting T cells was clearly observed 4 d after challenge . The GFP expression in the cytokine-treated cells remained stable for >2 wk (data not shown). However, the percentage of cells expressing GFP was lower as compared with VSV-G–pseudotyped viruses. This could be due to the relatively low titers of the R5 virus, which limit the infection to an MOI of 1–2 compared with 10–20 for VSV-G viruses. It is also possible that the infection is influenced by the expression of CCR5 on cytokine-stimulated versus TCR-activated T cells. Thus we examined CCR5 expression on resting T cells after culture in the presence of cytokines. The expression of CCR5 was modestly upregulated on IL-2 or IL-7 cultured resting T cells and more strikingly in the presence of IL-15 . The TCR- mediated stimulation induced CCR5 expression in nearly half of the cells . The expression of CCR5 was exclusively induced on the memory (CD45RO + ) subset of T cells , consistent with the expression pattern of CCR5 on PBMCs ( 52 ). The T-tropic HIV coreceptor CXCR4 is expressed on all the naive T cells and on most of the memory T cells (data not shown and reference 52 ); however, we also observed an approximately fivefold increase in CXCR4 expression levels in the presence of IL-4 (data not shown), similar to recent reports ( 53 – 55 ). One attractive feature of the retroviral system is its potential use for dissecting signaling pathways in primary cells. Because we are interested in costimulatory signals mediated by the CD28 molecule, we assessed the feasibility of performing a structure–function analysis of this molecule by expressing mCD28 on human T cells. CD4 + or CD8 + human T cells, expressing mCD28 , were stimulated at suboptimal concentrations of plate-bound anti-CD3, in the absence or presence of soluble antibodies to hCD28 or mCD28, and cell proliferation or IL-2 production were measured. T cells that expressed mCD28 proliferated equally well in response to antibodies against mCD28 or hCD28, in the presence of suboptimal TCR cross-linking . As a control, cells that stably expressed a mutant form of mCD28 bearing a frame-shift mutation in its cytoplasmic domain (at Asp188) displayed a severely impaired proliferative response to anti-mCD28 . It should be noted that the increase in proliferation observed with mutant mCD28 at higher concentrations of anti-CD3 was also observed with anti-mCD4 antibody when mCD4-expressing T cells were used, and thus may be the result of antibody-mediated clustering. Since one of the hallmarks of CD28 costimulation is upregulation of IL-2 production ( 56 ), we also quantitated IL-2 synthesis in response to mCD28 costimulation. IL-2 secretion was increased by up to 50-fold upon treatment of CD4 + T cell lines that expressed mCD28 with anti-mCD28 or -hCD28 . As expected, mCD28 antibodies did not upregulate IL-2 production in mCD28 nonexpressing cells or in cells expressing the cytoplasmic mutant form of mCD28 . Furthermore, the costimulation through mCD28 was also partially resistant to Cyclosporin A, similar to what is observed upon endogenous CD28 costimulation (data not shown). To exclude the possibility of heterodimer formation between mCD28 and hCD28, we used CD8 + T cell lines that stably express mCD28 but have completely downregulated hCD28 because of repeated stimulations during in vitro culture. These cells produced high levels of IL-2 in response to mCD28 costimulation, but, as expected, did not respond to treatment with anti-hCD28 antibodies . Similar studies were also possible in cytokine-cultured resting T cells expressing mCD28 (data not shown). These experiments clearly demonstrate that it will be feasible to perform a thorough structure–function analysis of CD28 and possibly other signaling molecules in primary human T lymphocytes. We have shown here, using an HDV system as well as replication-competent HIV-1, that cytokines can stimulate infection of resting T lymphocytes with these viruses. Previous studies have demonstrated a block in reverse transcription of HIV-1 in resting T cells. Cytokine stimulation appears to overcome this block, resulting in proviral integration and expression. This result may have major implications in understanding mechanisms of HIV-1 transmission and pathogenesis. The HIV-1–based vector system described here can be used for efficient and stable introduction of genes into all subsets of activated human T cells as well as NK cells, macrophages, DCs, and cytokine-stimulated resting T cells. Efficient transduction of both resting and activated cells was achieved despite the deletion of the accessory genes vif , vpr , vpu , and nef in the HIV vector. The removal of these accessory genes avoids many unwanted effects in T cells. Indeed, we did not observe any aberrant function of primary T cells with regards to activation or proliferation after HDV- mediated transduction. The use of a heterologous envelope in pseudotypes and the deletion of accessory proteins important for virulence also makes it very unlikely that replication-competent recombinant virus can be generated. Using a sensitive assay ( 57 ), replication-competent viruses were not detected in any of the infected lines. We found transduction of mitogen-activated T cells with HDV to be highly efficient; however, the transduction of CD4 + T cells was always better than that of CD8 + T cells. This may be due to the secretion of various inhibitory factors such as IFNs by activated CD8 + T cells and/or the lack of factors that promote more efficient integration/ transcription of HIV. In contrast, cytokine-activated resting CD8 + T cells were infected very efficiently, even better than CD4 + T cells stimulated with cytokines or CD8 + T cells activated with mitogens . Additional studies will be needed to identify host factors induced by cytokine- versus TCR-mediated signaling that affect the efficiency of HIV infection. The lentivirus subfamily of retroviruses, which includes HIV-1, can infect nondividing cells because the HIV preintegration complex can exploit the cellular machinery to allow transport into the nucleus ( 11 – 17 ). Efficient infection of nondividing, terminally differentiated cells such as neurons and macrophages or quiescent human hematopoietic stem cells with HDVs has been reported ( 26 , 27 , 29 , 33 ). However, infection of quiescent T cells is blocked before integration, probably due to incomplete reverse transcription ( 18 , 58 – 60 ) or failure to transport the viral preintegration complex to the nucleus ( 19 , 21 , 23 , 61 ). Consistent with these data, the HIV vector system described here does not support expression of ectopic genes in resting human T cells. However, by treating resting T cells with IL-2, IL-4, IL-7, or IL-15 before virus challenge we were able to overcome this block. Remarkably, these cytokines were equally or even more effective in enhancing transduction of CD45RA + naive human T cells, especially when combined with IL-6 and TNF-α. Although some resting T cells progress through the cell cycle in response to cytokine-mediated signals, this was not a prerequisite for the infection of these cells . Unlike TCR stimulation, cytokine-mediated signals do not change the differentiation state of the cells, such that naive T cells remain phenotypically naive and there is very little expression of activation markers such as CD69 (with the exception of IL-15 stimulation) and HLA-DR. Introduction of exogenous genes into purified resting T cells stimulated with cytokines may have applications in clinical gene therapy where it may not be desirable or even possible (for example in some immunodeficiencies), to activate the cells through the TCR. In addition, this system can be used to address questions related to differentiation of naive and memory T cells, by enabling genetic manipulation of these cells in the absence of TCR-mediated activation. Infection of cytokine-stimulated resting T cells may have important physiological implications for HIV infection in vivo. Cytokines have been reported to have dramatic effects on HIV replication in infected cells ( 45 , 46 , 62 , 63 ). In vivo, resting T cells that carry integrated provirus can be detected even after highly active antiretroviral therapy, albeit at a very low frequency ( 23 , 64 – 66 ). Interestingly, in a recent study, the cytokine combination of IL-2, TNF-α, and IL-6 was shown to induce expression of HIV in latently infected resting T cells isolated from HIV-infected individuals ( 67 ). However, it is not clear how resting T cells in vivo become latently infected. It is generally thought that memory T cells that may have been infected while in an active state become quiescent before the virus replicates and induces cytotoxicity ( 68 ). We have shown that an R5 strain of HIV-1, with intact accessory genes, can also infect cytokine-activated resting T cells. Our data and those of others indicate that CCR5 cell surface expression is upregulated in cytokine-stimulated cells especially with IL-15 and at a lower level with IL-2 or IL-7. Although this may facilitate infection by R5 viruses, the effect of the cytokines clearly extends beyond viral entry in the resting T cells as demonstrated by the HIV (VSV-G)–pseudotyped viruses. Therefore, it is possible that infection with HIV-1 in vivo might occur in resting T cells that are continuously exposed to cytokines at sites of infection or in secondary lymphoid organs ( 45 ). This may be important for the establishment of infection and for viral pathogenesis. For example, infection of macrophages or DCs with HIV-1 may induce innate immune responses that result in production of the relevant cytokines that promote infection of resting T cells. It will be necessary to determine the efficiency of viral replication and whether latency can be established in cytokine-stimulated T cells. It is also tempting to speculate that cytokine-mediated bystander activation of resting T cells may facilitate the spread of the virus in vivo, thus contributing to the rapid viral turnover rates ( 70 , 71 ). How do cytokine-induced signals allow transduction of resting T cells? Identifying the molecular mechanisms by which these signals enhance proviral establishment in resting T cells will help in understanding which host cofactors are required during early stages of HIV infection. It is interesting to note that, except for IL-6, the cytokines that enhance the transduction of resting T cells all share the γ c chain in their receptors ( 72 ), suggesting a role for signals delivered through this receptor component. It has been reported that phosphorylation of components of the viral preintegration complex by kinases, such as a virion-associated, mitogen-activated protein kinase, appears to enable efficient transport of this complex to the nucleus ( 73 ). Thus, signals delivered from the cytokine receptors may activate host factors that are necessary for the translocation of the preintegration complexes into the nucleus. Our finding that primary human B cells are not detectably transduced is intriguing. We did not assess whether the block in B cells is at the level of integration or transcription. It may be that primary B cells lack factors required for integration/ transcription and that these are induced in EBV-transformed B cells. It is also possible that other signaling conditions may permit efficient transduction of B cells. The infection of activated primary or transformed mouse T cells also was very inefficient (1–2%), whereas transduction using an MLV-based retroviral system routinely transduced 10–60% of transformed mouse T cells (data not shown). Although we did not attempt to infect other primary mouse cell types, two murine cell lines, NIH 3T3 fibroblasts and, interestingly, the IL-2–dependent T cell line CTLL, were efficiently and stably transduced (data not shown). Elucidating these cell-type specific restrictions or requirements for HDV-mediated transduction may also be helpful in enhancing the susceptibility of murine models of HIV infection ( 74 ). The ability to introduce genes into primary T lymphocytes should greatly assist the studies of T cell differentiation and signal transduction in vitro. As a proof of this concept we have shown that structure–function analysis of the costimulatory molecule CD28 is feasible in primary lymphocytes and that mCD28 is functional in delivering costimulatory signals in human T cells. Previously, these studies have been limited to transformed cell lines, and it is likely that signaling pathways differ in subtle but critical ways in primary T cells. Indeed, our ongoing mutational analysis of CD28 cytoplasmic domain residues has revealed differences when compared with similar studies performed in the transformed human Jurkat T cell line ( 39 ) and identified functionally important regions of CD28 in primary T cells (Unutmaz, D., and S. Marmon, unpublished data). Similar studies can readily be applied to other signaling molecules expressed in primary lymphocytes or myeloid cells. Furthermore, dominant negative or constitutively active forms of intracellular molecules involved in these pathways can be expressed and analyzed using the HDV-IRES. GFP by sorting for GFP + cells. In conclusion, the HIV-1–based transduction system described in this study will be a valuable tool in studying many aspects of signal transduction in primary T cells and can have potential applications in somatic gene therapy. Here we have for the first time demonstrated that stimulation of resting T cells with cytokines is sufficient for HIV-1 infection of these cells while preserving their differentiation state. This finding may have major implications in understanding the mechanisms of virus infection of resting T cells in vivo. | Study | biomedical | en | 0.999997 |
10359578 | The anti-Flag M2 mAb, biotinylated anti-Flag M2 antibody, and the anti-Flag M2 antibody coupled to agarose were purchased from Sigma Chemical Co. Cell culture reagents were obtained from Life Sciences and BioWhittaker. Flag-tagged soluble human APRIL (a proliferation inducing ligand; residues K 110 – L 250 ) was produced in 293 cells as described ( 10 , 11 ). FITC- labeled anti-CD4, anti-CD8, and anti-CD19 antibodies were purchased from PharMingen . Goat F(ab′) 2 specific for the Fc 5μ fragment of human IgM were purchased from Jackson ImmunoResearch Laboratories. Secondary antibodies were obtained from either PharMingen or Jackson ImmunoResearch Laboratories and were used at the recommended dilutions. Human embryonic kidney 293 T cells ( 12 ) and fibroblast cell lines (see Table I ) were maintained in DMEM containing 10% heat-inactivated FCS. Human embryonic kidney 293 cells were maintained in DMEM-nutrient mix F12 (1:1) supplemented with 2% FCS. T cell lines, B cell lines, and macrophage cell lines (see Table I ) were grown in RPMI supplemented with 10% FCS. Molt-4 cells were cultivated in Iscove's medium supplemented with 10% FCS. Epithelial cell lines were grown in MEM-α medium containing 10% FCS, 0.5 mM nonessential amino acids, 10 mM Na-Hepes, and 1 mM Na pyruvate. Human umbilical vein endothelial cells were maintained in M199 medium supplemented with 20% FCS, 100 μg/ml of epithelial cell growth factor (Collaborative Research, Inotech), and 100 μg/ml of heparin sodium salt ( Sigma Chemical Co. ). All media contained penicillin and streptomycin antibiotics. PBLs were isolated from heparinized blood of healthy adult volunteers by Ficoll-Paque ( Amersham Pharmacia Biotech ) gradient centrifugation and cultured in RPMI, 10% FCS. T cells were obtained from nonadherent PBLs by rosetting with neuraminidase-treated sheep red blood cells and separated from nonrosetting cells (mostly B cells) by Ficoll-Paque gradient centrifugation. Purified T cells were activated for 24 h with phytohemagglutinin (1 μg/ml; Sigma Chemical Co. ), washed, and cultured in RPMI, 10% FCS, 20 U/ml of IL-2. CD14 + monocytes were purified by magnetic cell sorting using anti-CD14 antibodies, goat anti–mouse-coated microbeads, and a Minimacs™ device (Miltenyi Biotech), and cultivated in the presence of GM-CSF (800 U/ml, Leucomax ® ; Essex Chemie) and IL-4 (20 ng/ml; Lucerna Chem) for 5 d, then with GM-CSF, IL-4, and TNF-α (200 U/ml; Bender) for an additional 3 d to obtain a CD83 + , dendritic cell–like population. Human B cells of >97% purity were isolated from peripheral blood or umbilical cord blood using anti-CD19 magnetic beads (M450; Dynal ) as described ( 13 ). Northern blot analysis was carried out using Human Multiple Tissue Northern Blots I and II . The membranes were incubated in hybridization solution (50% formamide, 2.5× Denhardt's, 0.2% SDS, 10 mM EDTA, 2× SSC, 50 mM NaH 2 PO 4 , pH 6.5, 200 μg/ml sonicated salmon sperm DNA) for 2 h at 60°C. Antisense RNA probe containing the nucleotides corresponding to amino acids (aa) 136–285 of human BAFF (hBAFF) was heat denatured and added at 2 × 10 6 cpm/ml in fresh hybridization solution. The membrane was hybridized 16 h at 62°C, washed once in 2× SSC, 0.05% SDS (30 min at 25°C), once in 0.1× SSC, 0.1% SDS (20 min at 65°C), and exposed at −70°C to x-ray films. A partial sequence of hBAFF cDNA was contained in several expressed sequence tag (EST) clones derived from fetal liver and spleen and ovarian cancer libraries. The 5′ portion of the cDNA was obtained by 5′ rapid amplification of cDNA ends (RACE) PCR (Marathon-Ready cDNA; Clontech ) with oligonucleotides AP1 and JT1013 (5′-ACTGTTTCTTCTGGACCCTGAACGGC-3′) using the provided cDNA library from a pool of human leukocytes as template, as recommended by the manufacturer. The resulting PCR product was cloned into PCR-0 blunt (Invitrogen) and subcloned as EcoRI-PstI fragment into pT7T3 Pac vector ( Amersham Pharmacia Biotech ) containing EST clone T87299 . Therefore, full-length hBAFF cDNA was obtained by combining 5′ and 3′ fragments using the internal PstI site of BAFF. The sequence has been assigned EMBL/GenBank/ DDBJ accession no. AF116456 . A partial 617-bp sequence of murine BAFF was contained in two overlapping EST clones . A PCR fragment spanning nucleotides 158–391 of this sequence was used as a probe to screen a mouse spleen cDNA library (Stratagene, Inc.). The sequence has been assigned EMBL/GenBank/DDBJ accession no. AF119383 . Full-length hBAFF was amplified using oligos JT1069 (5′-GACAAGCTTGCCACCATGGATGACTCCACA-3′) and JT637 (5′-ACTAGTCACAGCAGTTTCAATGC-3′). The PCR product was cloned into PCR-0 blunt and resubcloned as HindIII-EcoRI fragment into PCR-3 mammalian expression vector. A short version of soluble BAFF (sBAFF/short, aa Q136–L285) was amplified using oligos JT636 (5′-CTGCAGGGTCCAGAAGAAACAG-3′) and JT637. A long version of sBAFF (sBAFF/long, aa L83–L285) was obtained from full-length BAFF using internal PstI site. sBAFFs were resubcloned as PstI-EcoRI fragments behind the hemagglutinin signal peptide and Flag sequence of a modified PCR-3 vector, and as PstI-SpeI fragments into a modified pQE16 bacterial expression vector in frame with an NH 2 -terminal Flag sequence ( 14 ). Constructs were sequenced on both strands. The establishment of stable 293 cell lines expressing the short soluble form or full-length BAFF, and the expression and purification of recombinant sBAFF from bacteria and mammalian 293 cells were performed as described ( 14 , 15 ). Total RNA extracted from T cells, B cells, in vitro–derived immature dendritic cells, 293 wild-type (wt) and 293-BAFF (full-length) cells was reverse transcribed using the Ready to Go system ( Amersham Pharmacia Biotech ) according to the manufacturer's instructions. BAFF and β-actin cDNAs were detected by PCR amplification with Taq DNA polymerase (steps of 1 min each at 94°C, 55°C, and 72°C for 30 cycles) using specific oligonucleotides: for BAFF, JT1322 5′-GGAGAAGGCAACTCCAGTCAGAAC-3′ and JT1323 5′-CAATTCATCCCCAAAGACATGGAC-3′; for IL-2 receptor α chain, JT1368 5′-TCGGAACACAACGAAACAAGTC-3′ and JT1369 5′-CTTCTCCTTCACCTGGAAACTGACTG-3′; and for β-actin, 5′-GGCATCGTGATGGACTCCG-3′ and 5′-GCTGGAAGGTGGACAGCGA-3′. 293 T cells were transiently transfected with the short form of sBAFF and grown in serum-free Optimem medium for 7 d. Conditioned supernatants were concentrated 20 times, mixed with internal standards catalase and OVA, and loaded onto a Superdex-200 HR10/30 column. Proteins were eluted in PBS at 0.5 ml/min, and fractions (0.25 ml) were precipitated with TCA and analyzed by Western blotting using anti-Flag M2 antibody. The column was calibrated with standard proteins: ferritin (440 kD), catalase (232 kD), aldolase (158 kD), BSA (67 kD), OVA (43 kD), chymotrypsinogen A (25 kD), and ribonuclease A (13.7 kD). Samples were heated in 20 μl of 0.5% SDS, 1% 2-ME for 3 min at 95°C, then cooled and supplemented with 10% NP-40 (2 μl), 0.5 M sodium phosphate, pH 7.5 (2 μl), and Peptide N -glycanase F (PNGase F; 125 U/μl, 1 μl, or no enzyme in controls). Samples were incubated for 3 h at 37°C before analysis by Western blotting. 293 T cells were transiently transfected with the long form of sBAFF and grown in serum-free Optimem medium for 7 d. Conditioned supernatants were concentrated 20 times, fractionated by SDS-PAGE, and blotted onto polyvinylidene difluoride membrane (Bio-Rad Laboratories) as described previously ( 16 ), and then sequenced using a gas phase sequencer (ABI 120A; Perkin Elmer ) coupled to an analyzer (ABI 120A; Perkin Elmer ) equipped with a phenylthiohydantoin C18 2.1 × 250 mm column. Data were analyzed using ABI 610 software ( Perkin Elmer ). Polyclonal antibodies were generated by immunizing rabbits (Eurogentec) with recombinant sBAFF/long. Spleens of rats immunized with the same antigen were fused to x63Ag8.653 mouse myeloma cells, and hybridomas were screened for BAFF-specific IgGs. One of these mAbs, 43.9, is an IgG2a that specifically recognizes hBAFF. Cells were stained in 50 μl of FACS buffer (PBS, 10% FCS, 0.02% NaN 3 ) with 50 ng (or the indicated amount) of Flag-tagged short soluble hBAFF for 20 min at 4°C, followed by anti-Flag M2 (1 μg) and secondary antibody. Anti-BAFF mAb 43.9 was used at 40 μg/ml. For two-color FACS ® analysis, peripheral blood lymphocytes were stained with Flag-tagged sBAFF/long (2 μg/ml), followed by biotinylated anti-Flag M2 (1:400) and PE-labeled streptavidin (1:100), followed by FITC-labeled anti-CD4, anti-CD8, or anti-CD19. PBLs were incubated in 96-well plates (10 5 cells/well in 100 μl RPMI supplemented with 10% FCS) for 72 h in the presence or absence of 2 μg/ml of goat anti–human μ chain antibody ( Sigma Chemical Co. ) or control F(ab′) 2 and with the indicated concentration of native or boiled sBAFF/long. Cells were pulsed for an additional 6 h with [ 3 H]thymidine (1 μCi/well) and harvested. [ 3 H]Thymidine incorporation was monitored by liquid scintillation counting. In some experiments, recombinant sBAFF was replaced by 293 cells stably transfected with full-length BAFF (or 293 wild-type [wt] as control) that had been fixed for 5 min at 25°C in 1% paraformaldehyde. Assay was performed as described ( 17 ). In further experiments, CD19 + cells were isolated from PBLs with magnetic beads, and the remaining CD19 − cells were irradiated (3,000 rads) before reconstitution with CD19 + cells. Proliferation assay with sBAFF was then performed as described above. Purified B cells were activated in the EL-4 culture system as described ( 13 ). In brief, 10 4 B cells mixed with 5 × 10 4 irradiated murine EL-4 thymoma cells (clone B5) were cultured for 5–6 d in 200 μl medium containing 5% vol/vol of culture supernatants from human T cells (10 6 /ml) which had been activated for 48 h with PHA (1 μg/ml) and PMA (1 ng/ml). B cells were then reisolated with anti-CD19 beads and cultured for another 7 d (5 × 10 4 cells in 200 μl, duplicate or triplicate culture in flat-bottomed 96 well plates) in medium alone or in medium supplemented with 5% T cell supernatants, or with 50 ng/ml IL-2 (a gift from the former Glaxo Institute for Molecular Biology, Geneva) and 10 ng/ml each IL-4 and IL-10 ( PeproTech ), in the presence or absence of sBAFF. The anti-Flag M2 antibody was added at a concentration of 2 μg/ml and had no effect by itself. hBAFF was identified by sequence homology as a possible novel member of the TNF ligand family while we screened public databases using an improved profile search ( 18 ). A cDNA encoding the complete protein of 285 aa was obtained by combining EST clones (covering the 3′ region) with a fragment (5′ region) amplified by PCR. The absence of a signal peptide suggested that BAFF was a type II membrane protein that is typical of the members of the TNF ligand family. The protein has a predicted cytoplasmic domain of 46 aa, a hydrophobic transmembrane region, and an extracellular domain of 218 aa containing two potential N -glycosylation sites . The sequence of the extracellular domain of BAFF shows highest homology with APRIL (33% aa identity, 48% homology), whereas the identity with other members of the family, such as TNF, FasL, LTα, TRAIL (TNF-related apoptosis-inducing ligand), or RANKL (receptor activator of NF-κB ligand) is <20% . The mouse BAFF (mBAFF) cDNA clone isolated from a spleen library encoded a slightly longer protein (309 aa) due to an insertion between the transmembrane region and the first of several β strands which constitute the receptor binding domain in all TNF ligand members ( 19 ). This β strand–rich ectodomain is almost identical in mBAFF and hBAFF (86% identity, 93% homology), suggesting that the BAFF gene has been highly conserved during evolution . Although TNF family members are synthesized as membrane-inserted ligands, cleavage in the stalk region between transmembrane and receptor binding domains is frequently observed. For example, TNF and FasL are readily cleaved from the cell surface by metalloproteinases ( 20 , 21 ). While producing several forms of recombinant BAFF in 293 T cells, we noticed that a recombinant soluble 32-kD form of BAFF (aa 83–285, sBAFF/long), containing the complete stalk region and an NH 2 -terminal Flag tag in addition to the receptor binding domain, was extensively processed to a smaller 18-kD fragment . Cleavage occurred in the stalk region since the fragment was detectable only with antibodies raised against the complete receptor interaction domain of BAFF but not with anti-Flag antibodies (data not shown). This experiment also revealed that only N124 (located in the stalk) but not N242 (located at the entry of the F-β sheet) was glycosylated, since the molecular mass of the nonprocessed sBAFF/long was reduced from 32 to 30 kD upon removal of the N-linked carbohydrates with PNGase F, whereas the 18-kD cleaved form was insensitive to this treatment. Peptide sequence analysis of the 18-kD fragment indeed showed that cleavage occurred between R133 and A134 . R133 lies at the end of a polybasic region that is conserved between human (R-N-K-R) and mouse (R-N-R-R) BAFF. To test whether cleavage was not merely an artifact of expressing soluble, nonnatural forms of BAFF, membrane-bound full-length BAFF was expressed in 293 T cells . The 32-kD complete BAFF and some higher molecular mass species (probably corresponding to nondissociated dimers and trimers) were readily detectable in cellular extracts, but >95% of BAFF recovered from the supernatant corresponded to the processed 18-kD form, indicating that BAFF was also processed when synthesized as a membrane-bound ligand. Therefore, we engineered an sBAFF (Q136–L285, sBAFF/short) whose sequence started 2 aa downstream of the processing site . As predicted, the Flag tag attached to the NH 2 terminus of this recombinant molecule was not removed (data not shown), which allowed its purification by an anti-Flag affinity column. To test its correct folding, the purified sBAFF/short was analyzed by gel filtration where the protein eluted at an apparent molecular mass of 55 kD . We conclude that sBAFF/short correctly assembles into a homotrimer (3 × 20 kD) in agreement with the quaternary structure of other TNF family members ( 19 ). Finally, unprocessed sBAFF/long was readily expressed in bacteria, indicating that the cleavage event was specific to eukaryotic cells. Northern blot analysis of BAFF revealed that the 2.5-kb BAFF mRNA was abundant in the spleen and PBLs . Thymus, heart, placenta, small intestine, and lung showed weak expression. This restricted distribution suggested that cells present in lymphoid tissues were the main source of BAFF. Through PCR analysis, we found that BAFF mRNA was present in T cells and peripheral blood monocyte–derived dendritic cells but not in B cells . Even naive, nonstimulated T cells appeared to express some BAFF mRNA. A sequence-tagged site was found in the database which included the hBAFF sequence. This site maps to human chromosome 13, in a 9-cM interval between the markers D13S286 and D13S1315. On the cytogenetic map, this interval corresponds to 13q32-34. Of the known TNF ligand family members, only RANKL (Trance) has been localized to this chromosome ( 22 ) though quite distant to BAFF (13q14). For the ligand to exert maximal biological effects, it was likely that the BAFF receptor (BAFF-R) would be expressed either on the same cells or on neighboring cells present in lymphoid tissues. Using the recombinant sBAFF as a tool to specifically determine BAFF-R expression by FACS ® , we indeed found high levels of receptor expression in various B cell lines, such as the Burkitt lymphomas Raji and BJAB . In contrast, cell lines of T cell, fibroblastic, epithelial, and endothelial origin were all negative. Very weak staining was observed with the monocyte line THP-1, which, however, could be due to Fc receptor binding. Thus, BAFF-R expression appears to be restricted to B cell lines. The two mouse B cell lines tested were negative using the hBAFF as a probe, although weak binding was observed on mouse splenocytes (data not shown). The presence of BAFF-R on B cells was corroborated by analysis of umbilical cord and peripheral blood lymphocytes. While CD8 + and CD4 + T cells lacked BAFF-R , abundant staining was observed on CD19 + B cells , indicating that BAFF-R is expressed on all blood B cells, including naive and memory B cells. No evidence was obtained for a CD19 + , BAFF-R − population. Since BAFF bound to blood-derived B cells, experiments were performed to determine whether the ligand could deliver growth-stimulatory or growth-inhibitory signals. PBLs were stimulated with anti-IgM (μ) antibodies together with fixed 293 cells stably expressing surface BAFF . The levels of [ 3 H]thymidine incorporation induced by anti-μ alone were not altered by the presence of control cells but were increased twofold in the presence of BAFF-transfected cells . A dose-dependent proliferation of PBLs was also obtained when BAFF-transfected cells were replaced by purified sBAFF , indicating that BAFF does not require membrane attachment to exert its activity. In this experimental setup, proliferation induced by sCD40L required concentrations >1 μg/ml, but was less dependent on the presence of anti-μ than that mediated by BAFF . When purified CD19 + B cells were cocultured with irradiated autologous CD19 − PBLs, costimulation of proliferation by BAFF was unaffected, demonstrating that [ 3 H]thymidine uptake was mainly due to B cell proliferation and not to an indirect stimulation of another cell type (data not shown). The observed B cell proliferation in response to BAFF was entirely dependent on the presence of anti-μ antibodies, indicating that BAFF functioned as costimulator of B cell proliferation. To investigate a possible effect of BAFF on preplasma, germinal center–like B cells ( 13 ), purified peripheral or cord blood B cells were preactivated by coculture with EL-4 T cells in the presence of a cytokine mixture from supernatants of PHA/PMA-stimulated T cells ( 23 ). These B cells were reisolated to 98% purity and yielded a twofold increase in secreted Ig during a secondary culture in the presence of BAFF and activated T cell cytokines compared with cytokines alone. No significant effect was seen in the absence of exogenous cytokines, and an intermediate (1.5-fold) effect was observed in the presence of the recombinant cytokines IL-2, IL-4, and IL-10 . Here we report the molecular cloning, expression, and biological activity of a new member of the TNF ligand family. The human and mouse sequences exhibit the typical characteristics of this family, i.e., a type II membrane protein organization and the conservation of nine β sheets, which fold into a “jelly-roll” structure that trimerizes to form receptor interacting sites. The biochemical analysis of BAFF is also consistent with the typical homotrimeric structure of TNF family members. In this family of ligands, BAFF exhibits the highest level of sequence similarity with APRIL, which we have recently characterized as a ligand stimulating growth of various tumor cells ( 11 ). Unlike TNF and LTα, which are two family members with equally high homology (33% identity) and whose genes are linked on chromosome 6, APRIL and BAFF are not clustered on the same chromosome. APRIL is located on chromosome 17 (our unpublished data), whereas BAFF maps to the distal arm of human chromosome 13 (13q34). Abnormalities in this locus were characterized in Burkitt lymphomas as the second most frequent defect ( 24 ) besides the translocation involving the myc gene into the Ig locus ( 25 ). Considering the high expression levels of BAFF-R on all Burkitt lymphoma cell lines analyzed (see Table I ), this raises the intriguing possibility that some Burkitt lymphomas may have deregulated BAFF expression, thus stimulating growth in an autocrine manner. B cell growth was efficiently costimulated with recombinant sBAFF lacking the transmembrane domain. This activity is in contrast to several TNF family members that are active only as membrane-bound ligands, such as TRAIL, FasL, and CD40L. Soluble forms of these ligands have poor biological activity that can be enhanced by their cross-linking, thereby mimicking the membrane-bound ligand ( 15 ). In contrast, cross-linking Flag-tagged sBAFF with anti-Flag antibodies or the use of membrane-bound BAFF expressed on the surface of epithelial cells did not further enhance the mitogenic activity of BAFF, suggesting that it can act systemically as a secreted cytokine, like TNF does. This is in agreement with the observation that a polybasic sequence present in the stalk of BAFF acted as a substrate for a protease. Similar polybasic sequences are also present at corresponding locations in both APRIL and TWEAK (Apo-3L), and for both of them there is evidence of proteolytic processing (26; Holler, N., and J. Tschopp, unpublished observation). Although the protease responsible for the cleavage remains to be determined, it is unlikely to be the metalloproteinase responsible for the release of membrane-bound TNF, as their sequence preferences differ completely ( 21 ). The multibasic motifs in BAFF (R-N-K-R), APRIL (R-K-R-R), and TWEAK (R-P-R-R) are reminiscent of the minimal cleavage signal for furin (R-X-K/R-R), the prototype of a proprotein convertase family ( 27 ). The role of antigen-specific B lymphocytes during the different stages of the immune response is highly dependent on signals and contacts from helper T cells ( 28 ) and antigen-presenting cells such as dendritic cells ( 29 ). B lymphocytes first receive these signals early on during the immune response when they interact with T cells at the edge of the B cell follicles in lymphoid tissues, leading to their proliferation and differentiation into low-affinity antibody-forming cells ( 30 ). At the same time, some antigen-specific B cells also migrate to the B cell follicle and contribute to the formation of germinal centers, another site of B cell proliferation but also affinity maturation and generation of memory B cells and high-affinity plasma cells ( 31 ). Signals triggered by CD40L have been shown to be critical for the function of B lymphocytes at multiple steps of the T cell–dependent immune response ( 32 ). However, several studies clearly showed that CD40L–CD40 interaction does not account for all contact-dependent T cell help for B cells. Indeed, CD40L-deficient T cells isolated from either knockout mice or patients with X-linked hyper IgM syndrome have been shown to successfully induce proliferation of B cells and their differentiation into plasma cells ( 33 ). Likewise, studies using blocking antibodies against CD40L showed that a subset of surface IgD + B cells isolated from human tonsils proliferate and differentiate in response to activated T cells in a CD40-independent manner ( 34 ). Other members of the TNF family, such as membrane-bound TNF and CD30L, have also been shown to be involved in a CD40- and surface Ig–independent stimulation of B cells ( 33 , 35 ). Finally, CD40-deficient B cells can be stimulated to proliferate and differentiate into plasma cells by helper T cells as long as the surface B cell receptors are triggered at the same time ( 36 ). BAFF as well as CD30L and CD40L is expressed by T cells, but its uniqueness resides in its expression by dendritic cells as well as the highly specific location of its receptor on B cells in contrast to the wider expression patterns of CD40, CD30, and the TNF receptors ( 37 ). Hence, BAFF may uniquely affect B cells. In support of a role for BAFF in T cell– and/or dendritic cell–induced B cell growth and potential maturation, we found that BAFF costimulates proliferation of blood- derived B cells concomitantly with cross-linking of the B cell receptors. Moreover, using CD19 + B cells differentiated in vitro into preplasma, germinal center–like B cells ( 13 ), we observed a costimulatory effect of BAFF on Ig production by these B cells in the presence of cytokines from activated T cells. Thus, BAFF can induce signals in both naive B cells and germinal center–committed B cells in vitro. Whether this observation will translate during a normal immune response or not will have to be addressed by proper in vivo experiments. The biological responses induced in B cells by BAFF are distinct from that of CD40L, since proliferation triggered by CD40L occurred at a lower level independently of an anti-μ costimulus . Moreover, CD40L can counteract apoptotic signals in B cells after engagement of the B cell receptor ( 38 ), whereas BAFF was not able to rescue the B cell line Ramos from anti-μ–mediated apoptosis, despite the fact that Ramos cells do express BAFF-R (Table I ; MacKay, F., unpublished observations). Therefore, it is likely that CD40L and BAFF fulfill distinct functions. In this respect, it is noteworthy that BAFF did not interact with any of 16 recombinant receptors of the TNF family tested, including CD40 (Schneider, P., and J. Tschopp, unpublished observations). Several obscure zones remain in our understanding of an immune response. For instance, little is known about the mechanisms governing the differentiation of a B cell into a plasma cell versus a germinal center B cell. Similarly, aside from the possible involvement of the CD40 pathway shown in vitro ( 39 ), we have very little information about the signals deciding the differentiation of a germinal center B cell into a memory B cell or a plasma cell. It will be very interesting to investigate whether or not BAFF has any unique role to play in these critical checkpoint decisions. | Study | biomedical | en | 0.999996 |
10359579 | All media, including cystine-containing and cystine/methionine-free DMEM, were purchased from Biofluids, Inc. The mastocytoma cell line P815 (H-2 d ) and the thymoma cell line EL-4 (H-2 b ) were maintained in DMEM containing 10% fetal bovine serum (FBS), 1 5 × 10 −5 M β-ME, antibiotics, and 2 mM glutamine (DME-10). K d -transfected T2 (T2-K d ) and RMA-S (RMA-S/K d ) cells were cultured in RPMI 1640 containing 10% FBS and the above supplements (RP-10). CTL stimulation and maintenance were performed in RP-10 medium containing 10 U/ml of recombinant human IL-2. In some assays, cells were incubated with IMDM supplemented with 10% FBS (I-10). 8–10-wk-old female BALB/c mice or C57BL/6 mice were injected intraperitoneally with 1 ml of a 1:10 dilution of chicken egg allantoic fluid containing influenza A virus Puerto Rico/8/34 (PR8) or intravenously with 10 7 PFU of lymphocytic choriomeningitis virus (LCMV) WE strain. Splenocytes were stimulated with peptide-pulsed APCs for 7 d in vitro at least 3 wk after virus priming. Cells were stimulated in RP-10 with 10 U/ml IL-2 in 6-well plates. In brief, 3 × 10 7 splenocytes were stimulated with 6 × 10 5 virus-infected or peptide-pulsed (1 nM) APC, which were irradiated with 200 Gy before addition to cultures. After 4 d, live cells were recovered via a Ficoll–Hypaque gradient and recultured with fresh IL-2–containing medium. CTL activities were tested after 7 d in standard 51 Cr-release microcytotoxicity assays. In some cases, short-term CTL lines were used, which were restimulated weekly for up to 5–6 wk. For PR8 infection, log-phase cells were harvested and washed in serum-free Autopow MEM (Life Technologies), adjusted to pH 6.6, and resuspended in 200 μl of the same medium containing 100 μl PR8-containing allantoic fluid per 10 6 cells. For vaccinia virus (VV) infection, cells were infected at a multiplicity of infection of 10 in basal salt solution supplemented with 0.1% BSA (wt/vol) at a concentration of 5 × 10 6 cells/ml. Cells were incubated for 1 h at 37°C in a water bath before 2 ml of prewarmed complete medium was added. Cells were then further incubated for 1–2 h before 51 Cr labeling, unless indicated otherwise. All peptides were synthesized, HPLC-purified, and analyzed by mass spectrometry by the Biologic Resource Branch, National Institute of Allergy and Infectious Diseases. Peptides were dissolved at 1 mM in DMSO and stored at −20°C unless otherwise indicated. For analytical purposes, peptide masses were determined by matrix assisted laser desorption ionization with time of flight detection (MALDI-TOF) using a Hewlett Packard mass spectrometer and cyano-4-hydroxycinnamic acid as the matrix. Peptide binding to live cells was determined by protection of class I molecules to melting ( 6 ). In brief, T2-K d cells were cultured for 14–16 h at 26°C. Synthetic peptides, diluted in FBS-free DME in the presence or absence of tris (2-carboxyethyl) phosphine hydrochloride (TCEP; Pierce Chemical Co. ) as indicated, were added to cells, which were incubated at 37°C for 2 h to denature K d molecules not stabilized by peptide binding. Cells were then washed and stained with fluorescein-conjugated SF1-1.1 ( PharMingen ). Live cells were gated based on scattering and exclusion of ethidium homodimer (Molecular Probes, Inc.) present at 5 μg/ml for 5 min before the last wash. For each histogram, 10,000–20,000 cells were counted on a Becton Dickinson FACScan™, and live cells were analyzed using CELLQuest™ software ( Becton Dickinson ). Natural peptides were recovered and analyzed as previously described ( 7 ). In brief, cultures of P815 cells were expanded in roller bottles. 5 × 10 8 –10 9 cells were infected as described above and incubated for 6 h at 37°C before being pelleted, washed twice in PBS, lysed with ice-cold 0.33% TFA/ H 2 O, and further disrupted using a TenBroek tissue homogenizer on ice. At this stage, synthetic peptides were added to uninfected P815 cells as control. Lysates were sonicated and centrifuged at 10,000 g for 30 min, and the supernatants were passed through a 3K cutoff filter (Macrosep™ filtron 3K; Pall Filtron Corp.). Samples were dried to a volume >400 μl using a SpeedVac (Savant Instruments, Inc.) and fractionated on a C18 column (Deltapack; Waters) at 1 ml/min on TFA/acetonitrile gradient ( 7 ). Either 0.25- or 1-ml fractions were collected. Generally, 10 6 target cells were labeled with 100 μCi of Na 51 CrO 4 ( Dupont ) in minimum volume of medium at 37°C for 60 min. For some experiments, RMA-S/K d cells that had been cultured for 12–14 h at 26°C were labeled at 26°C for the same time period. After two washes, 10 4 cells were aliquoted into round-bottom, 96-well plates containing serial dilutions of effector T CD8+ . For testing HPLC fractions, target cells in 50 μl of either PBS or FCS-free medium were exposed to 5 μl of fractions for 30 min at 26°C before T CD8+ were added. In some experiments, TCEP was freshly dissolved in H 2 O and used at 200 μM, both at peptide-pulsing and microcytotoxicity assay stages. The radioactivity in supernatants collected after 4–6-h incubation at 37°C was determined using a gamma counter. The percent specific release was then determined as: % specific release = (CTL-induced release − spontaneous release)/(release by detergent − spontaneous release) × 100. We previously reported that in K d -restricted responses to PR8 influenza virus nuclear protein (NP), NP 147–155 is the immunodominant determinant, with NP 39–47 and NP 218–226 exhibiting subdominant status ( 8 ). This hierarchy is not accounted for by peptide affinity, as NP 147–155 binds to K d with the lowest efficiency, as determined by a K d “melting” assay performed either with RMA-S cells expressing K d from a transfected gene (data not shown) or T2 cells ( 8 ). We initially focused on T CD8+ avidity to explain the immunodominance of NP 147–155 , as 10-fold less synthetic NP 147–155 was usually required to sensitize target cells for lysis by T CD8+ lines raised to the individual peptides under conditions similar to those used for the peptide binding assay . This was observed using either short- or long-term lines stimulated in vitro by synthetic peptides derived from animals immunized either with PR8 or rVV expressing NP or cytosolic or endoplasmic reticulum (ER)-targeted minigene product versions of the determinants. Taking into account the lower efficiency of NP 147–155 binding to K d , the data in Fig. 1 suggest that <10% of K d -NP 147–155 complexes are required for T CD8+ triggering relative to K d complexed to either of the subdominant determinants. Several findings, however, suggested that matters might be a bit more complicated. Unlike NP 147–155 , the dose–response curves of NP 39–47 and NP 218–226 varied considerably between experiments, depending in part on the manner in which the assay was executed. We also experienced difficulties in stimulating and maintaining T CD8+ lines to these subdominant determinants, often observing slower growth after restimulation and morphological abnormalities of the cells, which were frequently larger than T CD8+ specific for NP 147–155 . This was not strictly related to the subdominant status of these determinants, as T CD8+ specific for other subdominant determinants behaved similarly to NP 147–155 -specific T CD8+ . A property shared by NP 218–226 and NP 39–47 is the presence of cysteine (Table I ). The report by Meadows et al. ( 4 ) demonstrating the dramatic effects of sulfhydryl modification of cysteine-containing residues on T CD8+ recognition prompted us to examine possible effects of cysteine modification on NP 39–47 and NP 218–226 binding and antigenicity. We first studied the properties of synthetic peptides in which cysteine is replaced by serine or alanine (the most conservative substitutions). For NP 39–47 , the cysteine→ serine substitution had no significant effect on peptide binding, whereas the cysteine→ alanine substitution increased peptide potency by ∼10-fold . For NP 218–226 , either substitution reduced peptide potency in stabilizing K d molecules by ∼10-fold . Each of the substitutions resulted in large increases in antigenicity relative to the wild-type peptides . For NP 218–226 , the substituted peptides were 1,000–10,000-fold more antigenic on a per-complex basis (assuming that complex formation at the endpoint of peptide titrations is proportional to peptide binding efficiency determined by the melting assay). These findings prompted us to study the effects of sulfhydryl modification on the antigenicity of NP 39–47 and NP 218–226 . In most experiments described below, the two peptides were studied in parallel. Because the results were highly similar, only results with NP 218–226 are shown for the sake of clarity and simplicity. Cysteine readily forms disulfide bonds at neutral or slightly basic pH in the presence of O 2 at atmospheric tension, and oxidation to the disulfide is stimulated by trace amounts of iron salts that are present in tissue culture media. The disulfide can be reduced to the original thiol form by exposure to reducing agents. To determine whether disulfide formation affected peptide antigenicity, synthetic NP 218–226 was added to cells in the presence of dithiothreitol or TCEP, and cells were tested for lysis by NP 218–226 -specific T CD8+ . Either of these reducing agents increased peptide potency by ∼10-fold . Reducing agents did not affect the potency of noncysteine-containing peptides, including NP 147–155 , an LCMV peptide (described below), or the cysteine→ serine- or cysteine→ alanine-substituted NP 39–47 peptides (not shown). Enhancement of cysteine peptide recognition is, as expected, dependent on the concentration of reducing agent, with TCEP being more effective on a molar basis than dithiothreitol (not shown). The optimal concentration for TCEP was 200 μM (used in additional experiments), as higher concentrations (1 mM) sometimes increased spontaneous release values in 51 Cr-release assays. The effect of TCEP on NP 218–226 antigenicity is particularly impressive when considered in view of the 100–1,000-fold decrease in peptide binding to K d in the presence of TCEP (described below). In additional experiments, we found that inclusion of reducing agents in the media used to stimulate and propagate T CD8+ specific for NP 218–226 or NP 39–47 greatly enhanced their growth and altered their appearance, to the extent that these T CD8+ were indistinguishable from T CD8+ raised to NP 147–155 or other immunodominant determinants. These findings indicated that sulfhydryl modification can have major effects on the antigenicity and immunogenicity of synthetic peptides, effects that can lead to erroneous conclusions regarding the nature of their interactions with class I molecules, the affinity of T CD8+ specific for the peptide, and the growth characteristics of the cells. To broaden these findings, we examined the effect of reducing agents on the in vitro antigenicity of three oft-studied, D b -restricted determinants from LCMV: two containing cysteine (GP 33–41 and GP 276–284 ) and a control determinant lacking cysteine (NP 396–404 ) (Table I ). As with the influenza virus determinants, the cysteine-containing peptides were recognized 10–100-fold less efficiently than the cysteine-free peptide in the absence of reducing agent , whereas TCEP had no affect on the antigenicity of NP 396–404 . Based on these findings, it is clear that sulfhydryl modification of cysteine-containing synthetic peptides can have major effects on T CD8+ growth and target cell recognition that must be taken into consideration in investigating the biological properties of the corresponding naturally produced determinants and the T CD8+ they induce. We turned our attention to why reducing agents enhance the antigenicity of synthetic NP 218–226 . The major possibilities were reduction of disulfide-linked peptide dimers and reduction of disulfide-bound species derived from culture media. The most abundant sulfhydryl-containing compound present in DMEM is cystine (cysteine–cysteine dimers). To determine whether NP 218–226 becomes cysteinylated in DMEM, freshly dissolved NP 218–226 was incubated in normal or cystine-free DMEM for 2 h. Peptides present in media were then separated by RP-HPLC, and the masses in peptide-containing fractions were determined by mass spectroscopy . After incubation in cysteine-free media, the only modification detected was a small amount of dimerization . By contrast, in cysteine-containing media, most of the monomer was converted to a separate eluting form representing cysteinylated peptide . This fraction also contained a minor species with an additional mass of 16 daltons that probably represents oxidation of the neighboring methionine residue in the peptide. The effect of cysteinylation on NP 218–226 antigenicity was examined by measuring the K d binding and antigenicity of an unmodified preparation of NP 218–226 incubated for 2 h in cystine-containing DMEM with or without TCEP before addition to cells. As seen in Fig. 6 A, the resulting cysteinylation was associated with an ∼10-fold increase in rescuing K d molecules from melting and an ∼10,000-fold decrease in capacity to sensitize target cells for lysis by T CD8+ induced by APCs pulsed with unmodified NP 218–226 . We also examined the effect of NP 218–226 dimerization on antigenicity and K d binding. Analysis of various stocks by RP-HPLC in conjunction with mass spectrometry revealed that a 1-yr-old stock of peptide in DMSO was >95% dimerized . Using this stock as an NP 218–226 dimer source, we investigated the effect of 2-h incubation at room temperature in cystine-free or cystine-containing DMEM. NP 218–226 dimers were stable under these conditions . Having identified a source of dimers and demonstrated the stability of dimers in DMEM, we could examine the K d binding and antigenicity of dimers , which revealed that dimers behaved similarly to cysteinylated NP 218–226 . We draw two conclusions from these findings. First, cysteinylation and dimerization of NP 218–226 is associated with enhanced K d binding yet greatly reduced antigenicity, using T CD8+ restimulated by the reduced peptide. Second, in normal DMEM, cysteinylation occurs preferentially to peptide dimerization, even when NP 218–226 is present at relatively high concentrations. As a second-order reaction, dimerization should be greatly disfavored at decreasing peptide concentrations, whereas cysteinylation continues at a first-order reaction rate. Therefore, cysteinylation is probably the major process for modifying cysteine-containing peptides at the concentrations used in K d -binding and 51 Cr-release assays. Given the potential for cysteine modification in vitro, we examined whether NP 218–226 produced by PR8-infected cells was modified in vivo. Low M r peptides present in acid extracts from whole cells were fractionated by RP-HPLC and tested for their abilities to sensitize target cells for lysis by T CD8+ raised to reduced NP 218–226 . TCEP was added to the fractions to reveal the presence of SH-modified forms of peptides rendered nonantigenic by the modification. As shown in Fig. 7 A, antigenic peptides were recovered in fractions eluting from 25–27 min, matching the elution times of cysteinylated (25 min) and unmodified (27 min) NP 218–226 . No activity was present in the 29-min fraction, where dimeric NP 218–226 elutes. The amounts of peptide recovered were well below that required for saturation , suggesting that a considerable fraction of NP 218–226 recovered from PR8-infected cells is cysteinylated. Cysteinylation of NP 218–226 might have occurred artefactually during the extraction process. To examine this possibility, cell homogenates were doped with synthetic, unmodified NP 218–226 and then processed identically to virus-infected cells (note that in this and subsequent experiments, to increase the chromatographic resolution, fraction size was reduced from 1 ml to 0.25 ml) . In this case, <1% of the antigenic activity (as determined by titrating fractions; data not shown) was recovered in the cysteinylated form. In this experiment, peptides were tested in the presence or absence of TCEP. Even unmodified NP 218–226 required TCEP treatment, which we attribute to rapid peptide cysteinylation during target cell sensitization. To examine themaximal potential for posttranslational modification of NP 218–226 , we infected cells with an rVV (VV-NP 218–226 ) that expresses the peptide in the cytosol as a minigene product (with NH 2 -terminal methionine to enable translation initiation). As reported previously ( 7 – 9 ), this greatly enhances the number of peptide–class I complexes generated by cells. RP-HPLC fractionation from minigene-expressing cells revealed the presence of material coeluting with unmodified NP 218–226 , as well as cysteinylated peptide . For the first time, dimeric peptide was also detected. These activities cannot be attributed to methionine-extended NP 218–226 , which, for each form, elutes slightly later than NP 218–226 (data not shown). Titration of the antigenic activities in the fractions (not shown) revealed a ratio of unmodified/cysteinylated/dimeric forms of ∼6:3:1. In an additional experiment (data not shown), we examined the K d dependence of NP 218–226 recovery in HPLC fractions after VV-NP 218–226 by using L929 cells and L929 cells expressing K d from a transgene. Peptides corresponding to cysteinylated and unmodified NP 218–226 were recovered from L929-K d cells but not L929 cells. Dilution of peak fractions revealed that expression of K d resulted in at least a 25-fold increase in the recovery of NP 218–226 . This confirms numerous prior studies demonstrating the MHC dependence of antigenic peptide recovery ( 1 ). The ultimate demonstration of the biological relevance of cysteinylated NP 218–226 is that specific T CD8+ are elicited in PR8-infected mice. We could show this by stimulating splenocytes derived from PR8-infected mice with RP-HPLC–purified, cysteinylated peptide. After three to four rounds of stimulation, we obtained T CD8+ that preferentially recognize cysteinylated peptide . In the same assay, the noncysteinylated peptide is preferentially recognized by T CD8+ induced in the standard manner . The recovery of T CD8+ specific for the cysteinylated peptide could not be attributed to in vitro stimulation of naive T CD8+ , as we failed to obtain any activity using splenocytes from nonimmunized mice (data not shown). Using T CD8+ stimulated by the cysteinylated NP 218–226 , it was possible to formally demonstrate that the 25-min fraction derived from minigene-expressing cells contained cysteinylated peptide . Indeed, now antigenicity was destroyed by TCEP exposure, in contrast to the enhancing activity observed with other peptides. We also detected an additional peak at 24.5 min. This probably represents the methionine-oxidized form of cysteinylated NP 218–226 , which, based on experience with other peptides, often elutes slightly faster than the nonoxidized form, providing evidence that this modification occurs in cells. In this paper, we confirm and extend the findings of Meadows et al. ( 4 ) that modification of the SH group of cysteine-containing peptides has important positive and negative effects on their antigenicity and immunogenicity in vitro and in vivo. Failure to consider these effects can have disastrous consequences for the accurate interpretation of several different types of experiments. In our own case, the use of synthetic peptides in the absence of reducing agents led us to erroneously favor the idea that the subdominant status of two cysteine-containing peptides was due to a greater number of complexes required for T CD8+ recognition and correlated with the atypical growth of T CD8+ in vitro. In other experiments (our unpublished results), it also led us to the incorrect quantitation of peptides recovered from virus-infected cells. A further potential methodological pitfall is that autooxidation of reducing agents can cause additional artifacts. This can be minimized by inclusion of a chelating agent, such as DTPA (diethylenetriaminepentaacetic acid), with the reducing agent. These errors are probably widespread. We demonstrate that two cysteine-containing LCMV determinants restricted by a different class I molecule are also modified in vitro through their sulfhydryl groups, with a resulting 10–100-fold loss in antigenicity. These determinants were the subject of a recent study ( 10 ) focused on the factors involved in the immunodominance hierarchy of the determinants. The failure to add reducing agents during peptide titration probably led to erroneous calculations of peptides present in virus-infected cells, particularly because the peptide titration curves were nearly identical to those we obtained in the absence of reducing agents. Results obtained in this study with in vivo transfer of T CD8+ lines must also be questioned, as the cells were propagated in vitro with synthetic peptides in the absence of reducing agents. In another recent study of the fine specificity of a T CD8+ clone for a cysteine-containing peptide, amino acids were substituted for cysteine, many of which enhanced the antigenicity of the peptide ( 11 ). Based on our findings, we would predict that simple reduction of the wild-type peptide would have a similar (or greater) effect. In addition to reducing antigenicity, modification of cysteine can result in the generation of T CD8+ specific for the modified determinant. We show that PR8-infected mice generate T CD8+ that prefer cysteinylated NP 218–226 . We also demonstrate that PR8-infected cells generate an SH-modified peptide that coelutes with cysteinylated NP 218–226 and is recognized by T CD8+ specific for cysteinylated NP 218–226 . This species almost certainly represents cysteinylated NP 218–226 , although definitive evidence requires mass spectroscopy. The recognition of posttranslationally modified peptides by T CD8+ adds to the already formidable challenge of understanding in vivo T CD8+ responses but can be ignored only at the peril of the investigator. We can only speculate where NP 218–226 is cysteinylated during its processing and presentation by virus-infected cells. It is theoretically possible that the cystine derives naturally from a disulfide bond present in NP. It is difficult, though admittedly not impossible, to imagine the proteolytic liberation of cysteinylated NP 218–226 . Given the highly reducing environment of the cytosol and nucleus, it also seems unlikely that cysteinylation would occur before peptide translocation into the ER. The ER provides a much more oxidizing environment and possesses resident proteins that catalyze thiol–disulfide interchange, including protein disulfide isomerase ( 12 ). There is evidence that exogenous homocysteine is added to HLA class I molecules in an early secretory compartment ( 13 ). If cysteinylation occurs in the ER, it may occur before peptide loading onto class I molecules. Alternatively, cysteinylation could occur after peptide binding, particularly in the case of NP 218–226 , as the SH must be directed away from the groove (so as to accommodate dimer binding). In this case, it could occur anywhere from the ER to the cell surface. Cysteine is thought to be the major reductant in the endosomal pathway, and although there is no evidence that functional K d molecules visit these compartments, this remains a possibility. It was previously reported that disulfide-linked homodimeric peptides could bind to class I molecules ( 14 ). We provide another example of this and further demonstrate that this can occur naturally in cells. Recovery of dimers required expression of NP 218–226 as a cytosolic minigene, which results in at least 10–1,000-fold overproduction of peptide–class I complexes relative to expression of peptide in its natural context ( 7 , 9 ), and the extent to which overexpression of peptide is required remains to be determined. As peptide cross-linking is expected to be a second-order reaction, it implies that NP 218–226 is present at a very high concentration intracellularly. It is hard to imagine this occurring outside of the ER, and it may occur in the vicinity of TAP, which is required for transport of NP 218–226 into the ER (our unpublished results). These findings have important clinical implications. First, for synthetic peptide vaccines (or other exogenous antigen vaccine preparations with vulnerable cysteine residues in antigenic peptides), modification of the cysteine in vitro or in vivo can obviously have major negative effects on immunogenicity. This can be avoided by modifying the side chain to a nonreactive form. For NP 218–226 and NP 39–47 , this is achieved simply by substitution with alanine or serine, which did not detrimentally affect peptide binding or T CD8+ triggering. This strategy will probably work for most peptides. For others, it is possible that chemical modification of the SH group (e.g., treatment with an alkylating agent or a heavy metal) will do the job. Second, if increased cysteinylation is associated with a disease process, this could lead to autoimmune recognition of cysteinylated self peptides. | Study | biomedical | en | 0.999997 |
10359580 | BALB/c mice, 4–6 wk of age, were purchased from The Jackson Laboratory . Transgenic mice expressing the DO11.10 TCR (DO11) ( 36 ), specific for the chicken OVA peptide OVA 323–339 in the context of the MHC class II molecule I-A d , were obtained from Dr. D. Loh (Hoffmann-LaRoche, Nutley, NJ). The DO11 mice were bred in our pathogen and viral antibody–free facility in accordance with guidelines of the Committee on Animals of the Harvard Medical School, and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Washington, D.C.). The mice were typed for the DO11 TCR by staining peripheral blood cells with antibodies against CD4 and V β 8 as previously described ( 37 ). Murine IL-4 was obtained from the culture supernatant of the I3XL6 cell line (obtained from Dr. Abul Abbas, Brigham and Women's Hospital), which constitutively expresses a stably transfected murine IL-4 gene. Recombinant murine IL-12 was a gift of Dr. Stan Wolf (Genetics Institute, Cambridge, MA). IL-2–containing supernatants were obtained from the X63-IL2 cell line, which expresses a stably transfected murine IL-2 gene (obtained from Dr. Fritz Melchers, Basel Institute of Immunology, Basel, Switzerland). The hybridoma cell line producing anti–IL-4 (clone 11B11) was obtained from the American Type Culture Collection. Neutralizing anti–mouse IFN-γ (XMG1.2) was purchased from PharMingen . Human E- and P-selectin human IgG chimeric proteins ( 38 , 39 ) were provided by Dr. R. Camphausen (Genetics Institute). OVA 323–339 was produced by the peptide synthesis facility of the Center for Neurologic Diseases, Brigham and Women's Hospital. Murine recombinant IP-10/CRG-2 was obtained from R&D Systems. Naive CD4 + T cells were purified from pooled lymph nodes and spleen cells from DO11 mice by using CD4 + Dynal beads and Detach-a-bead reagent ( Dynal A.S., Norway) according to the manufacturer's instructions. More than 95% of the cells were CD4 + as assessed by flow cytometry using FITC-conjugated anti–mouse CD4 + mAbs. The CD4 − fraction of the DO11 spleen and lymph node suspensions, or whole spleen cell suspensions from BALB/c mice, were treated with mitomycin C for 30 min at 37°C and used as APCs for DO11 T cells. T cell differentiation was induced by culturing 2 × 10 5 purified CD4 + TCR transgenic T cells in 1 ml of RPMI 1640 with 10% FCS with 2 × 10 6 APCs, 1 μM OVA 323–339 , and Th0 condition (no cytokines), Th1 condition (10 ng/ml IL-12 plus anti–IL-4 [11B11 hybridoma supernatant, 25% vol/vol]), or Th2 condition (1,000 U/ml IL-4 plus anti– IFN-γ [XMG 1.2, 1 μg/ml]). The cultures were fed with fresh medium containing 10 U/ml IL-2 after 4 d, and harvested for adoptive transfer on day 6. In some experiments, suspensions of BALB/c mouse lymph node and spleen cells were differentiated in vitro by stimulation with 2 μg/ml SEB ( Sigma Chemical Co. ) in the presence of cytokine and antibody reagents used for Th1 and Th2 differentiation, as described above. 6 d later, cells were harvested, centrifuged over Ficoll-sodium metrizoate (Cappel), labeled with a fluorescent tracker dye (see below), and used in adoptive transfer studies. Freshly prepared suspensions of whole lymph node and spleen cells from DO11 mice were used as a source of naive cells for adoptive transfer, as previously described ( 37 ). In vitro differentiated DO11 T cells were harvested at day 6 and centrifuged over Ficoll-sodium metrizoate before adoptive transfer. The number of T lymphocytes expressing the DO11 TCR in each population was measured by flow cytometric analysis of cell suspensions stained with the clonotypic antibody, KJ1-26 ( 40 , 41 ) (see below). Naive or differentiated T cells (1.5–2.0 × 10 7 in 500 μl PBS) were adoptively transferred into unirradiated BALB/c recipients by tail vein injection. 6 h later, mice were given intraperitoneal injections of 500 μg OVA protein or PBS emulsified in IFA (Difco) or with IP-10/CRG-2 in PBS. 5, 48, or 72 h after adoptive transfer, spleen and lymph nodes (inguinal, axillary, and mesenteric) were removed and peritoneal cells were harvested by PBS lavage. Cell suspensions were prepared and analyzed by flow cytometry for surface antigen expression. In some experiments, mice received naive DO11 T cells by tail vein injection, and were immunized 24 h later with OVA (100 μg) emulsified in CFA (Difco) through footpad injection. Draining popliteal and inguinal lymph nodes were harvested at varying times after immunization, and cell suspensions were prepared and were analyzed by flow cytometry. SEB-stimulated T cell cultures were harvested at day 6, centrifuged over Ficoll-sodium metrizoate, and then incubated with 1 μM Cell Tracker Green (5-chloromethylfluorescein diacetate [CMFDA]; Molecular Probe) diluted in RPMI 1640 with 10% FBS for 20 min at 37°C. Cells were centrifuged and incubated for an additional 30 min at 37°C in fresh medium to allow elution of unreacted dye before transfer. Labeled cells (1.5–2.0 × 10 7 ) were then adoptively transferred into BALB/c mice through tail vein injection. 6 h later, mice were given intraperitoneal injections of 500 μl of PBS-IFA. 72 h after transfer, spleen, lymph nodes, and peritoneal cells were harvested and analyzed by flow cytometry. In DO11 adoptive transfer studies, suspensions of cells from peritoneum, lymph node, or spleen were first incubated with 5 μg/ml anti-CD16/CD32 ( PharMingen ) to block Fc receptors. The cells were then stained with 1 μg/ml PE-labeled anti-CD4 antibody ( PharMingen ), and 1 μg/ml biotinylated KJ1-26 clonotypic antibody followed by Cy-Chrome– streptavidin ( PharMingen ), as previously described ( 37 ). These cells were also stained with 1 μg/ml P-selectin–human IgG chimera ( 38 , 39 ), followed by FITC-conjugated anti–human IgG (Southern Biotechnology Associates). SEB-stimulated T cells were stained with 1 μg/ml biotinylated anti-V β 8 antibody ( PharMingen ) followed by Cy-Chrome–streptavidin ( PharMingen ). SEB-stimulated cells were also stained with 1 μg/ml PE-labeled anti-CD8 or anti-CD4 antibodies ( PharMingen ), as well as with soluble P-selectin–IgG and FITC-conjugated anti-IgG, as described above for DO11 cells. More than 95% of the CD4 − V β 8 + T cells in the SEB-stimulated populations were CD8 + . We therefore chose to analyze CD4 + V β 8 + and CD4 − V β 8 + T cells in the same staining tubes as a convenient way to compare CD4 + and CD8 + populations while still being able to use two additional fluorochromes for V β 8 and P-selectin ligand analysis. All analyses were performed on a FACScan ® flow cytometer ( Becton Dickinson ). In some studies, CMFDA (green tracker dye)-labeled T cells were detected by direct fluorescence in the FL1 channel on the flow cytometer. We have recently shown that, in contrast to antigen-stimulated DO11 T cells, naive DO11 T cells fail to roll on selectins under flow conditions in vitro, nor do they bind soluble P- or E-selectin Ig chimeric molecules ( 18 ). To determine whether naive T cells are capable of entering a peripheral inflammatory site, we directly transferred spleen and lymph node cells from normal DO11 mice into BALB/c recipients and then further challenged the mice intraperitoneally with IFA. After 3 d, the recipient mice were assayed for the number of adoptively transferred cells in the peritoneum as well as in peripheral lymph node and spleen. As has been previously reported, naive DO11 T cells homed to lymph nodes, and to a lesser extent to spleen . In contrast, DO11 CD4 + T cells were not detectable (<0.2%) in the peritoneum of either the control or IFA-treated mice . The numbers of DO11 CD4 + T cells in both spleen and lymph nodes were comparable in control and IFA-treated mice, indicating that roughly equal number of cells were adoptively transferred into the circulation of each mouse. The T cells we isolated from normal DO11 mice express high levels of L-selectin and low levels of CD25 and CD44, which is a typical phenotype for naive T cells . After 6 d in culture with APCs and OVA peptide (see Material and Methods, Th0 condition), DO11 T cells had acquired a typical activated phenotype: L-selectin low CD25 high CD44 high . Upon restimulation with OVA peptide, these activated CD4 + T cells produced both IFN-γ and IL-4, as assayed by ELISA (data not shown). After they were transferred into BALB/c mice, the activated DO11 CD4 + T cells were recruited to the IFA-treated peritoneum . The frequency of CD4 + KJ126 + cells in the peritoneum increased from 0.9 ± 0.1% to 3.4 ± 0.3% ( n = 2 experiments) in the absence and presence of IFA, respectively. In contrast, the frequency of CD4 + KJ126 + cells in the spleens remained comparable at 4.1 ± 0.4% and 3.9 ± 0.3% ( n = 2 experiments) in the control and IFA-treated mice, respectively. Thus, we conclude that although both activated and naive CD4 + T cells are capable of entering secondary lymphoid tissues, only the activated subset is recruited to the inflamed peritoneum. To compare the recruitment of Th1 versus Th2 cells to an inflammatory site, we first generated differentiated Th1 and Th2 populations of DO11 T cells in vitro as described in Materials and Methods. Th1 cells produced abundant IFN-γ but no IL-4, whereas Th2 cells produced abundant IL-4 but little IFN-γ, as judged by cytokine ELISA assay and intracellular cytokine staining (reference 18 and data not shown). DO11 Th1 or Th2 cells were adoptively transferred into syngeneic BALB/c recipient mice, the mice were challenged with intraperitoneal injection of IFA and the number of adoptively transferred CD4 + KJ126 + cells in spleen, lymph nodes or peritoneum was determined 3 d later. Results of a typical experiment are shown in Fig. 3 . In that experiment, adoptively transferred Th1 cells comprised 3.1% of total peritoneal cells, whereas adoptively transferred Th2 cells comprised only 0.7%. In contrast, Th1 and Th2 cell recruitment into spleen or lymph nodes was approximately equal. In four experiments, the frequency of adoptively transferred Th1 cells in IFA-treated peritoneum was 3.2 ± 1.0 times the frequency of adoptively transferred Th2 cells. When OVA and IFA were injected into the peritoneum, and cells were recovered 3 d later, the percentage of DO11 T cells was significantly enhanced compared with exudates from animals treated with IFA alone. Furthermore, there was a greater difference between the numbers of Th1 and Th2 cells in the peritoneum when OVA was present than when it was absent . In the experiment shown, adoptively transferred Th1 cells comprised 19% of the total peritoneal cells in the IFA plus OVA-treated peritoneum, whereas Th2 cells comprised only 3.1%. The absolute numbers of DO11 T cells recruited in the peritoneum at various time points during this experiment are shown in Table I . With IFA alone at day 3, 10 6 DO11 Th1 cells were recruited to the peritoneum, which was five times more than the number of DO11 Th2 cells recruited under identical conditions. In the presence of IFA plus OVA, 3.2 × 10 6 of DO11 Th1 cells were present in peritoneum at day 3, but only 4 × 10 5 DO11 Th2 cells were recruited under identical conditions. The increased number of Th1 cells in the peritoneum when OVA is present may be due to increased recruitment and/or local proliferation in response to antigen after recruitment. When DO11 Th1 cells were labeled with tracker dye before transfer, the day 3 recovered peritoneal cells from OVA plus IFA treated mice had significantly less tracker dye than cells recovered from IFA-treated mice . The loss of tracker dye correlates with cell division and dilution of the intracellular label among progeny cells ( 37 , 42 – 44 ). This indicates local proliferation accounts for at least some of the accumulation of Th1 cells when antigen is present. To determine if the cytokine secretory phenotype of the adoptively transferred Th1 and Th2 cells remained stable in vivo, the recovered peritoneal cells were stimulated with OVA peptide and culture supernatants were assayed for IFN-γ and IL-4 production. Consistent with previous findings ( 45 ), DO11 Th1 and Th2 cells maintain their effector cytokine profile in vivo (data not shown). To dissect the mechanisms for differential recruitment of T cell subsets into the inflamed peritoneum, we first examined the role of P- and E-selectins and their ligands on T lymphocytes. T cells constitutively express PSGL-1, the best described receptor for P-selectin, which becomes functional only after posttranslational modification ( 46 , 47 ). The P-selectin binding status of T cells was examined by FACS ® analysis using soluble P-selectin–human IgG fusion protein. As shown in Fig. 6 A, naive CD4 + T cells failed to bind P-selectin. After in vitro differentiation with peptide and specific cytokines, Th1 cells bound much more P-selectin than Th2 cells did. To further investigate whether binding to P- and E-selectin is essential for the selective recruitment of CD4 + T cells to the inflamed peritoneum, we studied the migration of Th1 cells under the influence of antibodies against P- and E-selectin. As shown in Fig. 6 B, combination of antibodies against P- and E-selectin effectively blocked Th1 cells from entering IFA-treated peritoneum. The percentage of DO11 T cells in the peritoneum dropped from 7 to 1.6%, whereas the number of DO11 T cells in spleens remained comparable in these two groups of mice. This data indicates that the interaction of P- and E-selectin with their ligands is essential for maximal recruitment of Th1 cells into sites of inflammation. Chemokines activate integrins on leukocytes and trigger firm interaction of leukocytes to endothelium ( 48 – 50 ). Recently, human CXCR3, receptor for IP-10, was shown to be highly expressed in Th1 but not in Th2 cells ( 26 – 28 ). To demonstrate a possible role of chemokines in mediating selective recruitment of T cell subsets in vivo, we injected the mouse IP-10 homologue, CRG-2, into the peritoneum of mice after adoptive transfer of either DO11 Th1 or Th2 cells. In response to CRG-2 treatment, DO11 Th1 cells were recruited to the peritoneum more efficiently than Th2 cells. In the experiment shown in Fig. 7 , Th1 cells comprised 7.9% of the total peritoneal population in CRG-2 treated mice, whereas Th2 cells comprised 2.3% in similarly treated mice. The total numbers of Th1 and Th2 cells harvested from the CRG-2–treated mice were 2.5 × 10 5 and 4 × 10 4 respectively. In two experiments in which CRG-2 treatment was performed, the frequency of adoptively transferred Th1 cells in the peritoneum was 3.7 ± 0.4 times the frequency of adoptively transferred Th2 cells. Therefore, differential expression of chemokine receptors and functional P/E-selectin ligand(s) contribute to differences in recruitment of T cell subsets. In vitro stimulation of BALB/c spleen cells with SEB for 6 d gave rise to activated CD4 + and CD8 + T cells. Over 80% of this mixed population was V β 8 + (data not shown) and CD8 + T cells comprised from 50 to 66% of the V β 8 + cells. Under the influence of Th1 promoting cytokines (IL-12 plus anti–IL-4), a majority of CD4 + V β 8 + and CD8 + V β 8 + T cells bound P-selectin . The ratio of CD4 + /CD8 + cells propagated under these condition was ∼1:2. On the other hand, under the influence of Th2 promoting cytokines (IL-4 plus anti– IFN-γ), both CD4 + V β 8 + and CD8 + V β 8 + populations failed to bind P-selectin . The CD4 + /CD8 + ratio of the cells propagated under these conditions was 1:1. To demonstrate in vivo recruitment of CD4 + and CD8 + SEB-stimulated T cells, the cells were labeled with green tracker dye (CMFDA) before adoptive transfer. As predicted based on P-selectin binding status shown above, a selective recruitment was clearly observed. After 3 d of IFA treatment, IL-12–treated cells were recruited to peritoneum more efficiently than those IL-4–treated cells. As shown in Fig. 8 B, IL-12–treated cells represented 16% of total peritoneal population, whereas IL-4–treated cells only represented 1.5%. In four experiments, the frequency of adoptively transferred cells in the peritoneum that were originally differentiated under Th1 conditions was 6.5 ± 2.8 times greater than the frequency of adoptively transferred cells originally differentiated under Th2 conditions. Furthermore, the CD4 + /CD8 + ratio of the V β 8 + cells recovered from the peritoneum was always the same as in the initial adoptively transferred population (data not shown). This indicates that conditions that promote CD4 + Th1 differentiation also act on CD8 + T cells to induce functional selectin ligands and the ability to migrate into inflammatory sites. We have demonstrated that naive T cells stimulated with antigen and IL-12 in vitro differentiate into P-selectin binding cells that are capable of entering an inflammatory site. To determine if naive T cells upregulate functional P-selectin ligand in response to antigen in vivo, we adoptively transferred naive DO11 T cells into BALB/c recipient mice. After 24 h, the mice were immunized by footpad injection with OVA peptide and CFA to provide specific antigen as well as a stimulus for IL-12 production. Draining lymph nodes were removed after 1, 2, and 3 d and cells were stained for CD4, DO11 TCR (KJ126), and P-selectin binding. As shown in Fig. 9 A, the DO11 CD4 + KJ126 + T cells expanded in lymph nodes in response to OVA stimulation. The percentage of CD4 + KJ126 + T cells increased from 3.1% to 8.7% by day 2 and further increased to 13% by day 3. At day 2 after immunization, there was a significant increase in the number of CD4 + KJ126 + cells that bound P-selectin . Interestingly, there was no increase in P-selectin binding by the CD4 + KJ126 − population indicating that cytokines in the lymph node cannot induce P-selectin ligand expression on T cells not directly stimulated by antigen. Forward scatter analysis indicated that the P-selectin binding cells were the largest, presumably most activated cells among the CD4 + KJ126 + population (data not shown). In this study, we examined recruitment of different subsets of T lymphocytes into a peripheral inflammatory site. Using the adoptive transfer approach, we were able to follow specific populations of cells with distinct phenotypic characteristics. The peritoneal inflammatory model was chosen because recruitment of T cells can be easily quantified by flow cytometric analysis of cells harvested from the cavity, the recruitment process requires transmigration across an endothelial barrier, and different inflammatory stimuli can be administered by intraperitoneal injections. The injection of IFA into the peritoneum induces a mixed inflammatory infiltrate that changes over time, but includes activated neutrophils and lymphocytes at day 3 (data not shown). Although there are many studies supporting the hypothesis that effector and naive T cells have different migratory patterns in vivo ( 3 , 4 , 51 ), there is little data directly comparing the abilities of effector and naive T cells to enter T peripheral inflammatory sites. The adoptive transfer of TCR transgenic T cells is a powerful method with which to address homing patterns of T cells. It has been used extensively to study homing of antigen specific T cells to secondary lymphoid tissues ( 52 ), but this technique has been applied only in a limited way to the study of effector T cell homing to inflammatory sites ( 35 , 53 ). Our finding that naive DO11 T cells fail to enter the IFA-treated peritoneum, whereas activated effector DO11 T cells do, provides important direct evidence for the concept that effector T cells preferentially home to peripheral inflammatory sites but naive T cells do not. We have also found that Th1 cells are more efficiently recruited to the inflamed peritoneum than are Th2 cells. This is consistent with previous studies indicating that Th1 cells enter dermal delayed-type hypersensitivity sites to a greater degree than Th2 cells ( 16 ). In our experiments, both Th1 and Th2 cells migrated to spleen and lymph node in equivalent numbers. This indicates that Th2 cells were as efficiently transferred into the circulation as Th1 cells, and that there is no intrinsic defect in the ability of in vitro– generated Th2 populations to transmigrate out of blood vessels into tissues. Furthermore, others have shown that Th2 cells can also migrate into lung tissue in mouse models of allergic airway inflammation ( 16 , 54 ). On the other hand, in a murine model of autoimmune gastritis, activated Th2 cells are found in draining lymph nodes but not gastric mucosa, whereas Th1 cells are abundant in the mucosa ( 55 ). Thus, Th2 cells appear to be capable of entering only a subset of inflammatory sites. Differential recruitment of Th1 but not Th2 cells into inflammatory sites has been attributed to the expression of higher levels of functional selectin ligands on Th1 cells than Th2 cells ( 16 – 18 , 20 , 56 ). Consistent with those studies, we have found that the adoptively transferred Th1 cells express more P-selectin ligand than do the Th2 cells, and that anti–P- plus anti–E-selectin antibodies diminish Th1 recruitment into the peritoneum . Unlike previous reports, we have examined the effect of the presence of antigen on the relative accumulation of antigen-specific Th1 and Th2 cells at the site. We have found that the difference between Th1 and Th2 cell accumulation seen without antigen is amplified when antigen is present . The tracker-dye data indicate that the increased number of Th1 cells in the peritoneum in the presence of antigen is at least partially attributable to local proliferation. Thus the combination of preferential recruitment and then antigen-induced expansion of recruited cells appears to markedly polarize the phenotype of the helper T cell responses to peripheral antigen. It is not clear if there are adhesion molecules that mediate selective recruitment of Th2 cells into inflammatory sites, such as in the asthmatic lung. A possible candidate is very late activation antigen (VLA)-4 on T cells interacting with vascular cell adhesion molecule (VCAM)-1 on inflamed endothelium. We have shown previously that an active form of VLA-4 that is capable of binding to VCAM-1 under physiological shear stress is expressed on memory but not naive human helper T cells ( 13 ). In addition, the Th2 cytokine, IL-4, can induce VCAM-1 expression on endothelial cells ( 57 – 59 ). The expression of functional P-selectin ligand on CD4 + ( 13 , 47 , 56 , 60 ) and CD8 + ( 61 ) T cells has been described. An important finding in this study is that the conditions used to drive CD4 + Th1 differentiation in vitro, namely antigen, IL-12, and anti–IL-4, also induce functional P-selectin ligand expression on CD8 + cells, whereas Th2 differentiation conditions, namely antigen, IL-4, and anti–IFN-γ generate CD8 + T cells that do not bind P-selectin. Furthermore, P-selectin ligand expression on CD8 + T cells correlates with their ability to home to the inflamed peritoneum. It is not clear whether the effector functions of these two CD8 + populations are different or not, although Tc1 and Tc2 subsets of CD8 + T cells producing different cytokines have been described ( 31 , 34 , 62 ). Recent evidence does suggest that adoptively transferred influenza-specific Tc1 cells are better able to clear pulmonary influenza infection then are Tc2 cells, even though both populations have comparable cytolytic activity ( 35 ). This may reflect different recruitment kinetics between the two populations or the effects of the different cytokines they produce. Our data suggest that the microenvironmental conditions in which naive CD8 + T cells are differentiated into CTLs are likely to influence not only the effector functions of these cells, but also their selectin-dependent homing patterns to peripheral inflammatory sites, just as with CD4 + helper T cells. The data showing induction of P-selectin ligand on lymph node T cells in vivo indicate one mechanism by which recruitment of effector T cells at inflammatory sites can be biased toward antigen-specific cells. We have shown that functional P-selectin ligand is induced on antigen-specific naive T cells in lymph nodes draining a site of exposure to the antigen, but not on bystander T cells that cannot recognize the antigen but are otherwise exposed to the same local cytokine milieu. Induction of P-selectin ligand on lymph node T cells has been reported recently, but that study did not use TCR-transgenic T cells and the antigen specificity of the ligand induction could not be addressed ( 1 ). The in vivo activated CD4 + DO11 T cells described in this paper appear to be Th1-type as they produce abundant IFN-γ but no IL-4 upon restimulation with OVA peptide. Because survival and propagation of naive TCR transgenic T cells in vitro requires antigen stimulation, it has not been possible to distinguish the necessity of T cell receptor signals from cytokine signals for the induction of functional selectin ligands. In the in vivo study reported here, viable T cells that cannot bind the administered antigen fail to acquire the ability to bind P-selectin, even though the microenvironment includes the necessary cytokines. Although the acquisition of a selectin binding phenotype appears to distinguish effector T cell subsets and determines their capability of homing to peripheral inflammatory sites, the induction of other functional adhesion molecules on T cells during effector differentiation are also likely to be important in peripheral homing. For example, CD44–dependent hyaluronate binding increases specifically on V β 8 + lymph node T cells in SEB-treated mice, and these hyaluronate-binding T cells are selectively recruited to SEB-containing peritoneum in a CD44-dependent manner ( 63 ). It is possible that CD44 or other adhesion molecules may also contribute selective recruitment of effector T cell subsets. In summary, the results presented here support the following model. When naive T cells encounter antigen in the lymph node, they become activated, undergo clonal expansion, and they differentiate into one or another subset of effector cells depending on the local cytokine milieu. During this process of activation and differentiation, specific adhesion molecules, including functional selectin ligands, and chemokine receptors are induced on the surface of activated T cells, in subset-specific patterns. Therefore, after entering circulation, the T cells are capable of migrating to particular peripheral sites where appropriate adhesion molecules and chemokines (ligands to the receptors on the T cells) are expressed or displayed by endothelium. The requirement that both antigen and cytokine signals be provided in the draining lymph nodes in order to stimulate the expression of functional selectin ligands may contribute to relative enrichment antigen–specific T cells at peripheral inflammatory sites. Maintenance of a functionally polarized T cell response at a peripheral site appears to require subset-specific recruitment, but the polarization is amplified by antigen-driven proliferation of the recruited T cells. | Study | biomedical | en | 0.999996 |
10359581 | An expression plasmid, pEFBOS ( 15 ), and p55IgκLuc, a reporter construct with three tandemly repeated κB motifs upstream of a minimal IFN-β promoter ( 16 ), were gifts from Drs. Shigekazu Nagata (Osaka University Medical School, Osaka, Japan) and Takashi Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), respectively. The human kidney cell line 293T was provided by Dr. T. Hirano (Osaka University Medical School, Osaka, Japan). LPS and lipid A were purchased from Sigma Chemical Co. The rat mAb against mouse CD14 ( 17 ) was provided by Dr. Shunsuke Yamamoto (Oita Medical University, Oita, Japan). An mAb against the flag epitope M2 was purchased from Sigma Chemical Co. , and a rat mAb against the protein C epitope was established in our own laboratory. The Est clone encoding human MD-2 was purchased from Genome Systems, Inc. Sequencing was conducted with an ALFexpress DNA sequencer and a Thermo Sequenase cycle sequencing kit ( Amersham Pharmacia Biotech , Ltd.). Total RNA was extracted from various cell lines or tissues with Isogen (Nippon Gene) and subjected to agarose electrophoresis (20 μg/lane). After transfer to a sheet of nylon membrane (Hybond N + ; Amersham Pharmacia Biotech , Ltd.), RNA was hybridized to a probe that had been labeled by random priming of a cDNA clone. The hybridization buffer consisted of 10% dextran sulfate ( Pharmacia Biotech ), 1 M NaCl, 1% SDS, and 50 mM Tris/HCl, pH 7.5. Hybridization was conducted at 65°C for 20 h. Washing was carried out in 2× SSC and 0.1% SDS at 65°C. Radioactive signals were visualized with an image analyzer . The same membrane was reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The IL-3–dependent line Ba/F3 ( 18 ) was transfected with the pEFBOS expression vector encoding human TLR4. Expression of TLR4 was confirmed by intracellular staining for the flag epitope, which had been attached to the COOH terminus of TLR4. The established line was then transfected with the p55κB reporter construct for measuring NF-κB activity with luciferase activity ( 16 ). Cell lines expressing TLR4 and the reporter construct were screened by measuring spontaneous luciferase activity, which was expected to be high due to TLR4 expression ( 11 , 19 ). One of the selected lines was further transfected with the expression vector encoding human MD-2 with either the flag epitope or another epitope tag, protein C ( Boehringer Mannheim ) at the COOH terminus. Cells expressing human MD-2 on the cell surface were screened by staining corresponding epitope tags. We also transfected human MD-2 with the flag epitope into the original Ba/F3 line, and transfectants expressing MD-2 were selected by permeabilized cell staining of the flag epitope. Cells were incubated with indicated mAbs. After washes with staining buffer (PBS containing 2% FCS and 0.1% azide), goat anti–mouse IgG–FITC (Chemicon International, Inc.) or goat anti–rat IgG–PE (Southern Biotechnology Associates, Inc.) was added. Propidium iodide was included in the second incubation to exclude dead cells. Cells were analyzed on a FACScan™ ( Becton Dickinson ). Permeabilized cell staining was conducted as described by Veis et al. ( 20 ). In brief, staining was conducted with the staining buffer containing saponin detergent (0.03%; Sigma Chemical Co. ). The transfectant line expressing TLR4 and MD-2 was stained as above with the mouse anti–human TLR4 mAb and the rat anti–protein C mAb, which recognized MD-2. The second Abs were goat anti–mouse IgG–FITC for the HTA125 mAb and goat anti–rat IgG–PE for the anti-protein C mAb. These second reagents did not react with the other first reagent, as judged on a FACScan™ (data not shown). Stained cells were viewed on a scanning confocal microscope system, Fluoview ( Olympus Corp. ). Cells were washed and lysed in lysis buffer consisting of 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 50 mM iodoacetamide, 1mM PMSF, 10 μg/ ml soybean trypsin inhibitor, 2 mM MgCl 2 , and 2 mM CaCl 2 . After 30-min incubation on ice, lysate was centrifuged and nuclei were removed. The N -hydroxysuccinimide-activated Sepharose 4FF beads ( Amersham Pharmacia Biotech , Ltd.) coupled with HTA125 (4 mg/ml) were added to cell lysate and rotated for 2 h at 4°C. Beads were washed in the lysis buffer, and bound proteins were subjected to SDS-PAGE (9% acrylamide under the nonreduced condition) and Western blot analysis. TLR4 and MD-2 were detected with the anti-flag mAb, M2 ( Sigma Chemical Co. ) and Supersignal ® chemiluminescent substrate ( Pierce Chemical Co. ). Transient transfection was conducted according to the report by Kaisho et al. ( 21 ). The human kidney cell line 293T was plated onto a 24-well plate at 1.5 × 10 5 cells/ well on the day before transfection. DNA was diluted in 100 μl deionized water, and 2 M calcium chloride (14 μl) was added to DNA. An equal amount (114 μl) of 2× Hepes-buffered saline (280 mM NaCl, 50 mM Hepes, and 1.5 mM sodium phosphate, pH 7.05) was added slowly in a dropwise manner. After 30-min incubation at room temperature, coprecipitates were added to 293T cells. Medium was changed on the following day, and the cells were cultured for another day. Cells were harvested with PBS containing 1 mM EDTA and used for luciferase assays. In transient transfection, the pEFBOS expression vector encoding β-galactosidase (a gift from Dr. Masato Ogata, Osaka University Medical School, Osaka, Japan) was used as an internal control for transfection. After transient transfection, cells were lysed in 50 μl lysis buffer, and luciferase activity was measured using 10 μl lysate and 50 μl luciferase substrate (Nippon Gene). The luminescence was quantitated by a luminometer (Berthold Japan). Luciferase activity was normalized with the β-galactosidase activity, which was measured with 2-nitro-phenyl-β- d -galactopyranoside ( Boehringer Mannheim ). Stable transfectants were inoculated onto 48-well plates (2 × 10 5 /well). LPS and lipid A were added together. After 4 h of stimulation, cells were harvested and lysed in 50 μl lysis buffer, and luciferase activity was measured as above. BALB/c mice were immunized with the Ba/F3 line expressing TLR4, and spleen cells were fused with SP2/0 myeloma cells. An mAb was chosen that stained the line used for immunization. A more detailed description of the mAbs and expression/function of TLR4 will be published elsewhere. RP105 was cloned in our laboratory as the first LRR molecule expressed on lymphocytes, the extracellular LRR of which had similarity to Drosophila Toll ( 22 , 23 ). We have recently isolated MD-1 as a molecule that is physically associated with RP105 on the cell surface ( 24 ). MD-1 itself is a secretory molecule but tethered to the cell surface by coexpressed RP105. MD-1 is likely to interact with LRR of RP105, which constitutes an entire region of the extracellular portion of RP105. As the extracellular LRR of RP105 is similar to Drosophila Toll or homologous TLRs, we hypothesized that TLRs might also be associated with a molecule like MD-1. The hypothesis prompted us to seek a molecule with similarity to MD-1. A computer search using the MD-1 amino acid sequence retrieved a human cDNA clone from an expressed sequence tag database . The whole nucleotide sequence was determined and is shown in Fig. 1 A. After an in-frame stop codon, the longest open reading frame codes for 160 amino acids, including a 16-residue, NH 2 -terminal hydrophobic stretch that may function as a signal peptide. We refer to a mature molecule as MD-2. Significant similarity (23% identity) to MD-1 was observed over the mature polypeptide . Notably, five out of seven cysteines of MD-2 are shared by MD-1. Expression of the MD-2 transcript was examined with Northern hybridization . The size of the transcript was ∼0.7 kb, which is consistent with the size of the cDNA clone. The transcript was demonstrable in all human lines studied . Three B lymphoma cell lines showed relatively high expression. We also studied distribution of the MD-2 transcript in mouse tissues, using the mouse MD-2 probe that was cloned in our laboratory (Shimazu, R., and K. Miyake, unpublished data). Mouse MD-2 was similar to a human homologue in the size of mRNA. The transcript was also ubiquitously observed in all mouse tissues examined, among which spleen and kidney showed pronounced expression . We then studied interaction of MD-2 and RP105 or TLRs. RP105 seemed unlikely to interact with MD-2, as transfection of MD-2 with RP105, contrary to the case with MD-1, did not result in cell surface expression of MD-2 (data not shown). We next studied interaction with TLRs, among which TLR4 is of particular interest, because it most resembled RP105 in the extracellular LRR domain, and cells expressing the TLR4 transcript were also positive for MD-2 mRNA . The expression vector encoding MD-2 was transfected into the Ba/F3 line. Although the precursor was demonstrable inside the cell , MD-2 was not detectable on the cell surface . In sharp contrast, MD-2 appears on the cell surface in a stable line expressing TLR4 as well as MD-2 and seemed to be colocalized with TLR4 on a scanning confocal microscope (data not shown). These results are consistent with membrane anchoring of MD-2 via physical association with TLR4. To confirm the association, immunoprecipitation experiments were conducted using the transfectants expressing TLR4 and MD-2 with the newly made mAb to TLR4 (HTA125; see Materials and Methods). It should be noted that the HTA125 mAb was established by immunizing cells expressing TLR4 alone and recognizes TLR4 but not MD-2 . Coprecipitation of MD-2 with the HTA125 mAb therefore demonstrates physical interaction of MD-2 with TLR4. We used two different transfectants, one of which expressed the flag epitope on both TLR4 and MD-2 . The other line, which had the flag epitope on TLR4 but not on MD-2, was used as a control . Precipitates were detected with the anti-flag mAb. TLR4 was specifically precipitated from either line with HTA125 as a 120-kD band, which is consistent with a previous report ( 19 ). The signal just below presumably represents an intracellular precursor. Another species of ∼25–30 kD was detected from the transfectant expressing MD-2 with the flag epitope . The size is similar to that of MD-1 ( 24 ) and is within a range expectable from the MD-2 amino acid sequence, consisting of 160 amino acids with two N -glycosylation sites . On the other hand, this signal was not observed in the control precipitate, in which MD-2 did not bear the flag epitope . MD-2 is thus physically associated with TLR4. We next explored a possible role for MD-2 in TLR4-dependent signaling. As shown in Fig. 5 , expression of TLR4 alone conferred the triggering of NF-κB activation in 293T cells, which confirmed previous reports ( 11 , 19 ). Interestingly, expression of MD-2 enhanced TLR4-dependent activation of NF-κBs by 2–3 fold. Transfection of MD-1 with TLR4 did not have such an effect. Physical association of MD-2 therefore influences the signaling via the transmembrane TLR4 molecule. Preliminary studies suggested that MD-2 forms a homodimer or a larger complex on the cell surface. Such a complex may have multiple binding sites for TLR4 and facilitate cross-linking of TLR4, leading to higher NF-κB activation. Further studies are underway. TLR4 may be an LPS receptor, as its gene is mutated in low-responder mice C3H/HeJ and C57BL/10ScCr ( 12 , 13 ). Transfection of TLR4, however, did not confer LPS responsiveness on recipient cell lines ( 11 ), suggesting a requirement for another molecule that linked TLR4 to LPS signaling. We hypothesized that MD-2 might be such a link, as it interacts with TLR4 and influences the signaling of TLR4. To address this possibility, we studied LPS responsiveness of stable transfectants expressing TLR4 alone or with MD-2 by measuring NF-κB activity (see Materials and Methods). The stable transfectant line that expressed TLR4 alone did not respond to LPS from Escherichia coli 0111:B4, E. coli 055:B5 or Salmonella minnesota Re595 or to lipid A , which is consistent with the report by Kirschning et al. ( 11 ). The Ba/F3 line, like the 293 cell line ( 11 ), might lack a molecule indispensable for LPS signaling via TLR4. The mRNA of a candidate molecule MD-2 was not expressed in either cell line (data not shown). The stable line expressing TLR4 and MD-2 was then examined. Transfection of MD-2 conferred on the line strong NF-κB responses to either LPS or lipid A at concentrations as low as 0.1 ng/ml . No response was observed to detoxified LPS from which the fatty acid side chains of the lipid A moiety were removed (data not shown). Receptor activity acquired by introducing MD-2 was triggered through TLR4, as the anti-TLR4 mAb HTA125 specifically inhibited the responses . MD-2 thus confers LPS signaling on TLR4. We found, by reverse transcriptase (RT)-PCR expression of the transcript of TLR2, another LPS receptor in the parental line Ba/F3. TLR3, TLR4, and TLR5 were not detected by RT-PCR or Northern hybridization (data not shown). In spite of the expression of the TLR2 transcript, stable transfectants expressing the NF-κB reporter gene alone (data not shown) or with TLR4 did not show any significant LPS response. The amount of the cell surface TLR2 protein, if any, may be too small to sense the presence of LPS, or mouse TLR2 may not respond to LPS as effectively as its human counterpart. Taken together with specific inhibition with the anti-TLR4 mAb, LPS responses in stable transfectants expressing TLR4 and MD-2 are mediated by the cell surface complex of TLR4–MD-2 but not by TLR2. The TLR4–MD-2 receptor complex efficiently senses the presence of bacterial endotoxin. CD14, another LRR molecule capable of binding to LPS, is able to enhance LPS signaling via TLR2 ( 10 , 11 ). Mouse CD14 was not demonstrable by cell surface staining of the Ba/F3 line (data not shown), but it is still possible that soluble CD14 in FCS of culture medium contributes to LPS signaling via TLR4–MD-2. Further study is of importance and underway concerning a role of soluble and membrane CD14 in LPS signaling of TLR4–MD-2. Another finding with the new receptor complex TLR4– MD-2 is that it has broader specificity than that recently described for TLR2 ( 10 ). TLR2 recognizes the LPS from S. minnesota Re595 much better than that from E. coli 055:B5. On the other hand, the TLR4–MD-2 complex responded equally to the two different types of LPS . LPS is a complex glycolipid composed of hydrophilic polysaccharides of the core and O-antigen structures, as well as a hydrophobic domain called lipid A. Lipid A is a common component, whereas considerable diversity of structure is noted among the O-antigens. Because both TLR2 and TLR4/MD-2 receptors responded well to lipid A , the core and O-antigen from E. coli 055:B5 must selectively affect recognition by TLR2. Studies using stable transfectants expressing each TLR would reveal further difference in recognition specificity of each TLR. MD-2 might associate with other TLR family members and confer the ability to respond to a broader spectrum of pathogens, including gram-positive bacteria and fungi. Such fundamental information concerning innate recognition of pathogens may also suggest new treatments for infectious diseases and endotoxin shock. | Study | biomedical | en | 0.999997 |
10359582 | Human recombinant Trx, its C32S/C35S mutant [Trx(SGPS)], and human recombinant glutaredoxin were prepared as previously described ( 20 – 22 ). Goat anti–human Trx neutralizing antibody ( 23 ) was from Imco (Sweden). Control antibody (goat antibody against Schistosoma japonicum glutathione S -transferase [GST]) was from Pharmacia . Spirulina Trx was from Sigma Chemical Co. Human Trx was measured in culture supernatants by ELISA, as previously described ( 18 ). Chemotactic activity for human monocytes, PMNs, and T cells was evaluated using 48-well micro Boyden chambers (Neuro Probe, Inc.), as previously described ( 24 , 25 ). The filters were stained and chemotactic activity was expressed as the average number of cells migrated in five oil immersion fields counted. Air pouch formation was induced by injecting 4 ml of air 7 and 3 d before the experiment. Then, 1 μg of Trx was injected into the air pouch in 1 ml of sterile, pyrogen-free saline, containing 0.5% (wt/vol) carboxymethylcellulose (USP grade; Sigma Chemical Co. ) to avoid a rapid diffusion of Trx from the site of injection. Control mice were injected with this vehicle. To determine the proportion of different leukocyte subsets, cells from Trx-treated and control mice were stained with antibody markers to identify granulocytes, monocyte/macrophages, and lymphocytes. Reagent preparation and staining methods were as previously described ( 26 ). Cells from six individual Trx-treated mice (4 h) were analyzed. Six untreated control mice were pooled in order to obtain sufficient cell numbers for analysis. Samples were analyzed on a flow cytometer modified to simultaneously detect 10 fluorochromes ( 27 ). Propidium iodide exclusion identified live cells. Granulocytes were identified as GR-1 + (RB6-8C5). Monocyte/ macrophages were identified as GR-1 − , CD11b + (M1/70), F4/80 + . Lymphocytes were identified as negative for the above-mentioned markers and confirmed by forward and side scatter characteristics. Animal studies were performed in accordance with Institutional guidelines and approved by the Institutional Review Board. Adherent monocytes or PMNs on coverslips were loaded with FURA-2AM ( Sigma Chemical Co. ), washed, and incubated at 37°C with the different stimuli. Fluorescence was monitored using an epifluorescence microscope equipped with fluorescence optics and dichroic mirror appropriate for FURA-2 fluorescence. FURA-2 was excited at 350 and 380 nm every second and the emitted fluorescence was filtered between 510 and 530 nm and monitored using a CCD camera (Dage MTI) and a Georgia Instrument Image Analyzer. Regions of interest corresponding to individual cells were identified in each experiment, and average fluorescence was recorded and stored as individual data files. Fluorescence intensity was converted into intracellular free Ca 2+ ([Ca 2+ ] i ), as previously described ( 28 ). Representative experiments are shown as fluorescence tracings of individual cells. Results from several experiments are also summarized as number of responsive cells (when the stimulus-induced increase of [Ca 2+ ] i was twice the SD over the mean of baseline values) or mean Ca 2+ increase of responsive cells. Testing human recombinant Trx for chemotactic activity towards PMNs, monocytes, and T lymphocytes in a standard in vitro chemotaxis assay with micro Boyden chambers , we found that Trx is chemotactic for all three cell populations. The optimal concentration (0.1–2.5 nM; 1–30 ng/ml) and degree of response are comparable with that of an optimal concentration of a reference chemokine (IL-8 for PMNs, monocyte chemotactic protein [MCP]-1 for monocytes, and RANTES for T cells, respectively). Trx showed the typical bell-shaped dose–response curve reported for all chemokines. To evaluate the possibility that the chemotactic activity of Trx was specific and not due to a contaminant we used different approaches. First, Trx was heat inactivated by boiling, to rule out the possibility that the chemotactic activity might be due to endotoxin, which is heat stable, or to small contaminating peptides. As shown in Fig. 1 , boiled Trx was inactive towards all cell populations. Furthermore, a neutralizing anti-Trx antibody, but not a control antibody, neutralized the chemotactic activity of Trx for human monocytes but not the activity of MCP-1 . To assess whether Trx was indeed chemotactic or if it induced random cell migration, we performed checkerboard experiments using PMNs and monocytes. As shown in Table I , no migration was observed when the same concentration of Trx was added to both the upper and lower chambers, indicating that Trx had a true chemotactic, not chemokinetic, activity. To investigate the role of the protein disulfide oxidoreductase activity, we tested the chemotactic action of Trx from Spirulina algae (which has the same cys-gly-pro-cys [CGPC] active site and enzymatic activity as the mammalian Trx) of a C32S/C35C mutant where the CGPC active site was substituted by a ser-gly-pro-ser [SGPS] sequence, and of human recombinant glutaredoxin. As shown in Fig. 3 , Spirulina Trx was chemotactic for PMNs and monocytes at concentrations equivalent to those of human recombinant Trx, whereas glutaredoxin or Trx(SGPS) were inactive. Fig. 4 shows the trace of [Ca 2+ ] i in single monocytes or single PMNs stimulated with MCP-1, IL-8, or Trx. Consistent with previous evidence ( 28 , 29 ), IL-8 induced a rapid rise in intracellular Ca 2+ in PMNs and MCP-1 induced a slower, but nonetheless definitive, increase of Ca 2+ in monocytes . However, chemotactic concentrations of Trx did not induce any Ca 2+ response in either cell population. In several experiments analyzing up to 30 individual cells, we never observed an effect of Trx on intracellular Ca 2+ in a concentration range of 1–100 ng/ml (0.083–8.3 nM) (Table II ). We also studied the effect of the G protein inhibitor, pertussis toxin (PT), on Trx chemotactic activity on monocytes. As shown in Fig. 5 , the chemotactic activity of Trx on monocytes was not inhibited by PT, whereas a marked inhibition was observed on MCP-1 activity . We tested Trx in the murine air pouch model of inflammation ( 30 ). 4 h after injection of 1 μg Trx into a pouch (formed by prior subcutaneous injection of air, as described in Materials and Methods), the pouch was washed with medium, and infiltrated cells were counted, stained, and analyzed by flow cytometry. As shown in Fig. 6 , injection of Trx induced a marked infiltration of granulocytes, and, to a lesser extent, monocytes and lymphocytes. Although the absolute response to Trx varied somewhat in different experiments, granulocytes were consistently the most frequent cells in the infiltrate. Infiltration was higher at 4 h than at 24 h (data not shown), consistent with the hypothesis that Trx acts directly as a chemotactic agent and does not cause cell infiltration by inducing other chemokines. To investigate whether Trx could contribute to the chemotactic activity released by the HTLV-1–transformed MT4 cell line, we studied the effect of an anti-Trx antibody on the chemotactic activity of the MT4 supernatants. As shown in Fig. 7 , 50% of the chemotactic activity of the MT4 supernatant was inhibited by an anti–human Trx antibody. The Trx content of the MT4 supernatant, as measured by ELISA, was 6 ng/ml, which is consistent with the potency of Trx as a chemotactic factor. Our data show that Trx is a potent chemoattractant for PMNs, monocytes, and T cells. Several lines of evidence suggest that this activity is specific and not due to a contaminant. In particular, chemotactic activity was observed with different Trx preparations, including Trx from algae, it was inactivated by boiling and by an anti-Trx antibody. Our findings also show that Trx contributes significantly to the chemotactic activity released in HTLV-1–infected cells, that have long been known to secrete Trx ( 31 ). In particular, the HTLV-1–transformed MT4 cell line spontaneously releases chemotactic activity for monocytes that is not mediated by TNF, IL-8, or MCP-1 ( 32 ). We recently purified a major monocyte chemotactic factor from MT4-conditioned medium that we identified as MIP-1α/LD78 ( 33 ) and showed to be active on monocytes but only weakly chemotactic for PMNs ( 33 ). We show here that an anti-Trx antibody significantly inhibits the chemotactic activity of MT4 supernatants, an observation that might explain why the known chemokines produced by these cells could not account for all of their chemotactic activity. As far as the mechanism of the effect of Trx is concerned, we obtained convincing evidences that Trx does not act through a chemokine receptor. First of all, Trx does not induce an increase of intracellular Ca 2+ , which is observed with all chemokines, whose receptors are coupled to G proteins. Furthermore, chemotactic activity of Trx is not inhibited by PTX. Although chemotactic receptors have been shown to be able to couple with both PTX-sensitive and PTX-insensitive GTP binding proteins ( 34 , 35 ), only βγ subunits associated with Gαi are responsible for chemotactic receptor-mediated cell migration ( 36 ). The finding that Trx chemotactic response is PTX insensitive, along with the lack of effect of Trx on intracellular Ca 2+ , strongly argue against its direct interaction with a seven-transmembrane domain chemotactic receptor. In contrast, Trx may initiate signal transduction for chemotaxis by oxidizing and cross-linking appropriate cell surface molecules. Trx is mainly known as a protein disulfide–reducing enzyme; however, it can also act as protein disulfide isomerase in the formation of disulfides during protein folding ( 1 ), particularly when in an oxidative environment ( 37 ). Thus it is possible that, in an extracellular environment, Trx acts by oxidizing thiols of one or more membrane proteins or catalyzing isomerization reactions. However, since Trx's chemotactic activity is G protein independent, this putative oxidized receptor is likely to differ from any of the known chemokine receptors. In fact, the Trx target could behave like a “sensor”, in a fashion similar to the hemoprotein, which acts as the oxygen sensor in the induction of erythropoietin ( 38 ). Several lines of evidence suggest that the chemotactic activity of Trx is due to its enzymatic action on cell surface protein substrates. First of all, a C32S/C35S mutant of Trx where the cysteines of CXXC active site have been mutated, and that has lost enzyme activity ( 22 ), was not chemotactic. Furthermore, glutaredoxin, which differs from Trx in substrate specificity, has no chemotactic activity. In fact, although Trx catalyzes the oxidation/reduction of a wide range of inter- or intra-protein disulfides, the preferred substrates of glutaredoxin are mixed disulfides between proteins and glutathione, and the substrate specificity for disulfides is very different ( 1 , 2 , 21 ). A further support to this hypothesis is the chemotactic activity of Trx from Spirulina . Since algal Trx has only little (∼20%) similarity with human Trx ( 39 ), but has the same conserved CGPC active site human Trx, we think it is more likely that Trx may act through its enzymatic activity rather than by binding to a membrane receptor. Consistent with this, various investigators have been unable to identify specific binding sites for Trx on the cell membrane of various cell types ( 40 – 43 ). Our findings suggest two different hypotheses concerning the pathogenic role of Trx in infection and inflammation. First, the local release of Trx is likely to be important, in concert with other chemokines, in recruiting cells during infection and inflammation. Consistent with this hypothesis, Trx is secreted by IFN-γ– or endotoxin-stimulated macrophages or activated T lymphocytes ( 12 , 44 ) and has been measured locally in arthritic patients ( 19 ). Trx is also an acute-phase protein in that its production by the liver is increased in rats injected with LPS ( 45 ). In addition, Trx may also be a major chemoattractant in diseases associated with oxidative stress, such as ischemia/reperfusion, since Trx production is induced by oxidants ( 46 ) and Trx is elevated locally in cerebral ischemia or brain injury ( 47 , 48 ) and open-heart surgery ( 23 ). Thus, according to this hypothesis, Trx might function as a signal of oxidative stress that amplifies the cellular response at a site of inflammation. A second hypothesis on the significance of Trx in disease stems from the observation that Trx levels were found to be elevated (>30 ng/ml plasma) in a proportion of HIV- infected subjects with CD4 T cell counts <200/μl blood ( 18 ). Nearly all of the high-Trx subjects died within 18 mo of the Trx measurement, although none had active opportunistic infections or other signs of debilitating disease at the time of measurement. Deaths in an otherwise similar group of HIV-infected subjects with normal Trx levels were minimal during the same period (Nakamura, H., S. de Rosa, M. Roederer, J. Yodoi, A. Holmgren, P. Ghezzi, L.A. Herzenberg, and L.A. Herzenberg, manuscript in preparation). This survival difference may be directly traceable to the chemoattractant activity of the Trx present in circulation. Intravenous injection of IL-8 has been shown to inhibit PMN accumulation in response to local injection of IL-8 ( 49 ). Furthermore, leukocyte migration is impaired in transgenic mice overexpressing human IL-8 or MCP-1 in circulation ( 50 , 51 ). High levels of circulating Trx may similarly decrease the ability of leukocytes to migrate efficiently to a site of infection, either by counteracting the gradient of local chemoattractants or downregulating the chemokine receptors by desensitization (or both). Consistent with this, PMNs and monocytes from AIDS patients have been shown to be deficient in their ability to migrate ( 52 , 53 ). Thus, by potentially interfering with chemotaxis, elevated serum Trx levels in HIV patients could contribute to augmented susceptibility to bacterial or viral infections and hence constitute a serious threat to survival in the ensuing months. The possibility that a chemoattractant acts through its enzymatic activity, rather than through classical receptor binding, opens the way to new strategies aimed at inhibiting its action, using enzyme inhibitors rather than receptor antagonists or antibodies. | Study | biomedical | en | 0.999997 |
10359583 | The generation of the QM mouse has been previously described ( 14 ). C57BL/6 mice were obtained from the breeding colony of the Institut für Labortierkunde, Faculty of Veterinary Medicine, University Zürich-Irchel, Zürich, Switzerland. Experiments were carried out with age- and sex-matched animals kept under specific pathogen–free conditions. All animals were 8–16 wk old unless indicated otherwise. VSV-IND (vesicular stomatitis virus Indiana serotype; Mudd-Summers isolate) was originally obtained from D. Kolakofsky (University of Geneva, Switzerland), and was grown on BHK cells in MEM with 5% FCS to virus stocks containing 10 9 PFU/ml. Poliovirus (PV) stock solutions of serotypes I, II, and III were obtained from the Swiss Serum and Vaccine Institute (Bern, Switzerland). Lymphocytic choriomeningitis virus (LCMV)-WE was originally provided by F. Lehmann-Grube (Heinrich Pette Institut für Experimentelle Virologie und Immunologie, University of Hamburg, Hamburg, Germany), and was grown on L-929 cells for 48 h in MEM with 5% FCS after infection with an initial multiplicity of infection (moi) of 0.01. Mice were immunized intravenously on day 0 with a standard dose of 2 × 10 6 PFU of VSV-IND or 200 PFU of LCMV-WE as indicated. For PV experiments, 0.5 ml of PV vaccine, Salk (minimal content [min.] 30 D-antigen U type 1, min. 6 D-antigen U type 2, min. 24 D-antigen U type 3, phenoxyethanol; Poliomyelitis-Impstoff Berna, Swiss Serum and Vaccine Institute) was injected intravenously on days 0 and 14. Blood was collected at various time points as indicated. Serial twofold dilutions of serum samples (previously diluted 1:40) were mixed with equal volumes of VSV or PV (PV serotype II was used) containing 500 PFU/ml, and the mixtures were incubated for 90 min at 37°C in an atmosphere containing 5% CO 2 . 100 μl of the mixture was transferred onto Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. Monolayers were overlaid with 100 μl of DMEM containing 1% methylcellulose, incubated for 24 h (up to 48 h for PV) at 37°C, then the overlay was removed and the monolayer was fixed and stained with 0.5% crystal violet dissolved in 5% formaldehyde, 50% ethanol, 4.25% NaCl. The dilution reducing the number of plaques by 50% was taken as titer ( 17 ). To determine IgG titers, undiluted serum was treated with an equal volume of 0.1 M 2-ME in MEM tissue culture medium for 1 h at room temperature. LCMV neutralization in vitro was determined as described previously ( 18 ). In brief, serial 2-fold dilutions of 10-fold prediluted sera were incubated with LCMV for 90 min at 37°C in a 96-well plate. MC57G mouse fibroblasts were added, and incubated for 4 h to allow cells to settle and be infected by nonneutralized virus; cells were then overlaid with 1% methylcellulose in DMEM. After 48 h, cell monolayers were fixed with 4% formalin and infectious foci were detected by intracellular LCMV staining of infected cells with the rat anti-LCMV mAb VL-4. For LCMV neutralization by total Ig, sera were tested under nonreducing conditions. For LCMV neutralization by IgG, sera were first reduced by addition of 2-ME (final concentration 0.05 M) for 60 min. All samples were heat inactivated at 56°C for 30 min to destroy complement. To detect LCMV nucleoprotein– specific Abs, 96-well plates were coated overnight at 4°C with recombinant baculovirus–derived LCMV nucleoprotein ( 19 , 20 ), washed three to five times with PBS 0.05%/Tween 20, and blocked with 1% BSA in PBS for 2–3 h at room temperature. Sera diluted in PBS containing 0.1% BSA were added and incubated for 90 min. Horseradish peroxidase–labeled specific goat anti–mouse IgG Ab (1:1,000; Zymed Laboratories ) was added for 1 h, and the reaction was developed by addition of 0.4 mM ABTS ( Boehringer Mannheim ), in NaH 2 PO 4 , pH 4.0, 0.01% H 2 O 2 . The OD was read at 405 nm using a microplate reader . Positive titers were defined as 3 SD above the mean value of negative controls. To measure the different IgG isotype-specific anti-VSV Abs, ELISA plates were coated with 10 μg/ml of polyethylene glycol (PEG)-precipitated VSV-IND, and the assay was performed as described above. Phage display libraries were constructed as described previously ( 21 ). In brief, mRNA was isolated from 10 7 spleen cells using the QuickPrep mRNA purification kit ( Amersham Pharmacia Biotech ) and reverse transcribed using random hexamer primers according to the manufacturer's instructions (first strand synthesis for RT-PCR; Amersham Life Science). The V region of the heavy chain (V H ) was amplified using the VHback and VHfor mixes of degenerate primers, and the light chain V region (V L ) was amplified using the λ-specific primers LBλ and LFλ as described previously ( 21 ). The V H and V L PCR products were purified and assembled in a single-chain Fv (scFv) configuration using splicing by overlap extension PCR. The full-length scFv fragment product was gel-purified, digested with SfiI, and ligated into the phage display vector pAK100 (gift of Dr. Andreas Plückthun, University of Zürich-Irchel, Zürich, Switzerland). The ligation mixes were electroporated into XL1-Blue electrocompetent cells (Stratagene, Inc.) and plated. Colonies were scraped off the plates, and the scFv displaying phages were rescued by infection with the VCS-M13 helper phage (Stratagene, Inc.). Phage particles were PEG precipitated twice and resuspended in PBS. VSV-binding phages were selected by panning using immunotubes (Maxisorp; Nunc, Inc.) coated with 100 μg/ml PEG-precipitated VSV-IND particles by overnight incubation at room temperature. Tubes were blocked with 4% dried skim milk powder in PBS and washed 20 times with PBS containing 0.1% Tween and 20 times with PBS. Bound phages were eluted with 0.1 M glycine/HCl, pH 2.2, for 10 min and immediately neutralized with 2 M Tris/HCl, pH 8.5. Eluted phages were used to infect XL1-Blue cells that were plated, rescued as described above, and subjected to another round of panning before isolated clones were analyzed by ELISA. Isolated colonies from the second round of panning were grown in 96-well plates and rescued with VCS-M13 helper phage. After overnight incubation, 100 μl of the phage-containing supernatant was added to VSV-coated plates (10 μg/ml) and incubated for 90 min. The wells were washed five times with PBS containing 0.1% Tween, and peroxidase-labeled anti-M13 antiserum ( Amersham Pharmacia Biotech ) was then added and the plates incubated for another 90 min. The plates were again washed five times with PBS/0.1% Tween, and the bound peroxidase was revealed by incubation with O -phenylenediamine ( Sigma Chemical Co. ) and hydrogen peroxide. The reaction was stopped with 1 N HCl, and the absorbance was read at 490 nm using a Bio-Rad 3550 microplate reader. VSV is a cytopathic RNA negative strand virus of the Rhabdoviridae family closely related to rabies virus ( 22 ). After VSV infection, virus-neutralizing Abs mediate protection against a progressive paralytic disease ( 23 , 24 ). Most VSV-IND–specific mAbs are directed against overlapping subsites clustered within one major antigenic site of the viral glycoprotein ( 25 – 27 ). The early IgM response (days 3–5) can be induced in the absence of T cell help ( 28 , 29 ), whereas the switch to IgG (days 6–8) is T cell help dependent ( 30 ). QM and C57BL/6 mice were immunized with 2 × 10 6 PFU of VSV-IND intravenously, and neutralizing serum Abs were monitored for 150 d. VSV total Ig neutralizing titers were measurable in QM mice 4 d after infection, but these titers were ∼30-fold lower than in C57BL/6 control mice . The switch to the IgG subclass was delayed by ∼6 d in QM mice: VSV-neutralizing IgG Abs appeared ∼12 d after infection, whereas in control mice they were first detected at about day 6 . About 40 d after infection, total anti-VSV neutralizing Abs reached similar levels in QM and control mice, and remained elevated for the duration of the 150-d observation period . Similar kinetics were observed after infection with 10 2 or 10 4 PFU of VSV-IND (data not shown). Despite the delayed kinetics of Ab production, QM mice were able to control the viral infection and survived. However, the LD 50 was decreased 10-fold from 2 × 10 8 in C57BL/6 control mice to 2 × 10 7 in QM mice (data not shown). Determination of the isotypes of the VSV-specific IgG Abs revealed that IgG2a was more abundant in infected QM mice than in control mice . In contrast, IgG1 titers were higher in the control animals than in QM mice, while IgG2b and IgG3 Ab titers were approximately the same . Similarly to infected QM mice, naive QM mice also show elevated IgG2a and IgG2b levels, which may indicate a certain bias towards Th1 cell development in QM mice ( 14 ). The delayed kinetics of the VSV-neutralizing Ig response in QM mice was not due to the absence of kappa light chains because Cκ-deficient mice ( 31 ) mounted virtually normal anti-VSV Ig responses (data not shown). The delayed kinetics observed in QM mice is likely to be attributable to the time required for the small number of anti-VSV B cell precursors present in naive QM mice to be activated or expanded and to produce sufficient serum Abs to be detected in the neutralization assay. It has previously been reported that the amount of NP-specific serum IgG1 decreased from 13.1% of total IgG1 in 4–5-wk-old QM mice to 2.8% in 20–24-wk-old mice, which is consistent with the idea that B cells with variant receptors are preferentially expanded as QM mice age ( 15 ). This could lead to increased numbers of VSV-specific B cell precursors and an accelerated VSV-neutralizing Ab response in older mice. To test this hypothesis, 9- and 56-wk-old QM mice were infected with 2 × 10 6 PFU of VSV-IND. The anti-VSV neutralizing Ab response in both 9- and 56-wk-old QM mice displayed similar kinetics, suggesting that there was no significant increase in the number of VSV-specific B cell precursors in QM mice older than 9 wk . To analyze V region sequences of VSV-specific neutralizing Abs generated in QM mice, phage display libraries were constructed from splenic mRNA isolated from naive QM mice and from QM mice after primary and secondary VSV-IND infections. In a screening ELISA, 37% of the phages isolated from the naive library and ∼57% of the phages isolated from QM spleens after primary or secondary VSV infections bound VSV. Thus, it can be concluded from this result that VSV-specific B cells were present in QM mice before infection. The serum of the mouse used to generate the naive library was tested for the presence of VSV-specific Abs and was found to be negative. In addition, it must be emphasized that mice kept in our facilities for prolonged periods of time have been tested repetitively during the last 10 yr, and in no circumstances have we found the presence of VSV-IND–specific Abs, which could indicate accidental spreading of virus. Individual clones showing strong positive signals in the screening ELISA were concentrated by PEG precipitation and tested for their relative binding potential to VSV in a second ELISA. As shown in Fig. 2 a, scFv-displaying phages showed a specific dose-dependent binding to VSV-coated plates. To identify clones specific for the viral glycoprotein (VSV-G), binding of phages to VSV-infected EL4 cells expressing VSV-G on their surface was analyzed cytofluorometrically. From 48 tested clones, 15 showed binding to VSV-infected EL4 cells (data not shown). These 15 glycoprotein-specific clones were tested in a VSV-neutralization assay, which revealed that 12 out of 15 clones neutralized viral infectivity in vitro. The neutralizing titers of six independent scFv clones (see below) are shown in Fig. 2 b. The V regions of the 12 VSV-neutralizing scFv Ab fragments were sequenced and aligned to the 17.2.25 V H sequence expressed in QM mice. From the 12 clones analyzed, 6 independent sequences were identified, 2 from each of the three libraries . Clones 0B1 and 0G6 were isolated from the naive library, clones 1A10 and 1E12 from the primary library, and clones 2F7 and 2H5 from the secondary library. The other six clones were multiple isolates of clone 0G6 (four copies) and of clone 1A10 (two copies); such repeated isolation is often the consequence of the amplification of binding clones during the panning procedure. All six independent clones used a V L fragment belonging to the λ1 family. In addition, they all showed evidence for V H gene replacement. Instead of the original 17.2.25 V H gene segment, all clones used a different V H gene segment belonging to the VH1 (J558) family that has previously been shown to play a role in secondary and hyperimmune responses against VSV in BALB/c mice ( 32 ). All six analyzed clones underwent secondary rearrangements by recombining a rearranged V H D segment with the D element of the rearranged V H DJ H segment of the QM mouse . This type of V H D to V H DJ H rearrangement has previously been observed in naive QM mice in the majority of idiotype-negative sorted B cells ( 14 , 15 ). However, unlike the events described here, a small proportion of idiotype-negative B cells did show signs of canonical V H to V H DJ H secondary rearrangements ( 14 , 15 ). Therefore, it is possible that such V H D to V H DJ H rearrangements are preferred to the canonical secondary rearrangements because they generate greater diversity by drastically varying the length of CDR3, which might in turn increase the chances of generating new specificities. It is unclear whether the heptamer motif embedded at the 3′ end of the V H gene segment of the 17.2.25 V region and most other V H gene segments was involved in these rearrangements because the recombination breakpoint cannot be unambiguously determined. In addition, N regions flanking the inserted D elements were present in all analyzed clones , which argues for the involvement of the normal rearrangement machinery including terminal deoxynucleotidyl transferase (TdT), which is expressed in bone marrow B cells and has recently been shown to be also reexpressed in germinal center B cells ( 13 ). Furthermore, the 6 VSV-specific clones sequenced showed extensive hypermutation in the remaining DSP2.10 and JH4 segments of clone 17.2.25 with a high frequency of 3.8% (15 mutations in 393 bp cumulated from the 6 scFv sequences). Interestingly, mutations were present in clones isolated from the naive library, confirming the results obtained in B cells isolated from the peripheral blood of unimmunized QM mice that also showed a high frequency of somatic mutations ( 14 ). Since germline Abs can already efficiently neutralize VSV infection in vitro and a relatively restricted B cell repertoire can mount an efficient early anti-VSV response ( 27 ), it is possible that by chance the right V H /V L pairing in QM mice could give rise to VSV-specific but not other virus-specific Abs. Therefore, in addition to the VSV-specific Ig response, the responses of QM mice to LCMV and PV were analyzed. LCMV is a noncytopathic ambisense RNA virus of the Arenaviridae family for which the mouse is the natural host. Acute LCMV infection is predominantly controlled by CTLs in a perforin-dependent manner ( 33 , 34 ). Early after infection, at about day 8, a strong Ab response specific for the LCMV nucleoprotein is mounted ( 20 ) that does not exhibit virus-neutralizing capacity ( 35 , 36 ). Late after infection, between days 30 and 60, LCMV glycoprotein–specific neutralizing Abs develop ( 20 , 37 , 38 ) that have been shown to play an important role in protection against reinfection ( 39 – 41 ). QM and C57BL/6 mice were immunized intravenously with 10 2 PFU of LCMV-WE, and the LCMV nucleoprotein–specific ELISA Ab and the LCMV-neutralizing Ab responses were monitored. QM mice mounted a delayed LCMV nucleoprotein–specific Ab response at about day 12 that was initially reduced by ∼10-fold in comparison with control mice, but which eventually reached similar levels by day 32 . Neutralizing Abs to LCMV normally appearing late after infection ( 20 , 37 , 38 ) followed the same slow kinetics in QM and control mice . Low neutralizing Ab titers were first detected after 30 d of infection, and by day 70 a distinct neutralizing Ab response was measured in both QM and C57BL/6 mice . The long time period required to mount a neutralizing response against LCMV could well have permitted the generation of sufficient LCMV-specific precursors to mount a neutralizing Ab response comparable to control animals. On the other hand, the reason for the long period of time needed to generate LCMV-neutralizing Abs is not well understood and could be dependent on factors other than low B cell precursor frequency (e.g., immunopathology ). PV is a cytopathic positive strand RNA virus of the Picornaviridae family. The viral surface contains four regularly ordered proteins (VP1–4) against which neutralizing Abs are directed ( 43 ). In BALB/c mice, PV induces an early T cell–independent IgM response followed by a T cell–dependent IgG response ( 44 ). Early after immunization with formalin-inactivated PV (Salk vaccine), total neutralizing Ig titers in QM mice were similar to control animals . The IgG response was initially delayed, but 20 d after infection titers reached the levels observed in C57BL/6 control mice. Interestingly, in the memory phase after 28 d of infection, serum titers in QM mice showed a three- to fourfold reduction compared with controls . This could reflect differences in the numbers or in the expansion potential of the PV-specific B cell clones generated in QM mice. In addition, the inactivated PV vaccine used in this experiment might have provided a weaker stimulus for the selection and expansion of PV-specific QM B cells. Taken together, our experiments have shown that V H replacement and hypermutation generated the VSV-specific immune response observed in QM mice and demonstrated the surprisingly great diversification potential of the QM B cell repertoire, establishing that this phenomenon is not restricted to one particular viral antigen. Where was V H replacement taking place in QM mice, and what triggered it? The current model for the role of receptor editing in B cell development incorporates two different events ( 45 ). During the early phase of B cell development in the bone marrow, editing is induced by the interaction between a self-antigen and the receptor of a developing B cell ( 2 – 4 ). At a later phase, weak interaction between antigen and the B cell receptor of a mature peripheral B cell would induce editing, whereas strong binding turns it off ( 12 , 13 ). Evidence for the occurrence of V H replacement in both bone marrow and spleen of nonimmunized QM mice was recently reported ( 16 ). However, a slight accumulation of idiotype-negative cells in the spleen compared with the bone marrow suggests that the spleen might be a privilege site for ongoing secondary rearrangement ( 16 ). In addition, a higher proportion of idiotype-negative B cells was also observed in B cells isolated from the peritoneum when compared with peripheral blood ( 15 ). Thus, it would appear that there is a constant need for diversification of the QM mouse B cell repertoire throughout B cell development and in different tissues. The molecular events involved in the induction of secondary rearrangements are still not clearly defined, but different triggering mechanisms could be envisaged. First, B cells could have a certain intrinsic capacity to undergo receptor editing in an antigen-independent fashion through help from T cells, cytokines, or other stimuli. Although this scenario cannot be ruled out, there is enough evidence supporting the notion that B cell receptor triggering is required for induction of secondary rearrangements both in the bone marrow ( 7 , 8 ) and in the periphery ( 10 – 12 ) to make this unlikely. The B cell receptor in QM mice would not be expected to bind any other antigen than NP with high affinity. However, it could be argued that weak cross-reactive interactions between NP-specific B cells and different antigens could induce V gene replacement and generate virus-specific B cells. Second, it was proposed that environmental antigenic pressure could be the driving force behind the diversification of the V gene repertoire of naive QM mice ( 15 ). Recent data suggested that secondary rearrangements in peritoneal B-1 cells might contribute to the development of autoreactive Abs ( 46 ). In this study, the authors proposed that frequent exposure of B-1 cells to LPS may lower the threshold for activation of secondary rearrangements resulting in a rapid shift in the Ab repertoire. In view of our findings, this mechanism could generate not only autoreactive B cells but also potentially useful pathogen-specific Abs. On the other hand, de novo V H replacement could have been triggered by cross-reactive binding of the NP-specific B cell receptor to the viral antigens used in this study. Although viral antigens might play a role in the expansion and maintenance of the virus-specific B cells in the infected mice, these interactions may be excluded as initial triggering events because VSV-specific scFv Abs were isolated from a phage display library constructed from a naive QM mouse. Therefore, virus-specific clones were generated in QM mice even before the introduction of viral antigens. In addition, nonimmunized QM mice undergo frequent V H replacement ( 14 , 15 ). It is difficult to address the role of these events in the complexity of a normal Ab response. However, new evidence is emerging in favor of the frequent involvement of secondary rearrangements during normal B cell development ( 47 ). Moreover, sequence analysis of human heavy and light chain V domains suggested that receptor editing occurs in human peripheral B cells ( 48 ). Taken together with our results, these experiments suggest that, together with hypermutation, secondary rearrangement could participate in the shaping of the natural B cell repertoire. In conclusion, our data illustrate the potential of V H gene replacements in the diversification of a restricted repertoire and definitely show that this expanded repertoire is functional. | Study | biomedical | en | 0.999998 |
10359584 | BALB/c mice were purchased from Harlan Sprague-Dawley, Inc. MRL- lpr/lpr and MRL+/+ mice were purchased from The Jackson Laboratory . VH3H9 Tg mice have been described previously ( 4 ). The VH3H9 Tg mice have been backcrossed onto the BALB/c and MRL backgrounds for at least 9 and 17 generations, respectively, and have been bred and maintained in the animal facility at The Wistar Institute. VH3H9 MRL- lpr/lpr mice deficient in the JH locus were obtained from M. Shlomchik (Yale University, New Haven, CT ). In all cases, age-matched BALB/c mice and/or Tg − littermates were used as controls. The presence of the VH3H9 Tg was determined by PCR amplification of tail DNA with primers specific for VH3H9 ( 4 ). The presence of antinuclear Abs (ANAs) in serum was detected using permeabilized HEP-2 cells as the substrate (Antibodies Incorporated). Manufacturer's instructions were followed with serum samples tested at a 1:100 dilution. Anti-homogeneous nuclear (HN) ANA binding and ANA titers were detected with a combination, defined as total Ig, of FITC-conjugated goat anti–mouse Ig(H+L) and anti–mouse IgM reagents (Southern Biotechnology Associates). To test for the presence of λ ANAs, an FITC-conjugated goat anti–mouse λ reagent (Southern Biotechnology Associates) or LS136-biotin (anti-λ1; gift of Garnett Kelsoe, Duke University, Durham, NC) followed by streptavidin (SA)-FITC (Fisher Biotech) was used. The samples were then visualized under a fluorescent microscope and scored blind, without knowledge of age or genotype of the mice. The results using the monoclonal LS136 and polyclonal anti-λ reagents were comparable. Sera were tested from six litters of mice. These mice were bled once a week between 6 and 16 wk of age. These serial samples were tested, in the protocol described above, for the presence of total Ig anti-HN and Igλ ANAs. The first positive bleed was considered the seroconversion time point. The anti-HN seroconversion time points of a subset of Tg − and VH3H9 MRL- lpr/lpr mice were examined for total Ig ANA titers. In each case, the time point was 10 wk of age. These samples were tested at an initial 1:100 dilution and at serial 10-fold dilutions. The serum titer was defined as the reciprocal of the last dilution at which positive staining was seen. Bone marrow (BM), spleen, and LN cells were removed from VH3H9 Tg and Tg − mice. Single-cell suspensions were prepared and, where necessary, erythrocytes were removed by hypotonic lysis. Because the VH3H9 H chain Tg has been shown to be a good excluder of endogenous H chain rearrangement in the BALB/c background, we have followed the fate of anti-dsDNA B cells in VH3H9 Tg mice using anti-λ–specific reagents ( 4 ). Additionally, the restricted use of VH3H9 by λ + cells was confirmed using JH −/− MRL- lpr/lpr mice ( 28 ). Several different reagents were used to track λ + and λ1 + B cells (LS136, R11-153, JC5, and R26-46). Using these reagents and flow cytometry we have shown that the majority of λ + B cells in VH3H9 Tg mice are λ1 ( 10 ). Therefore, we are able to follow VH3H9/Vλ1 B cells in MRL mice using anti-pan λ reagents. Cells (5 × 10 5 ) were surface stained according to standard protocols ( 29 ). The following Abs were used: RA3-6B2–FITC, –PE, or –biotin (anti-B220), R11-153–FITC (anti-Vλ1), R26-46–FITC (anti-Vλ total), R8-140–PE (anti-Igκ), 1D3-FITC or -biotin (anti-CD19), 7G6-FITC (anti-CD21/35), Cy34.1-FITC (anti-CD22), B3B4-FITC (anti-CD23), 3/23-FITC (anti-CD40), Mel-14–FITC (anti-CD62L, L-selectin), M1/69-FITC (anti–heat-stable antigen [HSA]), and IM7-FITC (anti-CD44) ( PharMingen ); 7E9-FITC (anti-CD21/35; gift of A. Naji, University of Pennsylvania, Philadelphia, PA); LS136-biotin (anti-Vλ1; gift of G. Kelsoe) and JC5.1-PE (anti-Vλ total; gift of J. Kearney, University of Alabama, Birmingham, AL); polyclonal anti-IgM–PE and SBA-1–PE (anti-IgD; Southern Biotechnology Associates); SA-Red670 ( GIBCO BRL ); SA-FITC (Fisher Biotech); and SA-PE (Vector Laboratories). All samples were analyzed on a FACScan™ flow cytometer ( Becton Dickinson ) using CellQuest software. 15,000–40,000 events were collected for each sample and gated for live lymphocytes based on forward and side scatter. In analyzing the cell surface phenotype, it became apparent that SA-Red670 binds to B cells from MRL- lpr/lpr mice. The reagent does not bind to conventional T cells in MRL- lpr/lpr mice, but does bind to ∼10% of the B220 + CD3 + CD4 − CD8 − (double-negative [DN]) T cells that accumulate. This binding segregates with the mouse strain: BALB/c and C57BL/6 B cells do not bind to SA-Red670, whereas MRL- lpr/lpr and MRL+/+ B cells do bind (data not shown). SA-Red670 binding is not due to the SA in the conjugate because neither SA-FITC nor SA-PE binds to MRL-derived cells (data not shown). Because Red670 is composed of PE plus a Cy5 residue ( 30 ), this suggests that Cy5 mediates the binding. It is unclear what Red670 binds on the B cell, but it does not appear to be mediated through Ig since the Ig low cells have the same amount of Red670 binding as the Ig high cells (data not shown). Because all B cells in MRL mice and only a small fraction of DN T cells bind to SA-Red670, in some experiments we have used SA-Red670 as a marker to enrich for B cells. To identify B cells specifically, a second B cell marker that is absent on DN T cells, such as CD19 or Ig, was used to identify the B cells. Spleens were suspended in OCT, frozen in 2-methyl-butane cooled with liquid nitrogen, sectioned, and fixed with acetone. The spleen sections were stored at −20°C and then stained according to the protocol described ( 31 ). In brief, the sections were blocked using PBS/5% normal goat serum ( Sigma Chemical Co. )/0.1% Tween-20 and then stained with GK1.5-biotin (anti-CD4), 53-6.7–biotin (anti-CD8), 30H12-FITC (anti–Thy-1.2) (grown as supernatants), Cy34.1-FITC (anti-CD22), 281-2 (anti–syndecan-1) ( PharMingen ), MOMA-1 (anti–marginal metallophilic macrophages; Bachem), and/or anti-Igλ–alkaline phosphatase (AP) (Southern Biotechnology Associates). Streptavidin– horseradish peroxidase (HRP) or -AP (Southern Biotechnology Associates), polyclonal anti–rat-HRP (Jackson ImmunoResearch Laboratories), MAR18.5-biotin (anti–rat Ig, grown as supernatant), and anti-FITC–AP ( Sigma Chemical Co. ) or anti-FITC–HRP (Chemicon) were used as secondary Abs. HRP and AP were developed using the substrates 3-amino-9-ethyl-carbazole (3-AEC) and Fast Blue BB base ( Sigma Chemical Co. ), respectively. Splenic B cells were plated at 4 × 10 5 cells/well and diluted serially 1:4 in Multiscreen HA mixed cellulose ester membrane plates ( Millipore ) coated with unlabeled goat anti–mouse total Ig (Southern Biotechnology Associates). The Ig secreted by the plated cells was detected by AP-conjugated goat anti–mouse total Ig or Igλ as secondary Abs (Southern Biotechnology Associates) and visualized using NBT/BCIP substrate (nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate; Sigma Chemical Co. ). Statistical significance was determined using an unpaired nonparametric test and Instat software. In this study, we have used the VH3H9 Tg to track a specific population of anti-dsDNA B cells, in the context of a diverse repertoire, and compare their fate in nonautoimmune and autoimmune environments. Transfection and hybridoma analyses have shown that the germline Vλ1 L chain pairs with the VH3H9 H chain to generate an anti-dsDNA ANA + Ab ( 25 , 32 ). Using anti-λ–specific reagents, we have followed B cells of this specificity in terms of serum Ab expression, surface phenotype, splenic localization, and ability to differentiate into antibody-forming cells (AFCs). As a first step in studying VH3H9 MRL- lpr/lpr mice, we examined the overall rate at which ANAs appeared in the serum. An age-matched cohort of Tg − and VH3H9 MRL- lpr/lpr mice was bled weekly from age 6 to 16 wk. The sera were tested for the presence of ANAs using reagents to detect total Ig, and scored as positive when they showed an HN staining pattern. This ANA pattern is found in a high frequency of SLE serum and correlates with the presence of anti-dsDNA, antihistone, and antichromatin Abs ( 33 ). Fig. 1 A shows that Tg − MRL- lpr/lpr mice became serum HN ANA + at approximately the same time (10.1 ± 3.0 wk) as VH3H9 MRL- lpr/lpr mice (9.1 ± 2.0 wk; P = 0.2). To test if the VH3H9 Tg alters the amount of ANA in the serum, ANA titers were determined for a subset of the mice . Again, no difference was detected between Tg − and VH3H9 MRL- lpr/lpr mice. Likewise, Fig. 1 C shows that the VH3H9 MRL- lpr/lpr mice seroconvert to Igλ ANA + at an average of 9.2 ± 1.4 wk, the same time when total HN ANAs appear in the serum (9.1 ± 2.0 wk). Within the time-frame analyzed (ages 6.0–43.5 wk), the VH3H9/λ1 specificity was not detected in serum from MRL+/+ mice (data not shown). The fact that most Tg − MRL- lpr/lpr mice failed to seroconvert to Igλ ANA + is consistent with data showing that the overwhelming majority of ANAs in Tg − MRL- lpr/lpr mice use the more abundant Igκ L chains ( 34 ). Given that VH3H9/Vλ1 ANAs arise with the same kinetics as other ANAs in both Tg − and Tg + MRL- lpr/lpr mice, VH3H9/Vλ1 B cells appear to be a good model for following the fate of ANA B cells in general. Having established the kinetics of seroconversion, we proceeded to compare the phenotype of anti-dsDNA B cells in mice that are serum ANA + (>9 wk MRL- lpr/lpr ) with mice that are ANA − (<9 wk MRL- lpr/lpr , MRL+/+, and BALB/c). Using λ-specific reagents and flow cytometry, we have previously shown that anti-dsDNA B cells are present in the periphery of nonautoimmune (BALB/c) mice with a decreased level (four- to fivefold) of surface Ig relative to Tg − B cells ( 10 ). Likewise, Igλ + B cells are also present in the spleen and LNs of VH3H9 MRL- lpr/lpr and VH3H9 MRL+/+ mice, with the same four- to fivefold decreased level of surface Ig compared with Igλs in Tg − mice . Interestingly, when Ig levels are compared for Igλ + cells in the BM, the Ig level is also reduced, and to a similar extent, in VH3H9 MRL- lpr/lpr, VH3H9 MRL+/+ , and VH3H9 BALB/c mice ( 10 ). We and others have interpreted a low level of Ig to be an indication of Ag encounter ( 3 , 10 – 12 , 35 – 38 ). Additionally, the extent of receptor downmodulation has been correlated with the available concentration of self-Ag ( 39 , 40 ). Thus, the VH3H9/Igλ B cells in all backgrounds tested encounter their Ag, and this interaction first takes place in the BM. We next were interested in determining how encounter with Ag would affect the phenotype of the autoreactive cells from MRL+/+, MRL- lpr/lpr , and BALB/c mice. However, before the analysis of VH3H9 Tg anti-dsDNA B cells, we noted differences among the three background strains of mice in terms of B cell expression levels of several cell surface markers. Although a similar pattern of expression for all sets of mice was observed for the majority of markers, including B220 , HSA, CD22, and CD44 (data not shown), there were notable exceptions in L-selectin, CD23, and CD21/35. The proportion of L-selectin low B cells is increased in MRL+/+ and MRL- lpr/lpr mice compared with BALB/c mice . Decreased levels of L-selectin can indicate activation, suggesting that activated B cells are accumulating in MRL+/+ and MRL- lpr/lpr mice ( 41 , 42 ). Additionally, as has been previously reported, CD23 levels are also decreased on MRL- lpr/lpr B cells and this decrease is more apparent as the mice age . A decrease in CD21/35 expression has been reported in MRL- lpr/lpr mice ( 44 ) as well as SLE patients ( 45 – 47 ). We also observe a twofold decrease in CD21/35 expression on a portion of the B cells in MRL- lpr/lpr mice compared with BALB/c and MRL+/+ mice. However, in our analysis, the most striking difference in the MRL mice both with and without the lpr mutation when compared with the BALB/c mice is the dramatic increase in CD21/35 high cells . This peak of CD21/35 high cells, which are also IgM high (data not shown), accumulates with age in the MRL- lpr/lpr mice. Because CD23 low CD21/35 high IgM high is reminiscent of the phenotype assigned to marginal zone (MZ) B cells ( 48 ), we are in the process of quantitating whether MRL mice have an exaggerated MZ area. With differences among the three background strains noted, the developmental status of the anti-dsDNA B cells in VH3H9 MRL+/+, VH3H9 MRL- lpr/lpr , and VH3H9 BALB/c mice was compared using flow cytometry and a panel of cell surface markers. Previously, we have shown that anti-dsDNA B cells in VH3H9 BALB/c mice are developmentally arrested and show signs of activation ( 10 ). To examine the cell surface phenotype of splenic anti-dsDNA B cells, the relative expression levels of B220, HSA, CD21/35, CD22, CD23, and CD44 ( 29 , 49 – 53 ) on VH3H9/λ B cells were compared with those on Tg − B cells in BALB/c , MRL+/+ , and MRL- lpr/lpr mice. The λ + B cells in Tg − MRL+/+ , MRL- lpr/lpr , and BALB/c ( 10 ) mice have equivalent levels of all surface markers tested, suggesting that there is nothing inherently different about Igλ + B cells. In contrast and as previously reported, anti-dsDNA B cells from VH3H9 Tg BALB/c mice appear developmentally arrested in that they express a low level, relative to mature splenic B cells, of B220 (decreased twofold), CD21/35 (decreased threefold), CD22 (decreased twofold), and CD23 (decreased twofold), an intermediate level of HSA (increased twofold), and an elevated level of CD44 (increased fourfold) . Importantly, VH3H9/λ B cells on the MRL+/+ and MRL- lpr/lpr background are no longer developmentally arrested: they express mature levels of B220, HSA, CD22, and CD44 . Additionally, VH3H9/λ MRL+/+ B cells express mature levels of CD23 ; however, in the MRL- lpr/lpr mice, CD23 is not a useful marker for maturation, given that the level of CD23 is dramatically reduced on all MRL- lpr/lpr B cells as the mice age . The surface phenotype of VH3H9/λ B cells was also compared in MRL- lpr/lpr mice both before and after seroconversion to determine if autoantibody production is marked by a change in cell surface phenotype. Igλ + B cells from both ANA − (age 4–7 wk) and ANA + (age 8–14 wk) VH3H9 MRL- lpr/lpr mice are phenotypically mature . One drawback of this analysis is that most of the markers tested are lost on B cells that have differentiated into AFCs. Thus, although we may be missing AFCs in the flow cytometry analysis, the majority of the VH3H9/λ B cell population is indistinguishable between ANA − and ANA + MRL- lpr/lpr mice. While several cell surface markers suggest that anti- dsDNA B cells in MRL+/+ and MRL- lpr/lpr mice are no longer developmentally arrested, CD21/35 levels are decreased on VH3H9/λ + B cells, although not to the extent they are in the BALB/c background . The reduced level of CD21/35 is not due to complement binding that in turn could block the Ab binding site, because staining with two different anti-CD21/35 Abs (7G6, which binds to the complement-binding site, and 7E9, which does not ) gave similar results (data not shown). Why CD21/35 levels remain decreased on VH3H9 MRL+/+ and MRL- lpr/lpr λ + B cells, despite normal levels of other developmental markers (B220, HSA, and CD22) is not clear. One possibility is that only those B cells expressing low levels of CD21/35 persist after negative selection, given that CD21/35 is a coreceptor for signals through the Ig receptor ( 56 ). A similar explanation has been described for the reduced levels of the CD8 coreceptor seen on self-reactive anti-HY TCR Tg T cells present in the periphery of male mice ( 57 ). Another possibility is that reduced CD21/35 levels reflect activation due to Ag encounter ( 44 ). Ig Tg B cells specific for model Ags, where Ag exposure can be controlled, may help to determine if Ig downmodulation in the presence of Ag is accompanied by a decrease in CD21/35. One scenario that could account for the more advanced maturation of VH3H9/λ + B cells in MRL as opposed to BALB/c mice is that they encounter Ag at a later developmental stage in the BM and thus their maturation goes unimpeded. As an indication of Ag encounter, the level of surface Ig on the λ + B cells in the BM of VH3H9 and Tg − BALB/c, MRL+/+, and MRL- lpr/lpr mice was compared. As is shown in Fig. 3 D, the level of Ig on Tg − B cells increases as the B cells mature from a CD22 low to a CD22 high stage. If the VH3H9/λ + B cells in the different genetic backgrounds are encountering Ag at a later stage, we would have predicted that the level of Ig would be higher on the less mature (CD22 low ) B cells in the VH3H9 Tg MRL mice and then decrease once they have encountered Ag. However, the level of Ig on VH3H9/λ + B cells from all three backgrounds is low at the CD22 low stage and remains low at the CD22 high stage, suggesting that these B cells have all encountered Ag at an immature stage. This encounter with Ag does not halt the maturation of the B cells: in BALB/c mice, the VH3H9/λ + B cells continue to develop to a slightly more mature stage as indicated by intermediate levels of HSA, B220, CD22, and CD23 , whereas in MRL mice (+/+ and lpr/lpr ), the splenic B cells appear fully mature . To confirm that the cells we are following are using the VH3H9 Tg, we have repeated our experiments using VH3H9 MRL- lpr/lpr mice deficient at the JH locus (JH −/− ). As B cells in VH3H9 JH −/− mice are unable to rearrange endogenous H chain loci, the only H chain they express is the VH3H9 Tg. The results from VH3H9 MRL- lpr/lpr mice with and without an intact JH locus were indistinguishable in terms of phenotypic analysis of BM and splenic VH3H9/Vλ B cells (data not shown). Given the changes in developmental maturity of anti-dsDNA B cells in BALB/c versus MRL mice, we next looked to see what effect these changes have on the B cells' splenic localization. For the most part, B and T cells are located in discrete areas in the spleen, called the B cell follicle and periarteriolar lymphoid sheath (PALS), respectively. Previously, using Ig Tg mice, we and others have shown that anergic B cells localize to the T–B interface of the splenic follicle ( 10 , 27 ). One hypothesis to account for this localization is that anergic B cells continually encountering Ag are at a competitive disadvantage for follicular space ( 27 , 58 ). Another hypothesis is that the location of the B cells is dependent on the amount of Ag exposure, not competition with other naive B cells ( 39 , 40 ). In both models, B cells encountering Ag move to the T–B interface, where they can interact with T cells. Immature B cells are also reported to first appear at the T–B interface in the spleen ( 59 ). Given that the VH3H9/λ BALB/c B cells that localize to the T–B interface are both immature and have encountered Ag, we have been unable to distinguish which of these features is responsible for their localization . However, on the MRL+/+ and MRL- lpr/lpr backgrounds, VH3H9/λ B cells are no longer developmentally arrested, providing an opportunity to separate maturation status from Ag encounter. Igλ + B cells in Tg − mice from autoimmune and nonautoimmune backgrounds are scattered throughout the splenic follicle . However, similar to their counterparts in the BALB/c background, VH3H9/λ B cells from MRL+/+ mice localize to the T–B interface . Therefore, follicular localization is not dependent on the developmental status of the autoreactive B cells, at least in MRL+/+ mice; rather, we suggest it is a consequence of Ag encounter. In striking contrast to the VH3H9/λ MRL+/+ B cells, VH3H9/λ + B cells in the MRL- lpr/lpr background are not localized to the T–B interface; instead, they are located within the B cell follicle . This pattern of localization is seen in mice as early as 4 wk of age, before their specificity is detectable in the serum. Identical results for localization of anti-dsDNA B cells were obtained using VH3H9 MRL- lpr/lpr mice with and without an intact JH locus, confirming that the cells we are following use the VH3H9 Tg (data not shown). Thus, while indistinguishable by developmental status, anti-dsDNA B cells in VH3H9 MRL+/+ and VH3H9 MRL- lpr/lpr mice exhibit different splenic localizations. Disruptions in the splenic architecture of MRL- lpr/lpr mice have been known for some time and have been attributed to the accumulation of CD3 + B220 + CD4 − CD8 − (DN) T cells. It has been reported that these DN T cells are located between the T and B cell areas in the follicles of older MRL- lpr/lpr mice ( 60 , 61 ). To confirm this, MRL- lpr/lpr spleen sections were stained with anti-B220 and anti-CD3, and coexpressing cells were identified by immunofluorescence. In agreement with previous studies, the B220 + CD3 + cells were located in discrete areas between the PALS and what remained of the B cell follicle (data not shown). In addition to the disruption in splenic architecture caused by DN T cells, we have identified a novel histological feature of MRL- lpr/lpr mice that is evident even before the emergence of DN T cells. In contrast to the usual segregation of B and T cells, CD4 T cells in VH3H9 MRL- lpr/lpr mice do not localize exclusively to the T cell area: a subpopulation of CD4 T cells are in the B cell follicle . To examine this altered CD4 localization more closely, spleen sections were stained with MOMA-1, a marker for MZ macrophages, and either CD22, CD4, or CD8 . In both BALB/c and MRL+/+ spleens, with and without the VH3H9 Tg, distinct B and T cell areas are present with few to no CD4 or CD8 T cells in the B cell area . However, in VH3H9 MRL- lpr/lpr spleens, although distinct B and T cell areas are present, there is also a subpopulation of CD4 T cells scattered throughout the follicle . CD4 T cells have been found in the B cell follicle in the context of germinal centers (GCs ). However, the CD4 T cells in the follicles of MRL- lpr/lpr mice are not like those described in GCs as they express Thy-1 (data not shown), a marker absent on GC T cells ( 62 ). CD8 T cells remain tightly compacted around the central arteriole, and neither CD4 nor CD8 T cells are present in the MZ of VH3H9 MRL- lpr/lpr mice . To determine if the altered localization of CD4 T cells is a general feature of MRL- lpr/lpr mice, or is unique to the VH3H9 Tg, spleen sections from Tg − MRL- lpr/lpr mice were examined . Unlike BALB/c and MRL+/+ spleens, the Tg − MRL- lpr/lpr spleens have CD4 T cells scattered throughout the B cell follicle , showing that this disrupted architecture is not limited to VH3H9 mice. The fact that MRL- lpr/lpr and not MRL+/+ mice show disrupted architecture suggests that this alteration is attributable to a block in the Fas/FasL pathway. At the time points examined, CD4 infiltrates were not found in other organs such as the liver or kidney (data not shown). Although the presence of the VH3H9 Tg is not necessary for the appearance of CD4 T cells in the B cell follicle, it does accelerate the process. Age-matched VH3H9 and Tg − MRL- lpr/lpr mice were analyzed for CD4 T cell infiltrates by immunohistochemistry . Alterations in T/B architecture are seen as early as 4–5 wk in VH3H9 MRL- lpr/lpr spleens ; however, Tg − MRL- lpr/lpr spleens show no disruption until 6–8 wk and it becomes more pronounced at 10–12 wk . VH3H9 BALB/c and VH3H9 MRL+/+ mice have normal splenic architecture, suggesting that the lpr mutation, not the VH3H9 Tg, is necessary for the disruption . Therefore, in VH3H9 MRL- lpr/lpr mice, although increasing the frequency of autoreactive B cells does not accelerate the kinetics or titers of serum ANAs, it does accelerate the kinetics of T–B mixing in MRL- lpr/lpr mice. In addition to noting differences in anti-dsDNA B cells in nonautoimmune versus autoimmune mice, we also examined VH3H9 MRL- lpr/lpr mice before and after seroconversion to determine if any architectural alterations correlate with the emergence of serum Ab. Although no difference by flow cytometry was detected between ANA + and ANA − mice , a difference was observed by immunohistochemistry . In ANA + mice, many darkly staining Igλ + cells are present in the PALS as well as in the bridging channels to the red pulp . These cells are not obvious in ANA − mice (data not shown). To determine if these darkly staining cells are AFCs, serial sections were stained for syndecan-1, a cell surface proteoglycan that is expressed on B cells that have differentiated into AFCs ( 63 ). The dark Igλ staining cells colocalize with syndecan-1 staining, indicating that they are AFCs. Importantly, many Igλ + /syndecan-1 − cells remain in the B cell follicles even in ANA + animals, suggesting that not all dsDNA B cells have differentiated into AFCs (data not shown). Syndecan-1 + cells are also found in the PALS in Tg − MRL- lpr/lpr mice, although in this case the vast majority are not Igλ + , consistent with Igκ + Igλ − ANAs in the serum . This localization of AFCs to the inner T cell area, as opposed to the bridging channels to the red pulp (also referred to by others as the red pulp area adjacent to the T cell zone ), is similar to that reported by Marshak-Rothstein and colleagues for rheumatoid factors in MRL- lpr/lpr mice ( 61 , 66 ). As an alternate approach to quantitate the number of AFCs in the various mice, ex vivo enzyme-linked immunospot assays (ELISpots) were performed . Using reagents to detect all AFCs, few to no AFCs were detectable in spleens from young (4–6 wk) Tg − and VH3H9 MRL- lpr/lpr , MRL+/+, and BALB/c mice, but became more pronounced in aged (10–12 wk) MRL- lpr/lpr mice . To follow anti-dsDNA AFCs in particular in VH3H9 MRL- lpr/lpr mice, Igλ AFCs were quantitated . Igλ AFCs were readily detectable in older VH3H9 MRL- lpr/lpr mice. By ELISpot, only 5–10% of the Igλ + cells in VH3H9 MRL- lpr/lpr mice are generating AFCs. The fact that not all Igλ + cells are secreting Ab as shown by histology and ELISpots supports the idea that it is a breakthrough of a fraction of the anti-dsDNA B cells and not an overall breakdown of tolerance that leads to serum ANAs in MRL- lpr/lpr mice. Several studies have documented alterations in total B and T cell populations in autoimmune mice and SLE patients ( 43 , 44 , 61 , 67 ). The unique aspect of the data presented here is that specific autoreactive B cells have been followed, both in nonautoimmune mice and in autoimmune-prone mice, as they develop autoantibodies. To do this we made use of the VH3H9 H chain Tg, which increases the frequency of anti-dsDNA B cells and allows their fate, in the presence of nonautoreactive B cells, to be compared in BALB/c, MRL+/+, and MRL- lpr/lpr mice. This model has allowed for the analysis not only of endpoints such as serum autoantibodies, but also of the B cells themselves and the factors that influence them during the process of seroconversion. In this study, we have described two novel observations regarding the breakdown of tolerance in MRL- lpr/lpr mice. The first is that MRL mice exhibit a defect in maintaining the developmental arrest of anti-dsDNA B cells. The second is that a deficiency in Fas allows these autoreactive cells to enter the B cell follicles from which they are normally restricted. Combining these observations, we propose that the transition of anti-dsDNA B cells from tolerized B cells on the BALB/c background to autoantibody-producing cells in the MRL- lpr/lpr is a multistep process that includes overcoming both developmental arrest and follicular exclusion. It has been clearly established that autoimmunity in MRL mice is polygenic, yet the mechanisms by which these different loci influence autoantibody production have not been defined ( 68 – 71 ). Although anti-dsDNA B cells are tolerized in BALB/c mice as manifested by their developmental arrest and accumulation at the T–B interface of the splenic follicle ( 10 ), anti-dsDNA B cells on the MRL+/+ and MRL- lpr/lpr backgrounds are developmentally mature. One possibility that could account for this difference is timing of initial Ag exposure. For example, if anti-dsDNA B cells in MRL mice are not exposed to the tolerizing Ag during their development, maturational arrest would not occur. However, the data presented are inconsistent with this scenario: for anti-dsDNA B cells in both MRL and BALB/c mice, the level of surface Ig is low at the same developmental stage in the BM. A second possibility is that although anti-dsDNA B cells in both BALB/c and MRL mice initially encounter their Ag at the same time in development, an absence of continual Ag stimulation in the MRL mice allows the B cells to subsequently progress to maturity. In the HEL B cell tolerance model in the context of bcl-2, the removal of Ag allows the development of the anergic B cells to progress ( 38 ). However, in all the mice studied here, anti-dsDNA B cells are uniformly Ig low in the spleen as well as in the BM, suggesting that constant Ag exposure is occurring in all cases. A third possibility, and one we favor, is that a defect in MRL mice allows autoreactive B cells to survive a selection checkpoint that would normally lead to developmental arrest. This is important in that the maturation state of a B cell may translate to significant changes in function in terms of B cell receptor–associated intracellular signaling capabilities and subsequent B cell responsiveness ( 72 ). Mapping studies by various groups have identified several autoimmunity-associated loci in MRL mice ( 68 – 71 ). It will be important to determine if any of these loci, together or in isolation, control the developmental maturation of autoreactive B cells. We observe in this study a previously undefined connection between Fas and the entry of cells to the B cell follicle. Comparing MRL mice with and without the lpr defect in Fas, we find that despite their mature status, anti-dsDNA B cells in MRL+/+ mice remain at the T–B interface, whereas in MRL- lpr/lpr mice, they are in the B cell follicle. Exclusion from the B cell follicle has been correlated with the decreased survival of B cells ( 58 ). However, studies using bcl-2 Tgs have shown that this correlation is not absolute, as simply increasing a B cell's life span does not alone alter the localization of anti-HEL B cells from the T–B interface ( 27 ). The data from the present study indicate that Fas can play an important role in determining follicular entry; what remains to be discovered is the mechanism by which it exerts this effect. A deficiency in Fas could act directly on the survival of the anti-dsDNA B cells. Alternatively, lack of Fas could have an indirect effect through the shaping of the peripheral T and B cell repertoires. The lack of Fas could translate into an alteration in the availability of T cell help. It has been clearly established that T cells are required for the generation of autoantibodies in MRL- lpr/lpr mice ( 73 – 75 ). Additionally, it has been suggested that Ag-experienced B cells, such as the anti-dsDNA B cells followed here, are held at the T–B interface in the absence of T cell help ( 39 , 58 ). In light of this idea, one possible interpretation of our results is that Fas normally plays a crucial role in eliminating the autoreactive T cell population. In its absence, a fundamental shift occurs in the T cell repertoire such that autoreactive T cell help is now available, and autoreactive B cells are given the signals they need to proceed past the T–B interface. The data presented here argue against this possibility. The anti-dsDNA B cells are found peppered throughout the B cell follicle and appear not to be proliferating (data not shown). If they had received T cell help, we would have expected to observe either (a) the compaction of B cells in follicular GCs, or (b) the formation of AFCs ( 31 ). Although at later time points (mice >8 wk) we do see evidence of AFCs, at the early time points (4–7 wk) dsDNA B cells appear exclusively in the follicles. Thus, the effect of Fas on follicular entry seems unlikely to be mediated solely through an effect on T cell help. Alternatively, Fas could regulate follicular entry through its functions on the B cell itself. For example, Rathmell and colleagues have shown that anergic B cells, far from being rescued by T cell help, are killed in a Fas-dependent manner ( 76 , 77 ). In the absence of Fas, then, it is possible that anti-dsDNA B cells would not die and thus would be able to proceed into the B cell follicle. One prediction from this is that anti-dsDNA B cells in Fas wild-type animals would have a reduced life span. Experiments are currently underway to determine if the life span of anti-dsDNA B cells is reduced in MRL+/+ compared with MRL- lpr/lpr mice. Again, however, if T cell help were present in the absence of Fas, we would expect to see evidence of further differentiation of the anti-dsDNA B cells in young MRL- lpr/lpr mice. Interestingly, experiments using TCR −/− mice, in the context of the HEL B cell tolerance model, have shown that T cells are not required to maintain follicular exclusion ( 78 ). Therefore, if a signal through Fas on the B cell is what restricts it to the T–B interface, other FasL-expressing cells would be required to deliver that signal. A scenario that is consistent with our data is that the deficiency in Fas is affecting the proportion of autoreactive B cells in the peripheral B cell repertoire and, as such, alters B cell localization. Studies in the HEL system indicate that changes in the B cell repertoire can affect the localization of autoreactive B cells. In mice where anergic anti-HEL B cells make up the vast majority of the B cell population, they are present in the B cell follicle; however, when nonautoreactive B cells are also present, the anergic B cells are excluded from the B cell follicle ( 27 , 58 ). If Fas plays a role in eliminating autoreactive B cells, then the lpr mutation could affect the proportion of autoreactive B cells in the periphery such that, instead of the majority of competing B cells being nonautoreactive, the anti-dsDNA B cells are in an environment with mostly other autoreactive B cells. Therefore, anti-dsDNA B cells in MRL- lpr/lpr mice would be present in the B cell follicle due to lack of competition and not because they are missing a signal through Fas to restrict their entry. Altering the composition of the B cell repertoire by transfer experiments of dsDNA B cells from VH3H9 MRL- lpr/lpr mice into MRL- lpr/lpr mice where the B cells express a nonautoreactive Ig Tg ( 23 ) should allow us to discern if the addition of competitive B cells now results in follicular exclusion of the anti-dsDNA B cells. An additional component that must be considered is the role of Fas in Ag composition. The role of Ag in MRL- lpr/lpr autoantibody production remains controversial. BM chimera and allophenic experiments, mixing lpr/lpr and Fas wild-type cells, have suggested that intrinsic B and T cell defects in lpr/lpr mice, and not alterations in Ag milieu, are responsible for autoantibody production ( 79 – 82 ). However, the interpretation of these results is complicated. lpr/lpr T cells are known to express elevated levels of FasL ( 83 , 84 ), and it is distinctly possible that the autoreactive non- lpr/lpr B cells in the chimeras, susceptible to Fas-mediated death upon activation, are simply triggered to die before they can contribute to autoantibody levels. Therefore, it remains a possibility that eliminating Fas-mediated death alters the antigenic make-up of MRL- lpr/lpr mice. Consistent with this, studies by Rosen and colleagues have shown that some of the nuclear Ags targeted in SLE are packaged into discrete blebs at the surface of apoptotic cells ( 85 ). They have further demonstrated that triggering apoptotic pathways by different stimuli generates unique substrate fragments that can then be clustered in these blebs ( 86 ). Preferential use of one pathway over another, as could occur in Fas-deficient MRL mice, would potentially alter the ability to produce and display tolerogenic or immunogenic epitopes. The fact that dsDNA B cells from both MRL+/+ and MRL- lpr/lpr mice are uniformly Ig low suggests that both are encountering a similar amount of Ag. However, it may be that a difference in antigenic context determines whether a B cell enters the follicle, as in MRL- lpr/lpr mice, or is held at the T–B interface, as in MRL+/+ mice. Because of the potential significance of this issue, experiments are currently underway to explore the nature of the in vivo Ag recognized by these anti-dsDNA B cells. In examining the localization of anti-dsDNA B cells, we have identified a unique histological feature of CD4 T cells in both Tg − and VH3H9 MRL- lpr/lpr mice. A subpopulation of CD4 T cells, which normally are located in a discrete area around the central arteriole, have infiltrated the B cell follicle. The lack of this infiltrate in Tg − and VH3H9 MRL+/+ mice suggests that the absence of a Fas–FasL interaction is the determining factor. In VH3H9 MRL- lpr/lpr mice, the T cell splenic architecture is disrupted as early as 4 wk of age (before we see evidence of DN T cells) and becomes more apparent as the mice age. Interestingly, increasing the frequency of autoreactive B cells by the presence of the VH3H9 Tg, while not accelerating seroconversion, does accelerate the appearance of T–B mixing. Why a subpopulation of T cells resides in the B cell follicle in VH3H9 MRL- lpr/lpr mice is unclear, but the follicular localization of CD4 T cell infiltrates occurs in mice that also have follicular anti-dsDNA B cells. Our working model is that the increased frequency of autoreactive B cells in the VH3H9 Tg mouse plays a role in accelerating the influx of CD4 T cells. There is precedent for B cells, in general, affecting the activation status of T cells in MRL- lpr/lpr mice: B cell–deficient MRL- lpr/lpr mice do not have as many activated/memory T cells as B cell– sufficient MRL- lpr/lpr mice ( 28 ). To explore this idea further, we are examining the spleens of MRL- lpr/lpr mice carrying Ig Tgs in the absence of their cognate Ag ( 23 ). We would predict that the infiltration of T cells would occur at a decreased rate in mice with a B cell repertoire skewed away from autoreactivity. Alternatively, it may be the infiltrating CD4 T cells that allow the anti-dsDNA B cells to enter the follicle. To independently regulate the follicular entry of CD4 T cells and dsDNA B cells, we are breeding the VH3H9 Tg onto MHC class II–deficient MRL- lpr/lpr mice ( 74 ). These mice will allow us to determine if the altered localization of VH3H9/λ B cells is dependent on the presence of CD4 T cells. This study has allowed us to compare the phenotype of the anti-dsDNA B cells in MRL- lpr/lpr mice before and after seroconversion. Even before seroconversion, the anti-dsDNA B cells are developmentally mature and in the B cell follicle. Furthermore, using a decreased level of surface Ig to indicate Ag encounter ( 3 , 10 – 12 , 35 – 38 ), anti-dsDNA B cells are Ig low in the BM, spleen, and LNs, suggesting continual Ag exposure, beginning in the BM. The consistent histological difference between VH3H9 MRL- lpr/lpr mice before and after seroconversion is the presence of Igλ AFCs in serum Igλ ANA + animals. We note two striking characteristics of this AFC formation. The first is that, while mice immunized with model Ags, such as (4-hydroxy-3-nitrophenyl)acetyl (NP), form AFCs in the bridging channels to the red pulp ( 31 , 64 , 65 ), the VH3H9/Igλ AFCs are, in addition to being in the bridging channels, also located in the PALS. This altered localization of AFCs is consistent with that previously reported by Marshak-Rothstein and colleagues for rheumatoid factor AFCs in MRL- lpr/lpr mice ( 61 , 66 ). Whether this localization of AFCs to the PALS is restricted to autoreactive B cells or extends to AFCs in response to foreign Ags is currently under study. The second difference is that even in serum Igλ ANA + animals, not all of the Igλ B cells become AFCs, suggesting a breakthrough of only some autoreactive B cells and not a global breakdown that would affect the population as a whole. Therefore, we have segregated initial exposure of Ag and entry into the follicle from terminal differentiation into AFCs. In summary, the factors that are required to transform an autoreactive B cell population from a tolerant one to one producing Ab are clearly complex. This study has compared the phenotype and splenic localization of a population of anti-dsDNA B cells in nonautoimmune mice with anti-dsDNA B cells in autoimmune MRL+/+ and MRL- lpr/lpr mice. BALB/c mice actively regulate anti-dsDNA B cells as manifested by their developmental arrest, retention at the T–B interface, and lack of anti- dsDNA Ab in the serum. In MRL- lpr/lpr mice where anti-dsDNA Ab is present in the serum, the anti-dsDNA B cells are no longer developmentally arrested or excluded from the B cell follicle. Anti-dsDNA B cells in MRL+/+ mice are also not developmentally arrested; however, they remain at the T–B interface. This allows us to assign a defect in maturational arrest to the MRL genetic background. The maturation state of B cells may have profound effects on their life span and ability to be reactivated. Additionally, in this model, a defect in Fas is needed for the B cells to enter the follicle. However, even Fas-deficient B cells require an additional factor to become AFCs, as young VH3H9 MRL- lpr/lpr mice, where the λ + B cells are mature and in the follicle, are serum − . Changes in T cell help or Ag presentation may be required for seroconversion. Interestingly, a defect in Fas also leads to an infiltration of CD4 T cells into the B cell follicle. What role this infiltrate plays in the production of autoantibodies is unknown. Given the requirement for CD4 T cells in the production of autoantibodies in MRL- lpr/lpr mice ( 73 – 75 ), it will be important to determine the specificity of these infiltrating CD4 T cells. | Study | biomedical | en | 0.999995 |
10359585 | HUVECs were purchased from Cell Systems/ Clonetics and cultured in endothelial basal medium supplemented with hydrocortisone (1 μg/ml), bovine brain extract (3 μg/ml), gentamicin (50 μg/ml), amphotericin B (50 μg/ml), epidermal growth factor (10 μg/ml), and 10% FCS until the third passage. HUVECs (5 × 10 5 cells) were incubated for the time indicated with TNF-α, and homogenates were obtained as described previously ( 3 ); Western blots were performed with anti–Bcl-2 antibody ( Boehringer Mannheim ) or anti-myc antibody ( Santa Cruz Biotechnology ). For detection of phosphorylated extracellular signal–regulated kinase (ERK), proteins were isolated as described previously ( 32 ), and blots were probed with phospho-specific ERK1/2 ( New England Biolabs ). For immunoprecipitation, proteins (3 mg) were incubated with anti-myc antibody (1 μg/ml; Santa Cruz Biotechnology ) at 4°C for 4 h and bound to A/G-PLUS–agarose beads ( Santa Cruz Biotechnology ). Western blot analysis was performed using antiubiquitin antibody ( Sigma Chemical Co. ). Human Bcl-2 was amplified by PCR with oligonucleotides containing EcoRV and HindIII restriction sites and cloned into the respective sites of pcDNA3.1(−)Myc-His (Invitrogen) under the transcriptional control of the CMV promoter. MAP kinase phosphatase (MKP)-3 was cloned after PCR amplification with oligonucleotides containing EcoRV and BamHI restriction sites into the pcDNA3.1(−)Myc-His vector. Bcl-2 mutants and the MKP-3 mutant were obtained by PCR-directed mutagenesis or by site-directed mutagenesis (Stratagene). Clones with verified sequences were transfected in HeLa cells or HUVECs. HeLa cells were transfected with plasmids encoding wild-type Bcl-2 or mutated Bcl-2 by the lipofectamine procedure ( GIBCO BRL ) and selected with 0.5 mg/ml G-418 for 6 d before stimulation of apoptosis. Heterogenous populations of the stably transfected cells were used to avoid any possible clonal variations. HUVECs were transfected with 3 μg pcDNA3.1 plasmid encoding the corresponding sequence as described previously ( 32 ). For the detection of apoptosis, HUVECs were cotransfected with β-galactosidase. In brief, 150 μl medium was mixed with 3 μg plasmids (1 μg pcDNA3.1-lacZ and 2 μg pcDNA3.1–Bcl-2) and 30 μl Superfect (Qiagen) and incubated for 10 min at room temperature. During the incubation time, medium was removed from the cell culture plates, and HUVECs were washed twice in medium without FCS. 1 ml medium was then added to the plasmid-Superfect mixture, and HUVECs were incubated with this mixture for 3 h at 37°C. After the incubation, culture medium was removed, 3 ml fresh complete medium was added, and HUVECs were incubated for 36 h to allow protein expression. Transfected cells were identified by β-galactosidase staining ( 32 ). Viable versus dead stained cells were counted by two blinded investigators, and results were expressed as (dead cells/viable cells) ×100. In addition, necrotic cell death was excluded by measuring lactate dehydrogenase (LDH) release, thus indicating that the cell death of the transfected cells is caused by apoptosis. The transfection efficiency with 3 μg pcDNA3.1– β-galactosidase was 24 ± 4%. HUVECs were starved in RPMI medium without methionine and cysteine for 1 h, then metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine for 3 h. HUVECs were then chased in nonradioactive medium for the time periods indicated, in the presence or absence of TNF-α. Cells were washed twice with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Tris-HCl, pH 8) at 4°C for 20 min. Samples containing equal amounts of protein were precleared with anti–rabbit IgG for 30 min at 4°C, immunoprecipitated with an anti-myc antibody, and resolved on 12% SDS-PAGE. The gel was dried and exposed to x-ray film. pcDNA3.1–Bcl-2 wild-type or mutants were in vitro transcribed/translated using the T7 polymerase kit ( Promega Corp. ) in the presence of [ 35 S]methionine. Degradation was then determined by incubation of 35 S-labeled Bcl-2 with TNF-α–treated HUVEC homogenates (70 μg) at 37°C. DNA fragmentation was demonstrated by the typical DNA laddering and quantified by an ELISA specific for histone-associated DNA fragments as described previously ( 3 ). For morphological staining of nuclei, cells were centrifuged (10 min, 700 g ), fixed in 4% formaldehyde, and stained with 4′,6-diamidino-phenylindole (DAPI; 0.2 μg/ml in 10 mM Tris-HCl, pH 7, 10 mM EDTA, 100 mM NaCl) for 20 min. For detection of in vitro phosphorylation of Bcl-2 by MAP kinase, COS cells were transfected with myc-tagged Bcl-2 wild-type or mutant, and overexpressed proteins were immunoprecipitated with anti-myc antibodies. The immunoprecipitates were incubated at 30°C in 30 μl kinase reaction mixture containing 25 mM Tris (pH 7.5), 5 mM β-glycerophosphate, 0.1 mM Na 3 VO 4 , 2 mM dithiothreitol, 10 mM MgCl 2 , 50 μM ATP, 5 μCi [γ- 32 P]ATP with or without 20 ng activated MAP kinase ERK2 ( New England Biolabs ) for 30 min. The reaction was terminated by addition of SDS loading dye, and samples were subjected to a 12% SDS-PAGE and analyzed by PhosphorImager (Molecular Dynamics). Treatment of HUVECs with the proapoptotic stimuli TNF-α, staurosporine, and doxorubicin resulted in a profound time- and dose-dependent reduction of Bcl-2 protein levels . Reduction of Bcl-2 protein levels clearly preceded the induction of apoptosis . In contrast, TNF-α did not affect Bax or Bcl-X L protein levels . Reduced Bcl-2 protein levels in response to various apoptotic stimuli were also observed in HeLa cells, illustrating that the effects are not unique for endothelial cells (data not shown). Bcl-2 mRNA levels were essentially unchanged after TNF-α–induced apoptosis (data not shown), suggesting a posttranscriptional effect on Bcl-2 protein. Pulse–chase experiments demonstrated that an increased rate of Bcl-2 protein degradation rather than a reduced protein biosynthesis accounts for the decline of Bcl-2 protein levels . Thus, TNF-α, staurosporine, and doxorubicin appear to stimulate the selective degradation of Bcl-2 by a posttranscriptional mechanism. The ubiquitin-dependent proteasome pathway plays an important role in the degradation of regulatory proteins and thereby fulfills important functions in cell cycle regulation and signal transduction ( 30 , 31 ). The ubiquitin-dependent pathway requires the covalent conjugation of ubiquitin with ε-amino groups of lysine residues within the target proteins ( 24 ). Indeed, TNF-α stimulated the ubiquitination of transiently expressed myc-tagged Bcl-2 . In contrast, ubiquitination was significantly prevented in a Bcl-2 construct, where all four lysine residues were mutated to arginine . Moreover, the proteasome inhibitors lactacystin ( 33 , 34 ), carbobenzoxyl-leucinyl-leucinyl-leucinal-H (Z-LLL-H ), and N -acetyl-leucinyl-leucinyl-norleucinal-H (ALLN ) completely prevented TNF-α– as well as staurosporine-induced degradation of Bcl-2 in vivo . Similar results were obtained in an in vitro assay system in which the cleavage of 35 S-labeled Bcl-2 by homogenates of TNF-α–treated HUVECs was assessed . The proteolytic cleavage of 35 S-labeled Bcl-2 was abolished by the specific proteasome complex inhibitors . Moreover, the degradation of Bcl-2 was inhibited by ATPγS , which is a typical feature of the proteasome complex ( 35 ). Furthermore, mutating all four lysine residues (mt 1) precluding ubiquitination as shown in Fig. 1 E completely abrogated the degradation of Bcl-2 . Mutation of three out of four lysine residues (mt 2–5) led to a partial inhibition of Bcl-2 degradation . In contrast, caspase inhibitors (Ac-DEVD-CHO, Ac-YVAD-CHO) or inhibitors of serine-, aspartate-, or metalloproteases did not prevent Bcl-2 degradation . Mutation of the caspase cleavage site Asp 34 of Bcl-2 ( 36 ) did not confer resistance against TNF-α–mediated Bcl-2 degradation in vitro or in vivo . Additionally, mutation of the loop region (amino acid residues 35–79) of Bcl-2 did not affect stimulus-dependent degradation . These results indicate that the ubiquitin-dependent proteasome pathway is required for TNF-α–induced degradation of Bcl-2. To establish a functional role of stimulus-dependent degradation of Bcl-2 on apoptosis induction, the degradation-resistant Bcl-2 mutant (mt 1) was transfected into HUVECs. TNF-α–induced apoptosis was significantly reduced in cells expressing the corresponding Bcl-2 mutant, whereas overexpression of wild-type Bcl-2 provided less protection . Likewise, expressing the degradation-resistant Bcl-2 mutant in HeLa cells significantly reduced TNF-α–induced apoptosis by 71 ± 6% in Bcl-2 mutant transfected cells compared with 41 ± 0.7% reduction in wild-type Bcl-2 transfected cells ( P < 0.05), indicating that the observed effects are not unique to endothelial cells. In contrast, overexpression of the caspase cleavage–resistant D34A Bcl-2 mutant did not prevent TNF-α–induced apoptosis in HUVECs (19 ± 3 compared with 20.7 ± 4% apoptosis in Bcl-2 wild-type transfected cells). Moreover, apoptosis induced by staurosporine was dramatically inhibited by overexpression of the degradation-resistant Bcl-2 mutant, which rescued almost all cells . The different effects of the Bcl-2 mutants were not due to enhanced basal expression as demonstrated by Western blot analysis . Thus, these results establish inhibition of the ubiquitin- dependent degradation of Bcl-2 as an extremely potent mechanism to suppress stimulus-induced apoptosis induction. Targeting of proteins for ubiquitin-dependent degradation is often regulated by phosphorylation or dephosphorylation of the target protein ( 27 , 30 ). Since previous studies suggested that reduction of the activity of MAP kinases ERK1/2 may be linked to dephosphorylation of Bcl-2 ( 37 ), we investigated whether the MAP kinase pathway is involved in the Bcl-2 degradation process. Prolonged incubation with TNF-α induced a drastic dephosphorylation and deactivation of ERK1/2 . Moreover, inhibition of ERK1/2 activity by the MAP kinase/ERK kinase (MEK) inhibitor PD98059 triggered the degradation of Bcl-2 and induced apoptotic cell death (data not shown), suggesting that inhibition of ERK1/2 may trigger the degradation of Bcl-2. Furthermore, activated ERK induced phosphorylation of Bcl-2 in vitro . ERK-induced Bcl-2 phosphorylation was prevented by mutation of the three residues matching the consensus sequence for the putative MAP kinase sites (P-X-X-T/S-P) in Bcl-2 into alanine , demonstrating the specificity of the reaction. To test whether phosphorylation of Bcl-2 might prevent its degradation, we specifically mutated the three MAP kinase sites (P-X-X-T/S-P) into phospho-mimetic aspartic acid residues, which mimics continuous phosphorylation of the protein. Expression of the phospho-mimetic mutants completely prevented TNF-α–induced Bcl-2 degradation and apoptosis . Thus, mimicking phosphorylation of putative MAP kinase sites within Bcl-2 is associated with complete inhibition of TNF-α–induced degradation. Finally, we tried to more precisely characterize the role of ERK to interfere with Bcl-2 degradation. Therefore, we assessed the effect of the ERK-specific phosphatase MKP-3. Since MKP-3 is exclusively located in the cytosolic compartment of cells, whereas MKP-1 and MKP-2 are localized in the nuclei ( 38 , 39 ), MKP-3 may be one possible effector enzyme inducing the dephosphorylation of ERK. Overexpression of MKP-3 not only reduced ERK1/2 activity as described previously (data not shown; reference 38 ) but also induced degradation of wild-type Bcl-2 and subsequent apoptotic cell death . In contrast, the phospho-mimetic Bcl-2 mutant or the Bcl-2 construct lacking all four lysine residues was resistant to MKP-3–triggered degradation . Taken together, ubiquitin-dependent proteolytic degradation decreases Bcl-2 protein levels and thereby renders cells susceptible for apoptotic stimuli. Moreover, our data may suggest that dephosphorylation of the putative MAP kinase sites targets Bcl-2 for ubiquitin-dependent degradation, whereas simulation of continuous phosphorylation of the putative MAP kinase phosphorylation sites of Bcl-2 not only abolishes its degradation, but—more important—confers resistance to stimulus-induced apoptosis. Bcl-2 is believed to control the activation of the caspase cascade by participation in a multiprotein “apoptosome” ensemble involving Apaf-1, cytochrome C, and caspase-9 ( 40 ). Thus, degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome. This is further evidenced by the finding that antisense-induced selective downregulation of Bcl-2 triggers the release of cytochrome C from the mitochondria (data not shown). Thus, ubiquitin-dependent degradation of Bcl-2 may represent an alternative pathway to amplify the caspase cascade. The activation of the ubiquitin-dependent proteasome complex leading to selective degradation of Bcl-2 not only appears to be a key signaling pathway used by TNF-α to amplify its potency to induce apoptosis, but may be involved in apoptosis signaling in all cells, where the mitochondrial amplification loop is important. Moreover, since the endothelium plays a pivotal role as a gatekeeper during inflammation, the selective downregulation of the antiapoptotic Bcl-2 by inflammatory cytokines such as TNF-α may significantly affect endothelial integrity and thus the progression of inflammatory diseases such as atherosclerosis. Inhibition of the signaling pathways involved in Bcl-2 degradation may not only provide insights into the pathophysiological role of Bcl-2 degradation, but may also have important novel therapeutic implications in disease states with deregulated apoptosis. | Study | biomedical | en | 0.999997 |
10359586 | 6–10-wk-old, male B10.BR mice were purchased as specific pathogen free from The Jackson Laboratory and housed under reverse barrier conditions at the Duke University Vivarium until they were killed. Whole PCC ( Sigma Chemical Co. ) was diluted into PBS and mixed with the Ribi adjuvant system (Ribi Immunochem Research). Primary immunization of 400 μg of PCC was injected into 200 μl of adjuvant emulsion in two 100-μl doses subcutaneously on either side of the base of the mouse tail. PBS alone was used for the adjuvant-only controls. The memory challenge was designed as a second primary immunization to reduce the differences between the two responses (i.e., no dose differences to account for changed kinetics of the cellular response). Secondary challenge was a repeat of the primary regime including adjuvant, also at the base of the tail, 8 wk after the initial priming. Animals were killed on various days after immunization as indicated, and the draining LNs were harvested for analysis. Inguinal and periaortic nodes were collected and teased through 80-μm mesh screens into single-cell suspensions in 0.17 M NH 4 Cl solution for erythrocyte lysis before estimation of cell count using a hemocytometer. Cells were pelleted and resuspended in PBS with 5% FCS. All cells were stained for flow cytometry at 2.0 × 10 8 cells/ml with predetermined optimal concentrations of fluorophore (or biotin)-labeled mAb (FITC–RR8.1 [anti-Vα11; PharMingen ], allophycocyanin–KJ25 [anti-Vβ3], PE–Mel14 [anti-CD62L; PharMingen ], Cy5PE–6B2 [anti-B220; PharMingen ], Cy5PE–53-6.7 [anti-CD8; PharMingen ], Cy5PE– M1/70.15 [anti-CD11b; Caltag Labs.], or biotin–IM7 [anti-CD44; PharMingen ]) together with desired volume of cells on ice for 45 min. After being washed twice, cells were resuspended at the same cell concentration with avidin–Texas Red (TR; PharMingen ) on ice for 15 min, washed again, and resuspended in 2 μg/ml propidium iodide (PI) (for dead cell exclusion) in PBS with 5% FCS for analysis. Samples were analyzed using a dual laser, modified FACStar PLUS™ ( Becton Dickinson Immunocytometry Systems ) (an argon laser as the primary and a tunable dye laser as the secondary) capable of seven-parameter simultaneous collection (five log-amp detectors for fluorescence, one log-amp detector for obtuse light scatter, and a photo diode for forward light scatter). The Cy5 component of the duochrome Cy5PE is also excited by the dye laser and detected in the allophycocyanin channel. For all experiments described, the Cy5PE fluorescence collected after primary laser excitation was used for exclusion criteria alone , thereby operationally avoiding the signal overlap across the two lasers that could not be compensated for electronically. PI was also excluded in the Cy5PE detection channel. All analyses required the collection of two files for each sample. The first file was a 100,000-event file of PI − events to ascertain the frequency of Cy5PE − Vα11Vβ3-expressing cells in the total LN population. The second file contained 1,000 events of PI − Cy5PE − Vα11 + Vβ3 + cells to evaluate the fraction of cells that upregulated CD44 and downregulated CD62L. Files were acquired using CELLQuest™ software ( Becton Dickinson ) and analyzed using FlowJo software (Tree Star, Inc.). All profiles are presented as 5% probability contours with outliers. Total cell numbers were calculated using frequencies estimated by flow cytometry and total cell counts for the draining LNs of each animal. Draining LNs used for confocal microscopy were snap frozen in OCT embedding compound (Miles Labs., Inc.). Cryostat microtome (Leica, Inc.) cut, 6-μm-thick frozen sections were mounted on gelatin-coated slides, air dried, and acetone-fixed for 10 min at 4°C and stored at −80°C until use. Sections were rehydrated with PBS and blocked with PBS containing 10% FCS, 10% skim milk (wt/vol) powder, and 2.4G2 (anti-FcR) (50% vol/vol hybridoma supernatant) for 30 min at room temperature. Sections were stained with TR-11.26 (anti-IgD), allophycocyanin–KJ25 (anti-Vβ3), and either FITC–RR8.1 (anti-Vα11) or FITC–Mel14 (anti-CD62L) for 1 h at room temperature, then washed and mounted in VectorShield (Vector Labs., Inc.). FITC is excited at 488 nm and collected using a 515–545-band pass filter, TR is excited at 568 nm and collected using a 615–645-band pass filter, and allophycocyanin is excited at 633 nm and collected using a 670–810-band pass filter. Data was acquired on a Zeiss Axiovert LSM 410 microscope system ( Carl Zeiss, Inc. ), and each image was collected serially in the first detector, using LSM 3.95 software. Quantitation was achieved manually after digital rendering of the image for optimal signal-to-noise and overlay using Adobe Photoshop (Adobe Systems, Inc.). Single Vα11 + and Vβ3 + cells were first counted separately (and then overlapping), using a grid covering each field (acquired using the 40× objective lens), covering one full section of the LN. The IgD staining was then used to assign each cell's location—T zone (mainly IgD − ), B zone (mainly IgD + ), or GC (IgD − area within the B cell zone)—as well as exclude nonspecific staining (positive for all three signals). Sections containing both T zone and B zone areas were used for analysis. Single cells with appropriate surface phenotype were sorted for repertoire analysis using the automatic cell dispensing unit attached to the FACStar PLUS™ and Clone-Cyt™ software ( Becton Dickinson ). Each cell was sorted into an oligo d(T)-primed, 5-μl cDNA reaction mixture (4 U/ml murine leukemia virus–RT [ GIBCO BRL ] with recommended 1× RT buffer, 0.5 nM spermidine [ Sigma Chemical Co. ], 100 μg/ml BSA [ Boehringer Mannheim ], 10 ng/ml oligo d(T) [ Becton Dickinson ], 200 μM each dNTP [ Boehringer Mannheim ], 1 mM dithiothreitol [ Promega Corp. ], 220 U/ml RNAsin [ Promega Corp. ], 100 μg/ml Escherichia coli tRNA [ Boehringer Mannheim ], and 1% Triton X-100) set up in low profile, 72-well microtiter trays (Robbins Scientific), immediately held at 37°C for 90 min, and then stored at −80°C until further use. Single cells were only sorted into the center 60 wells of each tray, with the first and last well of each row serving as a negative control (processed together with all other samples throughout the experimental procedure). These negative controls are critical to ascertain the efficacy of the “nested” PCR to follow (one negative for each five samples). Single hybridoma cells can be sorted into medium in these trays and visualized under a phase-contrast microscope to test the accuracy of sorting into these small format wells and demonstrated an accuracy range of 60–80% single cells (doublets never seen). A nested RT-PCR for actin mRNA serves as a more sensitive positive control for sorting and produces a PCR product for 70–100% of wells with a sorted cell. 2 ml of cDNA from single-cell cDNA reactions were used for two separate, 25-μl amplification reactions, one for the TCRVα11 and one for the TCRVβ3, using primers specific for the V and constant regions of each chain (2 U/ml Taq polymerase with the recommended 1× reaction buffer [ Promega Corp. ], 0.1 mM of each dNTP [ Boehringer Mannheim ], and for [a], TCRVα11, 2 mM MgCl 2 , 1.2 μM primer Vα11.L1 [5′-ATGCAGAGGAACCTGGGAGC-3′] and 1.2 μM primer Cα.2 [5′-AATCTGCAGCGGCACATTGATTTGGGA-3′]; [b], TCRVβ3, 3 mM MgCl 2 , 0.4 μM primer Vβ3.L2 [5′-ATGGCTACAAGGCTCCTCTGGTA-3′], and 0.4 μM primer Cβ.2 [5′-CACGTGGTCAGGGAAGAA-3′]). Each reaction set begins with 95°C for 5 min; then 40 cycles of 95°C for 15 s, 50°C for 45 s, and 72°C for 90 s; and ends with 72°C for 5 min. 1 μl of the first PCR product was used for further 25-μl amplification reactions for each chain of the TCR, using primers nested medially to the primers used in PCR-a (2 U/ml Taq polymerase with the recommended 1× reaction buffer [ Promega Corp. ], 0.1 mM of each dNTP ( Boehringer Mannheim ), and for [a], TCRVα11, 2 mM MgCl 2 , 0.8 μM primer Vα11.L2 [5′-AATCTGCAGTGGGTGCAGATTTGCTGG-3′] and 0.8 μM primer Cα.ext [5′-GAGTCAAAGTCGGTGAACAGG-3′]; and [b], TCRVβ3, 4 mM MgCl 2 , 0.4 μM primer Vβ3.1 [5′-AATCTGCAGAATTCAAAAGTCATTCA-3′], and 0.4 μM primer Cβ.3 [5′-AATCTGCAGCACGAGGGTAGCCTTTTG-3′]). Each reaction set begins with 95°C for 5 min; then 35 cycles of 95°C for 15 s, 55°C for 45 s, and 72°C for 90 s; and ends with 72°C for 5 min. At least 2 negative cDNA samples were processed per 10 single-cell samples. The negatives were interspersed with positives to control for contamination during sample preparation. Frequency for obtaining a sequenceable PCR product from single cells varies between TCR-α and TCR-β (primarily due to lower abundance of TCR-α mRNA per cell), with some variation across different days in the response (Table I ). 5 μl of PCR-b product was run on a 1.5% agarose gel to screen for positives (single bands of the right size). PCR product was then separated from primers using a CL-6B Sepharose column ( Pharmacia LKB Biotechnology, Inc. ). The PCR product was then directly sequenced (3 μl of PCR product, 4 μl Dye Terminator Ready Reaction Mix [ Perkin Elmer Corp. ], 1.5 pmol primer [Vα11.seq, 5′-CAGGAACAAAGGAGAAT GGGAG-3′; Vβ3.seq, 5′-CTGTGCTGAGTGTCCTTCAAAC-3′]) using a linear amplification protocol for 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min on a 9600 GeneAmp PCR system ( Perkin Elmer Corp. ). Samples were separated on a 6.5% acrylamide gel after ethanol precipitation of sequencing reaction products, run on an ABI 373 sequencing system, and processed using ABI Prism sequence 2.1.2 for collection and analysis ( Perkin Elmer Corp. ). To purify the PCC-specific subset, we isolated Vα11Vβ3-expressing CD4 + cells that modulate surface CD44 and CD62L ( l -selectin) expression in response to Ag. Fig. 1 A outlines our flow cytometric strategy for purifying PCC-specific cells, using seven cellular parameters simultaneously. This new strategy significantly decreases background to allow more confident cell sorting, even at extremely low target cell frequencies (<1/10 4 cells on day 3). The initial background of Vα11Vβ3-expressing cells that are also CD44 high CD62L low is negligible before immunization . In the absence of protein Ag, not only on day 3 but also through to day 7, there is negligible appearance of Ag-responsive cells . There is a significant difference between adjuvant-only versus day 3 PCC-responsive cells ( P = 5.0 × 10 −4 ) . In addition, the response to an irrelevant protein, such as hen egg lysozyme, is similarly low (data not shown). These critical in vivo controls attest to the specificity of cells responding to PCC. The increase in frequency for Ag-responsive Vα11Vβ3 cells (CD44 hi CD62L lo ) is depicted in the probability contours of Fig. 1 B. It is important to note that the total cellularity of the draining LNs also changes over the course of the immune response. Therefore, it is more informative to consider the change in total cell numbers of Ag-responsive cells over the course of the response . We observed a 250-fold increase in cell numbers between days 0 and 7 of the primary response. Of course, the fidelity of the day 0 quantitation is limited by detection and not the actual precursor frequency in the preimmune repertoire. There is an apparent plateau in cell numbers from days 7 to 9 of the primary response and then a gradual decline. The extent of the cellular response to the secondary challenge with the same dose of Ag is very similar to the primary response. It is the accelerated kinetics of this cellular response that highlights one of the unique characteristics of a memory Th response ( 13 ). Using the flow cytometric strategy outlined above, we can isolate single PCC-responsive cells from the emergent phase of both the primary and memory response to Ag. We first focus our attention on the memory response. We had previously defined the expression of highly restricted TCR on day 6 of the memory PCC response ( 13 ). It was not known whether the restricted TCR expressed on day 6 were the result of clonal maturation following secondary challenge. Therefore, we isolated single cells from days 2, 3, and 4 for repertoire analysis, as described in detail in Materials and Methods. Regardless of the day after challenge, TCR expressed by memory response cells are highly restricted . The similarity between aa sequences from different cells can be easily seen; however, clonal relatedness can only be established by comparing DNA sequences of both TCR-α and TCR-β chains from single cells. Identical sequences for both chains were seen in only 6/46 TCR sequences from the memory response (across four separate animals). These repeat sequences were not amplification artifacts (which are rigorously scrutinized in the experimental design) and therefore represent examples of single cells from the same parent clone in vivo. Overall, these data demonstrate that the memory response to PCC emerges rapidly, using a broad array of memory–response precursor cells that already express highly restricted TCR. Using this more complete data set, we can define four preferred CDR3 features (in each chain of the TCR) that typify the PCC-specific memory Th compartment. In the TCR-α chain : (i) glutamic acid (E) at α93; (ii) CDR3 length of 8 aa; (iii) serine (S) at α95; and (iv) Jα 16, 17, 22, and 34. In previous studies, a threonine was also seen at α95, but it was not seen in this study (0/51 TCR-α chains). We can now assign Jα 16, 17, 22, and 34 as preferred, with each Jα used by >10% of the memory responders and together accounting for 70% of memory responders. These four Jα can produce a serine at position α95 given the appropriate V–J junction; however, the four Jα represent only a subset of total Jα segments that can create this serine (at least 10 others). Therefore, the Jα segments are preferred for reasons other than simply the creation of a serine at α95. The four preferred features in the TCR-β chain are: (i) asparagine (N) at β100, (ii) CDR3 length of 9 aa, (iii) alanine (A) or glycine (G) at β102, and (iv) Jβ1.2 and Jβ2.5. The β102 position is considered separately from the Jβ1.2, as most often, the alanine appears to be lost in D–J joining in the preimmune repertoire (4/13 Jβ1.2 expressing preimmune TCR retain the alanine, and only two of these express the alanine at the correct position). Therefore, Jβ1.2 is not the preferred motif but rather a Jβ1.2 that retains an alanine at position β102. A glycine is also found in the β102 position when Jβ2.5 is used. In these cases, the glycine is encoded by D region or N sequence insertions and is not present in the germline Jβ2.5. To evaluate when the dominant clonotype emerges, we next sorted single PCC responder cells from throughout the primary response. The dot plot displays in Fig. 3 , A–D and Fig. 4 , A–D summarize the CDR3 sequence information for either the TCR-α or TCR-β chains from over 500 single cells. Each dot represents the sequence from a single cell, and each of the eight CDR3 features from these cells are displayed separately. The preferred CDR3 motif is presented at the top of each panel, with alternative features displayed in order of prevalence. A summary for each chain in Figs. 3 E and 4 E combines all CDR3 features from individual animals to demonstrate when the repertoire narrows over the course of the developing immune response. Considering all eight preferred CDR3 features, only the glutamic acid at α93 preexists antigenic challenge to any significant extent . In preimmune and PCC-nonresponsive Th (Vα11 + Vβ3 + CD44 lo CD62L hi ), 68% express glutamic acid at position α93 ( n = 62). In the same population, the three other preferred TCR-α chain features are present in <30% of the cells . The glutamic acid at α93 is encoded by the last codon of the V region and may be lost on imprecise V–J joining. Its presence in the preimmune repertoire may be simply stochastic or the result of thymic selection pressures. Greater than 90% of all Vα11Vβ3-expressing T cells in the periphery of normal B10.BR mice are CD4 + , implicating thymic selection and MHC class II restriction as a defining characteristic of the preimmune repertoire for these particular T cells (data not shown) ( 13 ). Of 75 murine TCR-α regions listed by Arden et al. ( 29 ), only four can express a germline-encoded glutamic acid at α93. All four V regions are Vα11 subfamily members. Therefore, it seems likely that the presence of this one critical peptide contact residue that preexists antigenic challenge at very high frequencies in the Vα11Vβ3-expressing Th imposes the TCR-α chain V region bias of the I-E k –restricted PCC response. The remaining seven preferred CDR3 features are rapidly selected during the cellular expansion phase of the primary response (days 3–7; doubling time of the population was 17.5 h). Even by day 3 of the primary response, there is an accumulation of Ag-activated Vα11Vβ3 cells with many of the preferred CDR3 features . There is a large spread of Jα usage in the PCC-nonresponsive cells that has already narrowed by day 3 (35%) and narrows further (65%) to use the four preferred Jαs by day 5 . The CDR3 lengths of 8 and 9 aa are most prevalent in the PCC-nonresponsive cells; however, the preference for a length of 8 aa in the PCC-specific compartment is evident even by day 3 and maximal by day 5 . Selection for serine at α95 also appears maximal by day 5 of the primary response . Many PCC-specific hybridomas contain at least three out of the four CDR3 features described. Therefore, in Fig. 3 E, we consider the change in the frequency of cells that express ≥3 preferred CDR3 features in their TCR-α chains to assess the dynamics of clonal maturation. By day 3, there is a significant difference in cells that express ≥3 preferred features over the PCC-nonresponsive cells ( P = 0.01, 2-tail t test). There is a further increase in frequency of restricted TCR by day 5 (days 3–5, P = 0.01) but no significant difference over the course of the primary response. This was also true between the late primary response and the memory response. Furthermore, we found no evidence for somatic diversification of the TCR-α genes ( 30 ) (no mutations observed for 5,441 bases analyzed for the TCR-α chain from days 7, 9, and 11; n = 83 single cells, 40–90 bp upstream of the CDR3 in each case). A similar rapid progression of Ag-driven selection was apparent for the TCR-β chain. Very few PCC-nonresponsive cells express an asparagine at β100 (6%), with evidence for selection in the PCC-specific compartment by day 3 (20%) and clearly by day 5 (65%) . This preferred CDR3 feature appears further selected by day 7 (73% by day 7; 78% average over days 7–14), with still further selection in the memory response (90% average over days 2–4). Preferred Jβ usage (1.2 and 2.5) follows a similar course : a small increase in Jβ1.2 and 2.5 usage on day 3 (23% resting to 34% by day 3) that is clearly dominant by day 5 (77%), with a further increase by day 7 (88%; 88% average, days 7–14) and a slight increase in the memory response (94% average). CDR3 length restriction may be more rapid than the previous two features . The appearance of PCC-specific cells with a 9-aa length is close to maximal frequencies by day 5 (87%), with little further change in the primary (93% average days 7–14) and memory (96% average) responses. Appearance of alanine or glycine at β102 follows the kinetics of the first two TCR-β chain features presented . There is a small increase in prevalence noticeable by day 3 (33% resting to 46% by day 3) that is clearly dominant by day 5 (80%), with a further increase by day 7 (96%; 94% average, days 7–14) and little change in the memory response (97%). The summary in Fig. 4 E presents the change in frequencies of cells that express ≥3 preferred CDR3 features across multiple animals. There is a significant increase between the resting cells and PCC-specific cells by day 3 ( P = 0.04, 2-tail t test) and the greatest change between days 3 and 5 ( P = 0.01). Although there appear to be some differences between days 5 and 7 for the individual CDR3 features discussed above, when considered together in this summary, there was no statistically significant difference. There is also no apparent difference between day 7 and week 2 of the response nor any difference between these days and the memory response. Therefore, we conclude that a rapid maturation in the PCC-specific Th compartment for clones that express these preferred TCR-β CDR3 features is largely complete by day 5 of the primary response. We found no evidence for somatic diversification of the TCR-β chain ( 31 ) (no mutations observed for 3,406 bp analyzed for the TCR-β chain from days 7, 9, and 11; n = 104 single cells, 20–40 bp upstream of the CDR3 in each case). In Fig. 5 , we summarize sequence information from both chains of the TCR of single, Vα11Vβ3-expressing Th . In the dot plot display, we emphasize the emergence of PCC-specific cells that express ≥6 of the preferred CDR3 features described above as the change in their frequency over time. Only 1/47 resting Vα11Vβ3 cells expressed ≥6 preferred CDR3 features. By day 3, 40% of the PCC-responsive compartment (Vα11Vβ3CD44 hi CD62L lo ; n = 50) already expressed ≥6 preferred CDR3 features. By days 5–7, this frequency doubled to 83% ( n = 59; these days have been combined to present similar numbers in each group. There was no significant difference in frequency of cells with ≥6 preferred features between these two timepoints). There was no further change in frequency of these restricted TCR to day 14 of the primary response ( n = 43). After secondary challenge, 96% of PCC responders expressed ≥6 preferred CDR3 features ( n = 46). This further increase in restricted responders may indicate a separate phase of Ag-driven selection associated with the induction of a memory response. Overall, these data consider the complete TCR as the selecting unit and further attest to the rapidity of Ag-driven selection in this system. Fig. 6 displays a representative set of TCR sequences from PCC-responsive, Vα11Vβ3-expressing Th from day 3 of the primary response. These sequences can be divided into three groups based on the degree of restriction in their CDR3 regions. 40% of day 3 responders expressed ≥6 CDR3 features associated with the PCC response . The second group is designated as unrestricted (containing ≤5 preferred CDR3 features), but expressed TCR-α chains similar to those sequenced from PCC-specific hybridomas, PCC-specific cell lines, or binders of moth cytochrome c (MCC)/I-E k tetramers . Clones, such as the well-characterized 2B4, fall into this category, with a TCR-α chain that expresses none of the preferred CDR3 features we have found in the memory PCC response but is clearly specific for PCC. These first two groups account for 80% of the cells from day 3. Cells in the third group make up the remaining 20% of PCC responders from day 3 and expressed unrestricted TCR (≤5 preferred CDR3 features) that have not been previously associated with PCC specificity . It is important to note that not only was the cellular response on day 3 significantly above the adjuvant-only control (the main in vivo criteria for specificity), but the few events that were sorted from adjuvant-only controls ( n = 60) gave rise to a PCR product with a sixfold lower efficiency (see Materials and Methods for details). The few TCR sequenced from these control populations were as diverse in their CDR3 as their preimmune counterparts (data not shown). Overall, these data indicate that PCC responders initially recruited into the immune response express more diverse TCR. Subsequently, the Th with preferred TCR are selectively expanded, and a subset of these cells are preserved for the memory response. We have determined that Ag-driven selection in the draining LNs is largely complete by day 5 of the primary response. In Fig. 7 , we outline a quantitative analysis of the GC and non-GC distribution of PCC-specific Th over the course of the primary response. It should be noted that >90% of Vα11Vβ3-expressing T cells in the LN of B10.BR mice are CD4 + Th (by flow cytometric analysis; data not shown). In Fig. 7 A, we display an example of three-color laser scanning confocal microscopic (LSCM) imaging to localize Vα11Vβ3-expressing T cells (yellow). IgD staining is used primarily to delineate B cell zone (IgD + ) and T cell zone (IgD − ) but also to locate the IgD − regions within the B cell zones that indicate the presence of GC. In Fig. 7 B, we compare our quantitation of Vα11- and/or Vβ3-expressing T cells by LSCM analysis and flow cytometry. Quantitation was undertaken from either 100,000 event flow cytometric files or cell counts from entire cross-sections of LN tissue. The concordance for proportions of the single-positive (Vα11 or Vβ3) and double-positive (Vα11Vβ3) cells within the LN populations between LSCM analysis and flow cytometry is high . Reproducibility is also high across different animals . In Fig. 7 C, the GC and non-GC distribution of Vα11Vβ3-expressing T cells across days 5, 7, and 9 is presented, and the unadjusted graphical representation of this data is shown in Fig. 8 A. From these data, it is clear that the GC reaction is in its very early stages on day 5 of the LN response, increasing by day 7 and increasing further on day 9. Not all Vα11Vβ3-expressing T cells are PCC specific. Our flow cytometric analysis has focused on CD62L downregulation as one index for activation within the Vα11Vβ3-expressing compartment, and we can calculate the fraction of the total Vα11Vβ3 compartment that is CD62L lo at any stage of the response in vivo. Even at the peak of the primary response, only about half of the Vα11Vβ3-expressing cells in the draining LNs are PCC specific . Using LSCM analysis, we demonstrate that all Vβ3 + cells in the GC (the majority of which are Vα11 + ; data not shown) are also CD62L lo . Combining the flow cytometric and LSCM data, we can calculate the proportion of Vα11Vβ3 cells in the non-GC compartment that are not PCC specific. Fig. 8 B presents the adjusted distributions for PCC-specific cells over the course of the primary response and highlights the coincident decline in the non-GC compartment with the increase in the GC compartment. In Fig. 8 C, we present the expansion and decline of total PCC-specific cells and then apply the frequencies of GC and non-GC Vα11Vβ3 T cells calculated by the LSCM analysis to illustrate the emergence and decline of the total PCC-specific compartment in these distinct microenvironments. We find that the plateau phase of the cellular response demonstrated by flow cytometric analysis resolves into two peaks when the microenvironment is taken into account . The first peak indicates maximal non-GC cell expansion (day 7), and the second peak indicates GC cell expansion (day 9). Whether these two peaks are the result of migration alone or migration and then proliferation is not clear from our data. Nevertheless, with Ag-driven selection virtually complete by day 5 of the primary response, the more delayed kinetics of the GC T cell pathway strongly argue that Ag-driven selection is a non-GC activity. Our study documents the evolution of clonal dominance in vivo. We believe that these processes are fundamental to the development of highly specific Th–based regulation of primary immune responses. The PCC model allows experimental access to a Th response that becomes dominated by Ag-specific Th expressing highly restricted TCR. The dominant PCC-specific cells exhibit a bias in V region usage (Vα11Vβ3) and TCR with preferred CDR3 features that provide molecular indicators of TCR diversity. In this study, we demonstrate that 70% of all Vα11 + Vβ3 + Th express a critical PCC peptide contact residue (glutamic acid at α93), even before initial antigenic challenge. However, PCC-specific cells with all eight CDR3 features associated with the dominant clonotype only emerge to detectable levels after initial priming with PCC. Although there is some increase in the frequency of the dominant PCC-specific clonotype between the primary and memory responses (81–96%), the majority of Ag-driven selection occurs very rapidly during the first 5 d after initial priming. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with the “best fit” TCR. The TCR repertoire narrows before significant GC expansion, implicating Ag and the non-GC microenvironment as the principle selecting influences in vivo. Of all eight preferred CDR3 features used by the dominant clonotype, only the glutamic acid at α93 of Vα11 preexists antigenic challenge to any significant degree. The prevalence of this residue is likely to impose the Vα11 dominance associated with PCC specificity in I-E k –restricted animals. In PCC-specific hybridomas from many sources, Vα11 is more consistently expressed than Vβ3 ( 10 , 11 , 17 , 32 , 33 ). In studies of single chain TCR–transgenic animals, immunization with analogue peptides of MCC altered the V region dominance of PCC-specific responders ( 14 ). The Vβ3 dominance was more readily perturbed than Vα11, presumably due to modification of Ag-driven selection. Manipulations of the thymic selecting environment can also perturb V region dominance in the response to PCC. When wild-type or analogue peptides of MCC are introduced centrally, the Vβ3 dominance of MCC responders in the periphery is more noticeably affected than Vα11 ( 17 , 32 ). These central manipulations are most likely to alter the availability of particular clonotypes in the preimmune repertoire rather than directly affect Ag-driven selection. The presence of glutamic acid at α93 is not the only feature that predisposes Vα11Vβ3-expressing Th in the preimmune repertoire to bind PCC epitopes. Most Vα11Vβ3 Th on days 3 and 5 after initial priming that have not modulated CD44 or CD62L also express glutamic acid at α93. Therefore, the combination of Vα11 with the glutamic acid at α93 and any Vβ3 V region are not sufficient for PCC specificity. Furthermore, Vα11-expressing Th after PCC immunization that do not bind tetramers of MCC– I-E k also retain a predominance of glutamic acid at α93 (67% of D8 and D14; n = 12) (McHeyzer-Williams, L.J. and M.G. McHeyzer-Williams, unpublished data). Therefore, the glutamic acid at α93 may impose the Vα11 bias seen in PCC responders, but other particular TCR features are also clearly required for fine specificity. These other features are not easily recognized in the TCR of PCC responders initially recruited in the response (isolated on day 3 of the primary). It is possible that the early responders represent a stochastic selection from the Vα11Vβ3 Th subset of preimmune Th from which the dominant clonotype is then selected. In this latter scenario, subsequent Ag-driven selection events only focus on the cells initially recruited. This would explain why there is no obvious depletion of Vα11Vβ3 Th from the nonresponder population. To argue against this stochastic model, only a minute fraction of all preimmune Vα11Vβ3 Th (0.04%) are able to bind tetramers of MCC–I-E k (McHeyzer-Williams, L.J., J.F. Panus, J.A. Mikszta, J.D. Altman, M.M. Davis, and M.G. McHeyzer-Williams, manuscript in preparation). Overall, it is more likely that the TCR structural requirements for early recruitment into the PCC response are less stringent and, therefore, more difficult to identify. TCR specificity evolves rapidly after primary exposure to Ag in vivo. By day 5 of the primary response, >80% of PCC-specific Th express restricted TCR (≥6 preferred CDR3 features). The biochemical basis for Ag-driven selection in vivo is still not clear. Lanzavecchia and colleagues demonstrate the utility of having TCR with high off-rates to enable serial triggering of multiple receptors ( 34 , 35 ). In this model, lower affinity TCR receptors may be preferred and used for memory responses. In their recent study, Crawford et al. demonstrate a hierarchy of affinities for a series of PCC-specific hybridomas (using biacore analysis and correlated levels of MCC–I-E k tetramer staining) ( 36 ). The KMAC-92 hybridoma (6/8 preferred CDR3 features) has a K d of 29 μM ( 33 ), whereas the well-characterized 2B4 hybridoma (3/8 preferred CDR3 features) has a K d of 90 μM ( 37 , 38 ). Single cells with TCR similar to 2B4 can be found early in the primary response but do not appear to be selected into the memory compartment. Furthermore, the AD10 and TCR-transgenic cells in the study by Crawford et al. ( 36 ) both express eight preferred CDR3 features (similar to the 5C.C7 TCR). Tetramers of peptide–MHC complexes have been used for analysis of class I–restricted responses in conventional animals ( 22 , 23 , 39 – 43 ) and class II–restricted responses in single-double–chain transgenic animals and T cell hybridomas ( 36 , 44 ). These tetrameric reagents provide the means to assay affinity directly ex vivo in both class I– and class II–restricted responses. Ag-driven selection of the preferred clonotypes is enhanced by selective cellular expansion in vivo. The preferred clonotype is already present on day 3 of the primary response (40% prevalence). Whether the presence of the day 3 PCC-specific cells already represents cell expansion or simply recruitment from distant sites is not yet clear. Nevertheless, the day 3 PCC-specific compartment expands a further 20-fold before reaching a plateau on day 7. Although the maximal frequency of preferred clonotypes is reached by day 5 (80%), there is still further expansion of this already restricted cell population up to day 7 . Our data provides a glimpse of TCR structures that are initially recruited but are not further expanded (or preserved) in the response to PCC . Although the TCR from these early responders are less restricted than the dominant clonotype, similarly diverse CDR3 structures have been observed in the TCR-α chain of many PCC-specific hybridomas ( 11 , 17 , 32 , 33 ). The hybridomas have been selected in vitro, with excess amounts of specific Ag providing no selective pressure between PCC-specific clones. In contrast, there may be significant selection pressure between clones in vivo, as Ag depots recede over the course of the response. We and others have documented similar diversity in the TCR-α of T cells that bind tetramers of MCC–I-E k ( 36 ) (McHeyzer-Williams, L.J., J.F. Panus, J.A. Mikszta, J.D. Altman, M.M. Davis, and M.G. McHeyzer-Williams, manuscript in preparation). We see no downregulation of TCR during the PCC-specific response, as occurs in vitro after Ag stimulation ( 45 ). Although our isolation strategy clearly relies on TCR expression, there is no difference in levels of TCR between the PCC-specific cells and the nonresponder Vα11Vβ3-expressing population (data not shown). These data further argue that Ag may be limiting in vivo (at least by day 3 after initial priming). It is also possible that some Vα11Vβ3-expressing, PCC-specific clones recognize different peptide epitopes. The failure of particular clones to expand may correspond to the relative lack of availability of different epitopes over the course of the response, as suggested by Butz and Bevan for class I–restricted responses ( 46 ). It would be surprising if TCR specific for completely different epitopes used the same V region pair with similar CDR3 features. It is more likely that there may be subtle differences in the nature of the selecting peptide early in the response due to Ag processing or APC type (with different costimulatory molecules). Our data favor a simple model of preferential clonal expansion that conforms to the edicts of the clonal selection theory ( 47 ). In this model, there is an initial recruitment of cells expressing the appropriate V region genes with particular bias toward Vα11 usage as well as Vβ3. This initial set of PCC-specific cells has more diverse TCR than the dominant clonotype but is more restricted in its CDR3 loops than in the preimmune compartment. The clones expressing all preferred TCR structures are then selectively expanded from this initial pool and dominate rapidly through cellular expansion. Both the rapid kinetics of Ag-driven selection and the highly restricted memory response suggest that focusing of TCR specificity precedes memory cell development. There is a further increase in the frequency of restricted TCR in memory responders over the late primary responders (81– 96%) that could suggest another phase of selective expansion after secondary challenge. The highly restricted TCR of memory cells may underpin the rapid cellular expansion that typifies the response to secondary antigenic challenge. There were suggestions of clonal maturation in the Th compartment in our earlier study of the PCC response ( 13 ). The initial study suffered from two technical limitations that have been overcome in the current analysis. The first involves an emphasis on population analysis. The majority of the CDR3 sequence analysis was presented for the Vβ3 chain only, from populations of 1,000 cells as the starting point for RT-PCR. Whereas the same trends were apparent in the limited single-cell survey presented at that time ( n = 12 from each of the primary and memory response), single-cell resolution of this study was required to provide confidence in the changes in frequency of the dominant clonotype over time (combining both cellular and molecular analyses). The second technical difficulty was the very low frequency of PCC-specific cells at the early stages of the primary response. The addition of the seventh parameter in the flow cytometric analysis reduced the background at least 10-fold. The use of an exclusion channel (excludes not only cells outside the lineage of interest but also cells that nonspecifically bind antibodies). In addition, the use of CD44 and CD62L, together with the TCR-specific reagents, greatly clarified the day 3 and 5 selection of PCC-specific cells. With this new strategy, we extended our initial survey (two timepoints, day 6 of the primary and day 6 of the memory) to the extended timecourse needed to resolve the dynamics of clonal selection in vivo. The emergence of PCC-specific Th in the non-GC and GC microenvironments of the draining LNs of these animals is in general agreement with early studies of the splenic T cell response to this Ag ( 18 , 28 ). Our analyses of repertoire narrowing are similar to the studies of Zheng et al. ( 18 ); however, the rate and extent of selection in our current study appears far more rapid. The apparent slower rate may be due to differences between the splenic and LN microenvironments that regulated these processes. Alternatively, differences may be due to the phenotypic selection used for repertoire studies in each case. The splenic PCC response also appears more restricted in the GC environment than non-GC at the same timepoint of analysis. Zheng et al. imply that Ag-driven selection is occurring in the GC and demonstrate that GC T cells are highly susceptible to CD3-mediated apoptosis resembling thymic development and selection ( 18 ). In the LN, the vast majority of Ag-driven selection is over before significant expansion of the GC compartment. It appears unlikely that the GC reaction plays a role in the repertoire narrowing itself; it rather appears to be a site for migration of already restricted PCC-specific Th. The Ag-specific GC Th continue expanding in vivo ( 18 , 28 ) and differentiate into effector cells that support the development of B cell memory ( 48 , 49 ). Furthermore, we see no evidence for the somatic diversification of either chain of the TCR in this study, as previously reported ( 30 , 31 ). This was also true on day 9 of the primary response, when 75% of the PCC-specific compartment resides in the GC . Given the kinetics of cellular expansion in the LNs, early T zone proliferation associated with APC–Th conjugates is the most likely location for the selective expansion of preferred clonotypes ( 25 , 27 ). TCR specificity evolves rapidly through the preferential expansion of Ag-specific T cells well before the peak of the initial cellular response to Ag priming. These earliest events help to regulate the nature of effector cell function and shape the final specificity of the long-lived memory compartment. Here, we demonstrate not only the TCR structures of the preferred clonotypes and the kinetics of their selection, but also the TCR structures of clones initially recruited into the specific response but not expanded significantly for effector function or preserved into the memory compartment. These studies provide the framework for understanding the biochemical basis and functional consequences of maturation in the Th compartment. | Study | biomedical | en | 0.999998 |
10359587 | Using a 0.2-kb BstEII-Bsu36I restriction fragment from the 5′ end of human IKKβ cDNA as a probe, three murine IKKβ genomic fragments were isolated from a 129/SvJ mouse genomic library (Stratagene, Inc.). One of the clones contained at least the first three coding exons and was used to construct the targeting vector IKKβKO. A 1.4-kb SacI restriction fragment harboring part of the second exon was used as the short homology arm, and the long arm was a 5.5-kb EcoRV-XhoI restriction fragment containing part of the third intron. The two arms were inserted into the XmnI and SmaI sites, respectively, of pGNA, which contains the G418 resistance gene ( Neo r ) and LacZ ( 26 ). As a negative selection marker, a diphtheria toxin gene cassette ( DT ) was inserted into the KpnI site of pGNA. After cutting with PmeI, 20 μg of the linearized targeting vector was electroporated into 10 7 mouse embryonic stem (ES) cells (line GS from Genome Systems). After selection with G418 at 0.4 mg/ml, G418-resistant colonies were picked and screened by PCR. The genotype of the PCR-positive clones was confirmed by Southern blotting analysis. Homologous recombinants were karyotyped and analyzed for mycoplasma. Two homologous recombinant ES clones were injected into C57BL/6 blastocysts. Resulting male chimeras were crossed with C57BL/6 females, and germline transmission was scored by coat color. Heterozygous mice were identified by PCR and Southern analysis of mouse tail DNA. Embryos from intercrosses of heterozygous ( Ikk β +/− ) mice, as well as mouse embryonic fibroblasts (EFs), were genotyped by PCR and Southern analysis using DNA isolated from a piece of each embryo or a cell pellet, respectively. PCR was performed in the presence of 10% DMSO with Taq DNA polymerase using a Perkin-Elmer 9600 thermocycler programmed for denaturation at 95°C for 5 min, amplification for 35 cycles (94°C for 30 s, 55°C for 30 s, 65°C for 2 min), and elongation at 72°C for 10 min. Primers used were: P1 (5′-AGTCCAACTGGCAGCGAATA-3′) located outside of the homology arm and P2 (5′-CAACATTAAATGTGAGCGAG-3′) located within the LacZ gene. Southern blotting analysis was performed according to a standard protocol ( 27 ) except that hybridization was performed in phosphate-SDS buffer ( 28 ). Ikk β −/− , Ikk β +/− , and Ikk β +/+ ES and EF cells were treated with TNF-α or IL-1 at 20 ng/ml. Kinase assays and immunoprecipitations were performed as described ( 9 ). Immunoblotting was performed as described ( 14 , 16 ). Electrophoretic mobility shift assays (EMSAs) using the consensus κB and NF-1 sequences were performed as described ( 16 , 29 ). Mouse embryos or embryo livers were fixed in 10% buffered formalin and embedded in paraffin. After routine processing, the sections (5-μm thick) were stained with hematoxylin and eosin (H&E) for histological analysis. In situ TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling) assay was done using the in situ cell death detection kit according to the manufacturer's instructions ( Boehringer Mannheim ). For electron microscopy, embryonic day 13 (E13) embryos were removed and the livers were dissected out and fixed for 1 h in 2% formaldehyde and 2% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) at 4°C. The remainder of the embryos were placed in PBS for subsequent PCR and Southern analysis. After washing in cacodylate buffer, the livers were postfixed in 1% osmium tetroxide in cacodylate buffer for an additional 1 h. After postfixation, the samples were rinsed in double distilled water, dehydrated in a graded ethanol series, and infiltrated and polymerized in Durcupan ACM resin (Electron Microscopy Sciences). Sections 80-nm thick were stained with Sato lead and examined at 80 keV with either a JEOL 100CX or 2000EX transmission electron microscope. To create a strain of IKKβ-deficient mice, we used gene targeting technology ( 30 ). Mouse genomic Ikk β DNA was cloned from a 129 strain library and, after mapping and sequencing, was used to construct the targeting vector . To eliminate IKKβ kinase activity, part of the second and the entire third coding exon that specifies an essential part of the kinase domain were replaced with a DNA fragment encoding β-galactosidase ( LacZ ) and neomycin resistance ( Neo r ). Because the Neo r gene contains transcription termination and polyadenylation signals, the COOH-terminal three quarters of IKKβ including its protein interaction motifs are unlikely to be expressed from the targeted allele. After selection and screening by Southern blotting, six ES cell clones with homologous integration of the targeting vector into the Ikk β locus were isolated, and two of them were used to generate chimeric mice. Chimeric mice derived from these clones transmitted the targeted Ikk β allele to their progeny . Although Ikk β +/− male and female mice appeared normal and were fertile, upon intercrossing they did not give rise to live Ikk β −/− progeny. Analysis of protein extracts of Ikk β +/+ , Ikk β +/− , and Ikk β −/− cells revealed that, as expected, no IKKβ protein was expressed from the targeted allele . In addition, Ikk β +/− cells expressed approximately half the dose of IKKβ present in wild-type cells. No compensatory increases in IKKα, IKKγ, p65(RelA), or p50(NF-κB1) expression were observed. Given the expected importance of IKKβ for NF-κB activation and the embryonic lethality of RelA −/− mice ( 21 ), we suspected that the loss of IKKβ would result in a similar phenotype. Therefore, we analyzed embryos from timed pregnancies of Ikk β +/− intercrosses. Although Ikk β −/− embryos isolated at E11.5 were alive and had perfectly normal appearance (data not shown), Ikk β −/− embryos isolated at E13.5 were no longer alive and were rather anemic in appearance . Even external examination suggested that the liver of E13.5 Ikk β −/− embryos had degenerated. Notably, however, the limbs and head of Ikk β −/− embryos were normally developed, unlike those of Ikk α −/− E13.5 embryos ( 20 ). Histochemical examination of transverse sections of normal and mutant E13.5 mouse embryos stained with H&E revealed massive cell death in livers of Ikk β −/− embryos . Essentially, no viable hepatocytes could be detected, and the numbers of dead cells with highly condensed and fragmented nuclei were markedly increased. However, hematopoietic precursors retained their normal appearance in Ikk β −/− livers. TUNEL staining revealed that the observed cell death is most likely due to apoptosis, whose rate was increased manyfold . Examination of E13 Ikk β −/− embryos revealed close to normal external appearance (data not shown), but electronmicroscopic examination of ultrathin sections from their livers revealed massive numbers of dead hepatocytes with highly condensed nuclei characteristic of apoptotic cell death . The livers of Ikk β +/+ or Ikk β +/− littermates had perfectly normal appearance. We used two different approaches to determine the consequences of the loss of IKKβ expression on IKK and NF-κB activation. First, we prepared Ikk β −/− ES cell lines by subjecting Ikk β +/− ES cells to selection at higher G418 concentration. One Ikk β −/− cell line was identified. As shown in Fig. 5 A, stimulation of these cells with either TNF-α or IL-1 did not result in IKK activation, whereas a normal activation response was observed in Ikk β +/− cells. Note, however, that Ikk β +/− cells had ∼50% of the IKK activity of wild-type ( Ikk β +/+ ) ES cells, consistent with the reduced amount of IKKβ protein (data not shown). In addition to the defect in IKK activation, hardly any induction of NF-κB DNA binding activity was observed in Ikk β −/− cells after stimulation with either IL-1 or TNF-α . Even the basal level of NF-κB DNA binding activity was considerably reduced in Ikk β −/− cells, despite no detectable changes in p65(RelA) or p50(NF-κB1) abundance (data not shown). The second approach to evaluate the function of IKKβ was to prepare cultures of EFs from E11.5 mouse embryos of all three genotypes. As shown in Fig. 6 , essentially no induction of IKK or NF-κB activity could be detected in Ikk β −/− EF cells treated with either IL-1 or TNF-α. Interestingly, Ikk β +/− EF cells exhibited an ∼50% reduction in IKK activity (consistent with the reduction in IKKβ expression) but a much larger decrease in NF-κB DNA binding activity. The results described above indicate that IKKα, which is expressed in normal levels in Ikk β −/− cells, cannot be activated by either TNF-α or IL-1. To further examine this point, we cotransfected an HA epitope–tagged IKKα expression vector into Ikk β −/− ES cells in the absence or presence of an NIK expression vector. NIK is the most potent IKK activator identified to date ( 31 ) and was suggested to be a direct IKKα kinase ( 18 ). Recently, however, we obtained results that suggested that NIK-induced IKKα phosphorylation is not direct and is likely to be dependent on IKKβ ( 19 ). Consistent with this hypothesis, we found no increase in IKK activity towards IκBα(1-54) substrate upon coexpression of HA-IKKα with NIK in Ikk β −/− cells . Yet, when an IKKβ expression vector was included in these transfections, NIK elicited a clear increase in IKK activity. As shown previously, NIK coexpression efficiently stimulates IKKα-associated IKK activity in IKKβ-expressing cells ( 19 ). One reason for the inability of IKKα to respond to proinflammatory stimuli or NIK in the absence of IKKβ could be its inability to directly associate with IKKγ, the regulatory subunit of the IKK complex. Previous experiments indicate that IKKγ is essential for recruitment of upstream activators to IKK ( 16 ). In addition, using recombinant proteins, it was found that IKKβ directly interacts with IKKγ much more efficiently than does IKKα ( 16 , 17 ). Having available IKKβ-deficient cells, we reexamined the ability of IKKα to interact with IKKγ. In contrast to the results obtained with recombinant proteins, very efficient coprecipitation of IKKα by anti-IKKγ antibodies was observed using lysates of Ikk β −/− cells as a starting material . Therefore, the refractoriness of IKKα to IKK activators in IKKβ-deficient cells is not due to its inability to associate with IKKγ. The enzymatic activity of the IKK complex, composed of two catalytic subunits, IKKα and IKKβ, and one regulatory subunit, IKKγ, is rapidly stimulated by proinflammatory cytokines and LPS (for a review, see reference 12 ). Activated IKK phosphorylates the different IκBs at the two NH 2 -terminal serines that trigger their polyubiquitination and proteasome-mediated degradation. Once the IκBs are degraded, the freed NF-κB dimers migrate to the nucleus and activate target gene transcription. Based on their similar primary structures ( 11 , 13 , 14 ) and substrate specificities ( 15 ), IKKα and IKKβ were expected to play redundant and interchangeable roles in proinflammatory signaling to NF-κB. Therefore, it was rather surprising that only IKKβ was found to be involved in IKK activation. Alanine substitutions of the two serines in the activation loop of IKKβ, whose phosphorylation is stimulated by either TNF-α treatment or NIK overexpression, prevented IKK activation. Yet, the same mutations introduced into the activation loop of the IKKα subunit had no effect on the response of IKK to TNF-α or NIK ( 19 ). These results were confirmed by the analysis of IKKα-deficient cells and tissues which revealed no defect in IKK activation and IκBα degradation in response to TNF-α, IL-1, or LPS ( 20 ). However, it remained possible that the function of IKKα in IκB phosphorylation in response to proinflammatory stimuli can be fully replaced by IKKβ. The results described here indicate that IKKβ and IKKα have different physiological functions and that IKKα cannot substitute for IKKβ. To determine the physiological function of IKKβ, we generated Ikk β −/− knockout mice and cell lines. The loss of IKKβ results in embryonic death at mid-gestation due to massive hepatocyte apoptosis. This phenotype is remarkably similar to that of RelA knockout mice ( 21 ), with one exception: while Ikk β −/− embryos die around E13, RelA −/− embryos die around E15. The earlier death of Ikk β −/− embryos is likely to be due to a more extensive reduction in NF-κB activity, as embryos that are deficient in both the p65 (RelA) and the p50 (NF-κB1) subunits of NF-κB die at E12.5, the same time as IKKβ-deficient embryos, from massive hepatocyte apoptosis ( 32 ). Thus, IKKβ and RelA are genetically proven to be components of the same pathway. Accordingly, cells that lack IKKβ are completely defective in IKK and NF-κB activation in response to either TNF-α or IL-1. Therefore, the IKKβ subunit is absolutely essential for mounting a response to proinflammatory stimuli. This function is not replaced by IKKα, whose expression is not diminished in the absence of IKKβ. In addition, as indicated by the normal morphology of the head and limbs of E13.5 Ikk β −/− embryos, IKKα can carry out its developmental function ( 20 ) in the complete absence of IKKβ. Interestingly, a 50% reduction in IKKβ expression, as in Ikk β +/− cells, results in a similar decrease in IKK activity but a much more severe defect in NF-κB activation. These results underscore the importance of the IKKβ subunit and indicate that the NF-κB activation response does not follow a simple linear relationship to the magnitude of IKK activation. It also appears from these results that a low level of NF-κB activity may be sufficient for protecting the liver from TNF-α–induced apoptosis. One possible cause for the inability of IKKα to substitute for IKKβ was its relatively lower affinity to IKKγ, the regulatory subunit that is absolutely required for IKK activation ( 17 ). Using recombinant proteins, it was observed that IKKα does not form a stable complex with IKKγ in vitro, whereas IKKβ readily associates with IKKγ ( 16 , 17 ). However, immunoprecipitation experiments indicate that a similar amount of IKKα is precipitated by IKKγ antibodies from Ikk β −/− cells as from Ikk β +/+ cells. Despite its ability to associate with IKKγ in the absence of IKKβ, IKKα is refractory to upstream activators involved in proinflammatory signaling, including the most potent IKK activator identified so far, NIK, in IKKβ-deficient cells. These results underscore the differences in regulation of IKKα and IKKβ activities. In summary, together with the previous analysis of IKKα-deficient mice, the analysis of IKKβ-deficient mice, described here, indicates that the two catalytic subunits of the IKK complex, although similar in structure, have very different functions. Although IKKβ is responsible both for activation of the entire complex in response to proinflammatory stimuli, through phosphorylation at its activation loop, and for activation of NF-κB, through IκB phosphorylation, IKKα is assigned the control of epidermal and skeletal morphogenesis. Although the stimuli that activate IKKβ and the substrates that mediate its biological activity are known, the stimuli and the relevant substrates for IKKα remain to be identified. | Study | biomedical | en | 0.999996 |
10359588 | Material from a previously described ( 5 , 6 ) male patient lacking neutrophil-specific granules was studied. Research was conducted with informed consent under the guidelines of a National Institutes of Health (NIH) Internal Review Board– approved protocol, no. 92-I-99. The patient died from complications of pneumonia at age 20. Peripheral blood neutrophils were isolated as described ( 28 ), cryopreserved with dimethylformamide ( Sigma Chemical Co. ), and maintained at −140°C. Cell proteins were extracted as described ( 29 ). DNA extraction from cryopreserved fibroblasts proceeded as described ( 30 ). RNA was extracted from patient bone marrow aspirate using RNAzol reagent (Teltest) as per manufacturer's protocol. Normal human bone marrow RNA was purchased from Clontech . PCR reaction was performed using Platinum taq DNA polymerase (Life Technologies) per manufacturer's instructions and cycled as follows: 96°C for 12 min, followed by a three-step cycle—94°C for 30 s, 60°C for 30 s, and 72°C for 2 min—for 35–40 cycles. PCR products were gel purified and recovered using Gene Clean (Bio101). Products were sequenced with an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit ( Perkin-Elmer Corp. ). Primers were chosen from a published sequence available from EMBL/GenBank/DDBJ under accession no. U48865 . Primer sets (upstream, downstream): B, 5′-AGC GGC CAT GCA AAA GGA AAG ACA, 5′-TCC ACC TAC CCC CAA GAG AAA GTT ; C, 5′-CCC ACG GGA CCT ACT ACG A, 5′-GGG CTG GCC TGC TCT TAC ; F, 5′-CTC CCC GGC TGG CCC CTT ACA C, 5′-GCC AAC AGT CCC AAC ACC CAG TCA ; G, 5′-GGA GGT GGG GCT ACA AAA GAA ACT, 5′-TCA GGG AGG GGC AGG ACA ; H, 5′-ACA GGA GTG GGT GAC AGA GGA GAC, 5′-GGG CCG AAG GTA TGT GGA GGG TAG ; I, 5′-CCA TGC CCC CTC CTC TTG TTT CTC, 5′-ACT GCC TTC TTG CCC TTG TGT AA ; K, 5′-AAC TTT CTC TTG GGG GTA GGT GGA, 5′-TCG TAG TAG GTC CCG TGG . Homozygosity was determined by hybridization of the PCR product fragment C to a [ 32 P]γdATP-endlabeled internal oligonucleotide (H. downstream; sequence above). Labeled oligo was mixed with hybridization buffer (75 mM NaCl, 5 mM EDTA) in a ratio of 1:10, and 10 μl was added to 30 μl PCR product. Hybridization was cycled in a thermal cycler: 95°C for 5 min and 55°C for 10 min. Reactions were immediately placed on ice. Products were resolved on 4–20% Tris/borate/EDTA polyacrylamide gel (Novex) at 250 V. 10 μg of total RNA isolated from the patient's bone marrow and 0.25, 0.5, and 1 μg of control polyadenylated (pA)-mRNA was electrophoretically separated, blotted, hybridized, and washed as described ( 27 ). The membrane was stripped by boiling and stored at −20°C. Protein quantitation was performed using a BCA Protein Assay kit ( Pierce Chemical Co. ) according to the manufacturer's instructions. 10–100 μg protein extracts were electrophoretically separated, transferred to nitrocellulose, and incubated with primary antibody as described ( 29 ). Primary antibody was generated in rabbits by Research Genetics, Inc., using a synthetic peptide encoded in exon 2 of C/EBPε, downstream of the SGD deletion (DPRAVAVKEEPRGPEGSR). The membranes were washed, incubated with anti–rabbit horseradish peroxidase conjugate antibody (ECL Western blot kit; Amersham Pharmacia Biotech, Inc. ), and developed according to the manufacturer's instructions. Membranes were stripped and reblotted with anti–mouse human β actin antibody ( Boehringer Mannheim ) to control for protein loading. The patient's mutation was introduced into the pCMV-C/EBPε 32 expression vector using a Stratagene QuikChange site-directed mutagenesis kit per manufacturer's instructions using a complementary oligonucleotide (PAGE purified; purchased from Genosys Biotech) containing the deletion (5′-CCA CTA CTT GCC GCC CTC GGC CCT TTG CCT ACC). Presence of the mutation and maintenance of the vector sequences was verified by sequencing and restriction enzyme digestion, respectively. HeLa cells were maintained in DMEM (BioWhittaker) supplemented with 10% heat-inactivated FBS (Life Technologies, Inc.) and penicillin/streptomycin at 37°C and 5% CO 2 . Cells were plated in 6-well plates and transfected within 24 h, at 30–50% confluency. Transfections, using the Mammalian Transfection System (Stratagene), were performed using 5 μg reporter plasmid (G-CSF receptor promoter-luc); 1, 2, or 5 μg inducer plasmid (pCMV-C/EBPα, pCMV-C/EBPε 32 isoform, pCMV-C/EBPε 14 isoform, or pCMV-C/EBPε 32 -SGD, described above); and 0.5 μg pCMVβ, as described ( 23 , 31 ). The DNA content of transfections was normalized, and transfection was performed according to the manufacturer's instructions, with 300 μl transfection solution applied to the cells. Samples were harvested 24 h after transfection. Luciferase and β-galactosidase activities were measured using a Dual-Light kit (Tropix, Inc.), according to the manufacturer's protocol, on a Turner 20/20 Luminometer. Samples were read for 15 s after a 3-s delay. Sequencing of PCR products from genomic DNA detected a 5-bp deletion, TGACC, in exon 2 of the patient's C/EBPε sequence. Fig. 1 A shows sequence data from one normal control (top sequence) and the SGD patient (bottom sequence). The mutation predicts a frameshift and a premature termination of the encoded C/EBPε 32 isoform . The missense code after the frameshift results in the loss of the critical DNA binding domain and leucine zipper region required for C/EBP dimerization and function. C/EBPε transcripts encoding the shorter 27- and 14-kD isoforms are predicted to be unaffected, based upon the splice donor and acceptor and translational start sites ( 23 ). Homozygosity of the deletion was determined by PCR amplification of the affected region and resolution of the DNA fragments on a 4–20% polyacrylamide gel . DNA from one normal control and the SGD patient were mixed before amplification and electrophoresis (lane 3), showing bands from both affected and normal alleles. In comparison, PCR products from the SGD patient (lane 4) and normal controls (lanes 1 and 2) show only one fragment, indicating homozygosity for their respective alleles. RNA blot analysis of the SGD patient's bone marrow total RNA showed decreased amounts of C/EBPε transcripts in comparison with control human bone marrow pA-RNA . Hybridization with a [ 32 P]dCTP- labeled actin probe (provided by L. Perera, National Cancer Institute, NIH) showed that 10 μg of SGD patient bone marrow total RNA was equivalent to 1 μg of normal bone marrow pA-mRNA and verified the stability and quality of the patient's RNA preparation. Specific loss of C/EBPε transcripts in the SGD patient is likely due to mRNA instability secondary to the frameshift and the premature termination codon, as seen in other similar gene mutations ( 32 , 33 ). Residual C/EBPε message is likely comprised by C/EBPε 14 and C/EBPε 27 transcripts, which are unaffected by the 5-bp deletion and similar in size to the C/EBPε 32 transcript. Transcripts of C/EBPα were present in normal amounts. C/EBPα has a more proximal role in the myelopoietic pathway and specifically induces expression of C/EBPε ( 31 , 34 , 35 ). As expected, message for lactoferrin was not detected in the SGD patient's bone marrow RNA. As predicted from the C/EBPε transcript maps , immunoblotting detected C/EBPε 27 and C/EBPε 14 isoforms, but not C/EBPε 32 , in neutrophils from the SGD patient . All three isoforms were seen in the normal control. The antibody used is specific for a peptide sequence immediately downstream of the 5-bp mutation and should not bind the C/EBPε 32 -SGD protein. Transient transfection assays in HeLa cells, using the G-CSF receptor promoter driving the luciferase gene ( 31 ), compared the transactivation potentials of the inducer genes C/EBPα, C/EBPε 32 , C/EBPε 14 , and C/EBPε 32 -SGD . C/EBPε 32 has been shown to transactivate the G-CSF receptor promoter, whereas the C/EBPε 14 isoform lacks transactivating function ( 23 ). Transient transfection of these plasmid constructs showed a significant loss of transactivation with the C/EBPε 32 -SGD isoform ( P = 0.02, Mann-Whitney U test). The demonstrated in vitro data, as well as the in vivo SGD phenotype, mark the full length, 32-kD isoform as the major transactivator encoded in the C/EBPε locus. The temporal link between granule protein production and myeloid lineage differentiation is well described: primary granule proteins are synthesized in myeloblasts and promyelocytes, secondary granules are produced in myelocytes and metamyelocytes, and tertiary granule proteins are generated in band and segmented neutrophils ( 36 ). Previous work suggested that C/EBPε functions at the terminal stages of myeloid differentiation ( 23 , 26 ). However, the total absence of patient neutrophil secondary granules and the selective loss of primary granule defensins marks an early myelopoietic block at the promyelocyte transition . Further evidence for this conclusion comes from in vitro differentiation experiments using C/EBPε-deficient stem cells, which do not proceed beyond the promyelocyte stage ( 26 ). Other functional defects seen in mouse and human C/EBPε-deficient neutrophils, such as loss of tertiary granule gelatinase ( 27 ) and abnormalities in chemotaxis and cytokine expression ( 6 , 27 ), may occur secondary to the block at the promyelocyte or later stage. Functional analysis of the previously developed C/EBPε knockout mouse model ( 26 , 27 ) was critical for the interpretation of the C/EBPε mutation in SGD. The apparent multiplicity of C/EBPε target genes at different cell stages suggests that C/EBPε transactivates a set of early cell stage– specific genes, inducing normal promyelocyte differentiation and granule development. Additional evidence supporting these conclusions comes from recent observations suggesting that C/EBPε is induced by and transduces the G-CSF signal in neutrophils early in myelopoiesis ( 37 ). Absence of secondary granules, defensins, eosinophil cationic protein, eosinophil-derived neurotoxin ( 12 ), and platelet α granule high-molecular-mass von Willebrand factor ( 14 ) in SGD demonstrates a critical role for C/EBPε in the development of granules and their contents in multiple myeloid lineages. | Other | biomedical | en | 0.999997 |
10366586 | Human primary lymphocytes were stimulated with phytohemagglutinin; both lymphocytes and lymphoblasts (46XY) were grown in RPMI + 10% FCS. Human primary fibroblasts (less than passage 10) and HT1080 cells were grown in DMEM + 10% FCS. To identify cells in S phase, BrdU was added to cultures at 0.1 mM. To irreversibly inhibit transcription, actinomycin D (AMD) was added 2 h before harvest to 5 μg/ml. To reversibly inhibit RNA pol II cells were cultured with 50 μg/ml 5,6-dichloro-β- d -ribofuranosylbenzimidazole (DRB) for 5 h . Inhibition was relieved by culturing the cells for a further 1 h in the absence of drug. To inhibit histone deacetylase activity, cells were cultured in the presence of 10 ng/ml Trichostatin A (TSA) for 2 h . Cells were fixed in 3:1 methanol/acetic acid (MAA) using standard procedures. Human lymphoblasts, swollen in 0.075 M KCl, were also cytocentrifuged onto slides and fixed with 4% paraformaldehyde (pFa) made up in 120 mM KCl, 20 mM NaCl, 10 mM Tris HCl, pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100. To preserve three dimensional (3D) nuclear structure, HT1080 cells and primary fibroblasts were grown on slides and fixed with 4% pFa for 10–20 min. An exponential culture of lymphoblastoid cells was labeled with 0.1 mM BrdU for 45 min before harvest. 1.25 × 10 8 cells in 10 ml of PBS, 1% FCS, 0.3 mM EDTA, 0.1% glucose were loaded at 11 ml/min into a 5-ml elutriation chamber of a JE5.0 centrifugal elutriator (Beckman) at 2,500 rpm. Fractions were taken at increasing flow rates up to a maximum of 60 ml/ min; the last fraction was collected at 60 ml/min, 2,000 rpm. Fractions were assayed by FACS ® after staining with propidium iodide (PI), by nuclear size, and by their pattern of BrdU incorporation. Interphase nuclei from lymphoblasts, prepared in polyamine buffer, were incubated in extraction buffer containing increasing concentrations of NaCl (0.5, 1.0, 1.2, and 1.8 M). Slides were then fixed twice in MAA before FISH. Chromosome paints for HSA18 and 19 were prepared in a variety of different ways and their specificity was confirmed by FISH to mitotic chromosomes. A chromosome 19 paint was prepared by human-specific inter-Alu PCR from a Chinese hamster–human monochromosome 19 hybrid cell line GM10449A and labeled by nick translation. To test that this probe detected the entirety of HSA19, total DNA from the hybrid cell line was also nick translated and a commercially available HSA19 paint, produced by both inter-Alu and inter-LI PCR (Oncor) was also tested. Chromosome paints prepared by the amplification of total DNA from microdissected HSA19p and q arms were also used . A total chromosome 18 paint was prepared from FACS ® -sorted chromosomes digested with MseI, and ligated to catch-linkers (5′-TACCGTTAAGCGTCAATCATGG-3′ [CH18-1] and 5′-CCATGATTGACGCTTAACGG-3′ [CH18-2]), provided by S. Cross. CH18-2 was used as a primer for PCR in the presence of biotin- or digoxigenin-labeled dUTP for 35 cycles. Total DNA from the Chinese hamster–human somatic cell hybrid GM10110, a commercially available chromosome 18 paint (Oncor) and microdissected 18p and q arm paints were also tested. The area taken up by FISH signal with paints from different sources was assessed in 50 nuclei. No significant differences were found between paints for the same whole chromosome prepared in different ways but detected with the same fluorochrome. However, when signals from the same paints labeled with both biotin and digoxigenin and cohybridized were compared, Texas red (TR) signals occupied a larger proportion of the nuclear area than did FITC signals. Therefore, it was important that comparisons made were with the same fluorochrome. FISH on 3:1 fixed nuclei was as described previously . Where flattened cells had been fixed in pFa for 15 min, slides were treated with 0.07 M NaOH-EtOH (2:5) for 5 min before denaturation. 3D-preserved cells were fixed in 4% pFa for 10 min, washed in PBS, and then permeabilized with 0.5% saponin, 0.5% Triton X-100 in PBS for 10 min at room temperature. The slides were incubated in 20% glycerol-PBS for 30 min and then subjected to <5 repeated freeze–thaw cycles in liquid N 2 . Before hybridization these slides were treated with 0.1 M HCl for 5 min at room temperature and with 100 μg/ml RNase A at 37°C for 20 min. Slides were then denatured at 75°C in 70% formamide, 2× SSC, pH 7.0, for 3 min followed by 1 min in 50% formamide, 2× SSC, pH 7.0 . 150 ng of chromosome paint and 10–30 μg CotI DNA, or 200 ng labeled P1-BAC DNAs and 3 μg Cot 1 DNA, were used per slide. Biotinylated probes were detected using fluorochrome-conjugated avidin (FITC or TR) (Vector), followed by biotinylated antiavidin (Vector) and a final layer of fluorochrome-conjugated avidin. Digoxigenin-labeled probes were detected with sequential layers of FITC-conjugated antidigoxigenin (BCL) and FITC-conjugated anti–sheep (Vector). To combine immunofluorescence with FISH in cells fixed with 4% pFa, after detection of the hybridization signals, slides were incubated for 1 h at room temperature, or at 4°C overnight, with a 1:10 dilution of rabbit anti-pKi67 MIB-1 antibody (Dianova) and a monoclonal antibody LN43.2 (gift of B. Lane) directed against B-type lamin. These were detected with TR anti–rabbit and AMCA anti–mouse secondary antibodies (Vector). Slides were counterstained with either 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) or 0.2 μg/ml PI and examined either using a Zeiss Axioplan fluorescence microscope, equipped with a triple band-pass filter , or with a BioRad-MRC 600 confocal laser scanning microscope fitted with an argon laser and a dual filter set (FITC and PI). For random selection of nuclei for analysis, images were taken of consecutive nuclei that presented in a spiral scan pattern from the center of the slide and which did not touch adjacent nuclei. Gray scale images from the Axioplan were collected with a CCD camera (Photometrics), pseudocolored, and merged using Digital Scientific SmartCapture extension to IPLab Spectrum. Using IPLab Spectrum software, scripts were written to analyze the data from flattened specimens. In the first, most applicable to the analysis of circular (spherical) nuclei, the DAPI image was segmented and the area and centroid coordinates calculated. The mean FITC and TR pixel intensities within the area of the DAPI segmented nucleus were calculated and subtracted from the FITC and TR images to remove background. A region of interest was then defined manually around the signal. The signal within this region was then segmented and the area and signal intensity weighted centroid coordinates calculated. The area of the signal was normalized for nuclear size by dividing by the nuclear area (see Table II ). The DAPI image was converted to binary form. Using the coordinates of the signal weighted centroid as the center, an appropriately sized segmentation disc was adjusted by dilation and erosion until a single pixel with zero intensity was determined. This was taken as the nearest edge of the nucleus to the signal. The signal segment was converted to binary form, a chord was drawn from the centroid of the signal to the nearest edge of the nucleus, and the coordinates established for the first pixel with zero intensity. This was taken to be the edge of the hybridization signal closest to nuclear periphery. These coordinates were used to determine the relative distances between either the edge of the hybridization signal, or the weighted center of the signal, and the nearest edge of the nucleus and also between the center of the signal and the center of the nucleus (see Table I ). The erosion analysis script segmented the DAPI signal and recorded the area and centroid coordinates. The area was divided into concentric shells (1–5) of equal area from the periphery of the nucleus to the center. Background was removed from the FISH signal by subtraction of the mean signal pixel intensity within the segmented nucleus, as described above. The proportion of FISH signal and DAPI fluorescence was then calculated for each shell . For confocal analysis nuclei were scanned and imaged using BioRad Comos software. Images from four high resolution scans were averaged using a Kalman electronic filter and 0.5 or 1 μm optical sections were taken. The positioning of chromosomes in relation to the nuclear periphery was assessed manually from the PI staining using display software in IP LabSpectrum. The area of hybridization signal in optical sections was measured as a proportion of the nuclear area as defined by the counterstain. Chromosome paints for HSA18 and 19 were used in FISH to two-dimensional (2D) preparations of nuclei, swollen in hypotonic, and fixed in MAA, from asynchronous cultures of two primary and two transformed human cell types , and also to nuclei swollen in hypotonic and then fixed with 4% pFa . In all of these cases HSA18 and 19 were seen to adopt different positions within the nucleus, and when nuclei were cohybridized with differently labeled paints for HSA18 and 19 signals for both chromosomes 18 appeared to be in a more peripheral location than those of HSA19. Using flattened specimens enabled the capture and analysis of images from large numbers of nuclei (∼50 in each case) hybridized either with biotinylated HSA18 or HSA19 and subsequent statistical evaluation of the relative nuclear positions of these two chromosomes. In the circular (spherical) nuclei of lymphocytes and lymphoblasts the distances between both the edge, and the signal intensity weighted center of the chromosome territory, to the edge of the nucleus were measured, as was the distance between the center of the nucleus and the signal intensity weighted center of the chromosome territory (Table I ). Signals for both chromosomes 18 were significantly closer to the nuclear periphery in lymphocytes and lymphoblasts than those of HSA19 and, conversely, chromosomes 19 were located significantly closer to the center of the nuclear area than HSA18s. These data were independent of whether total PCR amplified chromosome 19p and q arm DNA was used as a probe or whether the DNA was labeled by inter-Alu PCR (Table I ). The nuclei of fibroblasts and fibrosarcoma cells are ellipsoid, so the analyses in Table I were not appropriate. 2D nuclei from four cell types were examined by erosion. The nuclear area was divided into concentric shells (1–5) of equal area from the periphery of the nucleus to the center. The proportion of FISH signal and DAPI fluorescence was calculated for each shell. The absolute values of these are influenced by the geometric properties of the nucleus in each cell type and the definition of the nuclear periphery afforded by the DAPI counterstain, but a comparison of the proportion of the DAPI stain or hybridization signal from HSA18 and 19 paints in each concentric nuclear shell indicates that chromosome 18 is more peripheral than HSA19 within the diploid nuclei of all the cell types and in nuclei fixed with either MAA or pFa. The proportion of hybridization signal from HSA18 in the outermost shell (shell 1) was always greater than, or similar to, the proportion of DAPI stain located there, whereas the proportion of HSA19 located in this shell was always significantly less than the proportion of the DAPI stain. Conversely, in the most central shell (shell 5) the proportion of HSA19 signal found there was always greater than that of the DAPI stain. The same relative positions of HSA18 and 19 were also seen in fibrosarcoma cells carrying trisomies for either HSA18 or 19 (the population of these cells has a variable karyotype) . Extrapolation of data from 2D preparations assumes that the relative organization of the intact nucleus is not significantly perturbed. In Drosophila , a comparison of data collected from hypotonically treated and squashed nuclei, with those collected from 3D-preserved nuclei, has confirmed the validity of this assumption . However, we still considered it important to confirm our observations in 3D human nuclei that had not been subject to hypotonic swelling. Also, signals that appear to be in the center of flattened nuclei might be located close to the periphery on either the top or bottom surface of the nucleus. Therefore, hybridization signals for HSA18 and 19 were examined in optical sections through the nuclei of primary fibroblasts and HT1080 cells that had been fixed with pFa, using confocal laser scanning microscopy . In 94% of cells ( n = 78 cells, 156 chromosomes), a substantial amount of the signals from both chromosomes 18 was coincident with the nuclear periphery, as defined from the DNA counterstain . By contrast, in 16% of cells hybridized with an inter-Alu HSA19 paint ( n = 56 cells, 112 chromosomes) and sectioned at 0.5-μm intervals, and in 20% of cells hybridized with total HSA19 DNA ( n = 35 cells, 70 chromosomes) (1-μm sections) we could not detect any signal from either chromosome 19 adjacent to the nuclear periphery at this level of resolution . Hence our observations in 3D-preserved cells, that had been fixed with pFa and not subjected to hypotonic swelling, are consistent with the conclusions of our analysis of 2D specimens. It was also interesting to note that in the z sections of 3D-preserved fibroblasts, 65.5% of chromosomes 18 were close to the lateral edge of confocal midsections . Proximity of chromosomes 18 to the upper or lower edges of the nucleus could be seen . Where chromosomes 19 did appear close to the nuclear periphery this was often at the top or bottom surface of the nucleus. The demands of preserving nuclear structure yet allowing hybridization to denatured target DNA are conflicting. However, it has been shown that in cells fixed with pFa, permeabilized, and then processed for FISH using the method described here, that centromeres remain in the same spatial position before and after the FISH procedure . To confirm that some components of nuclear compartments and the nuclear periphery were not disrupted by this procedure, FISH with HSA18 and 19 paints was combined with immunofluorescence for a B-type lamin, a component of the nuclear lamina , and for pKi-67, a protein that locates from satellite DNA to the nucleolus from mid-late G 1 . Between 50 and 100 nuclei were examined and the majority of chromosomes 18, but not 19, were located close to the nuclear lamina, as detected with anti–lamin B. The pKi-67 antigen coats chromosomes in mitosis and for most of interphase (from mid G 1 through to G 2 ) it accumulates in the nucleolus. However, in very early G 1 pKi-67 is distributed in nuclear foci that correspond with blocks of heterochromatin . In combined immunofluorescence-FISH of 3D preparations of human fibroblasts HSA18 was located close to the lamina both in cells with a nucleolar pattern of pKi-67 distribution and also in those cells with a pKi-67 staining pattern indicative of early G 1 . At these same stages human chromosome 19 appeared to be in a more internal region of the nucleus . Hence, the difference in subnuclear compartmentalization of HSA18 and 19 is established very early in the cell cycle, probably as cells exit telophase. HSA18 and 19 territories were also examined in the 2D nuclei from exponentially growing human lymphoblasts, pulsed with BrdU, and fractionated by centrifugal elutriation . 50 nuclei from each cell cycle stage fraction were examined. The relative positions of HSA18 and 19 were maintained throughout the cell cycle. Chromosome 18 was more peripheral than HSA19 even in cells that, on the basis of their fractionation and pattern of BrdU staining, appeared to be in late S phase . This suggests that the relative nuclear positions of chromosomes 18 and 19 are maintained before, during, and after chromosome replication. Paralleling their DNA content, chromosomes 18 at C-metaphase have a mean area 10% greater than chromosomes 19 (Table II ). However, HSA19 hybridization signal appeared to occupy a larger proportion of nuclear area in 2D preparations than did HSA18 . The normalized area of hybridization signals for both homologues of HSA18 and 19 were measured in lymphocytes and lymphoblasts . HSA18 occupied a significantly smaller fraction of nuclear area than HSA19, in flattened preparations of both MAA- and pFa-fixed nuclei, independent of the method of labeling of HSA19. The differences in the proportion of nuclear area occupied by whole chromosome paints for HSA18 and 19 were also seen when the signals from chromosome paints specific for the p or q arms of the chromosomes were compared . For example, although the q arm of the submetacentric chromosome 18 is estimated to contain 65 Mb of DNA, it occupied the same proportion of the nuclear area as the estimated 37 Mb of DNA in the q arm of metacentric chromosome 19 (Table II ). Variance in the proportion of nuclear area occupied by the two chromosomes 18 within a single nucleus was similar to that of chromosomes 18 in different nuclei (0.007 vs. 0.010, P < 0.079). However, for HSA19 territories there was significantly less variance between homologues within the same nucleus (0.016) than between chromosomes in different nuclei (0.040, P < 0.001) indicating that chromosome 19 compaction may vary between cells that, for example, are at different stages of the cell cycle to a larger extent than chromosomes 18. In elutriated fractions of lymphoblasts, areas of HSA18 signals were always smaller than those of chromosome 19 and the proportion of nuclear area that they occupied decreased from G 1 to G 2 . In contrast, HSA19 occupied the largest proportion of nuclear area in early S phase (Table II ). Signals from chromosome 19 also appeared to be more dispersed and irregular than those of HSA18 in both flattened nuclei and in optical sections of 3D nuclei . We do not have the facilities necessary to directly compute the volumes of HSA18 and 19 in 3D reconstructions. However, we analyzed the sum of the areas occupied by segmented hybridization signal from each chromosome as a proportion of the sum of nuclear areas in 1-μm sections through 10 nuclei (20 chromosomes 18 and 19 each). By this analysis, chromosomes 18 and 19 occupied 3.4 ± 0.3% and 5.0 ± 0.3%, respectively, of the summed nuclear areas of these 3D-preserved cells . The proportion of the summed nuclear areas occupied by signals from chromosomes 19 is significantly larger ( P < 0.001) than that occupied by chromosome 18, consistent with the analyses performed on flattened specimens (Table II ). To analyze the effects of transcription on the territories of HSA18 and 19, lymphoblasts were treated with AMD, an irreversible inhibitor of RNA pols I and II. To assess the drug's effectiveness a sample of cells, before and after treatment, was examined by immunofluorescence with an antibody against snRNPs (data not shown) . In AMD-treated cells the chromosome 19 territory, but not that of HSA18, occupied a significantly smaller proportion of the nuclear area than in untreated cells . The adenosine analogue DRB is a reversible and specific inhibitor of RNA polymerase II. The effect of DRB, and its removal, on lymphoblast and fibroblast cells was assessed by the redistribution of nuclear and nucleolar antigens by immunofluorescence (data not shown) . In the presence of DRB HSA19 territories occupied a smaller proportion of the nuclear area in flattened specimens than those of HSA18 and this compaction of HSA19 was partially reversed after removal of DRB for 1 h . This was confirmed by analyzing the proportion of nuclear area occupied by signal from each territory summed across confocal 1-μm optical sections of 3D-preserved cells fixed in pFa. Whereas in untreated cells the summed chromosome 19 territory areas were 1.5 times larger than those of HSA18, in DRB-treated cells chromosome 19 appeared to be only 0.33 times the size of chromosome 18 ( n = 10 cells each) . No significant differences between the summed areas of chromosomes 18 within treated and untreated cells were detected ( P < 0.3), however, chromosomes 19 in DRB-treated cells appeared to be significantly smaller than those in untreated controls ( P < 0.000). By contrast, treatment of cultures with the histone deacetylase inhibitor TSA, to concentrations known to influence replication timing within lymphoblast cells , enhanced the differences in HSA18 and 19 areas . There was no change in the relative chromosome positions within the nucleus when transcription was inhibited with AMD or DRB, or in the presence of TSA (Table I ). Subnuclear localization of specific human chromosomes may result from differences in DNA sequences distributed along the chromosome arms, or at the centromeres or telomeres of each chromosome. To determine whether both arms of chromosome 18 lie close to the nuclear periphery, and whether both 19p and q are equidistant from the center of the nucleus, paints specific for either 18p or q and 19p and q were cohybridized to flattened lymphoblast nuclei . Both 18p and q appeared in association with the edge of the nucleus and both 19p and q were found in a more central location . Examination of the distances between the edge of the nucleus and the edge or intensity weighted signal center for the p and q arms, and between the center of the nucleus and the intensity weighted signal centers (Table I ) confirmed that the relative subnuclear localization observed for HSA18 and 19 reflects properties of both the p and q arms equally. To establish the orientation of chromosome 18 with respect to the nuclear periphery, HSA18 paint was also cohybridized with P1 52M11 and BAC 75F20, specific for 18pter and 18qter, respectively (gift of J. Fantes). Most (63–65%) territories in flattened nuclei fixed in MAA had the p or q telomere at one or the other end of the territory ( n = 98). In most other cases the telomere appeared to be on the surface of the territory facing the nuclear interior; localization to the surface of the territory adjacent to the nuclear envelope was rare (7–11%) . This was also the case in 3D-preserved nuclei and suggests that HSA18 might contact the nuclear periphery through the bulk of its chromosome arms, not specifically through either telomeric end. Are the positions of HSA18 and 19 within the nucleus therefore confined to the intact chromosomes or can they be conferred by subchromosomal regions? Chromosomes and nuclei in standard cytogenetic preparations from an asymptomatic individual with a balanced reciprocal translocation t(18;19)(p11;p13) were analyzed. We estimate that 20 Mb of material from 18p (24% of HSA18) and 15 Mb of 19p (23% of HSA19) have been exchanged in this translocation . The nuclear disposition of both the normal and derived chromosomes was analyzed . Although the derived chromosome 18 tended to be less peripheral than the normal 18, this difference was not statistically significant (Table I , P < 0.059), neither was the apparently more central location of the normal 19 over that of the derived 19 significant ( P < 0.11) . However, there was a striking difference in the relative orientations of the two derived chromosomes. The translocated portion of 19p was more central than the remainder of the derived 18 in almost 80% of nuclei. Conversely, nearly 90% of translocated 18ps were peripheral to the derived 19 material. Hence the characteristic subnuclear localization of HSA18 and 19 can be conferred by 15–20-Mb subchromosomal regions and is not dependent on the centromeric heterochromatin from each chromosome. As no viable cells are available from this t(18;19) individual we could not analyze the localization of the derived chromosomes in 3D-preserved cells. The nuclear scaffold and matrix are nuclear substructures that include the lamina, residual nucleoli, and a proteinaceous network pervading the nuclear volume and that are left after extraction of soluble nuclear proteins with the detergent-like salt lithium diiodosalicylate (LIS) or with high salt . Nuclei were extracted with increasing concentrations of NaCl before fixation in MAA and hybridization with HSA18 and 19 paints . At low salt concentrations (0.5 M) no substantial release of DNA from the nucleus was seen. At 1 M NaCl, residual nuclei surrounded by halos of released DNA were revealed by DAPI staining and FISH exposed striking differences in behavior of HSA18 and 19. Most chromosome 18 DNA was released up to 3 μm away from the nuclear remnants, whereas the detectable chromosome 19 DNA remained in the center of the nucleus. As salt concentration increased up to 1.8 M, release of HSA18 became more exaggerated, extending for 7 μm away from the confines of nuclei, within which chromosome 19 was still tightly restrained . These differences in chromosome behavior were unchanged in nuclei from cells that had been treated with AMD to inhibit transcription. In this paper we have examined and compared the subnuclear localization of human chromosomes 18 and 19. In different primary and transformed human cells, and in both flattened specimens and in cells fixed to preserve 3D structure, we observed distinct dispositions toward either the interior or periphery of the nucleus for both the p and q arms of chromosomes 19 and 18, respectively . Specific parts of chromosomes may adopt different orientations during the cell cycle , but it was not known whether entire mammalian chromosomes move or change their state of condensation. Measurements of chromatin movements in vivo suggest that significant passive diffusion of chromatin could occur during the course of interphase but predict that whole chromosomes are largely constrained within a limited subregion of the nucleus . Therefore, specific subnuclear localization of DNA segments might be established most effectively as cells exit mitosis and before interphase nuclear architecture is fully formed. The distinctive arrangements of HSA18 and 19, that we have described here, are established early in the cell cycle and are maintained throughout interphase . However, the difference in spatial localization between HSA18 and 19 is at its smallest during late S phase (Table I ). This may reflect some movement of HSA18 to a more internal location accompanying the replication of its DNA . In addition to the differences between HSA18 and 19 in subnuclear localization, we have also demonstrated differences in the proportion of nuclear area that these two human chromosomes occupy in 2D preparations. The larger proportion of nuclear area occupied by HSA19, as compared with that of HSA18, is in contrast to the larger physical size (in bp) of the latter chromosome and its greater size in metaphase preparations (Table II ). Since much of HSA18 has the characteristics typical of G-band chromosome regions, whereas HSA19 is more R-band–like in its properties , our data are consistent with the regional differences in chromatin compaction at the 0.1–1.5-Mb level that have been recorded between G- and R-band regions of the human genome in nuclei prepared in similar ways to those described here . The proportion of nuclear area occupied by HSA19 reached a peak in early S phase. This might reflect HSA19 chromatin decondensation preceding its replication or merely the doubling of DNA content for this gene-rich chromosome before most of the rest of the genome. The apparently larger area of HSA19 within flattened nuclei is also seen in 3D-preserved nuclei . While sophisticated computational algorithms are necessary to accurately compute chromosome volumes within the nuclear space , our simplified approach of adding together the chromosome signal area and nuclear area in series of optical sections taken through nuclei suggests that chromosome 19 may occupy a larger volume within the human nucleus than chromosomes 18 and hence be less condensed. Hybridization signals seen with paints for HSA19 also appeared to us to have a more irregular and scattered character than those from HSA18 . The chromosome territory of the active X (Xa) is similarly more rutted in appearance than that of Xi ; however, the volumes calculated for the territories occupied by Xa and Xi in optical sections are, in fact, the same despite the more compact appearance of Xi in flattened preparations . It remains to be determined whether the actual nuclear volumes occupied by HSA18 and 19 are different from each other. We have also shown here that transcription affects the topology of chromosome territories since the larger apparent size of HSA19 is only seen in the presence of transcription by RNA polymerase II , i.e., in untreated cells or in cells in which the inhibition of transcription by DRB has been relieved. In the absence of transcription (AMD or DRB treatments) HSA19 occupies a compact territory similar in size, or smaller, than that of HSA18 . We do not see the gross disruption of chromosome territories in the presence of DRB that was reported previously , except in a small minority of both treated and untreated cells that we believe are dying cells. We have no explanation for this discrepancy, but different cell types may have different tolerances and responses to the same concentrations of drugs. The enhanced differences in areas of HSA19 and 18 we recorded when histone deacetylation was inhibited with TSA suggest that levels of steady-state histone acetylation influence the gross architecture of chromosome territories. However, chromosome position within the nucleus is independent of transcription and histone acetylation activities (Table I ). More genomic sequences partitioning with the operationally defined nuclear matrix (MARs) or nuclear scaffold (SARs) derive from HSA18 than from 19 . This, together with the proximity of HSA18 to the lamina , a component of the nuclear matrix, lead us to expect that there might be a tight association of this chromosome with the matrix. However, in Drosophila , sequences isolated as SARs do not correspond with loci at the nuclear periphery and so the relationship between MARs-SARs and sequences that visibly remain inside of the residual nucleus after extraction, rather than in the surrounding halo of DNA loops, is not clear. Indeed, we saw very little retention of chromosome 18 DNA within nuclear matrices in contrast to the retention of chromosome 19 within the bounds of residual nuclei . The degree of extension of chromosome 18 sequences varied between nuclei, but within individual nuclei the two homologues behaved similarly . It has been reported that ∼16 kb of inactive DNA can decondense to cover ∼5 μm in extracted nuclei ; therefore, the 85 Mb of chromosomes 18 extruded from nuclei with high salt retain a substantial degree of higher order structure that is independent of interactions with the nucleus. RNA is an important component of the nuclear matrix, and active genes associate with residual nuclei and not with the nuclear halo . However, retention of HSA19 within the residual nucleus and release of HSA18 was seen in the absence of transcription (AMD treatment). The more central location of chromosome 19 in the human nucleus may be mediated by substantive and transcription-independent association with, as yet unidentified, nuclear proteins that resist extraction from the nucleus with high salt. Jackson and Pombo have demonstrated that early replicating DNA is retained within the residual nucleus of salt-extracted human cells and, indeed, the bulk of HSA19 replicates earlier in S phase than does HSA18 . Subnuclear localization of HSA18 and 19 is not determined by the centromeres of the chromosomes since distinctive localization is retained by regions (<20 Mb) of the chromosome arms of HSA18 and 19 that are translocated to the reciprocal-derived chromosome . We also find no evidence that the telomeres of the chromosomes are attached at the nuclear periphery of human nuclei as has been observed in simpler eukaryotes . We conclude that differences in the overall composition of bulk chromosome 18 and 19 DNA sequences may play a direct role in the nuclear destiny of these two chromosomes and the genes placed upon them. It has been argued that lack of phenotypic abnormalities in individuals with balanced translocations is evidence that spatial arrangement of different chromosomes in the nucleus is not functionally important. This was based on the assumption that such translocations disrupt the normal nuclear location of chromosome domains . However, we have shown that this is not necessarily the case to any significant degree . The diffusion constraints on chromatin movement in vivo mean that the physical proximity of different chromosomes in interphase, and their interactions with nuclear substructure, may be important in determining the likelihood of any two chromosomes meeting and exchanging material in a translocation . The most recurrent chromosome rearrangements in humans are Robertsonian translocations between the acrocentric rDNA–carrying chromosomes that are known to be physically close to one another in both interphase and metaphase . We surveyed two large databases cataloguing balanced translocations in humans ( http://www.hgmp.mrc . ac.uk/ local-data/Cad_Start.html and http://mendel.imag.fr/BACH/transloc/carto/ ) and found that t(18;19) is indeed very rare in the human population when compared with other translocations among small chromosomes. The segregation of different chromosomes of the karyotype with different functional characteristics, described here, is reminiscent of the extreme genome separation seen in plant hybrids . What are the biological consequences of this type of compartmentalization? The chromosomal and nuclear position of a gene can influence its activity and the position of a gene within the nucleus can be dictated by the sequences it is joined to on the chromosome . The edge of the nucleus is also a place where genes are repressed in many eukaryotes . Condensed heterochromatin and later replicating DNA tend to concentrate toward the nuclear periphery in many vertebrates , whereas early replicating DNA and poly(A) RNA partition toward the nuclear interior . At the level of individual loci some, but not all, active mammalian genes have been found predominantly within the nuclear interior, whereas inactive genes have been located at the nuclear and nucleolar peripheries . The number of gene-based markers that has been assigned to HSA18 is small in comparison to those located on HSA19 . Because of their location within the nuclear space, the relatively small number of genes located on human chromosome 18 might be habituated to very different types of transcriptional regulation to those on HSA19. Flexibility of expression of exogenous genes placed into these two different chromosome environments may also differ. This might impose constraints on the chromosomal position of genes through evolution. | Study | biomedical | en | 0.999997 |
10366587 | Human normal primary fibroblasts were obtained from a skin biopsy performed on a healthy female donor. They were grown in RPMI medium supplemented by 10% fetal calf serum and 100 μg/ml ampicillin. For in situ analysis, cells were grown directly on two-chamber glass slides (Labtek). Heat treatment was performed by immersing the slides or the flasks in a waterbath set up at 42 or 45°C. Cadmium was used at a final concentration of 75 μM for 4 h and azetidine was used at a final concentration of 10 mM for 2 h. The pH 2.3 genomic probe covering the entire coding sequence (2.3 kb) of the human hsp70 gene was used to detect hsp nuclear transcripts . The hsp70 gene was detected using the cosmid clone 12HI which contains a portion of the coding sequence of hsp70 (kindly provided by Dr. R.D. Campbell, University of Cambridge, Cambridge, UK). cDNA probes specific for hsp90α (pHS 801) and hsp90β (pHS 811) genes were obtained from Dr. E. Hickey (University of Nevada, Reno, NV). pHS 801 and 811 probes contain, respectively, 1.3 and 0.9 kb of the coding region . All probes were labeled by random priming with biotin-14-dATP ( GIBCO BRL ). The mouse monoclonal antibody specific for the non-snRNP splicing factor SC35 ( Sigma ) was used at a dilution of 1:250 for immunofluorescence . The mouse monoclonal Y12 directed against the Sm protein of snRNPs was obtained from Dr. J.A. Steitz (Yale University, New Haven, CT) and used at 1:250 . The mouse monoclonal antibody against the U2B″ splicing component (Cappel) was used at 1:50 . The mouse monoclonal POL3/3 antibody against RNA Pol II was obtained from Dr. E.K. Bautz (University of Heidelberg, Heidelberg, Germany) and used at 1:200 . The mouse monoclonal CC-3 antibody against RNA Pol II was obtained from Dr. M. Vincent (University of Laval, Quebec, Canada) and used at 1:500 . The mouse monoclonal MARA3 antibody against RNA Pol II was obtained from Dr. B.M. Sefton (Salk Institute, La Jolla, CA) and used at 1:200 . 100 ng of the cDNA probe or 100 ng of the cosmid probe was precipitated with 30 μg of salmon sperm DNA. 3 μg of human Cot I competitor DNA ( GIBCO ) was also added to the cosmid probes. The pellets were resuspended in 50% formamide/10% dextran sulfate/2× SSC, and denatured for 5 min at 75°C. Cosmid probes were incubated 1 h at 37°C to allow suppression of repeated sequences . Immunofluorescence combined to FISH was performed as described previously . FISH was performed first to enhance the efficiency of hybridization. Briefly, cells were fixed in 4% formaldehyde/PBS. A first permeabilization step was performed by three successive incubations of 5 min each in 0.5% saponin/0.5% Triton X-100/PBS. After an equilibration step in 20% glycerol/PBS for 20 min, cells were freeze-thawed three times successively by briefly dipping in liquid nitrogen as a second permeabilization step. Cells were subsequently dehydrated through sequential incubations in 70%, 90%, and 100% ethanol baths for 5 min each. The denatured probe was applied to the dry slide and hybridization was allowed to run overnight. After hybridization, probes were detected using avidin-FITC ( Sigma ). After postdetection washes in 4× SSC/0.1% Tween 20, a 45-min blocking step in 10% FCS/0.3% Triton X-100/PBS was performed, followed by incubation for 90 min at 37°C with the anti-SC35 or the Y12 antibody. Antibody staining was revealed using an anti–mouse-TRITC antibody ( Sigma ) and nuclei were counterstained with 250 ng/ml DAPI (4′, 6-diamidino-2-phenylindole/2 HCl) ( Sigma ) diluted in an antifading solution consisting in 90% glycerol, 20 mM Tris-HCl, pH 8.0, 2.33% DABCO (1–4 diazabicyclo octane) ( Sigma ). Immunofluorescence was performed first in this case. After detection with the secondary antibody, cells were rinsed three times with PBS, and subsequently denatured by a 3-min incubation in 70% formamide/2× SSC at 75°C, followed by a 1-min incubation in 50% formamide/2× SSC at the same temperature. The denatured cosmid probe was then applied to the slide. After hybridization, cells were washed three times for 5 min in 50% formamide/2× SSC at 45°C, and three times for 5 min in 0.5× SSC at 60°C. Detection was then performed as described above for nuclear transcript detection. BrUTP incorporation was performed according to a protocol derived from Wansink et al. . Briefly, cells were rinsed once with PBS for 3 min, and once with a glycerol buffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 25% glycerol, 0.5 mM PMSF, 0.5 mM EGTA) for 3 min. Cells were then permeabilized by incubation for 3 min in the same buffer complemented with 0.05% Triton X-100 and 10 U/ml RNAsin, and subsequently incubated with the transcription cocktail (100 mM KCl, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 0.5 mM EGTA, 25% glycerol, 1 mM PMSF, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.2 mM BrUTP, 160 μM S -adenosyl-methionine, 25 U/ml RNAsin) for 15 min at room temperature. At the end of the reaction, cells were rinsed once in PBS containing 0.05% Triton X-100 and 5 U/ml RNAsin, once in PBS added with 0.5 mM PMSF and 5 U/ml RNAsin, and subsequently fixed in 4% formaldehyde in PBS. Incorporation sites were revealed using a mouse anti-BrdU antibody ( Sigma ) and anti–mouse FITC ( Sigma ). Images were acquired using a confocal laser scanning microscope ( Zeiss LSM 410) using a 63×, 1.25 NA oil immersion objective. Confocal images were analyzed for the relative distribution of the speckles and hsp transcription sites using software developed at the University of Grenoble . Deconvolution was used to revert the distortion of fluorescent signals due to the point spread function of the microscope which allowed our ability to define the limits of the speckles. Transcription sites were defined as associated with a speckle when no pixels were separating the two fluorescent signals. Percentages were determined based on the analysis of 100 nuclei which corresponds to 200 sites of gene transcription. The RT-PCR reaction was performed as described in Wang et al. . The reaction was internally controlled by including known amounts of internal control transcripts corresponding to the same genes carrying small deletions to distinguish from wild-type, thus allowing us to precisely quantify the levels of transcripts . Total RNAs were extracted using the procedure described by Gough . Briefly, cells at 80% confluency were scraped and spun down. The cell pellet was resuspended in 200 μl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 2.5 mM DTT), spun again, and the supernatant was added to 200 μl of ice-cold buffer B (7 M urea, 0.35 M NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, 1% SDS). 400 μl of phenol/chloroform (1:1) was then added, and RNAs were precipitated as described. Specific sense and antisense primers for hsp70, hsp90α, and hsp90β transcripts ( GIBCO BRL ) were designed using the MacVector program as follows (the numbers indicate positions on the wild-type transcripts): hsp70 sense: 5′-TTCCGTTTCCAGCCCCCAATC-3′ (nucleotides [nt] 435–455); hsp70 antisense: 5′-CGTTGAGCCCCGCGATCACA-3′ (nt 993–974); hsp90α sense: 5′-AAAAGTTGAAAAGGTGGTTG-3′ ; hsp90α antisense: 5′-TATCCACAGCATCACTTAGTA-3′ ; hsp90β sense: 5′-AGAAGGTTGAGAAAGGTGACAA-3′ ; hsp90β antisense: 5′-AAGAAGTTAGAGAGGGAATAAA-3′ . The expected sizes of PCR products are 641, 625, and 558 bp for wild-type hsp90β, hsp90α, and hsp70 transcripts, respectively, and 531, 499, and 438 bp for in vitro transcribed hsp90β, hsp90α, and hsp70 transcripts, respectively. The reaction was performed in a total volume of 20 μl. 2 μg of total RNAs was incubated for 1 h at 37°C with 3.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 10 mM DTT, 0.5 mM dNTP, 20 pmol of each antisense primer, 30 pg of each internal control transcript, 400 U RNAsin, and 400 U of Moloney murine leukemia virus reverse transcriptase ( Pharmacia ). PCR reactions were performed in a final volume of 50 μl. To the 20 μl of the reverse transcription reaction were added (to final concentrations): 3.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 20 pmol of each sense primer, 0.5 mM dNTP, 1 μCi [α- 32 P]dATP, 0.01 μg/μl DNase-free RNAse A, and 50 U Taq polymerase ( Pharmacia ). The reactions were performed in a Pelletier effect thermal cycler (MJ Research, PTC100) for 35 cycles (each cycle: 1 min at 92°C, 1 min at 56°C, 1 min at 72°C) with an initial denaturation of 1 min at 94°C and a final extension at 72°C for 10 min. PCR products were analyzed on a 4% acrylamide, 42% (wt/vol) urea denaturing gel. The levels of wild-type hsp70, hsp90α, and hsp90β transcripts were quantified using the PhosphorImager analyzer system (Molecular Dynamics) and normalized by the amount of the corresponding internal control transcripts. Run-on transcription reactions were performed with isolated cell nuclei in the presence of 50 μCi of [α- 32 P]UTP ( Amersham ) as described previously . After precipitation, radioactive RNA was hybridized to DNA probes for the human hsp70 gene (pH 2.3), human hsp90α (pHS 801), pBR322 as a control for nonspecific hybridization, and the rat gapdh gene as a normalization control for transcription. The intensities of radioactive signals were quantitated using the PhosphorImager analyzer system (Molecular Dynamics). We investigated the relative distribution of SC35 splicing factor and sites of hsp70 or hsp90α genes in normal human fibroblasts. Our rationale for selection of the hsp90α and hsp70 genes was based on three criteria: (a) both genes are transcribed at a low basal rate in cells at normal growth temperatures; (b) the transcription rates of both genes are induced to high levels upon exposure to heat shock and other stresses; and (c) the hsp90α gene contains 10 introns whereas the hsp70 gene is intronless. The relative distribution of hsp70 or hsp90α transcription sites and SC35 splicing factor was analyzed by using a procedure combining immunofluorescence for the detection of splicing factors and FISH for the detection of hsp nuclear transcripts . We have demonstrated previously that hsp70 and hsp90α gene expression is induced by heat shock and other stresses . At 37°C, hsp90α transcripts are constitutively detected whereas hsp70 mRNAs were undetectable . This corresponded, by transcriptional run-on analysis, to a very low basal rate of hsp90α gene transcription at 37°C while hsp70 gene transcription was repressed . Within the nucleus of diploid fibroblasts, hsp transcripts detected by FISH appear as two foci . Because hsp90α transcription rate is low, the foci detected by FISH may correspond partially to nascent transcripts which are retained at the site of transcription, as has been shown for hsp70 transcripts . Codetection of the transcripts and the corresponding gene by FISH showed a complete overlap of the two hybridization signals at the level of light microscopy (data not shown). For the hsp70 gene whose constitutive expression is too low , we chose to detect the gene itself by FISH. In control cells, SC35 speckles were associated with 30% of the hsp90α transcription sites, averaging over 200 transcription sites . In contrast, only 10% of the signals corresponding to the hsp70 gene were associated with SC35 speckles . No differences in the size or intensity of associated versus nonassociated transcription sites were observed. Nuclear speckles were found by quantitative digital imaging analyses to occupy 5–17% of the nuclear volume whereas hsp transcripts occupy <1% of the nuclear volume. The low percentage of association between SC35 speckles and hsp70 transcription sites consequently reflects random distribution, whereas the 30% association with hsp90α transcription sites is significant and likely reflects the higher basal transcription rate of the hsp90α gene. To address whether the distribution of SC35 speckles and hsp genes is a reflection of introns or of the transcription rate, we exposed the cells to a heat shock at 42°C or 45°C, conditions which result in a dramatic elevation of heat shock gene transcription . The analysis of hsp90α and hsp70 gene transcription rates, by nuclear run-on analysis, revealed that both genes were induced strongly following a 42°C or 45°C heat shock (5- and 12-fold induction for the hsp90α gene, and 14- and 35-fold induction for the hsp70 gene at 42°C and 45°C, respectively) . Quantification of the mRNA levels by RT-PCR revealed a 11.4-fold (42°C) and 29.7-fold (45°C) induction of hsp70 mRNA levels and a 1.5-fold (42°C) and 2.5-fold (45°C) induction of hsp90α mRNA. As expected, the fold-induction of hsp90α transcripts determined by measuring mRNA levels was lower than for hsp70 due to the higher basal levels of hsp90α transcripts in control cells. hsp70 or hsp90α transcripts were detected together with the SC35 splicing factors in heat-shocked cells. As shown in Fig. 3 , 92% of hsp90α transcription sites were observed to be adjacent to SC35 speckles in the 42°C treated cells and 94% in the 45°C treated cells . Likewise, 92% and 93% of the chromosomal sites of hsp70 transcription were associated with a SC35 speckle in cells exposed to 42°C and to 45°C , respectively. Identical results were obtained in cells exposed at 42°C or 45°C for only 10 min (data not shown), attesting that the association of splicing factors with transcribing genes is a very rapid process, directly correlated to the transcriptional activity of the gene and not due to major rearrangements of the nuclear architecture as a consequence of heat shock. The high degree of spatial coincidence between SC35 speckles and sites of hsp gene transcription following activation of the heat shock response reveals that a key feature of recruitment of SC35 splicing factors relates to the dynamics of transcription. A similar spatial association of the heat-activated hsp90β gene with the speckles has been reported previously . Our results demonstrate, however, that the splicing factors do not distinguish between intron-containing and intronless genes. Activated hsp genes were not found to be preferentially associated with larger speckles as has been observed for fibronectin transcripts , perhaps reflecting a gene specificity in the pattern of association with the speckles. To what extent do our observations reflect features of SC35 which are not general to splicing complexes? To ensure that our results were not limited to the SC35 splicing factor or due to the fact that the anti-SC35 antibody only recognizes a phosphoepitope of the protein, we performed the same experiments on control and heat-shocked cells with the Y12 antibody to detect snRNPs or with an anti-U2B′′antibody (data not shown). The results obtained for both antibodies revealed the association of both hsp70 and hsp90α transcription sites (data not shown) with the speckles only in cells exposed to 42°C. To exclude that the redistribution of splicing factors following heat shock was due solely to the effects of elevated temperatures on nuclear organization, heat shock gene transcription was activated by exposure to azetidine or cadmium . Both treatments resulted in an increase in hsp70 and hsp90α mRNA levels comparable to those induced by a 42°C heat shock (i.e., a 10.9- and 12.1-fold increase in hsp70 mRNA levels, and a 1.4- and 1.5-fold increase in hsp90α mRNA levels in azetidine- and cadmium-treated cells, respectively) . This was corroborated by transcriptional run-on assay showing that both genes were actively induced in cadmium- and azetidine-treated cells (data not shown). As shown in Fig. 5 , 94% and 95% of the hsp90α transcripts were associated with SC35 speckles in azetidine and cadmium-treated cells , respectively. Similarly, 90% of the hsp70 transcription sites were associated with SC35 speckles in azetidine-treated cells and 93% in cadmium-treated cells . These observations confirm and extend the results of the heat-induced association of hsp70 and hsp90α transcription sites with the speckles, and demonstrate that the dynamic relocalization of splicing factors is not caused by the thermal effects of heat shock but is primarily a reflection of the elevated rates of transcription of both gene loci. Human primary fibroblasts are relatively resistant to heat shock and display a very low percentage of cell death following a 1-h heat shock at 45°C . In addition, general features of nuclear morphology visualized by light microscopy do not appear to be altered by heat exposure . To address whether the different inducers of heat shock gene expression employed here caused a transcriptional arrest, we monitored general transcriptional activity by visualizing the sites of BrUTP incorporation into nascent transcripts . As shown in Fig. 6 , there was no detectable change in the transcriptional pattern in cells treated with either heat shock, cadmium, or azetidine when compared to control cells. We next investigated the distribution of RNA Pol II for its presence within the speckles and to determine whether the various stress conditions influenced its distribution. Since substantial variations in Pol II distribution depending on the cell type and the specific antibody used have been reported , we used three characterized antibodies recognizing different epitopes on the RNA Pol II. As previously reported for other cell types, the POL3/3 antibody, which recognizes an epitope outside of the COOH-terminal domain (CTD) of Pol II and is independent of the phosphorylation state of the enzyme, shows a diffuse nucleoplasmic staining at 37°C . In contrast, the CC-3 antibody, which recognizes a phosphoepitope in the CTD, stains a subpopulation of Pol II concentrated in speckles . The MARA3 antibody, which recognizes a phosphoepitope in the CTD different from CC-3, stains both a diffuse population and a subpopulation of the RNA Pol II concentrated in the speckles . As previously shown by others, these different patterns correspond to different subpopulations of RNA Pol II. In our human primary fibroblasts, at least two distinct hyperphosphorylated forms of RNA Pol II appear to concentrate in the speckles; however, whether these subpopulations represent active forms of the enzyme is still unknown. None of these patterns were altered by a 42°C or 45°C heat shock , or by cadmium and azetidine treatments (data not shown). These observations showed that in human fibroblasts at least a subpopulation of RNA Pol II was localized to the speckles and that the overall distribution of the enzyme was not affected dramatically by stress. As RNA splicing is affected by heat shock in many organisms , we chose to examine whether the differential distribution of hsp genes relative to SC35 speckles during stress was a consequence of a potential stress-induced arrest in RNA splicing. In human cells, heat shock results in a redistribution of snRNPs from the speckles to a diffuse nucleoplasmic pattern . It has also been shown that extracts from HeLa cells heat-shocked at 43°C or higher temperatures are unable to form a functional spliceosome; however, the putative factor(s) inactivated by heat remains unidentified . To examine whether the association of hsp transcription sites with speckles was due to a heat-induced retention of unprocessed hsp transcripts at the sites of transcription, we analyzed the transcripts of the two intron-containing hsp90α and hsp90β genes from cells exposed to heat shock at 42°C or 45°C, cadmium, or azetidine by RT-PCR using primers surrounding an intron. As shown in Fig. 1 a, the hsp90α and hsp90β transcripts detected under all conditions corresponded only to the expected processed species with no detection of the predicted precursor species. This study is the first to investigate, in mammalian cells, the relative distribution of splicing factors and endogenous intronless or intron-containing genes with relation to their inducible transcriptional activity. At 37°C the inactive hsp70 gene was associated randomly relative to the distribution of nuclear SC35 speckles, whereas the hsp90α gene was weakly associated with splicing speckles, consistent with a low but detectable basal transcription. When the cells were exposed to various stressors which resulted in the inducible transcription of the hsp genes, both hsp70 and hsp90α genes became associated with the speckles. The association of splicing factors with the new sites of transcription is a rapid process, occuring immediately upon gene activation. In addition, at least two subpopulations of hyperphosphorylated RNA Pol II were found to concentrate in the speckles, and this distribution was unaffected by stress. Altogether our data demonstrate that the association of specific genes with splicing factors is a reflection principally of the transcription rate of the endogenous cellular gene and does not depend upon the presence of introns in the primary transcript. These observations complement the recent findings by Smith et al. that some intron-containing pre-mRNAs are poorly associated with increased concentrations of SC35, both demonstrating a disconnection between the presence of introns and the spatial association with splicing factors. An important conclusion of our results is that the association of splicing factors with the sites of transcription of endogenous genes is best indicated by the level of transcriptional activity. Under control conditions where heat shock genes are either repressed or transcribed at low basal levels, they are randomly distributed in regards to splicing factors or weakly associated with them, whereas upon gene activation, splicing factors accumulate rapidly at the sites of abundant nascent transcripts. The significance of the 30% association of hsp90α transcription sites with the speckles at 37°C is uncertain. Since we did not observe a significant difference in the fluorescent intensity of associated versus nonassociated transcription sites, this may reflect variation in the relative rate of transcription at the chromosomal loci. Alternatively, this could reflect variation in the local concentration of splicing factors and the limitations of light microscopy. Finally, high concentrations of splicing factors may not be required at the sites of transcription, which may at least in part correspond to sites of accumulation of full-length mature nascent transcripts rather than growing RNA molecules . Our results are in good agreement with several previous studies, showing a redistribution of factors involved in transcription, pre-mRNA processing, and RNA packaging in response to changes in gene activity. Indeed, splicing factors have been shown to relocalize in response to RNA Pol II inhibition , to viral infection , or to inhibition of pre-mRNA splicing . Moreover, the localization of splicing factors to Balbiani ring genes occurs in a transcription-dependent manner in the nuclei of Chironomus tentans , and splicing factors are associated with the loops of lampbrush chromosomes in amphibian germinal vesicles . In human cells, several in situ studies have shown that the nuclear speckles are associated predominantly with transcriptionally active cellular and viral genes . Other observations, however, indicate that certain highly spliced endogenous pre-mRNAs are poorly associated with increased concentrations of SC35 . Similarly, some viral transcripts display little or no association with speckles . Altogether, these findings suggest that transcription is not sufficient for the association of specific genes with nuclear speckles and reveal a more complex view of the nuclear compartimentation of transcription and splicing activities. The most likely hypothesis to integrate our observations in the context of other results is the existence of a gene specificity and/or cell type specificity in the distribution of splicing factors regions . Likewise since some active genes predominantly associate with larger speckles , we can imagine that other active genes preferentially associate with very low amounts of splicing factors which can be missed due to the limitations of microscopic resolution. The concentration of splicing factors associated with specific transcription sites may be gene-specific and may depend on the combined effect of several factors such as the size and complexity of the gene, its transcription rate, and its position in the nucleus and/or its chromosomal environment which can impose structural constraints, thus limiting the access of splicing factors to these regions . Our work now adds to this by demonstrating that the presence or absence of introns is not a determining factor for the association of active genes with splicing factor–rich regions. The functional significance of splicing factors organized into subnuclear structures has been uncertain, although much of the data in the literature are consistent with a role of splicing factor complexes associated with the transcription and processing of intron-containing genes. The association of splicing factors with intron-dependent sites of transcription was demonstrated in experiments transiently or stably expressing intronless and intron-containing genes in HeLa cells . The recruitment of splicing factors to sites of transcription based on a phosphorylation cycle which is tightly correlated with splicing activity also supports this conclusion . Consequently, how do we incorporate the observations of the present study, that splicing factors can also be associated with sites of transcription independent of introns? In our analysis, we examined the sites of transcription of endogenous cellular genes, in contrast to genes reintroduced by transfection or expressed following viral infection . The introduction of exogenous nucleic acids, by transfection, could potentially influence aspects of nuclear organization with consequences on endogenous transcriptional activities and a redistribution of splicing factors . Likewise, the genomic site of integration in stably transfected constructs or integrated viral genomes may also influence their expression . The association of the intronless hsp70 gene with speckles could be the result of the physical proximity on the chromosome of a distinct, highly transcribed, intron-containing gene, although this seems unlikely given that our data show a very high percentage of association between hsp70 transcription sites and the speckles. In addition, this association is strictly observed in stressed cells, suggesting that the putative neighboring gene would need to be stress-responsive. The only known hsp gene located in the vicinity of the hsp70 gene, which is located in the 6p21.3 region , is the hsp90β gene which maps to the 6p12 locus ; the physical distance between these two genes allows a clear discrimination between the two transcription sites by light microscopy . Thus, the association observed between the intronless hsp70 transcription sites and the speckles is significant. A more likely explanation for the different interpretations is that the distribution of transcription sites relative to nuclear speckles varies in a gene-specific manner. Indeed, differential distributions of viral or endogenous transcripts relative to nuclear speckles have been reported already, and they may reflect differences in the organization of nuclear RNAs derived from endogenous or from integrated viral genomes . For example, some transcriptionally active genes containing introns display little or no association with SC35 speckles , whereas the collagen gene which has numerous introns is associated with speckles independent of its transcriptional activity . In that respect, we cannot rule out the possibility of a gene-specific organization of transcription sites with respect to nuclear regions enriched in splicing factors. Of potential interest is the observation that active hsp transcription sites are found adjacent to the speckles rather than colocalizing with them, suggesting that transcription and splicing activities occur preferentially at the edges of the speckles where PFs seem to be localized as described by electron microscopy . A similar observation has been reported for several other genes . This may represent a mechanism to accelerate the release of nascent transcripts from the sites of transcription, which would indeed be affected if transcription and splicing were to take place in the core of the speckle where the concentration in RNA Pol II and processing factors may be elevated. Alternatively, this situation may simply reflect gene-specific differences in the association with speckles. Whatever the hypothesis, our data show that the distribution of a specific active gene as adjacent or overlapping with speckles does not rely on the presence of intronic sequences in the gene. Why are splicing factors present at sites in the nucleus where they are apparently not required? A first hypothesis to explain the recruitment of splicing factors to an intronless gene is that complexes containing splicing factors could have other functions at the site of transcription in addition to intron excision. This suggestion is supported by a recent work in which we have shown that full-length nascent hsp70 transcripts are retained at the site of transcription for a period of <15 min after their completion . Perhaps splicing factors could be involved in a primary step of the transcription/splicing process to scan nascent transcripts for the presence of introns. An alternative explanation is that active transcription sites would in fact associate with a subset of active RNA Pol II to which splicing factors are bound. This is supported by observations that splicing factors and other mRNA processing enzymes interact transiently with the hyperphosphorylated CTD of RNA Pol II . Even in mitosis when transcription is arrested, the association between splicing factors and RNA Pol II persists . At the cell biological level, at least a subset of RNA Pol II colocalizes with splicing factors within the speckles , as well as factors involved in the 5′ capping and in the 3′ mRNA processing . The corecruitment of splicing factors and RNA Pol II, together with the movement of genes towards speckles, could be beneficial for the cell in several ways. First, it would accelerate the splicing reaction. Second, by ensuring a sufficient amount of splicing factors at the site of transcription, this system would decrease the risk of producing unspliced transcripts which may generate aberrant and nonfunctional proteins. Third, this system would provide a feedback control mechanism to arrest transcription if splicing is interrupted . Fourth, it would minimize the information quantity required to displace the different factors to the sites of transcription, since the polymerase and splicing factors would all be displaced together as a single complex. This would suggest the existence of signals both in the transcription and processing apparatus and at the level of the chromosome and/or within the primary transcript, which determine the recognition between active transcription sites and proteins involved in RNA biogenesis. Such targeting and/or retention signals, if localized to the nascent transcripts, are not contained within the introns as demonstrated by our results. Identifying these signaling pathways will provide clues to understand the mechanisms of production and trafficking of RNAs within the nucleus. While a growing number of observations support the idea of a compartimentation of transcription and processing activities in the nucleus, we may still have a simplified view of a more complex biological situation generated by the combination of multiple factors varying in a gene-specific and/or cell type–specific manner, therefore proving to be invalid for certain transcripts. Of particular importance may be the chromosomal context of the gene, which could dramatically influence the supply of splicing factors to the gene because of physical constraints . We are convinced that further understanding of the complex organization of transcription and splicing activities within the cell nucleus will come from a large scale analysis of specific endogenous genes, in particular genes with distinctive transcriptional and splicing characteristics such as heat shock genes or genes expressed in a tissue-specific manner. | Study | biomedical | en | 0.999999 |
10366588 | A fragment corresponding to human ASF/SF2 RS domain (amino acids 198–248) was PCR amplified and inserted into BamHI and XhoI sites in either pGEX-5X-1 ( Pharmacia Biotech ) or pMal-c2 ( New England Biolabs ). This fragment was also cloned between EcoRI and SalI sites of pLexA ( Clontech, Inc. ) for the construction of the yeast two-hybrid library screening bait plasmid. The plasmid encoding human SC35 RS domain (amino acids 90–222) was made by ligation of the BamHI-XhoI fragment of SC35 RS domain amplified by PCR into pGEX-5X-1. The full-length of TRN-SR was amplified by PCR with Pfu DNA polymerase (Stratagene) and inserted as an EcoRI-XhoI fragment into pET28A (Novagen). Proteins were overexpressed in the BL21(DE3) Escherichia coli strain and were purified by methods that the manufacturers recommend. Glutathione-S-transferase (GST)-SV-40 T NLS, GST-IBB, and GST-M9 proteins were purified as described . His-tagged RanQ69L (GTP form) was purified as described previously . Nuclear import assays were performed as described . Rabbit reticulocyte lysate ( Promega ) was used as a cytosol source and prepared as described previously . The transport substrates were added at a concentration of 50 μg/ml. For competition experiments with maltose-binding protein (MBP) fusion proteins, competitors (1 mg/ml) were added to the complete transport mix except transport substrate, incubated on ice for 15 min, and then combined with transport substrate and nuclei. For the import experiments with recombinant receptor protein, recombinant His-tagged TRN-SR and His-tagged RanGDP were added at concentrations of 120 and 40 μg/ml, respectively. The HeLa MATCHMAKER LexA cDNA library, yeast strains, and cloning vectors were obtained from Clontech, Inc. All library screening and yeast manipulations were carried out as recommended by the manufacturer. Saccharomyces cerevisiae strain EGY48 was transformed simultaneously with pLexA-ASF/SF2 RS and the HeLa cell cDNA library. 2 × 10 6 transformants were plated onto 20 150-mm plates of X-gal–synthetic medium lacking histidine, uracil, tryptophan, and leucine. 32 Leu + growers that had shown blue color on those plates were isolated. Insert cDNAs were amplified by PCR on these yeast cells using the Advantage-HF™ PCR kit ( Clontech, Inc. ) and sequenced. The PCR fragment from clone 1-1 was used as a hybridization probe to screen the λ phage HeLa cell cDNA library ( Clontech, Inc. ). Several clones were isolated, and the clone that had the longest insert was sequenced and thus determined as the full-length coding sequence of TRN-SR. TRN-SR was produced by in vitro transcription-translation of His-TRN-SR, using a TNT kit ( Promega ) in rabbit reticulocyte lysate in the presence of [ 35 S]methionine ( Amersham ) according to the procedure that the manufacturer recommends. Purified recombinant GST and GST fusion proteins (5 μg each) were immobilized on 50 μl of glutathione-Sepharose ( Pharmacia ) in PBS for at least 1 h at 4°C. The resin was washed with 500 μl of binding buffer (50 mM Tris-HCl, 400 mM NaCl, 5 mM MgOAc, 2 μg/ml of leupeptin, 2 μg/ml of pepstatin, 1% aprotinin, and 0.05% [wt/vol] digitonin; Calbiochem ). In vitro translated TRN-SR was added and incubated with these immobilized proteins for 1 h at 4°C. For the experiments to check the effect of exogenous Ran protein, His-tagged RanQ69L (GTP form) was added at a concentration of 2 μM. The resin was washed with 500 μl of binding buffer five times and the bound fraction was eluted by boiling in SDS-PAGE sample buffer. The bound fraction was then analyzed by SDS-PAGE and visualized by fluorography. The binding experiments with recombinant proteins were done essentially as described above except 20 μg of His- and T7-tagged recombinant TRN-SR was used. Binders were analyzed by 12.5% SDS-PAGE, and detected by an anti-T7 monoclonal antibody (Novagen) and ECL system ( Amersham ). Purified SR proteins were kindly provided by Dr. Akila Mayeda prepared from HeLa cells as described previously . 10 μg of proteins was analyzed by SDS-PAGE and transferred to nitrocellulose membrane. Far Western blotting was performed as described previously by using either TRN-SR or TRN1 produced by a TNT kit ( Promega ) in rabbit reticulocyte lysate in the presence of [ 35 S]methionine ( Amersham ). To characterize the import pathway for SR proteins, we carried out in vitro nuclear import assays in digitonin-permeabilized HeLa cells . As a substrate we used recombinant GST fused to amino acids 198–248 of ASF/SF2 which corresponds to the RS domain (GST-ASF/SF2 RS) of this protein . Efficient nuclear import of GST-ASF/SF2 RS was observed in the presence of cytosol and an ATP-regenerating system . As no import was detected without addition of cytosol , this indicates that nuclear import of ASF/SF2 requires additional soluble factor(s). Efficient nuclear import of GST-ASF/SF2 RS was observed in the presence of an ATP-regenerating system and was reduced by incubation with apyrase , suggesting a role for NTPs in this process. The import of GST-ASF/SF2 RS was strongly inhibited by RanQ69L, a Ran mutant that cannot hydrolyze GTP at a significant rate , suggesting a role for RanGTP. GST-ASF/ SF2 RS import also has several characteristics of nuclear import that occur through NPCs. Both WGA and an importin β dominant-negative mutant (Impβ ΔN44), reagents which block active nuclear import through NPCs , completely abolished GST-ASF/SF2 RS nuclear import . To determine whether a specific and saturable factor(s) participates in RS domain–mediated nuclear import, we tested the effect of excess RS domain on the import of classical NLS, M9, and RS domain–bearing proteins. For these experiments we prepared an MBP fusion of the ASF/SF2 RS domain, termed MBP-RS, as a competitor. Nuclear import assays were carried out in the presence of a 20-fold molar excess of either MBP or MBP-RS. MBP itself had no effect on nuclear import; however, MBP-RS strongly inhibited GST-ASF/SF2 RS import, whereas import of other substrates, GST-SV-40 T NLS, GST-IBB, and GST-M9, was unaffected or only slightly reduced . These results suggest that a specific nuclear import receptor, distinct from importin β and TRN1, mediates RS domain nuclear import. To identify candidate mediator(s) of SR protein nuclear import, we carried out a yeast two-hybrid screening on a HeLa cell cDNA library using the COOH-terminal 51– amino acid region of ASF/SF2 as bait. This fragment contains the RS domain and is sufficient for complete nuclear localization of myc-tagged pyruvate kinase in HeLa cells (data not shown). Several positive interacting clones were isolated and characterized. One of these, clone 1-1, was isolated four times out of 16 clones, and its deduced amino acid sequence showed significant similarity to that of a putative importin β/transportin-related nuclear transport receptors. The 1-1 DNA insert was subcloned and used for hybridization screening of a λ phage HeLa cDNA library. A 3-kb clone that appears to contain the entire coding region was obtained. The predicted amino acid sequence of this protein, which we termed TRN-SR, because it turned out, like TRN1, to be a transport receptor of pre-mRNA/mRNA-binding proteins, is shown in Fig. 3 . TRN-SR is a 975– amino acid protein with a calculated molecular mass of 109,838 D and an estimated pI of 5.29. The amino-terminal domain of TRN-SR shows significant sequence similarity to other importin β/transportin family members, including a region required for RanGTP binding . The sequence of the original 1-1 clone isolated from the yeast two-hybrid screening starts at amino acid 590 of the TRN-SR sequence and contains the entire COOH-terminal domain. A BLAST homology search with full-length TRN-SR revealed three proteins that bear significant homology to TRN-SR in other species . The most similar of these, AF025464 of Caenorhabditis elegans , is 26% identical and 45% similar to TRN-SR. Another apparent homologue is AL022304 of Schizosaccharomyces pombe that is 25% identical and 46% similar, although this clone does not appear to contain the full-length protein sequence. These two sequences are the two closest orthologues of TRN-SR present in available databases. Of previously characterized proteins, the most significant similarity is found with the S . cerevisiae protein Mtr10p which has been shown recently to be a nuclear import receptor for Npl3p . Npl3p is an hnRNP protein in yeast . The amino acid sequences of TRN-SR and Mtr10p are 21% identical and 42% similar. To confirm that TRN-SR binds specifically to SR proteins, we carried out in vitro binding experiments using TRN-SR produced by transcription-translation in rabbit reticulocyte lysate. In the same experiments we also tested another RS domain, that of the SR splicing factor SC35 . TRN-SR binds to the RS domains of both ASF/SF2 and SC35, but not to IBB or to hnRNP A1 M9 . RanQ69L abolishes the binding of TRN-SR to RS domains , consistent with the possibility that it is a nuclear import receptor for these proteins. Since rabbit reticulocyte lysate contains many proteins, the binding of TRN-SR detected in Fig. 4 A could be indirect. To examine whether TRN-SR can bind to the RS domains directly, we carried out binding assays using purified recombinant TRN-SR. As shown in Fig. 4 B, bacterially produced TRN-SR binds to both GST-ASF/SF2 RS and GST-SC35 RS directly, but not to GST alone. These results strongly suggest that TRN-SR is a specific import receptor for SR proteins. To determine if TRN-SR is the nuclear import receptor of SR proteins, recombinant TRN-SR was used in in vitro nuclear import assays using either GST-ASF/SF2 RS or GST-SC35 RS as a substrate. Neither GST-ASF/SF2 RS nor GST-SC35 RS by itself accumulated in the nucleus . However, in the presence of ATP, an ATP-regenerating system and RanGDP, TRN-SR efficiently imported GST-ASF/SF2 RS and GST-SC35 RS into the nucleus . Thus, TRN-SR is a nuclear import receptor for ASF/SF2, SC35, and likely for other RS domain–containing proteins. Mammalian cells contain several SR proteins in addition to ASF/SF2 . Since TRN-SR binds the RS domains of both ASF/SF2 and SC35 , we examined whether it can also bind other SR proteins. The SR protein fraction was purified from HeLa nuclear extracts , resolved by SDS-PAGE, and immobilized on a nitrocellulose membrane. By Western blotting with the anti-RS domain antibody mAb104, these purified SR proteins show the typical pattern reported previously . The capacity of TRN-SR to bind these proteins was determined by far Western blotting using 35 S-labeled TRN-SR produced in rabbit reticulocyte lysate . TRN-SR bound several of these proteins, whereas TRN1 did not . In addition to proteins of ∼33 kD, that likely correspond to ASF/SF2 and SC35, proteins of ∼20, 46, and 55 kD also bound specifically to TRN-SR. This observed profile is similar to that detected by Western blotting with mAb104 , suggesting that TRN-SR is a common nuclear import receptor for many of the SR proteins. In this report we have identified a novel receptor, TRN-SR, as a nuclear import receptor for SR proteins. Of the known proteins currently present in the sequence databases, we note the considerable amino acid sequence homology of TRN-SR with the S . cerevisiae Mtr10p . Mtr10p has been shown to be a nuclear import receptor for the yeast pre-mRNA/mRNA-binding protein Npl3 . Npl3p, which is also referred to as Nop3p and Nab1p, is an hnRNP protein that contains within its carboxyl terminus an RGG-box within which are several serine-arginine (SR) dipeptides . The NLS of Npl3p has not been precisely delineated but is contained in this region of the protein . The SR dipeptides of Npl3p may be important for Mtr10p recognition, although this has not been determined. Two additional proteins, one in C . elegans and one in S . pombe , show similarity to TRN-SR . Several candidate SR proteins are found in the C . elegans database, and one SR protein has been recently cloned from S . pombe . Therefore, these TRN-SR homologues may be the import receptors of SR proteins in these organisms. TRN-SR binds to the RS domain of ASF/SF2 and of SC35, and these interactions are disrupted by RanQ69L . Furthermore, TRN-SR also binds other proteins enriched in an SR protein fraction . These results strongly suggest that TRN-SR is a general nuclear import receptor for SR proteins. However, we note that no binding of TRN-SR to SRp75 was detected by far Western blotting, although this protein is abundant in the fraction we tested . The reason for this is unknown, but it is possible that SRp75 may have a different receptor. There are additional SR proteins, including pre-mRNA splicing factors such as 9G8, U170K, U2AF35, and 65 , as well as two large SR proteins , and it remains to be determined whether TRN-SR also mediates the nuclear import of these proteins. Several abundant hnRNP proteins, including hnRNP A1, A2, and F, are imported by TRN1 . Thus, in mammalian cells there are at least two nuclear import pathways for pre-mRNA/ mRNA-binding proteins, one mediated by TRN1 and one by TRN-SR. The relative amounts of hnRNP proteins and SR proteins are important for alternative pre-mRNA splicing. For example, the ratio between hnRNP A1 and ASF/SF2 affects 5′ splice site selection . As both of these proteins shuttle between the nucleus and the cytoplasm , it is conceivable that their relative amounts in the nucleus may be controlled by regulating their rates of nuclear import. Thus, by modifying either the transportins themselves or the respective NLSs, M9 and RS, splice site selection could be modulated. Indeed, several protein kinases have been reported to phosphorylate serine residues in the RS domains of SR proteins . While overexpression of some of these SR protein kinases causes disruption of nuclear speckles , they do not disrupt the nuclear localization of SR proteins. However, overexpression of one SR protein kinase, Clk/Sty kinase, does cause cytoplasmic accumulation of ASF/SF2 in HeLa cells . More recently it was reported that overexpression of kinase-inactive mutant of SR protein kinase-2 causes cytoplasmic accumulation of ASF/SF2 . It will be interesting to determine the effect of RS domain phosphorylation on the SR proteins–TRN-SR interaction. The physiological function of the shuttling of SR proteins is not known. Both hnRNP A1/A2 proteins and SR proteins are associated with the same mRNAs as they are exported to the cytoplasm and it is thus possible that they both play a role in mRNA export. Nuclear export signals in the shuttling SR proteins have not been identified yet. The identification of nuclear export signals in shuttling SR proteins, if such exist, and of export receptors for them are issues of considerable interest that remain to be clarified. | Study | biomedical | en | 0.999996 |
10366589 | Yeast strains used in this study are listed in Table I . S . cerevisiae strains were grown in YEPD (1% Bacto yeast extract, 2% peptone, 2% glucose) or SD medium (2% glucose, 0.7% Difco yeast nitrogen base without amino acids, 0.07% amino acids solid mix). Growth supplements were added to SD medium when required. Escherichia coli was grown in LB medium (1% bactotriptone, 0.5% bacto yeast extract, 1% NaCl) supplemented with 100 μg/ml ampicillin. Standard methods were used for DNA manipulations and yeast genetics . Calcofluor resistance was tested by a plate assay in SD medium buffered with 50 mM potassium hydrogen phthalate, pH 6.1, and supplemented with different Calcofluor concentrations (50– 1,000 μg/ml; Bayer Industrial Corporation). Calcofluor staining was observed after growing cells in the presence of 75 μg/ml Calcofluor for 2 h at 30°C. Gene replacement was performed basically as described by Rothstein . The Δ chs7-1 disruption was constructed by replacing the 1.2-kb NdeI–NdeI fragment containing the CHS7 coding region (from nucleotide +24–297, downstream from the stop codon) with the HIS3 gene, thus creating pTM10. The NdeI downstream site is still 724 bp ahead of the next ATG, therefore this deletion should not affect the YHR143 open reading frame (ORF). Strains W303-1A and Y1306 were transformed with a linear 3.5-kb EcoRV–EcoRV fragment from pTM10, containing the chs7 :: HIS3 gene, giving rise to strains JAY6 and JAY27. The Δ chs7-2 disruption was constructed by replacement of the 1.2-kb NdeI–NdeI fragment with the HIS2 gene, affording pTM100. By digestion of pTM100 with SacI, a 4.1-kb fragment containing the chs7 :: HIS2 gene was used to transform strains HVY374 and HVY376, creating strains JAY25 and JAY26. The Δ chs6-1 disruption was constructed using a pGEM- CHS6 plasmid, in which the 1.6-kb BalI–EcoRI internal fragment of CHS6 was replaced with the URA3 gene, thus creating pTM12. By digestion of pTM12 with SalI–SphI, a 3.0-kb fragment containing the chs6 :: URA3 gene was used to transform strain Y1306, obtaining JAY28. Correct replacement of the CHS7 and CHS6 loci was determined by PCR analysis and tests of Calcofluor resistance. To construct a fusion gene encoding the full-length Chs7p fused to the green fluorescent protein , a NotI site was created by directed mutagenesis at the end of the CHS7 coding region to give pTM14. A 0.7-kb NotI–NotI fragment from pTM13 containing the GFP-encoding sequence was ligated into NotI-digested pTM14, thus creating pTM15 (pRS316:: CHS7-GFP ). CHS7 :: 3XHA construction (pTM16) was achieved by inserting a 115-bp NotI–NotI DNA fragment containing three copies of the hemagglutinin epitope (HA) at the same NotI site of pTM14. The functionality of the hybrid proteins was determined by complementation of the phenotypes associated with Δ chs7 . For intracellular localization of Sec63p, 15Daub cells were transformed with plasmid pRS315:: SEC63-MYC , kindly provided by R. Schekman . Multicopy plasmids pRS423:: CHS3 or pRS423:: CHS3-3XHA were obtained by inserting the EcoRI–SalI fragment of pHV7 that contains either CHS3 or CHS3-3XHA genes into pRS423 . pRS425:: CHS4 was done by cloning the CHS4 gene as a BamHI fragment into pRS425 . Similarly, for construction of pRS426:: CHS7 , CHS7 gene was cloned as a EcoRV–StuI fragment (pTM11; this work) into pRS426. Plasmids pRS423, pRS425, and pRS426 have been previously described . Chitin measurements were performed as described using chitinase from Serratia marcescens ( Sigma Chemical Co. ). GlcNAc release was determined colorimetrically by the method of Reissig et al. . Total amounts of chitin are expressed as nanomoles of GlcNAc liberated per 100 mg of cells. For CS activity assays, total cell membranes were prepared from 250 ml of exponentially growing cells (2 × 10 7 cells/ml) as described by Valdivieso et al. . CS activity was measured essentially as described in Choi and Cabib : CSI activity was assayed in 50 mM Tris-HCl, pH 6.5, and 5 mM magnesium acetate; CSII activity was assayed in 50 mM Tris-HCl, pH 8.0, and 5 mM cobalt acetate; and CSIII was assayed in 50 mM Tris-HCl, pH 8.0, with 5 mM cobalt acetate and nickel acetate. For the proteolytic activation step, 2 μl of trypsin (1–3 mg/ml) was added to the reaction medium and proteolysis activation was stopped after 15 min of incubation by adding 2 μl of soybean trypsin inhibitor solution. 1.1 mM UDP[ 14 C]GlcNAc (Nycomed Amersham ; 400 cpm/nmol) was used as substrate for the reaction. Newly synthesized chitin was determined by measuring the radioactivity incorporated into the insoluble material after the addition of 10% trichloroacetic acid and filtration through glass fiber filters . Specific activity is expressed as nanomoles of GlcNAc incorporated per hour per milligram of protein. Localization of Chs7p-GFP was observed in exponentially growing cells containing pTM15. After mounting, images were captured with a Zeiss laser-confocal microscope (LSM 510) with a 63× objective and processed with Adobe Photoshop software. Localization of Chs3p-3XHA by indirect immunofluorescence was carried out as described in Bähler and Pringle with slight modifications. Spheroplasts were obtained by treatment with 5 μg/ml Zymolyase 100T (Seikagaku) for 30–45 min at 30°C in PEMS (100 mM Pipes, 1 mM EGTA, 1 mM MgSO 4 , 1.2 M sorbitol, pH 6.9) buffer. Mouse HA.11 anti-HA mAb (Berkeley Antibody Co.) was used at a 1:100 dilution for 16 h at 4°C. Cy3-conjugated anti-mouse secondary antibody ( Sigma Chemical Co. ) was used at a 1:300 dilution for 45 min at 25°C. After mounting, images were captured with a Zeiss laser-confocal microscope (LSM 510) with a 63× objective. For these experiments, an epitope-tagged version of Chs3p in cells whose chromosomal locus had been replaced with CHS3-3XHA was used . All strains used for immunofluorescence were derived from Y1306. Sec63p-Myc was immunolocalized with a similar protocol, but using supernatants of 9E10 hybridome at 1:100 dilution as primary antibodies . For Northern blot analysis, total RNA was prepared as described by Sambrook et al. from cells grown under different conditions. 12.5 μg of total RNA was loaded per lane and after electrophoresis, transferred to Hybond membranes (Nycomed Amersham ). Hybridization was carried out as described using a 1.2-kb NdeI–NdeI fragment from CHS7 as a probe. For quantitative analysis, Northern blots were exposed to PhosphorImager screens . α-Factor treatment for Northern blot experiments was carried out in exponentially growing cells using YEPD medium supplemented with 5 μM α-factor . Cell lysates were prepared from a JAY25 strain transformed with pRS315:: SEC63-MYC and pRS316:: CHS7-3XHA plasmids. In brief, exponentially growing cells in SD medium (1.5 g wet wt), were resuspended in 5 ml of 17% sucrose (wt/vol) in 50 mM Tris-HCl, pH 7.5, and 1 mM EDTA containing the protease inhibitor cocktail (1 mM phenylmethanesulfonil fluoride and 1 mg/ml each of leupeptin, pepstatin, and aprotinin), and broken by vortexing with glass beads. Lysated cells were centrifuged at 1,500 g for 10 min. The cleared supernatant was layered on top of 33 ml of a linear sucrose gradient (10– 65% wt/vol) in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and centrifuged in a SW28 rotor at 25,000 rpm for 20 h at 4°C. 1.25 ml fractions were collected from the bottom using a peristaltic pump. Equal volumes of each fraction were examined for their content of Pma1p, Kre2p, Sec63p-MYC, and Chs7p-HA, determined by SDS-PAGE and immunoblot with their respective antibodies. Rabbit polyclonal antibodies (1:30,000 dilution; kindly provided by Dr. R. Serrano, Universidad de Valencia, Valencia, Spain) were used to detect Pma1p, a typical plasmatic membrane marker. Kre2p, a medial-Golgi compartment protein , was detected using polyclonal antibodies (1:1,000 dilution) kindly supplied by H. Bussey (McGill University, Montreal, Qúebec, Canada). Sec63-MYC, an ER marker, was detected using monoclonal anti-MYC antibodies (supernatants of 9E10 hybridome, 1:5,000 dilution). Mouse HA.11 anti-HA mAb (Berkeley Antibody Co.; 1:2,000 dilution) was used to detect Chs7p-HA. In all cases, Western blots were developed using ECL (Nycomed Amersham ). Chs3p-3XHA expression levels were determined in total cellular extracts by Western blot analysis as described in Cos et al. . Blotted proteins were incubated with mouse HA.11 anti-HA antibody (Berkeley Antibody Co.; 1:2,000 dilution) and developed using ECL (Nycomed Amersham ). Endoglycosidase H (EndoH) treatment was carried out on Chs3p-3XHA after immunoprecipitation under denaturing conditions as described by Cos et al. . The immunoprecipitated complex was subjected to Western blot analysis. Protein was measured by the method of Lowry et al. . Yeast strains were mated quantitatively as described in Santos et al. . The frequency of diploid formation was estimated as the number of diploids formed out of the total number of cells. Exoglucanase activity was assayed as described by San Segundo et al. ; the method is based on the release of reducing sugar groups from laminarin, which were quantified by the method of Somogyi and Nelson . The α-factor secretion assay was developed as described in Michaelis and Herskowitz . A new screening for S. cerevisiae mutants resistant to Calcofluor was carried out to identify new genes involved in chitin synthesis. Ethyl methanesulfonate-mutagenized JAY1 cells (∼20% of survival) were plated in YEPD media supplemented with 1 mg/ml Calcofluor exactly as described in Roncero et al. . After 4 d of incubation, 53 bona fide independent clones which were able to grow were selected (mutation frequency 1.1 × 10 −5 ). These mutants were back-crossed with previously known Calcofluor resistant and chitin-deficient mutants ( chs3 , chs4 , and chs5 ). Diploid analysis indicated that most mutants belonged to chs3 (83%), chs4 (5.7%), or chs5 (5.7%) complementation groups. Only three of them, cwr6 , cwr12 , and crw19 (Calcofluor white resistant), defined new complementation groups. Further genetic testing indicated that mutants cwr12 and cwr19 belonged to the same group. A preliminary characterization of these mutants indicated that all showed defects in chitin synthesis. The genes affected in cwr12 and cwr6 mutants were cloned by complementation of the Calcofluor resistant phenotype after transformation with a centromeric library . Complementation was confirmed to be dependent on the presence of plasmid and complementing plasmids were isolated in E . coli for further work. Originally isolated plasmids were subjected to endonuclease restriction mapping and the results obtained indicated that the plasmid complementing cwr12 contained the previously described CHS6/CSD3 gene . Therefore, its study was discontinued. The DNA fragment that complemented the cwr6 mutant contains several ORFs . Subcloning experiments indicated that the minimum fragment able to complement was the StuI–EcoRV fragment included in pTM11. With these results, we were able to obtain a preliminary identification of YHR0142w as the gene that complements cwr6 mutation. Due to the defect in CS observed in cwr6 mutant, we named this gene CHS7 following accepted nomenclature . CHS7 encodes a protein of 316 amino acids that contains six or seven putative transmembrane domains . It has no significant homology with any known protein, although the search programs showed limited homologies with other transmembrane regions. We made several chs7 null mutants by replacing the CHS7 ORF by different auxotrophic markers (Materials and Methods). Table II shows some of the characteristic phenotypes associated with this mutation. chs7 null mutants had only 13.6% of the wild-type chitin, a defect that was associated with a comparable decrease in CSIII activity. Due to this defect in chitin synthesis, chs7 cells were resistant to 1 mg/ml Calcofluor and they did not show enlarged septa after Calcofluor treatment. In addition, Δ chs7/ Δ chs7 diploids showed compact ascospores, a defect associated with the absence of the chitosan layer . chs7 mutations also reduced their mating efficiency to a level similar to that of chs3 mutants (Table II ). It should be noted that the original cwr6 mutants showed similar, but less pronounced phenotypes. As expected, wild-type CHS7 in a centromeric plasmid complemented the phenotypes observed in the null (Table II , last column) or point (not shown) chs7 mutants. Once we had constructed null mutants, we confirmed the allelism between YHR142w and CHS7 genetically. Diploid strain yhr142 :: HIS3 / cwr6 was resistant to Calcofluor and analysis of 18 tetrads in this cross showed a Calcofluor resistance segregation of 4:0. Therefore, CHS7 and YHR142w must be the same gene. To analyze CHS7 expression levels, we determined CHS7 mRNA levels in cells harvested after growth under different conditions. CHS7 was detected in all conditions as a single band of ∼1.1 kbp, in clear agreement with sequence data. Therefore, CHS7 is expressed constitutively. However, as shown in Fig. 2 A, α-factor treatment (mimicking the mating process) increased CHS7 expression 3.5 times after only 15 min; shortly after, vegetative levels were resumed. Calcofluor treatment also increased CHS7 expression twofold . Despite its constitutive expression during vegetative growth, CHS7 mRNA levels were strongly increased during sporulation, reaching a maximum (∼24-fold more) after 10 h of sporulation induction. Thereafter, CHS7 mRNA levels slowly decreased. The typical expression pattern observed resembled that of a middle sporulation gene . These results allowed us to conclude that the expression of CHS7 is transcriptionally regulated. In addition, CHS7 transcription increased under all growth conditions in which chitin synthesis was increased. Chs7p was tagged at the COOH terminus either with the 3XHA epitope or GFP (Materials and Methods) to determine the subcellular localization of Chs7p. Chimeric proteins Chs7p-3XHA or Chs7p-GFP complemented the Calcofluor resistance phenotype of chs7 null mutant (JAY6 strain). In addition, the chimeric proteins restored wild-type levels of chitin synthesis in this strain, as determined by chitin staining with Calcofluor . It can be concluded that Chs7p-3XHA and Chs7p-GFP are fully functional. Western blot analysis indicated that Chs7p-3XHA runs as a 35-kD protein that is localized in the particulate fraction (data not shown). Molecular size and subcellular localization are in clear agreement with sequence predictions. Logarithmically growing cells expressing Chs7p-GFP from its own promoter showed the fluorescent staining depicted in Fig. 3 A. Chs7p-GFP was uniformly localized in the nuclear periphery and in discrete patches associated with the cytoplasmic membrane. This pattern coincides with that of Sec63p-Myc , a typical ER marker . The vacuolar staining observed seems to be an artifact because it does not appear in all the cells and it does not show the typical green color of GFP (data not shown). A similar distribution in the ER was observed by indirect immunofluorescence in cells expressing Chs7p-3XHA (data not shown). No polarized distribution of Chs7p was observed in either case. To confirm the immunofluorescence results a subcellular fractionation experiment was carried out. Total cellular extract were loaded into the top of a linear sucrose density gradient and after centrifugation, Pma1p, Sec63p-MYC, Kre2p, and Chs7p-3XHA distribution was analyzed by Western blot as described in Materials and Methods. Fig. 3 C shows that membrane protein Pma1p is localized at the bottom of the gradient, mainly between fraction 1 to 4. Sec63p is localized from fractions 5 to 17, with a significant peak between the 11 to 15 fractions. Chs7p distribution is quite similar with that of Sec63, also showing maximum accumulation between 11 to 15 fractions. Golgi compartment distribution, marked by Kre2p , is displaced to lighter fractions. Therefore, it can be concluded that Chs7p is an ER membrane protein. Functional CSIII activity depends on the appropriate synthesis and transport of Chs3p, its catalytic subunit. Chs7p does not seem involved in the synthesis of Chs3p, since Chs3p levels in Δ chs7 mutants are similar to those observed in wild-type . Therefore, Chs7p could be involved in the transport of Chs3p, a process that is mediated by Chs4p, Chs5p, and Chs6p. Following a strategy similar to that used in the characterization of these genes, we localized an HA-tagged version of Chs3p in wild-type and chs7 -null mutants. As previously reported , Chs3p-3XHA localized in the base of the emerging bud in wild-type strains . However, in Δ chs7 mutants all the Chs3p-3XHA remained in the ER, showing a perinuclear localization with partial association with the plasma membrane . No polarized distribution of Chs3p-3XHA was observed in Δ chs7 mutants. Higher magnification indicated that the perinuclear staining was punctuated, suggesting possible Chs3p aggregation. In addition, it is important to notice that although retained in the ER, Chs3p-3XHA is correctly core-glycosylated in chs7 mutants . Therefore, it can be concluded that Chs7p is required for Chs3p export from the ER and Chs7p must be the initial step in the hierarchy of proteins required for CSIII activity. To confirm this hypothesis, we analyzed the localization of Chs3p-3XHA in Δ chs4 Δ chs7 , Δ chs5 Δ chs7 , or Δ chs6 Δ chs7 double mutants . In all cases, Chs3p was retained in the ER and did not show the localization described for Δ chs4 , Δ chs5 , or Δ chs6 single mutants. As previously reported , the distribution of Chs3p in Δ chs4 mutant was polarized . However, it was not as densely packed at the base of the bud as in the wild-type . In Δ chs5 and Δ chs6 mutants, Chs3p was localized in internal vesicles without polarized distribution . From these results, we conclude that the chs7 mutation is epistatic to the chs mutations and that Chs7p is necessary for Chs3p export from the ER. Chs7p could play a general role in secretion or protein sorting rather than being specific for chitin synthesis. To test this hypothesis, we analyzed the behavior of several secreted or sorted proteins in Δ chs7 mutants. α-factor and exo-β(1-3)glucanase are two extracellular S. cerevisiae proteins that follow the normal secretion route. Fig. 6 shows that the expression levels of these two proteins are unaffected in Δ chs7 mutants. Similarly, CSI and CSII, the other two CS activities present in S. cerevisiae , showed wild-type levels in Δ chs7 mutants. Apparently, Chs7p is not required for secretion or sorting of other cellular proteins. In addition to the regular secretion mechanism, S. cerevisiae contains several other mechanisms involved in the specific assembly and transport of certain proteins. Sorting of vacuolar ATPase (v-ATPase) or several amino acid permeases to cytoplasmic membrane has been shown to depend on specific mechanisms of export from the ER , raising the possibility of the involvement of Chs7p is such processes. However, chs7 null mutants grew in glycerol and did not show synthetic lethality with other amino acid auxotrophies, clear indications that v-ATPase and amino acid permeases function properly in Δ chs7 mutants. So far we have shown that Chs7p is required for Chs3p export. However, from Fig. 2 it is also apparent that CHS7 expression increases when more chitin is synthesized. These observations suggest that the level of expression of CHS7 could be involved in the control of CSIII activity. If so, this would explain why overexpression of CHS3 does not increase CSIII levels. To confirm this possibility, we measured CSIII activity in strains overexpressing CHS genes in different combinations. Overexpression of CHS3 or CHS7 alone had no significant effect on CSIII. As previously reported , overexpression of CHS4 increased CSIII activity and reduced its trypsin dependence. Joint overexpression of CHS4 and CHS3 or CHS4 and CHS7 did not alter the effect of CHS4 on CSIII . Cooverexpression of CHS3 and CHS7 increased total CSIII almost four times. However, basal activity was only increased 1.3 times and, as a consequence, the activation factor by trypsin treatment was almost five times, in comparison with 1.6–2 times, for the controls. Joint overexpression of CHS4 , CHS3 , and CHS7 did not further increase total CSIII activity. However, basal activity was increased more than twofold. Therefore, activation by trypsin treatment was reduced to 2.5 in this case. Taken together, these results indicate that the amounts of Chs7p and Chs3p act as limiting factors to total CSIII activity. In addition, Chs4p levels are also limiting in the reconstitution of an in vitro fully active CSIII. Overexpression of CHS3 , CHS4 , and CHS7 genes also induced a modest, but significant, increase in chitin synthesis in vivo because a strain overexpressing these three genes contained ∼68 ± 11% more chitin than the corresponding wild-type. This increase in chitin synthesis was not observed if only CHS3 and CHS7 were overexpressed (not shown). These results clearly suggest that the amount of Chs7p is a limiting factor in vivo for CSIII activity and therefore, could be the reason for the absence of an increase in CSIII activity after CHS3 overexpression. In fact, immunostaining of cells overexpressing CHS3-3XHA alone revealed a severe retention of Chs3p in the ER even in the presence of normal amounts of Chs7p . Cooverexpression of CHS3 with CHS7 dramatically reduced Chs3p accumulation in the ER and only residual staining was observed at ER level. In this case, Chs3p was mainly localized in discrete patches in the cytoplasm, which in some cells showed a polarized distribution . This distribution resembled an intermediate stage between Δ chs6 , Δ chs5 , and Δ chs4 mutants . In sum, Chs3p excess is not retained in the ER in the presence of high levels of Chs7p and hence, higher levels of functional CSIII activity are achieved. Chitin is a minor, but essential polymer in the S. cerevisiae cell wall. Despite its low abundance, three different CS activities have been described in this yeast . From a quantitative point of view, CSIII activity is the most important, since it is involved in the synthesis of 90–95% of cell wall chitin during vegetative growth . In addition, it is required for the synthesis of new chitin that takes place during the mating and sporulation processes . Recently, it has been shown that the transcriptional regulation of CHS3 , the gene that encodes the catalytic subunit of CSIII, is not the control mechanism for CSIII activity . Therefore, the identification of genes involved in the control of CSIII and chitin synthesis gains interest. Three proteins, Chs4p, Chs5p, and Chs6p, have been identified, so far, as factors involved in the control of CSIII activity. This report deals with the isolation of a new gene, CHS7 , also involved in the control of CSIII activity. CHS7 was isolated by complementation of a mutant resistant to Calcofluor, showing that this strategy still has potential, although most (83%) of the mutants isolated belong to the chs3 complementation group. Δ chs7 mutants have reduced levels of CSIII activity and chitin in their cell walls (Table II ). This defect is comparable to that observed in chs3 null mutants and stronger than that detected in chs4 , chs5 , or chs6 mutants , underscoring the relevance of this gene in the control of CSIII activity. CHS7 encodes a small protein with six or seven putative transmembrane domains . Repeated database searches using different algorithms did not reveal any protein with significant homology to this protein. Some Chs7p regions with similarity to other integral membrane proteins probably reflect the hydrophobicity of the membrane-spanning domains rather than protein conservation. Chs7p colocalized with Chs3p in a crude particulate cell fraction and we therefore expected to find colocalization of Chs3p and Chs7p in the plasma membrane. However, Chs7p was localized exclusively in the ER , showing the typical localization of other ER proteins such as Sec63p . This localization does not seem to be an artifact of the chimeric Chs7p-GFP because similar results were obtained with Chs7p-3XHA either in immunofluorescence (data not shown) or in subcellular fractionation experiments. A functional explanation to account for this localization, unique among Chs proteins, became apparent when we observed that Chs3p accumulates at the ER in the absence of Chs7p . However, the Chs3p protein accumulated in the chs7 mutant is correctly core-glycosylated , a clear indication that Chs3p is translocated and correctly localized in the ER compartment in this mutant. Therefore, Chs7p should be required for Chs3p export from the ER. Moreover, the chs7 mutation is epistatic over other chs mutations, suggesting that Chs7p is the initial element in the hierarchy of proteins involved in CSIII activity. These results explain the stronger defect in CSIII activity observed in Δ chs7 , as compared with Δ chs4 , Δ chs5 , or Δ chs6 mutants. It should be noted that Chs4p, Chs5p, or Chs6p is not required for Chs3p export from the ER . At this point, all the data points to the specific involvement of Chs7p in the export of Chs3p from the ER. However, the concomitant participation of other CHS products in this export cannot be ruled out. Despite this, preliminary evidence indicates that Chs5p and Chs4p are efficiently exported in Δ chs7 mutants (data not shown) and therefore, their participation is rather unlikely. To date, there is no clear relationship between the levels of expression of different CHS genes and chitin levels, although regulation of chitin synthesis has been observed during the yeast life cycle . It should especially be stressed that overexpression of CHS3 several times (∼30) did not increase CSIII activity or chitin levels and that Chs3p levels remain stable during vegetative growth . Under these circumstances, only Chs4p can be envisaged as a controller of CSIII activity . Might Chs7p also be involved in the control of CSIII? Fig. 2 shows some results in favor of this hypothesis, since CHS7 is the only CHS gene whose transcription is increased under all conditions in which chitin synthesis is seen to be increased: mating, Calcofluor treatment, and sporulation. The increase in CHS7 expression during sporulation is especially relevant and its pattern of induction resembles that of a middle-late sporulation gene . The timing of expression places CHS7 among the sporulation genes involved in spore wall formation . Further and more direct confirmation of this hypothesis was achieved after joint overexpression of CHS3 and CHS7 genes. CSIII activity rose dramatically after the overexpression , reaching values several-fold higher than those of the control strain. This increase depends on joint overexpression, suggesting the requirement of a balanced expression of both genes. Biochemically, we have shown that Chs7p acts as a limiting factor when Chs3p levels are increased, but the reason at an intracellular level for this limitation remains unknown. As shown in Fig. 8 A, the cells cannot process an excess of Chs3p and therefore, overexpression of CHS3 leads to Chs3p retention in the ER. However, if CHS7 is overexpressed in strains with high levels of Chs3p, this protein is released from the ER . In addition, the localization of Chs3p in this strain resembles that observed in Δ chs6 or Δ chs5 mutants (bright internal spots) with partial polarization , as in chs4 mutants . These results indicate that although Chs3p is efficiently exported from the ER, its transport to its physiological site of action is impaired, probably due to some other limiting factors. In this condition, the high amount of active CSIII detected suggests that the limiting factor, if it exists, should appear at the time of or after CHS6 participation, because mutations in genes that function early on the pathway show severe defects in CSIII activity . Chitin synthesis increases in S . cerevisiae during α-factor treatment and this process is associated with the increase in CSIII activity . Recently, it has been shown that during α-factor treatment Chs3p levels increase significantly . However, no Chs3p is localized at the ER . We have shown that the effect of increased Chs3p levels on chitin synthesis depends mainly on correct Chs3p export from the ER. Therefore, the increase in CHS7 transcription observed during α-factor treatment guarantees the export of excess Chs3p and the increase in chitin synthesis required during this process. Overexpression of CHS3 and CHS7 leads to a significant increase in CSIII activity. However, the activity obtained under these conditions was highly zymogenic. When we increased Chs4p levels in the same experiment, the CSIII dependence on trypsin decreased significantly. Therefore, it seems that Chs4p is also a limiting factor for this activity. We cannot be sure if Chs4p function is mediated by proteolytic processing of CSIII, but it does seem clear that in vitro trypsin treatment of CSIII resembles the activation carried out in vivo by Chs4p. Moreover, the strain carrying the triple overexpression also showed a significant increase in cellular chitin levels (68% higher than controls) indicating that these genes not only participate in the control of CSIII, but also in the in vivo control of chitin synthesis. We cannot expect a perfect correlation between CSIII and chitin levels, since there could be as yet undescribed cellular determinants participating in this process. In addition, problems of interference could arise in the replication of three independent plasmids that could mask quantitative interpretation of these data. By contrast, strains overexpressing only CHS3/CHS7 , although they showed high CSIII activity, did not show any increase in chitin levels. This result is the first experimental evidence that Chs4p is an in vivo regulator of chitin synthesis because until now, the Chs4p function has only been determined in CSIII measurements in vitro . With this evidence in mind, it can be concluded that the extent of CSIII activity in vivo depends on a delicate balance between the levels of Chs3p, Chs4p, and Chs7p proteins, a balance that ensures the level of chitin synthesis required at each moment of the S. cerevisiae life cycle, besides the specific roles of CSI and CSII activities. At this point, we are still unable to pinpoint the exact role of Chs7p in the export of Chs3p. However, we can compare this system with the assembly of v-ATPase or the transport of amino acid permeases, the two previously reported cases in S. cerevisiae that involve specific proteins in their exit from the ER . v-ATPase mature complex is assembled in the ER with the help of several proteins, such as Vma12p or Vma22p, which do not leave the ER and therefore do not participate in the active v-ATPase complex . Likewise, Shr3p is required for the release of several amino acid permeases from the ER . If this protein is not present, the amino acid permeases cannot be loaded into COPII vesicles . We can exclude the participation of CHS7 in any of these processes because chs7 mutants did not show phenotypes associated with the lack of v-ATPase or the amino acid permeases. The role of Chs7p in the export of Chs3p could be similar to that reported in any of these cases. In the absence of Chs7p, the CSIII complex, including Chs3p, is either not assembled or not loaded into secretory vesicles. There is indirect evidence suggesting that Chs7p could have a similar role to Shr3p. First of all, Shr3p and Chs7p, although lacking significant homology, have a very similar secondary structure with several transmembrane domains. This type of secondary structure is typical of resident ER proteins (outfitters) rather than proteins loaded into cargo vesicles . Chs7p also lacks the protein sequences involved in COPI mediated retrograde transport. In addition, we have been unable to detect Chs7p outside of the ER compartment. However, we cannot exclude that Chs7p is rapidly recycled between the ER and Golgi compartments. The answer to this question will have to await the description of the nature of the secretory vesicles involved in the transport of Chs3p. The results reported here indicate that CHS7 is part of a specific mechanism for CS export and its presence in vertebrates is therefore unlikely. This opens a new possibility in the design of an antifungal agent that selectively inhibits chitin synthesis. | Study | biomedical | en | 0.999998 |
10366590 | General methods are described by Brenner . The wild-type parent for all strains was C . elegans var. Bristol strain N2. Strains were grown at 20°C unless otherwise noted. Mutations used were: LG I: arIs12 [lin-12(intra)] ; LG III: unc-36(e251) , unc-32(e189) , lin-12(n676n930ts) , lin-12(n302) , lin-12(ar170) , glp-1 , glp-1(q415) ; LG V: dpy-11(e224) , rol-3(e754) , unc-23(e25) , sel-9(ar22, ar26) , mom-2(ne141) ; and extrachromosomal array arEx29[lin-12(+)] . At 25°C, glp-1 hermaphrodites produced inviable progeny; this phenotype is suppressed by sel-9(ar22) . Furthermore, glp-1; sel-9(ar22)/Df hermaphrodites also produce viable progeny (data not shown), suggesting that null alleles in principle may be obtained by complementation screening. EMS mutagenesis was performed as described by Brenner . glp-1; him-8; rol-3(e754) sel-9(ar26)/sel-9(ar22) unc-23(e25) males were mated to EMS mutagenized glp-1; dpy-11(e224) hermaphrodites at 15°C. The parents were transferred to fresh plates daily for 5 d. F1 progeny were grown at 15°C until the L4 stage. Non-Dpy cross progeny were picked to fresh plates and transferred to 25°C. 10 F1 animals were put on each plate and the total number of F1 cross progeny was counted while picking. After 4 d, plates at 25°C were screened for live F2 progeny. Eventually only one animal from each plate was kept as a candidate. Dpy animals were backcrossed at least twice before further analysis. sel-9 was previously mapped between rol-3 and unc-42 . We mapped sel-9 between rol-3 and unc-23 : 2/10 Rol non-Unc recombinants from heterozygotes of the genotype rol-3 unc-23/ sel-9 segregated rol-3 sel-9 . sel-9 was further mapped between rol-3 and mom-2 : from heterozygotes of the genotype rol-3 sel-9 unc-23/mom-2 , 1/23 Rol non-Unc recombinants harbored a rol-3 sel-9 mom-2 recombinant chromosome. Transgenic lines were generated by microinjecting lin-12(n676n930); sel-9(ar22) hermaphrodites with cosmid or plasmid DNA at a concentration of 10 μg/ml, along with the dominant rol-6 marker pRF4 at a concentration of 100 μg/ml . Stable Rol lines were reared at 25°C, and individual Rol hermaphrodites from each line were analyzed for the Egl defect. A line is considered rescued if >50% of the Rol hermaphrodites were Egl. Initial rescue was obtained with each of two overlapping cosmids, F21F8 and W02D7. The 20-kb overlapping region was further subcloned into plasmid vector pBS(SK + ) (Stratagene). Plasmid pSX2.8 contains the 2.8-kb DNA fragment that contains sel-9(+) activity in the antisuppression assay; this plasmid was also shown to be able to rescue the morphological defects caused by sel-9(ar173) . Standard molecular biology protocols were performed as described in Sambrook et al. . The DNA sequences of F21F8 and W02D7 were obtained from the C . elegans genome sequencing project . The exons of sel-9 were predicted by GENEFINDER ; we confirmed this prediction by sequencing a cDNA clone, yk371h2 (generously provided by Dr. Yuji Kohara, National Institute of Genetics, Japan). The lesions associated with all sel-9 mutations were found by sequencing the sel-9 coding region of the mutants. We amplified the sel-9 genomic region by PCR reactions from individual sel-9 mutant hermaphrodites. For each mutation, two independent PCR products were cloned into Bluescript(SK + ) (Stratagene). A lesion was considered confirmed if it appeared in two independent clones. Lesions associated with lin-12(n941) , lin-12(n676n930) , lin-12(ar170) , and lin-12(oz48) were found by sequencing most of the coding region of the mutants, as for sel-9 . lin-12(n941) corresponds to W400STOP. The n930 lesion corresponds to C138T. The ar170 lesion corresponds to G270R. The oz48 lesion corresponds to G449R. A PCR product containing the coding sequence of GFPS65T was cloned into a PmeI site at the end of the sel-9 coding region in pSX2.8 (see above). As a result, the stop codon of sel-9 was changed to Ser. The resulting SEL-9::GFP hybrid protein is nonfunctional in the antisuppression assay, but, as it is expressed under the control of sel-9 regulatory sequences, it was useful for determining that all somatic cells express SEL-9 (data not shown). Double-stranded RNA (dsRNA) 1 was prepared using the RNA transcription kit (Stratagene) and injected without dilution according to Fire et al. . lin-12(n676n930); sel-9(ar26) hermaphrodites, along with unoperated control animals, were kept at 15°C except for the period of laser microsurgery (20°C for ∼5 min). The nucleus of Z4 was ablated in newly hatched L1 larvae. The presence of an anchor cell (AC) was scored during the late L3 stage. Anti–GLP-1 staining of dissected hermaphrodite gonads was performed as described in Crittenden et al. . Phalloidin staining was performed as described in Strome . Worms were mounted on a 2% agarose pad with 3 μl of 10% N -propyl gallate and viewed with a Zeiss LSM410 laser scanning confocal attachment on a Zeiss Axiovert 100 microscope. Our analysis of sel-9 depends on genetic interactions between sel-9 and the lin-12 or glp-1 genes. Here, before we describe the genetic analysis of sel-9 , we summarize the relevant properties of mutations in lin-12 and glp-1 , which both encode receptors of the LIN-12/Notch family . We note that lin-12 and glp-1 are functionally redundant in some cell fate decisions , and that GLP-1 can fully substitute for LIN-12 when expressed under the control of lin-12 regulatory sequences . In our analysis, we have made consistent observations using both genes. Three morphological characteristics influenced by lin-12 were used for our analysis of sel-9 (Table I ): the number of ACs, vulval morphology, and egg laying. Wild-type hermaphrodites have one AC and a normal vulva, and are able to lay eggs. Reduced lin-12 activity causes an extra AC to be produced (the 2 AC defect), a variably abnormal vulva, and a defect in egg laying (Egl) . In this study, we focus on genetic interactions between sel-9 and two alleles that reduce, but do not eliminate, lin-12 activity: lin-12(n676n930) and lin-12(ar170) . Constitutive, elevated lin-12 activity causes the AC to be missing (the 0 AC defect) and, as a consequence, an egg-laying defect (the 0 AC-Egl defect) which is different from the Egl defect associated with reduced lin-12 activity . A greater elevation of lin-12 activity also causes extra vulval cells to be made (the Multivulva defect). Constitutive activity may result from missense mutations in the extracellular domain, collectively termed lin-12(d) , or by removing the extracellular domain, as in the lin-12(intra) transgene . The absence of glp-1 activity causes defects in germline proliferation and maternal effect lethality . The glp-1 allele used extensively in this study displays only the maternal effect lethal defect at 25°C . Other glp-1 mutations used are temperature-sensitive for both defects, and their properties are described where relevant below. Two sel-9 mutations were isolated as suppressors of the egg laying defect (Egl) of the partial loss-of-function (hypomorphic) allele lin-12(n676n930) . Genetic analysis suggested that these two sel-9 alleles, sel-9(ar22) and sel-9(ar26) , are not null alleles . We isolated new sel-9 mutations in noncomplementation screens that in principle could have yielded null alleles (see Materials and Methods). Five sel-9 alleles ( ar173 , ar174 , ar175 , ar176 , and ar178) were isolated after screening 12,000 mutagenized haploid genomes. We classified the existing sel-9 mutations into two groups, group A (weaker) and group B (stronger), based on their interactions with lin-12 hypomorphic alleles (Table II ). Mutations in group B appear to increase lin-12 activity to a greater extent than mutations in group A. This inference is based on two observations: in combination with lin-12(n676n930) , alleles of group B result in a Multivulva phenotype (Table II ), and in combination with lin-12(ar170) , alleles of group B suppress the 2 AC defect (Table II ). We note that the alleles in group B were all isolated in noncomplementation screens and could not have been isolated as suppressors of lin-12(n676n930) , since they cause lin-12(n676n930) to lack an AC, which is necessary for vulval development and egg laying. All sel-9 alleles show genetic interactions with several different missense mutations affecting the LIN-12 extracellular domain (see below). However, none suppress defects caused by lin-12(n941) , a lin-12 null allele associated with a stop codon at position 400 in the extracellular domain (Materials and Methods). Genetic analysis suggests that all sel-9 mutations are antimorphic (dominant-negative) . A heterozygous deficiency is unable to suppress the 2 AC defect of lin-12(n676n930) or the maternal effect lethality of glp-1 . In contrast, sel-9/+ heterozygosity suppresses these defects, implying that sel-9 alleles have gain-of-function character. The gain-of-function character appears to be antimorphic, because addition of wild-type alleles can reverse the suppression of lin-12(n676n930) and glp-1 by mutations in sel-9 . In addition, we have evidence that all sel-9 mutations cause reduction of sel-9(+) activity (see below). To examine the cell autonomy of sel-9 function, we examined its effect on the decision of two cells, Z1.ppp and Z4.aaa, between the AC and VU precursor cell fates. Normally, lin-12 –mediated interactions between Z1.ppp and Z4.aaa causes one to become the AC . When lin-12 is constitutively active, hermaphrodites lack an AC, because both Z1.ppp and Z4.aaa become VUs. The penetrance of the 0 AC phenotype reflects the degree of elevated activity . For example, lin-12(n676n930) behaves like a weakly activated allele at 15°C, in that ∼5% of hermaphrodites lack an AC. In the presence of sel-9(ar26) , the proportion of lin-12(n676n930) hermaphrodites lacking an AC is increased to 78%, suggesting that lin-12 activity is elevated by sel-9(ar26) . When either Z1 or Z4 (the precursors to Z1.ppp or Z4.aaa) is ablated with a laser microbeam, the fate of the remaining cell reflects its intrinsic level of lin-12 activity in the absence of signaling. In wild-type hermaphrodites, if Z1 (or Z1.ppp) is ablated, Z4.aaa always becomes an AC because lin-12 is not activated in the absence of ligand. However, in lin-12(d) hermaphrodites, if Z1 (or Z1.ppp) is ablated, Z4.aaa becomes a VU because of the ligand-independent activation of LIN-12(d) . If sel-9(+) functions in the receiving end of lin-12 –mediated cell–cell interactions, we would expect to see that sel-9(ar26) increases the intrinsic lin-12 activity in the absence of the signaling cell. The results of ablation experiments suggest that sel-9 can affect lin-12 activity cell autonomously (Table III ). As mentioned above, 78% of lin-12(n676n930); sel-9(ar26) hermaphrodites lack an AC. Similarly, in operated lin-12(n676n930); sel-9(ar26) hermaphrodites, when Z4 was removed at early L1 stage, Z1.ppp became a VU in 77% of the cases. These results demonstrate that the effect of sel-9 on lin-12 activity does not depend on the signaling cell, since sel-9(ar26) can increase the intrinsic level of lin-12 activity in the absence of the signaling cell. Although this experiment does not rule out an additional role for sel-9 in the signaling cell, the extent of enhancement of lin-12(n676n930) can be completely accounted for by sel-9 function in the receiving cell. sel-9(ar22) suppresses the Egl phenotype caused by lin-12(n676n930ts) at 25°C. Adding a copy of sel-9(+) allele [lin-12(n676n930); sel-9(ar22)/sel-9(ar22)/sel-9(+)] can partially reverse the suppression . Therefore, we were able to use reversal of suppression to assess the sel-9( + ) activity of microinjected DNAs . We mapped sel-9 between rol-3 and the cloned gene mom-2 , an interval of <0.1 map unit. The mom-2 gene resides on cosmid clone F38E1 . We tested cosmid clones to the left of F38E1 for their ability to complement sel-9(ar22) in a lin-12(n676n930); sel-9(ar22) background (see Materials and Methods). Arrays containing either one of two overlapping cosmid clones, F21F8 and W02D7, gave rescue in the antisuppression assay. The overlapping region was further subcloned and the 2.8-kb fragment in pSX2.8 was determined to contain sel-9(+) activity. The sequence of the 2.8-kb fragment is predicted to encode a single gene . A cDNA clone, yk371h2 (generously provided by Dr. Yuji Kohara), contains the DNA sequence of all predicted exons contained in pSX2.8. This predicted gene was confirmed to be sel-9 by finding that all sel-9 mutations contain molecular lesions in the coding region . The predicted SEL-9 protein sequence reveals that SEL-9 belongs to the p24 family of proteins . Multiple members of the p24 family are found in all eukaryotes, from yeast to mammals. Members of the p24 family are type I membrane proteins with a signal peptide at the amino terminus, a lumenal (extracytosolic) domain, a single transmembrane domain, and a short cytoplasmic tail. p24 proteins have a predicted lumenal coiled-coil domain, conserved amino acids in the transmembrane domain and cytoplasmic tail, and similar overall size and organization . They may be grouped into at least three subfamilies based on primary sequence . One subfamily comprises yeast Emp24p and mammalian p24A; SEL-9 appears to be a member of this subfamily. Another subfamily comprises yeast Erv25p and mammalian Tmp21, and the third subfamily comprises mammalian gp25L proteins. We searched the C . elegans genomic sequence database for additional p24 proteins and identified at least four other proteins with the hallmarks of p24 proteins . As described below, we have also performed a functional test of one of these, F47G9.1, a member of the Erv25/ Tmp21 subfamily . p24 proteins are major membrane components of COPI- and COPII-coated vesicles . Genetic studies in yeast and biochemical studies in yeast and mammalian cells have led to proposals for p24 protein function in cargo selectivity of ER to Golgi transport . Our analysis of the interaction of sel-9 with lin-12 and glp-1 is consistent with a role in cargo selectivity, as described further below and in the Discussion. We constructed a SEL-9::GFP hybrid protein in which green fluorescent protein (GFP) was fused in frame to the carboxy terminus of SEL-9 (see Materials and Methods). The hybrid protein appeared to accumulate intracellularly in all somatic cells and at all developmental stages (data not shown). This observation implies that SEL-9 is present in cells undergoing lin-12 –mediated cell fate decisions. Unfortunately, this SEL-9::GFP hybrid protein does not appear to function normally, as it was unable to reverse the suppression of lin-12(n676n930) by sel-9 at 25°C (data not shown), so we cannot meaningfully analyze its subcellular distribution. We sequenced all existing sel-9 mutations and found that none creates an early stop codon or deletion that would be a clear molecular null allele . sel-9(ar173) creates a stop codon at the beginning of the predicted transmembrane domain, and is predicted to result in a truncated SEL-9 protein lacking most of the TM domain and the entire cytoplasmic tail. All other sel-9 mutations contain missense changes in the predicted lumenal region of SEL-9. Six of the seven sel-9 alleles, corresponding to four different missense mutations, do not cause any obvious defects in an otherwise wild-type background. In contrast, sel-9(ar173) homozygous hermaphrodites are dumpy (Dpy), uncoordinated (Unc), and slightly roller (Rol) and egg-laying defective (but have normal vulval lineages; data not shown). These phenotypes are all complemented by an extrachromosomal array containing multiple copies of the sel-9(+) gene, and hence appear to result from the sel-9(ar173) mutation. However, sel-9(ar173)/sel-9(ar173)/ mnDp26 [sel-9(+)] hermaphrodites still display a similar spectrum of phenotypes as sel-9(ar173) , although they are less abnormal than sel-9(ar173)/sel-9(ar173) , suggesting that sel-9(ar173) is an antimorph (data not shown), perhaps interfering with the secretion of proteins other than LIN-12 and GLP-1. We postulate that the presence of the lumenal portion of SEL-9 interferes with secretion when an absent or nonfunctional carboxy terminus prevents association with vesicle coat proteins. In support of this interpretation is the observation that fusion of GFP in frame at the carboxy terminus causes the same spectrum of phenotypes as sel-9(ar173) (data not shown). Although we do not have demonstrable sel-9 null alleles, we note that in yeast, deletion of either emp24 or erv25 , or of both genes, has measurable effects on specific aspects of secretion without any significant deleterious effects . Thus, it is conceivable that the sel-9 null phenotype may, at a gross level, be wild-type. Below, we provide evidence suggesting that available sel-9 alleles, in addition to being antimorphs, also reduce or eliminate sel-9 activity. The technique of RNA-mediated interference (RNAi) may be used to investigate the effects of reducing gene activity . RNAi is based on the observation that injection of RNA, and, in particular, dsRNA , can produce specific phenotypes similar to loss or reduction of function of the target gene. We have used this method to investigate the effects of reducing sel-9 and F47G9.1 activity. The assay we used depends on the observation that at 15°C, lin-12(n676n930) behaves like a weak gain-of-function allele, and a small proportion of lin-12(n676n930) hermaphrodites lacks an AC and displays the 0 AC-Egl phenotype. We examined the ability of dsRNA to enhance the 0 AC-Egl phenotype of lin-12(n676n930) at 15°C. Double-stranded sel-9 or F47G9.1 RNA was injected into lin-12(n676n930) homozygous L4 hermaphrodites grown at 15°C, and injected hermaphrodites and mock-injected control hermaphrodites were maintained at 15°C. All hermaphrodites injected with sel-9 dsRNA or F47G9.1 dsRNA produced a markedly greater proportion of 0 AC-Egl progeny than did control hermaphrodites (see Table IV ). We also examined the ability of dsRNA to suppress the maternal effect lethal phenotype of glp-1 at 25°C, since maternal gene activity seems to be particularly sensitive to inhibition by this method. Double-stranded sel-9 or F47G9.1 RNA was injected into glp-1 homozygous L4 hermaphrodites grown at the permissive temperature. The injected hermaphrodites and uninjected control hermaphrodites were shifted to 25°C. Control hermaphrodites laid only dead eggs. In contrast, all hermaphrodites injected with sel-9 dsRNA or F47G9.1 dsRNA produced live progeny (see Table IV ). The suppression of glp-1 by sel-9 or F47G9.1 RNAi appears to be incompletely heritable for at least one additional generation, as has been observed in RNAi experiments for certain other genes. The sel-9 and F47G9.1 RNAi effects on lin-12(n676n930) and glp-1 indicate that a reduction in p24 activity appears to elevate lin-12 and glp-1 activity. These results also suggest that available sel-9 alleles reduce sel-9 activity, since all sel-9 mutations enhance lin-12(n676n930) and suppress the maternal effect lethality of glp-1 . We also injected F47G9.1 dsRNA into sel-9(ar174) , and did not observe any reduced viability or obvious effects on the egg-laying ability or vulval morphology of adult progeny (see Table IV ). This result is consistent with the observation that the phenotype of a Δemp24 Δerv25 strain is no more severe than either single mutant . sel-9 mutations do not suppress/enhance all lin-12 or glp-1 mutations. Rather, they appear to be specific for alleles of lin-12 and glp-1 that cause alterations in the extracellular domain . sel-9 appears to increase the activity of the partial loss-of-function mutations lin-12(n676n930) , lin-12(ar170) , and lin-12(oz48) , all of which have missense mutations in the extracellular domain . sel-9 also appears to increase the activity of gain-of-function lin-12(d) mutations such as lin-12(n302) : the penetrance of the Multivulva phenotype is increased in the presence of sel-9 mutations (Table V ). In contrast, sel-9 does not appear to increase the activity of arIs12[lin-12(intra)] (Table V ): the penetrance of the Multivulva phenotype is not increased in the presence of sel-9(ar174) . These results suggest that the interaction between sel-9 and lin-12 requires the extracellular domain of LIN-12 or that LIN-12 be a transmembrane protein. The ability of sel-9 to enhance the weak gain-of-function phenotype caused by a multicopy lin-12(+) transgene (Table V ) is consistent with this inference. We further investigated the possibility that sel-9 suppression is specific for mutations in the extracellular domain by taking advantage of a variety of available, sequenced alleles of glp-1 . All sel-9 alleles can suppress the maternal effect lethality caused by glp-1 , a missense mutation in the first EGF-like motif of the extracellular domain, but cannot suppress either the germline defect or the embryonic lethality caused by glp-1 and glp-1(q231) , missense mutations in the cdc10/SWI6 domain of GLP-1 (Table VI ). Since glp-1 and glp-1(e231) appear to lower glp-1 activity more than glp-1 , the ability of sel-9 alleles to suppress glp-1 but not glp-1 and glp-1(q231) may be explained in three different ways: sel-9 does not function in the germline; sel-9 mutations can only suppress mild loss of glp-1 activity; or sel-9 mutations interact only with specific glp-1 alleles. To distinguish among these possibilities, we examined the effect of sel-9 on glp-1(q415) (Table VI ). glp-1(q415) contains a missense mutation (Gly226→ Glu) in the fourth EGF-like repeat . At 15°C, the germline of glp-1(q415) proliferates normally but all embryos are dead; at 25°C, the germline proliferation of glp-1(q415) is defective . We found that sel-9(ar26) , a weaker sel-9 allele, can suppress the germline proliferation defect but not the maternal effect lethality of glp-1(q415) at 25°C. However, sel-9(ar174) , a stronger sel-9 allele, can suppress both the germline defect and the maternal effect lethality caused by glp-1(q415) at 25°C. Thus, sel-9 does function in the germline. In the germline, glp-1(q415) probably has a glp-1 activity level similar to that of glp-1 and glp-1(q231) , since all three mutants produce similar numbers of germ cells at the restrictive temperature . Thus, the ability of sel-9(ar26) to suppress glp-1(q415) but not glp-1 and glp-1(q231) suggests that the interaction between sel-9 and glp-1 depends on alterations in the extracellular domain of GLP-1. To determine if the effect of sel-9 is on LIN-12/GLP-1 trafficking in C . elegans , we examined the level or subcellular localization of wild-type and mutant GLP-1 proteins in a sel-9(+) and sel-9 mutant background using an antibody cocktail that recognizes GLP-1 in dissected gonads . In the germline of wild-type hermaphrodites, GLP-1 is visible mainly in the plasma membrane in the distal region in a honeycomb pattern corresponding to the membranes surrounding each germline nucleus . We saw no evidence for a change in the level or subcellular localization of GLP-1(+) in a sel-9(ar174) mutant background (data not shown). In the germline of glp-1(q415) hermaphrodites, the membranes are present in their typical honeycomb pattern, as shown by phalloidin staining; however, GLP-1(q415) does not display the honeycomb pattern, and instead accumulates within the cell, consistent with a defect in its transport to the plasma membrane. In contrast, in the germline of glp-1(q415); sel-9(ar174) hermaphrodites, plasma membrane accumulation of GLP-1(q415) is at least partially restored, as a honeycomb pattern is evident. These results suggest that the absence of sel-9 activity relieves the block on trafficking of mutant GLP-1(q415) protein to the plasma membrane. In this study, we have shown that SEL-9 is a member of the Emp24/p24A subfamily of p24 proteins. Reducing the activity of sel-9 and F47G9.1, which encodes a member of the Erv25/Tmp21 subfamily of p24 proteins, can increase the activity of certain mutations in lin-12 or glp-1 . The interaction between sel-9 and lin-12 appears to be cell autonomous. The common feature of the alleles that are affected by sel-9 is that they are missense mutations in the extracellular domain of LIN-12 or GLP-1; in contrast, the mutations that are not affected include an activated form of LIN-12 caused by truncation of the extracellular domain and transmembrane domain, and point mutations of GLP-1 that alter the intracellular domain. We examined the subcellular localization of the protein encoded by glp-1(q415) , one of the suppressed mutations, and found that it accumulates within the cell; however, a mutation in sel-9 enables GLP-1(q415) to accumulate in the plasma membrane. We discuss specific aspects of these results in the previous section. Here, we discuss how these results are consistent with a function for SEL-9 in cargo selection during transport of LIN-12 and GLP-1 to the cell surface. In eukaryotic cells, secretory protein trafficking is mediated by transport vesicles, which bud from a donor membrane of one compartment and fuse with a recipient membrane of a different compartment. Distinct vesicle coat protein complexes mediate different budding/fusion events. Anterograde transport from ER to Golgi is mediated by COPII-coated vesicles . Bidirectional transport between the ER and Golgi, and intra-Golgi transport, is mediated by COPI-coated vesicles . Endocytic trafficking is mediated by clathrin-coated vesicles . A key feature of vesicle-mediated trafficking is the net transfer of cargo from one compartment to another, while components of the donor compartment are selectively excluded from vesicles and/or recycled. Furthermore, there appears to be a quality control mechanism, so that misfolded or mutant proteins cargo proteins are not transferred . Little is known about how selective packaging or quality control occurs. Signals on cargo and the coat proteins appear to influence assembly of the COPII coat complex . However, other factors appear to influence selectivity: for example, null mutations which bypass the anterograde secretion block associated with the absence of Sec13p (one component of the COPII coat complex) also cause leakage of ER resident proteins and mutant invertase . One gene identified as a bypass suppressor of Δsec13 was EMP24 , a defining member of the p24 subfamily to which SEL-9 belongs. p24 proteins are transmembrane protein components of COPI- and COPII-coated vesicles , and interact with coat proteins via their transmembrane/carboxy-terminal domains . Strains lacking the p24 proteins Emp24p or Erv25p have similar secretion defects: there is reduced ER to Golgi transport of a subset of secretory proteins and leakage of ER resident proteins . Two different roles for p24 proteins have been proposed. One possibility is that p24 proteins are receptors/adaptors for lumenal cargo . Alternatively, Elrod-Erickson and Kaiser have proposed that the proteins encoded by EMP24 and other Δsec13 bypass suppressors are part of a quality control mechanism that prevents the premature formation of vesicles that have not properly segregated cargo from ER-resident proteins. The genetic interactions between sel-9 and lin-12 or glp-1 are consistent with an Emp24p-like role for SEL-9 in the transport of LIN-12 and GLP-1. SEL-9 and F47G9.1 may act during the sorting process to keep misfolded or mutant LIN-12 and GLP-1 proteins from transport vesicles, or, as proposed by Elrod-Erickson and Kaiser , as a general block to the progress of vesicles containing aberrant proteins. Our genetic data are more consistent with a role for p24 proteins in a quality control mechanism as opposed to a role in cargo reception. In our functional interactions, sel-9 behaves as a negative regulator. If sel-9 were principally to function as a LIN-12/GLP-1 cargo receptor, we might have expected it to behave as a positive factor: loss or reduction of a cargo receptor should reduce the amount of LIN-12 or GLP-1 at the cell surface. Instead, loss or reduction of sel-9 activity increases the amount of lin-12 or glp-1 activity, enhances the weak gain-of-function activity associated with overexpression of an essentially wild-type LIN-12 protein, and demonstrably increases the amount of a mutant GLP-1 protein at the cell surface. Our results, like those of Elrod-Erickson and Kaiser , are therefore more consistent with a major role for p24 proteins in quality control as opposed to cargo reception. All of the mutations that were affected by reducing sel-9 activity alter the extracellular domain of LIN-12 or GLP-1. These mutations may lead to general structural defects in the extracellular domain, since the mutations affect different subregions and have different effects (some elevate and some reduce activity). SEL-9(+) may directly or indirectly recognize the abnormal extracellular domains of the mutant LIN-12 or GLP-1 proteins and block their transport, thus effectively functioning to negatively regulate the amount of LIN-12/GLP-1 in the plasma membrane. In sel-9 mutants, however, abnormal LIN-12/GLP-1 proteins may instead be transported to cell surface, where they may be able to function. This inference is supported by our examination of the cell biology underlying these genetic interactions. When sel-9 activity is normal, the mutant GLP-1(q415) protein appears to be retained within the cell, and the hermaphrodites display a glp-1 mutant phenotype. In contrast, when sel-9 is mutant, the GLP-1(q415) mutant protein is found in the plasma membrane, and the hermaphrodites display a wild-type phenotype. We postulate that the effect of sel-9 on mutant LIN-12 or GLP-1 reflects a role for SEL-9(+) in the transport of LIN-12(+) and GLP-1(+). SEL-9(+) may inhibit the transport of aberrant LIN-12(+) and GLP-1(+) proteins, which may occur at some frequency due to misfolding or misprocessing. The finding that sel-9 mutations enhance the weak gain-of-function defect associated with overexpression of a tagged LIN-12 protein with a wild-type extracellular domain is consistent with this postulated role. One issue that deserves comment is the lack of a severe phenotype associated with reduced sel-9 activity. In yeast, Δemp24 causes only a moderate reduction of secretion of a select group of proteins and does not cause a marked visible phenotype . This lack of a visible phenotype might be attributable to functional redundancy among the multiple p24 proteins in yeast; however, particularly if p24 proteins depend on each other for stability , then perhaps elimination of all p24 protein activity might not result in a deleterious phenotype. In C . elegans , there also appear to be multiple p24 proteins. The sel-9 alleles we isolated appear to reduce sel-9 activity, but we do not know the sel-9 null phenotype with certainty: none of the existing mutations cause early stop codons or deletions of the coding region. Like Emp24p, SEL-9 may be involved in the transport of a select group of proteins, including LIN-12 and GLP-1. We note that if sel-9 activity were essential for all secretory protein transport, we might reasonably have expected to see some evidence for a phenotype caused by RNAi. The definitive answer to the question of the phenotype caused by a lack of p24 proteins will be most readily addressed in yeast, where it will be feasible to construct strains lacking multiple genes for p24 proteins. Our characterization of sel-9 emphasizes a link between the secretory apparatus and cell signaling during development. The characterization of other developmental genes is providing other linkages between secretion and cell signaling processes. For example, the establishment of dorsoventral polarity occurs during oogenesis and involves a signal from the oocyte to the follicle cells and a second signal from follicle cells back to the oocyte . The gene windbeutel , which is required for proper dorsoventral polarity, acts in the follicle cells and encodes an ER protein that has been proposed to chaperone a secreted signal produced in the follicle cells . Whether the linkages that have been found between the secretory apparatus and cell signaling processes reflect constitutive secretory functions or serve as points of regulation will be an issue for future study. | Study | biomedical | en | 0.999998 |
10366591 | HA11 mAb, used to detect the hemagglutinin epitope (HA) in an Axl2/ Bud10p-HA fusion protein, was from Berkeley Antibody Company. Polylysine, protease inhibitors (phenylmethylsulfonyl fluoride, benzamidine, antipain, leupeptin, and pepstatin), calcofluor white, and protein standards were from Sigma Chemical Co. HRP-coupled sheep anti–mouse antibodies were from Nycomed Amersham, Inc. Peptide N-glycosidase F (PNGase) and tunicamycin were from Boehringer Mannheim Corp. Strains are described in Table I , and plasmids in Table II . DNA and yeast genetic manipulations were performed as described . Synthetic low ammonium histidine dextrose (SLAHD) solid medium for assaying pseudohyphal growth was prepared as described by Gimeno et al. . Microcolony budding assays were performed as previously described . The HMR a gene of IH2531 (α ura3-52 ) was disrupted by introduction of pDR58 digested with EcoRV , creating SY77. The disruption was confirmed by Southern blot analysis. A culture of SY77 was grown to saturation in rich medium, resuspended in water, and UV-irradiated in separate aliquots to 0.3–24% survival. Mutagenized cells were plated on solid SLAHD medium to induce pseudohyphal growth and incubated at 30°C for 2 wk in the dark. Of ∼70,000 surviving colonies, 407 exhibited the pseudohyphal colony morphology. Microcolony budding assays were performed on the 107 colonies that retested for pseudohyphal growth. 23 of these mutants exhibited >30% bipolar budding and were studied further. Mutants were tested for single-gene segregation by analyzing the budding pattern of the meiotic progeny of crosses to IH2393 ( a Bud + ). Mutants whose progeny exhibited 2:2 axial/bipolar budding were studied further. Because a /α strains do not exhibit the axial budding pattern, dominance or recessiveness was assessed by analyzing mat a 1/MAT α diploid progeny (phenotypically α) formed by crosses between the mutants and a mat a 1 strain . Recessive mutations yielded an axial budding pattern in the mat a 1/MAT α diploids; dominant or semidominant mutations yielded a bipolar or partially bipolar phenotype. Mutants defective in known genes required for axial budding were identified by complementation tests using plasmids carrying BUD3 , BUD4 , or AXL1 , by mating to bud3 , bud4 , axl1 , or axl2 strains, and, for new BUD4 and AXL1 alleles, by linkage tests. Complementation tests among the mutants were performed by analyzing diploids (phenotypically α) formed by crossing mat a 1 and α mutant strains. If the resultant diploid exhibited axial budding, the two mutations were assigned to different complementation groups. If the resultant diploid exhibited bipolar budding, the two mutations were assigned to the same complementation group. The bud-site selection axial determinant BAD15/PMT4 gene was cloned by complementation of the pseudohyphal growth phenotype. SY167 (α bad15-1 ura3 ) was transformed with a low copy yeast library marked with URA3 . Approximately 10,200 transformants were patched onto SLAHD plates in two concentric rings of 25 (because the pseudohyphal phenotype in a and α cells was sensitive to position on the plate, as well as the number of colonies per plate). Plates were incubated for 2 wk at 30°C and scored for pseudohyphae by microscopic examination at 30×. Nine transformant colonies failed to exhibit pseudohyphal growth and were assayed for budding pattern. Plasmid DNA isolated from the three colonies that exhibited axial budding contained overlapping inserts by restriction analysis. The pmt4 Δ constructs replaced the entire PMT4 coding sequence with the LEU2 or TRP1 gene. The HindIII–SalI fragment of pSS47, which contains PMT4 and YJR142W, was cloned into the vector pBluescript (Stratagene), creating pSS48. All sequences of pSS48, except the PMT4 open reading frame (ORF), were amplified using PCR with divergent primers (OSS8 5′-CCGaggcctCTTTTTCTGTTCACTAACCACAAACGAACTG and OSS9 5′-CCGaggcctCTGTACTTCTGTGGACTGTCAGAAAATATCTTGG; a StuI restriction site is denoted in lower case) that hybridized just upstream of the PMT4 start codon and downstream of the stop codon. The resulting linear fragment was cleaved at the introduced StuI sites and circularized, yielding a plasmid (pSS49) that contained the PMT4 flanking regions separated by the StuI site. The LEU2 gene was then introduced into the StuI site to form pSS51, with the direction of LEU2 transcription opposite that of PMT4 . The resulting disruption fragment was excised from pSS51 by digestion with HindIII and SphI and used to replace the chromosomal PMT4 gene by one-step gene replacement . The presence of the pmt4 deletion was confirmed by colony PCR with the following oligonucleotides: OSS10, derived from LEU2 : 5′-TTAGACCGCTCGGCCAAACAACC; and OSS11, derived from the PMT4 3′ sequences not present in the pSS49 plasmid, 5′-GGCCATACCATGAAGTATACC; amplified a ∼0.8-kB fragment in pmt4 :: LEU2 cells. Control oligonucleotides: OSS7, derived from the PMT4 3′ flanking sequences, 5′-CCGAGGCCTCTGTACTTCTGTGGACTGTCAGAAAATATCTTGG; and OSS11 amplified a ∼0.5-kB fragment in Pmt + cells. To create a pmt4 :: TRP1 allele, the HincII–StuI fragment of YPP14, containing TRP1 , was introduced into the StuI site of pSS49. The resultant plasmid, pSS52, was digested with ClaI and SphI and used to disrupt the chromosomal copy of PMT4 by one-step gene replacement . The presence of the pmt4 deletion was confirmed by analyzing bud-site selection pattern. Yeast cells grown overnight and harvested in log phase were washed with 50 mM Tris-HCl, pH 7.5, 50 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and disrupted with glass beads in the same buffer containing 20 μg/ml antipain, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. The cell wall fraction was removed by centrifugation at 1,500 g . The resulting supernatant was centrifuged at 48,000 g to generate a membrane pellet. 5 to 10 μg of membrane protein was subjected to SDS-PAGE using 8 or 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked in TBS containing 0.1% Tween 20 and 4% nonfat dry milk, and decorated with a 1:1,000 dilution of HA antibodies in TBS containing 0.1% Tween 20 and 1% nonfat dry milk. Axl2/Bud10p-HA was visualized using the Nycomed Amersham EC L protein detection kit. Membrane pellets were resuspended in 1% SDS, 1% β-mercaptoethanol, 35 mM EDTA, and heated to 95°C for 3 min. 10 vol of incubation buffer (50 mM potassium phosphate buffer, pH 6.8, 35 mM EDTA, 1% β-mercaptoethanol, 0.55% octyl glucoside) was added, followed by a second incubation at 95°C for 3 min. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 20 μg/ml antipain, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) were added, and the reaction mixture was divided into two parts. PNGase was added to one aliquot, and both reaction mixtures were incubated at 37°C for 12 to 16 h. O- and N-linked sugars were removed by anhydrous hydrogen fluoride (HF) cleavage . Membrane fractions (50 μg protein) were lyophilized and dried in a desiccator containing solid P 2 O 5 . 250 μl HF was added and samples were treated for 80 min at 0°C. HF was subsequently removed by evaporation in a nitrogen stream. Yeast cells were grown overnight to early log phase, harvested, resuspended in fresh medium, and grown for 30 min at 30°C. Tunicamycin (10 μg/ml) was added, cells were incubated at 30°C for 60 min, and then subjected to membrane extraction as described in Membrane Extracts and Western Blot Analysis. To observe the autofluorescence of a fusion protein containing Axl2/ Bud10p and green fluorescent protein (GFP), cells in log phase were mounted on 0.5% agarose pads under coverslips sealed with pink or red nail polish. To observe bud scars, cells were stained with calcofluor as described by Pringle . Cells were visualized using a Zeiss Axioplan microscope with the FITC filter set and photographed with a three-chip cooled CCD camera. To identify genes required specifically for the axial budding pattern, we searched for mutants of α cells that exhibited a bipolar, rather than axial, budding pattern. This screening method took advantage of the relationship between pseudohyphal growth and budding pattern. Pseudohyphal growth is exhibited by cells with a bipolar budding pattern, such as a /α cells , as well as a bud4 and α bud4 mutants in the Σ1278b strain background . In contrast, cells that bud in the axial pattern, such as α strain SY77 (α ura3-52 hmr a :: URA3 ), do not exhibit pseudohyphal growth. We screened ∼70,000 colonies derived from UV-irradiated SY77 cells for the pseudohyphal growth phenotype. Budding patterns of the 107 mutants that displayed the pseudohyphal phenotype were assayed using a microcolony assay (see Materials and Methods). Ten BAD mutants exhibited bipolar budding patterns upon retesting and were shown to be defective in single genes by segregation analysis (see Materials and Methods). Of these, four were defective in BUD4 , one was defective in BUD3 , and one in AXL1 . Complementation tests revealed that the four remaining mutants, bad15 , bad43 , bad54 , and bad85 , defined three additional complementation groups. We show here that the complementation group identified by bad15 and bad43 corresponds to the previously identified gene PMT4 . bad15 mutant cells exhibited a novel budding pattern that is neither axial nor bipolar (Table III , line 3). In the axial pattern, cells bud adjacent to the previous division site . In the bipolar pattern, daughter cells typically bud distal to the previous division site, whereas mother cells bud either near the division site or distal to the previous division site . bad15 mutants (and to a lesser extent, bad43 mutants) exhibited a distinctive, unipolar budding pattern in which daughter cells budded distal to the division site, as in the bipolar pattern, and mother cells exhibited a typical axial pattern, budding adjacent to the previous division site (Table III , lines 3 and 5). The similar phenotypes of bad15 and bad43 mutants suggested that they might be defective in the same gene. Indeed, these mutants failed to complement (Table III , line 9). To determine whether the bad15 mutation affected bipolar budding, a /α bad15/bad15 diploids were constructed and analyzed. These strains exhibited normal bipolar budding (Table III , compare lines 2 and 4). These data indicated that the bad15 mutation affected only the axial budding pattern and primarily the behavior of daughter cells. This conclusion was supported by observing the budding patterns of bad15 cells over several generations. In one experiment in which eleven separate pedigrees were followed, 38/39 bad15 mother cells budded towards the previous division site, whereas 27/28 bad15 daughter cells budded away from the previous division site. Nine daughter cells in this experiment divided to become mother cells that divided again. In all cases, these cells budded towards the previous division site in the next cell cycle when they became mothers. These data demonstrated that the bad15 budding defect was exhibited only by daughter cells and not by mother cells. To determine whether bad15 mother cells exhibited normal axial budding, bud scars were analyzed by calcofluor staining. Since bad15 is an allele of PMT4 (see below), a complete deletion of PMT4 was used for this study. Cells with a single bud or bud scar and a visible birth scar were scored. Consistent with the microcolony data, which suggest that daughter cells budded incorrectly, only 29% of pmt4 cells placed the first bud or bud scar adjacent to the birth scar. Rather, 66% of pmt4 cells placed the first bud or bud scar opposite the birth scar ( n = 100), as is typical of the first bud in the bipolar pattern . In contrast, 94% of wild-type (WT) cells positioned a bud or bud scar adjacent to the birth scar ( n = 100), as is typical of the axial budding pattern . However, after the initial incorrect bud-site selection event, pmt4 mother cells budded in an axial pattern . 99% of the bud scars in both WT and pmt4 cells with two bud scars or a bud and a bud scar were adjacent ( n = 100 in both cases), as is typical of the axial budding pattern . In addition, 96% of the bud scars in WT and 92% of the bud scars in pmt4 cells with three or more scars were adjacent ( n = 300). The calcofluor staining pattern of WT and pmt4 cells with three or more bud scars was indistinguishable, suggesting that pmt4 cells bud axially after an initial incorrect bud-site selection choice. The BAD15 gene was cloned by screening for reversal of the pseudohyphal behavior of bad15 strain SY167. Only subclones containing the PMT4 gene were able to complement the bud-site selection defect of bad15 mutants . Pmt4p is one of seven yeast enzymes that initiate addition of O-linked oligosaccharides to proteins . BAD15 was confirmed to be allelic to PMT4 from the following experiments. First, pmt4 mutants failed to complement the bud-site selection defect of bad15 mutants, although both pmt4 and bad15 mutations were recessive (Table IV , lines 5, 6 and 7). Second, the same spectrum of proteins underglycosylated in pmt4 mutants was underglycosylated in bad15 and bad43 mutants (data not shown). Third, the bud-site selection phenotype of strains with mutations in PMT4 was identical to that of bad15 and bad43 mutants: all exhibited an axial budding defect that was daughter-specific, resulting in unipolar, rather than axial or bipolar budding (Table V , lines 8–13). Fourth, BAD15 and PMT4 exhibited tight genetic linkage: only two axial-budding recombinants were found in 13 tetrads (52 segregants) in crosses between α bad15 (SY167) and a pmt4 :: LEU2 (SY450). The seven yeast pmt enzymes have different substrate specificities: for example, O-glycosylation of Bar1p and Cts1p is severely affected by mutations in PMT1 and PMT2 , but not by mutations in PMT4 . In contrast, O-glycosylation of Kex2p and Gas1p/Gpg1p/gp115 is severely affected by mutations in PMT4 , but not by mutations in PMT1 and PMT2 . To determine if PMT4 is unique among PMT genes in being required for normal budding pattern, mutants defective in each of the PMT genes were analyzed. Of the six pmt mutants tested, only pmt4 mutants exhibited altered budding patterns (Table V , lines 1–7). The difference in penetrance of the pmt4 phenotype in the experiments shown in Tables III and V was due to differences in the strain backgrounds (data not shown). Since Pmt4p is a protein mannosyl transferase, we hypothesized that it was involved in O-glycosylation of a protein required for axial budding. Because PMT activity is confined to the ER, only proteins that pass through the ER can be glycosylated by these enzymes. Of all the proteins known to be involved in axial bud-site selection, only Axl2/Bud10p transits the secretory pathway and thus, is a candidate for O-glycosylation. We have used an epitope-tagged allele, AXL2/BUD10-HA , which complements axl2/bud10 mutations , to test whether Axl2/Bud10p is a substrate for Pmt4p. Because a lack of O-linked sugars might cause a change in the apparent molecular weight of Axl2/Bud10p, its mobility in pmt mutants was examined by SDS-PAGE . Full-length Axl2/Bud10p (∼205 kD) was found in all pmt mutants, with the exception of pmt4 , in which smaller fragments (∼43–59 kD) accumulated. Since the predicted molecular weight of the Axl2/Bud10p ORF is ∼91 kD, these data indicated that Axl2/Bud10p had undergone proteolytic cleavage. These 43–59-kD fragments, found exclusively in the membrane fraction (M. Gentzsch and W. Tanner, unpublished data), apparently lacked N-linked oligosaccharides, as their mobility did not change after treatment with PNGase . Fortuitously, studies of Axl2/Bud10p in the presence of tunicamycin revealed a stabilized form of the protein in pmt4 cells . We do not understand the nature of this tunicamycin-induced stabilization. In the presence of tunicamycin, Axl2/Bud10p from WT cells migrated at ∼125 kD. The absence of fully glycosylated (∼205 kD) Axl2/Bud10p suggests that its half-life is short since all is cleared within the 60 min tunicamycin treatment. Axl2/ Bud10p from pmt4 mutants migrated at ∼114 kD under the same conditions, indicating that Axl2/Bud10p in pmt4 mutants lacked a posttranslational modification. The modification was characterized by subjecting the Axl2/Bud10p species from tunicamycin-treated WT and pmt4 cells to treatment with HF to release remaining carbohydrates . HF treatment of WT extracts resulted in a further reduction in molecular weight of Axl2/Bud10p to ∼100 kD, indicating that it is an O-glycosylated protein . After treatment with HF, Axl2/Bud10p from pmt4 cells comigrated with the Axl2/Bud10p from WT cells at ∼100 kD . The shift in SDS-PAGE mobility of the 114-kD form of Axl2/Bud10p from pmt4 mutants indicated that it possessed some O-linked oligosaccharide . These observations indicate that Axl2/Bud10p from pmt4 mutants and from WT cells both contained O-linked oligosaccharides, but that Axl2/Bud10p received fewer O-linked oligosaccharides in pmt4 cells than in WT cells. Axl2/Bud10p received ∼25 kD of O-linked oligosaccharides: ∼13 kD are Pmt4p-independent; the remainder are Pmt4p-dependent. In contrast to its behavior in pmt4 strains, Axl2/Bud10p from tunicamycin-treated pmt1 , pmt2 , pmt3 , pmt5 , and pmt6 strains was almost indistinguishable in size from WT Axl2/Bud10p . Longer SDS-PAGE runs, which separated proteins of high molecular weight, showed that Axl2/Bud 10p was slightly smaller in pmt1 and pmt2 strains (data not shown). Addition of the Pmt4p-independent O-linked oligosaccharides present on Axl2/Bud10p in pmt4 mutants may be added by Pmt1p and Pmt2p. To determine the subcellular compartment in which proteolysis of Axl2/Bud10p occurred in pmt4 cells, Axl2/ Bud10p was analyzed in strains with temperature-sensitive mutations in sec18 and sec7 , which disrupt secretory traffic from the ER to the Golgi apparatus and through the Golgi apparatus, respectively . Axl2/Bud10p was degraded in pmt4 sec18 cells grown at permissive temperature . Incubation of cells at 37°C resulted in accumulation of full-length, nondegraded Axl2/ Bud10p . In contrast, Axl2/Bud10p was degraded at both 25°C and 37°C in the sec7 pmt4 strain . These data suggested that degradation of Axl2/Bud10p occurred after the secretory block imposed by sec18 and before the block imposed by sec7 . Thus, degradation of Axl2/Bud10p most likely occurred in the Golgi apparatus. If Axl2/Bud10p is degraded in the Golgi apparatus, its cleavage should be independent of vacuolar proteases. That prediction was borne out. Mutations in the genes encoding vacuolar proteases Pep4p, Prb1p, and Prc1p did not prevent degradation of full-length Axl2/Bud10p in pmt4 mutants . Considerably more of the 43–59-kD Axl2/Bud10p species was found in pmt4 pep4 prb1 and pmt4 pep4 prb1 prc1 mutants than in pmt4 mutants alone, suggesting that vacuolar proteases further degrade the 43– 59-kD fragments. Degradation of Axl2/Bud10p in pmt4 mutants was unaffected by mutation of UBC7 , which encodes a ubiquitin-conjugating enzyme that participates in degradation of ER proteins , again suggesting that the cleavage of Axl2/Bud10p does not occur in the ER . Localization of Axl2/Bud10p is dependent upon cell cycle stage . In early G1 cells, Axl2/Bud10p is found in a patch at the presumptive bud site; it is later found at the periphery of the bud. By the time of the G2/M transition, Axl2/Bud10p is observed on the bud side of the mother-bud neck and is subsequently found on both sides of the mother-bud neck. Axl2/ Bud10p persists at the neck region of mother and daughter cells past cytokinesis. We made similar observations in our Pmt + strain background using cells that produce a fusion of Axl2/Bud10p to the GFP . 97% of fluorescent WT cells in an asynchronous population exhibited strong staining that fell into one of three classes. Almost 50% of cells (202/407) exhibited staining at bud periphery . 30% of cells (121/407) exhibited staining predominantly at the bud side of the mother-bud neck . 17% exhibited strong staining that was symmetrically distributed with respect to the mother-bud neck . Though we detected no difference in the distribution of cells with respect to the cell cycle in pmt4 mutants versus WT cells, only 10% of pmt4 cells (37/369) exhibited staining that fell into the three classes defined by WT cells. Most mutant cells exhibited a strikingly different localization pattern : 58% (216/369) exhibited a punctate intracellular pattern that resembled the Golgi apparatus . Such staining was seen in only 3% of Pmt + cells. 13% of pmt4 cells with these punctate intracellular patches also exhibited relatively normal staining at the bud periphery . Many pmt4 cells (206/369) accumulated Axl2/Bud10p at the neck. However, unlike WT cells, the neck staining was enriched on, or was specific to, the mother side of the mother-bud neck or faint, but equally distributed on either side of the neck . None of the neck staining in WT cells was observed in either of these two categories. Localization of the Bud4 protein was unaffected by mutation of pmt4 (data not shown). If Axl2/Bud10p activity is decreased due to the lack of O-linked oligosaccharides, overexpression of AXL2/BUD10 might partially restore the axial budding pattern to pmt4 mutants. To test this possibility, pmt4 and WT strains were transformed with YEp24- AXL2 , which carries AXL2/ BUD10 on a high copy plasmid. This plasmid slightly decreased the axial budding of WT cells, from 93 to 76% axial. In contrast, YEp24- AXL2 partially rescued the unipolar budding pattern of pmt4 mutants to axial, increasing axial budding from 5 to 40%. Axl2/Bud10p is a transmembrane glycoprotein required for yeast cells to exhibit the axial budding pattern . Like Bud3p and Bud4p , it is proposed to function as an intracellular landmark: Axl2/ Bud10p localizes to both sides of the mother-bud neck for part of the cell cycle and is inherited by both mother and daughter cells at the position of bud emergence in the previous cell cycle. Through the isolation and characterization of mutants with altered bud-site selection, we have uncovered a requirement for O-linked glycosylation by the mannosyl transferase Pmt4p, which presumably acts directly on Axl2/Bud10p. Though O-linked glycosylation has been assumed to be important for protein function, identifying specific roles for this modification on particular proteins has been elusive. We suggest that O-linked glycosylation of Axl2/Bud10p promotes its movement through the secretory pathway, prevents its degradation, and allows Axl2/Bud10p to function on the cell surface to assemble components required for bud emergence. Axl2/Bud10p acquires O-linked oligosaccharides, some of which are dependent upon Pmt4p. Only the Pmt4p-dependent sugars appear to be important for Axl2/Bud10p function. Because Axl2/Bud10p in pmt4 mutants contains some O-linked oligosaccharide, it is clear that the simple presence of O-linked oligosaccharide is not sufficient to facilitate Axl2/Bud10p protein function. It appears that, as with protein phosphorylation, the amount of O-linked oligosaccharide or the specific sites of modification are important for function. Studies of Axl2/Bud10p provide an opportunity to identify the determinants for recognition by Pmt4p and other PMT proteins. Because proteins, such as Kex2p and Gas1p/gp115/Ggp1p are glycosylated primarily by Pmt4p , comparison of Kex2p, Gas1p/gp115/Ggp1p, and Axl2/Bud10p may help to identify consensus acceptor sequences for Pmt4p-dependent O-linked glycosylation. All three proteins have extracellular domains rich in serine and threonine residues that are close to the membrane . These domains are glycosylated in the case of Kex2p and Gas1p/ gp115/Ggp1p , and perhaps Axl2/Bud10p as well. Serine- and threonine-rich domains close to the membrane may be recognized specifically by Pmt4p. Pmt4p is required for the stability of Axl2/Bud10p. Axl2/Bud10p is a large, type I integral protein of the plasma membrane. In pmt4 mutants, Axl2/Bud10p is cleaved to produce stable membrane-associated fragments of ∼43–59 kD, which appear to be the cytoplasmic-transmembrane segment of Axl2/Bud10p. This structure is inferred from three lines of evidence. First, the fragments are recognized by antibodies specific for an epitope tag at the COOH terminus of Axl2/Bud10p. Second, as these fragments are membrane-associated, they presumably also contain the transmembrane domain. Third, these fragments lack both N- and O-linked oligosaccharides, which should be present solely in the extracellular domain. Roemer et al. determined that Axl2/Bud1p is a type I membrane protein by demonstrating that the COOH terminus of Axl2/Bud10p is refractory to degradation by proteases added extracellularly. The apparent molecular weight of the protease-resistant fragments (a collection of fragments 50–55 kD) is similar to that of the fragments we observe in pmt4 mutants (43–59 kD), adding further credence to the notion that the fragments contain the COOH terminus of Axl2/Bud10p joined to the transmembrane domain. We do not know the fate of the remaining ∼40% of Axl2/Bud10p, which includes the NH 2 -terminal segment. Two lines of evidence suggest that the initial degradation of Axl2/Bud10p in pmt4 mutants occurs in the Golgi apparatus. First, proteolytic cleavage of Axl2/Bud10p occurs in sec7 pmt4 , but not in sec18 pmt4 mutants, suggesting that cleavage requires transit to the Golgi apparatus and occurs even when further transport is inhibited. Consistent with this notion, cleavage of Axl2/Bud10p to the ∼55-kD fragments does not require vacuolar proteases. The accumulation of these ∼55-kD fragments in pep4 prb1 and pep4 prb1 prc1 cells suggested that once generated, these fragments are degraded in the vacuole. Previous studies have revealed an effect of O-glycosylation on protein stability. Axl2/Bud10p is stabilized in pmt4 mutants grown in the presence of the N-linked glycosylation inhibitor, tunicamycin. We do not know the reason for this stabilization. One possibility is that in the absence of N-linked sugars, Axl2/Bud10p cannot reach the Golgi apparatus, where its initial degradation occurs. However, Axl2/Bud10p localization in pmt4 mutants appears the same in the presence versus absence of tunicamycin (data not shown). We favor a second possibility, that the conformation of Axl2/Bud10p lacking its N-linked sugars makes it a poorer substrate for proteolytic degradation. Several proteins, including the low density lipoprotein (LDL) receptor, decay accelerating factor, major envelope protein of Epstein-Barr virus, Kex2p, and neurotrophin receptor are less stable when they lack O-linked oligosaccharides . Not all O-linked oligosaccharides are important for protein function, however: Gas1p/Ggp1p/gp115 and human chorionic gonadotropin lacking O-linked oligosaccharides are indistinguishable in stability from their glycosylated counterparts. Instability of Axl2/Bud10p and its failure to accumulate efficiently at the cell surface without modification by Pmt4p are probably coupled, though it is unclear which is the primary consequence of the O-glycosylation defect. Perhaps unmodified Axl2/Bud10p is recognized as a misfolded protein and targeted for destruction by a quality control system of the Golgi apparatus. Resulting proteolytic fragments might be inefficiently targeted to the plasma membrane. Another possibility is that absence of Pmt4p-dependent modifications delays movement of Axl2/ Bud10p through the secretory pathway, increasing the probability of cleavage by a resident Golgi protease. Some Axl2/Bud10p does assemble at the bud periphery and neck in pmt4 cells, though the amount is reduced with respect to WT cells. As for the LDL receptor , the lack of O-linked oligosaccharide may also affect Axl2/Bud10p stability once it reaches the plasma membrane. Axl2/Bud10p seems to be inherently unstable. Because cells defective in PMT4 exhibit a bud-site selection defect, we initially predicted the existence of a Pmt4p substrate protein important for accurate budding. Our studies indicate that Axl2/Bud10p is the Pmt4p substrate relevant to bud-site selection: Axl2/Bud10p is O-glycosylated, unstable and mislocalized in pmt4 mutants, and its overproduction can partially rescue the pmt4 phenotype. The requirement for Pmt4p only in daughter cells might be explained in a variety of ways. In principle, Axl2/ Bud10p might be required only in daughter cells, or its glycosylation might be necessary only in daughter cells. Because Axl2/Bud10p is required for bud-site selection in both mother and daughter cells , the first explanation appears to be incorrect. Therefore, we entertain a second possibility, that O-linked glycosylation of Axl2/Bud10p by Pmt4p is required only in daughter cells. Perhaps O-glycosylation is not needed to stabilize Axl2/Bud10p in mother cells. This explanation is unlikely, since a single, partially degraded population of Axl2/Bud10p is detected by Western blot , and intracellular patches of Axl2/Bud10p accumulate in both mother and daughter cells . Another possibility is that mother cells can accommodate the reduction of Axl2/Bud10p activity in pmt4 mutants, whereas daughter cells cannot. We favor this third possibility. The differences between mother and daughter cells in the requirement for PMT4 may arise from differences in the path by which Axl2/Bud10p reaches the mother-bud neck . Axl2/Bud10p is thought to become localized to the bud side of the mother-bud neck by reorganization of protein in cells with a small bud . Localization of Axl2/Bud10p at the mother side of the mother-bud neck is hypothesized to occur subsequently by new synthesis within the mother cell . After cytokinesis, both mother and daughter cells inherit a patch of Axl2/Bud10p, which promotes utilization of axial bud sites. In pmt4 mutants, we propose that Axl2/Bud10p is at a reduced level, so that the activity in the daughter side of the mother-bud neck is inadequate . In contrast, newly synthesized Axl2/Bud10p, which becomes localized to the mother side of the mother-bud neck , is proposed to be adequate for promoting utilization of axial bud sites after cytokinesis . In other words, Axl2-Bud10p used for axial budding in daughter cells must persist from S phase until the next G1 phase, whereas that in mother cells need persist only from late mitosis until the next G1 phase. That daughter cells exhibit a longer G1 phase than do mother cells might also make it harder for Axl2/Bud10p to persist until it is needed again in daughter cells. Support for the proposal that Axl2-Bud10p is differentially affected in mother and daughter cell compartments comes from the observation that Axl2-Bud10p can be observed preferentially on the mother side of the mother-bud neck in a substantial fraction of pmt4 mutant cells (21%), a distribution that is rare in WT cells (0.25%). For the 33% of pmt4 cells that exhibited faint Axl2/Bud10p staining on both sides of the mother-bud neck, we argue that this level of Axl2/ Bud10p is sufficient for bud-site selection in mother cells, but is inadequate in daughter cells. Several functional differences between mother and daughter cells have been described previously. Mother cells can switch mating type, whereas daughter cells cannot ; mother cells specifically inherit plasmids containing autonomously replicating sequences (ARS) and lacking centromere sequences, whereas daughter cells do not ; and mother cells budding in the bipolar pattern may bud distal or proximal to the birth site, whereas new daughter cells bud primarily at distal sites . O-glycosylated cell-surface molecules certainly contribute to selection of bud sites in a /α cells: both Bud8p and Bud9p appear to be O-glycosylated . Here, we have observed that the integrity of an O-glycosylated landmark protein can differentially affect the behavior of mother and daughter cells and generate an asymmetric cell division. Asymmetrically located landmarks that are anchored to the cell wall by O-glycosylated proteins might contribute to the asymmetric switching of mating type and plasmid inheritance as well. | Other | biomedical | en | 0.999995 |
10366592 | Frozen rat lungs were purchased from Pel-Freez Biologicals. Ampholines pI 3–10 and DEAE-Sephacel were from Pharmacia . Agarose bound or biotinylated Griffonia simplicifolia I lectin (GS I) and melibiose were from either Vector Laboratories or EY Laboratories. PVDF (polyvinyldifluoride) membrane was purchased from Millipore and nitrocellulose membrane from MSI. Protogel (30% acrylamide solution) was obtained from National Diagnostics. The rat lung expression library and the rat multiple tissue Northern Blot™ was purchased from Clontech . Protran 82 nitrocellulose circles were purchased from Schleicher & Schuell. Bacto-Tryptone and Bacto-Agar were from Difco and ampicillin, agarose, and Trizol™ from Life Technologies. Restriction enzymes were from New England Biolabs and cloning vectors pBluescript SK (−) and pBluescript II KS (+), pfu DNA polymerase, and QuickHyb™ hybridization solution were from Stratagene. pCR 2.1 vector and TACloning™ kit were from Invitrogen Corp. pQE-30 cloning vector and QIAprep™ plasmid DNA miniprep kits were from Qiagen. Luria-Bertani broth (LB-broth) and Luria-Bertani agar (LB-agar) were purchased from Bio101. MaxiScript™ and RPA II™ kits were purchased from Ambion. [ 32 P]dUTP and BSA were from ICN Biomedicals. Hybond-N + nylon membrane and [ 32 P]dCTP were from Amersham . All other reagents were either from Sigma Chemical Co. or Fischer. Buffers were as follows: Hepes-buffered sucrose: 250 mM sucrose, 20 mM Hepes, pH 7.2, supplemented with 5 mM MgCl 2 and protease inhibitors cocktail (10 μg/ml each leupeptin, pepstatin, o -phenantrolin, E-64, and 1 mM PMSF); Hepes-buffered saline (HBS): 150 mM NaCl in 10 mM Hepes, pH 7.2; Hepes-buffered saline/Triton X-100 (HBS-T): 0.2% Triton X-100, 150 mM NaCl in 10 mM Hepes, pH 7.2; lectin binding buffer: 0.2% Triton X-100, 150 mM NaCl in 10 mM Hepes, pH 7.2, supplemented with 1 mM each of CaCl 2 , MgCl 2 , and MnCl 2 . Fig. 1 a gives the schematic of the PV-1 isolation procedure. Frozen rat lungs were weighed, minced on ice in Hepes-buffered sucrose (1:4 wt/vol), and homogenized in a motor driven Thomas type “C” Teflon pestle-glass homogenizer by 15 strokes at 1,800 rpm. The homogenate was filtered through a 53-μm nylon mesh and separated into a nuclear pellet and a postnuclear supernatant by centrifugation (10 min, 1,800 g , 4°C) in a Beckman GPR centrifuge. The postnuclear supernatant was resolved into a total membrane pellet and a soluble supernatant by centrifugation (2 h, 100,000 g , 4°C) in a SW28 rotor. Next, the rat lung total membrane pellet was resuspended in 25 ml of ice-cold 0.1 M Na 2 CO 3 , pH 11, by low speed homogenization using a Thomas type “B” homogenizer followed by incubation (15 min, 4°C) with gentle agitation. The insoluble material was collected by centrifugation (1 h, 45,000 rpm, 4°C) in a Ti70 rotor and the supernatant discarded. The pellet was further resuspended by gentle homogenization in 20 ml ice-cold 2% Triton X-100 in 5 mM Hepes, pH 7.2, supplemented with protease inhibitors followed by a 1-h incubation at 4°C with end-over-end rotation. The Triton X-100 extract was clarified by centrifugation (1 h, 45,000 rpm, 4°C) in a Ti70 rotor. The supernatant, containing the Triton X-100 soluble proteins, was further adjusted to 4% (wt/vol) urea, 2% (vol/vol) ampholines, pI 3–10, 1% (vol/vol) Triton X-114, and 5% (wt/vol) glycerol to a final volume of 35 ml and subjected to isoelectric focusing using a Rotofor chamber (Bio-Rad) as per manufacturer's instructions. 20 fractions of ∼1.7 ml were collected, subjected to 8% SDS-PAGE, and analyzed for the presence of PV-1 by Western blotting using the anti–PV-1 21D5 mAb and by silver staining for visualization of the protein content. The fractions containing the antigen (usually fractions 11–14 counting from the acidic end of the gradient −pH 3) were pooled and dialyzed (4 h, 4°C) against 4,000 volumes of HBS-T using a 50-kD cut-off dialysis membrane. The dialysate was diluted 10× with HBS-T and next adjusted to 1 mM each CaCl 2 , MgCl 2 , and MnCl 2 . The resulting mixture was incubated (10–12 h, 4°C) with 2 ml (settled gel) of GS I-agarose with gentle rotation (GS I lectin specificity: Gal/GalNAcα1,3 .). The beads were collected by centrifugation (10 min, 500 g , 4°C) and washed 3 × 10 min with 45 ml lectin binding buffer. GS I bound proteins were eluted twice by incubation with 2.5 ml elution buffer (HBS-T supplemented with 0.2 M melibiose and 5 mM EDTA) for 1 h and the two eluates pooled. The eluted glycoproteins were further incubated with 2 ml (settled gel) DEAE-Sephacel (previously equilibrated with HBS-T) for 4 h at 4°C. The flow-through was collected and subjected to 15% TCA precipitation for 1 h on ice. The precipitated proteins, representing the purified material, were solubilized in 2× reducing sample buffer, resolved by 8% SDS-PAGE, and transferred to either PVDF (for NH 2 -terminal sequencing) or nitrocellulose (for internal sequencing) membrane. The PVDF membrane containing SDS-PAGE resolved proteins was stained with 0.1% Coomassie brilliant blue G 250 in 40% methanol and 10% acetic acid and the PV-1 band was excised and used for Edman degradation. The NH 2 -terminal sequencing was done on samples obtained from two separate experiments by the protein sequencing facility at the University of California, San Diego. The SDS-PAGE resolved proteins transferred to nitrocellulose membrane were stained with 0.1% Ponceau S in 1% acetic acid and the PV-1 band was excised and digested with trypsin. Two of the resulting peptides were purified, sequenced, and the sequence confirmed by mass spectrometry by the protein sequencing laboratory at the Worcester Foundation for Biomedical Research (Shrewsbury, MA). We used the anti–PV-1 21D5 mAb to screen a rat lung expression library cloned into the bacteriophage λgt11 as per manufacturer's instructions. Briefly, 500,000 phages were plated and induced with 10 mM IPTG (isopropyl β- d -thiogalactopyranoside) to express the proteins encoded by their inserts. The proteins were transferred to nitrocellulose membranes which were probed by Western blotting with the anti–PV-1 21D5 mAb. The positive plaques were purified to homogeneity by three more screening rounds. The four longest inserts were either subcloned into pBluescript SK(−) vector or PCR amplified using λgt11- specific primers (sense: 5′TCCTGGAGCCCGTCAGTATCGGCG3′ and antisense: 5′ATGGTAGCGACCGGCGCTCAGCTG3′) and the PCR product inserted into pCR 2.1 vector. The resulting clones were sequenced in both directions which led to a partial sequence of PV-1 message. To obtain the full length cDNA we designed a 428-bp DNA probe in the 5′ region of the message obtained by screening with the antibody. This probe was 32 P-labeled using PrimeIt™ kit (Stratagene) and used to screen another 500,000 phages. 24 positive phage clones were purified to homogeneity and the 5 longest inserts were sequenced after subcloning them into pBluescript II KS (+) vector. The sequencing of these later inserts yielded the full length PV-1 message. DNA sequencing was performed on an ABI Prism Sequencer (model 373XL) by either the Core Facility for AIDS Research at the University of California, San Diego or the Sequencing Facility at the Scripps Research Institute (La Jolla, CA). The resulting sequences were analyzed using the MacVector release 6.0 software from Oxford Molecular Group, Inc. A premade rat multiple tissue Northern blot containing 2 μg mRNA/lane from different rat tissues was probed with a 32 P-labeled 428-bp PV-1 cDNA fragment for detection of the PV-1 message. The hybridizations were done using QuickHyb™ hybridization solution as per manufacturer's instructions. A 283-bp fragment containing the nucleotides 1–283 of the full length PV-1 cDNA was PCR amplified, and the PCR product was gel purified and inserted into pCR 2.1 vector using the TACloning™ kit. The cloned insert was checked by DNA sequencing and a 32 P-labeled complementary RNA probe was synthesized with T7 RNA polymerase using the MaxiScript™ kit. Total RNA from rat lung, spleen, kidney, and liver were purified using the Trizol™ reagent as per manufacturer's instructions. 20 μg of total RNA from the above tissues was used in an RNase protection assay carried out using RPA II™ kit and following the manufacturer's standard protocol. The reactions were resolved by 5% denaturing polyacrylamide gel electrophoresis and the gel exposed to autoradiography film for 6–24 h. The PV-1 cDNA was directionally subcloned in the NcoI and PstI sites of the pQE-30 vector and the construct used to transform competent Escherichia coli (M15 strain). Positive clones were cultured for 6 h at 37°C until the OD 600 ≈ 0.7 and the recombinant protein production was induced by the addition of IPTG to 1 mM final concentration. Aliquots of noninduced (control) and induced were collected by centrifugation at 10,000 g and solubilized in reducing SDS-PAGE sample buffer. The solubilized bacterial proteins were resolved by 10% SDS-PAGE, transferred to PVDF membrane, and immunoblotted using the 21D5 mAb. A 14 mer peptide (KGPPLVNPAVPPSG single letter amino acid [aa] code) corresponding to the 12 last COOH-terminal amino acids of rat PV-1 (to which a lysine and a glycine were added to facilitate the coupling and to improve solubility) was synthesized by SynPep Corp. The peptide was coupled to BSA via glutaraldehyde and this conjugate was used as antigen for polyclonal antibody production in chickens at Lampire Biologicals using the company's standard immunization protocol. Total chicken IgY was purified from eggs laid by the immunized chickens using a modified procedure of Polson et al. ( 26 ). Briefly, egg yolks devoid of the amniotic sack were diluted 4× in PBS and brought to 3.5% polyethylene glycol (PEG). The precipitated lipoproteins were centrifuged for 25 min at 5,000 g in a JA-14 rotor and the supernatant was saved. The pellet was redissolved in 2× initial yolk volume of PBS and a second round of 3.5% PEG precipitation was performed as before. The two supernatants were pooled, filtered through four layers of cheesecloth, and centrifuged for an additional 30 min at 7,500 g. The supernatant was filtered through Whatman No. 4 filter paper, its volume measured (Z ml) and mixed with 0.085 × Z grams of PEG to precipitate IgY. The precipitate was collected by centrifugation at 5,000 g for 25 min. The pellet was solubilized in PBS (2.5× initial yolk volume) and IgY was again precipitated using 3.5% PEG. The precipitate was collected by 5,000 g centrifugation for 25 min and redissolved in PBS (0.25× initial yolk volume). This IgY solution was chilled on ice for 10 min and mixed with an equal volume of 50% ethanol which had been chilled at −20°C. The precipitated IgY was collected by 30 min of centrifugation at 10,000 g , redissolved in PBS (0.25× initial yolk volume), and dialyzed overnight against 20–40 vol of PBS to yield a total IgY fraction from egg yolk. The PV-1 COOH-terminal peptide used for immunizations (PV-1C peptide) was solubilized in 0.1 M Hepes, pH 8.0, at a final concentration of 20 mg/ml and coupled to AffiGel-10™ beads (4 ml settled gel) (Bio-Rad) by incubation (16 h, 4°C) with gentle agitation. After quenching (1 h, room temperature [RT]) the remnant active sites with 0.2 M ethanolamine, pH 8.2, the matrix was packed into a column and washed with 100 bed vol of PBS. Total IgY fraction from egg yolk was incubated (12–14 h, 4°C) with the column with the help of a peristaltic pump. The column was washed with 100 bed vol of PBS and the bound antibodies were eluted with 10 bed vol of 0.1 M glycine, pH 2.5. Fractions of 1.4 ml were collected onto 100 μl of 1.5 M Tris, pH 8.8, and promptly mixed for antibody activity preservation. Each fraction was monitored for protein content by absorbance at 280 nm and for antibody specific activity by ELISA assays using PV-1C peptide–coated microwell plates. The fractions containing the antibody were pooled, concentrated, and dialyzed at 4°C against 2 changes (4 h each) of 100 vol of PBS using a 100 kD cut-off dialysis membrane. Affinity purified anti–PV-1C pAb was checked for specificity and activity in an ELISA assay using serial dilutions of the antibody on 10 ng PV-1C peptide/plate well. The bound anti–PV-1C antibody was detected using an anti–chicken IgY HRP-conjugated reporter antibody (Biodesign) and TMB (3, 3′, 5, 5′ tetramethylbenzidine) substrate (KPL) for colorimetric reaction. Either preimmune IgY or an irrelevant peptide was used as negative controls. Whole rat lung lysate proteins (200 μg) were resolved by preparative 10% SDS-PAGE and transferred to a PVDF membrane which was subsequently blocked (30 min, RT) in 5% nonfat dry milk in PBS and 0.1% Tween 20. Strips, containing ∼20 μg protein, were cut and incubated (1 h, RT) with either serial dilutions of the anti–PV-1C pAb or 21D5 mAb or preimmune IgY (as positive and negative controls, respectively). The bound antibody was detected by incubation (30 min, RT) with a rabbit anti–chicken IgY HRP-conjugated antibody (Biodesign) and enhanced chemiluminescence (Super Signal™; Pierce). In the case of the peptide competition, 1-μg aliquots of anti–PV-1C pAb were incubated (1 h, RT) with different amounts of the peptide prior to the incubation with the strips. Rat lung membranes were extracted for 1 h on ice in immunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, containing 1% NP-40, 0.4% deoxycholate, 0.1% SDS, 300 mM NaCl, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail). The extract was clarified by centrifugation at 100,000 g for 1 h in a TLA45 rotor. The extracted proteins (200 μg) were incubated (14 h, 4°C) with 5 μg of anti–PV-1C pAb with gentle agitation. This mixture was further incubated (4 h, 4°C) with anti–chicken IgY antibodies insolubilized onto agarose beads to precipitate the antigen-antibody complexes. The beads were collected by centrifugation (500 g , 5 min) and washed (3 × 5 min) with immunoprecipitation buffer followed by one final wash in 50 mM Tris, pH 6.8. The beads were boiled in nonreducing SDS-PAGE sample buffer (2.3% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8) and collected by centrifugation. The supernatant containing the solubilized antigen-antibody complexes was saved, adjusted to 5% 2-mercaptoethanol, and boiled again for 2 min. The solubilized proteins were resolved by 8% SDS-PAGE, transferred to PVDF membrane, and immunoblotted using the anti–PV-1 21D5 mAb. For visualization of the immunoprecipitated protein, the solubilized antigen-antibody complexes were resolved in nonreducing conditions on 12% SDS-PAGE and silver stained. Control experiments were carried out by replacing the anti–PV-1C pAb with preimmune total IgY or by omitting it. Total membranes from different rat organs or tissues were prepared as in the case of the lung and high-pH extracted. The high-pH insoluble material (containing PV-1) was extracted in 0.5% SDS in 50 mM Tris, pH 6.8, and protease inhibitor cocktail, clarified by centrifugation for 30 min at 12,000 g , and the protein content determined. Equal amounts of protein (300 μg) from different rat tissues and 10 μg in the case of the lung were separated by 8% SDS-PAGE, transferred to PVDF, and immunoblotted using the anti–PV-1 21D5 mAb. Preembedding immunocytochemistry was performed as previously described in Predescu et al. ( 28 ). Briefly, the tissue was flushed free of blood by a 10-min perfusion with Hank's balanced salt solution, then fixed in situ by a 30-min perfusion of paraformaldehyde-lysine-sodium metaperiodate (PLP) fixative. The tissue was excised, cut into small blocks (∼3 × 3 mm), further fixed in fresh PLP for 1 h at RT, and then fixed overnight at 4°C (with a fresh PLP change). Fixed specimens were cryoprotected by infiltration (12–16 h at 4°C followed by 1 h at RT) with a solution containing 1.5 M sucrose, 50% polyvinylpyrolidone in PBS, and stored frozen in liquid nitrogen. Thick cryostat sections (∼45 μm) cut from the fixed blocks were rinsed (5 × 5 min) and incubated overnight at 4°C in 10% goat serum in PBS, quenched in 1% BSA in PBS (PBSA) for 30 min at RT and incubated overnight at 4°C with the anti– PV-1C pAb diluted (1:50–1:250) in PBSA. The sections were washed 3 × 30 min in PBSA at RT, incubated with the rabbit anti–chicken IgY 5 nm gold-conjugated antibody (1:100 dilution in PBSA) for 12–16 h at 4°C. After final washes as above the antibody-antigen complexes were stabilized by fixation (1 h at RT) in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, postfixed in 1% OsO 4 in acetate veronal buffer, pH 6.8 (1 h on ice), stained in the dark (1 h at RT) with Kellenberger uranyl acetate, dehydrated through graded ethanols, and finally embedded in Epon 812 resin. Tissue blocks were cured for 48 h at 90°C, and ∼50-nm sections, cut on a Reichert microtome, mounted on formvar-coated nickel grids, and stained with 2% uranyl acetate and saturated lead citrate. The stained sections were examined and photographed in a Philips CM10 electron microscope. This approach for immunolabeling relying on the diffusion of the antibodies along vascular and perivascular spaces provides adequate sampling of morphologically well preserved vascular endothelia. Small blocks (1 × 1 mm) from PLP fixed lungs, cryopreserved and stored as above, were sectioned on a Reichert ultramicrotome equipped with a F4 cryoattachment. Ultrathin cryosections transferred to nickel grids were stained and then washed by floating the grids, specimen down, on drops of filtered solutions (41). After quenching (30 min at RT) with 1% BSA and 0.01 M glycine in PBS, the sections were incubated (1 h, RT) with anti–PV-1C pAb diluted 1:100 in PBSA, washed (3 × 15 min) in PBSA, and further incubated with a rabbit anti–chicken IgY antibody conjugated to 5 nm colloidal gold (diluted 1:100 in PBSA) for 2 h at RT, then washed as above. The immune complexes were stabilized (30 min at RT) in 2% glutaraldehyde in PBS, postfixed in 1% OsO 4 in acetate-veronal buffer, pH 7.6, absorption-stained (5 min, RT) with 0.002% lead citrate in 2.2% polyvinylalcohol, and finally examined and photographed in a Philips CM10 electron microscope. This procedure has the disadvantage of a limited yield of useful sections and suboptimal structural preservation, but the advantage of providing information on the intracellular distribution of the antigen. The protein content was determined by the BCA method (Pierce) against either BSA or IgY standards made in appropriate buffers. The membranes were blocked (30 min, RT) in 5% nonfat dry milk in PBS and 0.1% Tween 20 (blocking buffer), incubated (1 h, RT) with the first antibody diluted in blocking buffer, washed (3 × 5 min) in PBS containing 0.1% Tween 20, incubated (30 min, RT) with a HRP-conjugated reporter antibody, washed again as above, and the signal detected by using ECL reagents. As the monoclonal antibody 21D5 is not a useful reagent for affinity purification of PV-1, we explored alternative approaches taking advantage of the already known properties of this protein (molecular weight, occurrence as a dimer, and resistance to high-pH and high-salt extraction). Pilot experiments as Triton X-100 differential solubility, pI determination, lectin binding and elution, and anion exchange chromatography were carried out using rat lung total membranes to assess the behavior of PV-1 in any of the isolation steps. Based on the pilot experiments, the isolation of PV-1 from rat lungs was carried out according to the protocol depicted in Fig. 1 a and detailed in Materials and Methods. Aliquots of different fractions throughout the purification procedure were monitored for presence of PV-1 by immunoblotting using the 21D5 mAb. The frozen rat lungs were minced, homogenized, and resolved by centrifugation in a tissue debris and nuclei pellet and a postnuclear supernatant. The latter was used for obtaining a total membrane fraction containing PV-1. As documented in Fig. 1 b, PV-1 distributes exclusively in the total membrane fraction. However, the anti–PV-1 21D5 mAb recognizes another band of ∼45–50,000 molecular weight which we determined to be a soluble rat blood plasma protein which is efficiently eliminated by the centrifugation step used to separate membranes from cytosol. To eliminate the membrane associated proteins, the rat lung total membranes were high-pH extracted, the insoluble material which contained PV-1 collected by centrifugation, and the supernatant discarded. This high-pH insoluble material was further extracted in 2% Triton X-100 and the extract clarified by centrifugation. Fig. 1 d shows the efficiency of PV-1 extraction which was essentially complete. The clarified Triton X-100 extract was further subjected to isoelectric focusing in a Rotofor chamber. PV-1 was recovered in 3–4 fractions of the pI gradient corresponding to a pI interval 6.85–7.9. These fractions were pooled, dialyzed, and incubated with GS I lectin bound to agarose. After washing nonspecifically interacting proteins the bound glycoproteins were eluted by competition with melibiose and EDTA . GS I lectin was chosen for the isolation procedure for two reasons. First, the binding efficiency and especially elution efficiency were the highest compared to other lectins tested (our unpublished data); and second, the binding sites for this lectin have been documented at the electronmicroscopic level in rat lung by previous work done in our laboratory ( 2 ). This study has shown some of the binding sites of GS I to be located in the caveolae of the rat lung endothelium and endothelia of other microvascular beds. The eluate from GS I column was further subjected to DEAE-Sephacel chromatography and the flow-through was collected, as PV-1 binds very weakly to this anion exchange column. We introduced this step because DEAE binds proteins in the 50–70 kD range, thereby improving the separation of PV-1 from other bands and making possible its excision free of contaminants. The proteins from the flow-through were precipitated by TCA, solubilized in 2× reducing sample buffer, and resolved by 8% SDS-PAGE to obtain maximum resolution in the 50–60,000 molecular weight range . The resolved proteins were transferred to either a PVDF or a nitrocellulose membrane, the PV-1 band identified, excised, and sent for NH 2 -terminal or internal sequencing. The sequences of three peptides, one representing the NH 2 terminus of PV-1 (NH 2 -terminal) and the other two representing internal sequences (Internal 1 and Internal 2) were obtained (Table I ). Mass spectrometry analysis was carried out on the internal peptides for confirmation of the sequence. Searches of protein databases and mass spectrometry databases showed that these sequences are novel, as very little homology with other known proteins was found. The anti–PV-1 21D5 mAb was used for screening an oligo-dT and random primed rat lung expression library cloned in the bacteriophage λgt11 and the inserts of the positive phages were sequenced as described in Materials and Methods. Only 1,274 nucleotides of the PV-1 full length message were obtained by this approach. Additionally, we were able to narrow down the region where the epitope of the 21D5 mAb is located as the overlap of the cloned phage inserts amounted to a sequence of 39 nucleotides which encodes for the amino acid residues 405–417 of the PV-1 translated protein sequence . To obtain the sequence of the full length message, the same library was rescreened with a 32 P-labeled DNA probe . Upon sequencing of seven longest inserts isolated through the second screening we obtained an 1968-nucleotide message . The size of the message was confirmed by Northern blotting of mRNA from different rat tissues using the same DNA probe that had been used for screening of the library . Along with the full length PV-1 cDNA we found one alternatively spliced form lacking the region in between residues 116–252. An RNase protection assay (see Materials and Methods) with a probe appropriate for Δ116–252 form showed that >99% of the naturally occurring message consisted of the full length RNA in rat lung, kidney, spleen, and liver (our unpublished results). The longest region of the full length PV-1 cDNA which encodes for protein is 1,341 bases long and contains all three peptides obtained by protein sequencing of the purified PV-1 . Even though there is no stop codon before the initiator ATG and the consensus Kozak sequence is incomplete (G is present in the position +4 but no A or G is found in position −3) we set the beginning of the coding region at residue 25 of the PV-1 full length cDNA based on the sequence obtained by NH 2 -terminal sequencing of the purified PV-1 (Table I ). The full length PV-1 cDNA encodes for a protein with a calculated 50 kD mass and an estimated pI ∼9.0. Hydrophilicity plot shows only one hydrophobic region (aa residues 25–50) which could qualify for a transmembrane domain. As no signal peptide could be found, the hydrophobic region (aa residues 25–50) could act as a signal and anchoring peptide thus defining PV-1 as a type II membrane protein with its NH 2 terminus intracellular and COOH terminus extracellular. Searches of protein databases with the translated protein sequence show little homology with known proteins. No known long protein patterns and few protein motifs were detected. Noteworthy features of PV-1 are the four consensus N-glycosylation sites (arginines 82, 88, 112, and 150), an odd number (nine) of cysteines in the extracellular domain which would imply the possibility of intermolecular disulfide bonds, a short proline-rich region (residues 397–411) at the COOH terminus, and two casein kinase consensus phosphorylation sites (the serine 4 [SMD] and threonine 13 [TGD]) in the cytoplasmatic domain. No consensus O-glycosylation sites were found. A striking feature of PV-1 is the regular pattern formed by the spacing of the cysteines in the extracellular domain starting with the cysteine in position 117 (C-23X-C-10X-C-24X-C-20X-C-24X-C-44X-C-67X-C-38X), whose significance, if any, is not known. A schematic of PV-1 monomer and the representation of different features is given in Fig. 2 c. To prove that this cDNA encodes indeed for PV-1, we expressed recombinant PV-1 as a His-tagged protein in bacteria (see Materials and Methods). The 21D5 mAb detected by immunoblotting a single band at ∼50 kD (the size of the unglycosylated PV-1 monomer) only in the induced transformant bacterial clones but not in the noninduced (our unpublished results). The presence of the PV-1 mRNA in several rat tissues was checked by Northern blotting (see Materials and Methods). As PV-1 was implied to be an endothelial antigen ( 10 ) we screened tissues containing several endothelial types (e.g., continuous, fenestrated, and sinusoidal). We found the PV-1 message to be present mostly in the lung and at much lower levels in kidney, spleen, liver, heart, and muscle (in decreasing order of the signal intensity) . A very weak signal, if any, of the PV-1 full length mRNA along with very low levels of two shorter mRNA species was found in the brain only after long exposure of the blot to the film . No message was detected in testis. Since we found that the 21D5 mAb was not a useful reagent for immunoprecipitation or immunolocalization of PV-1 at the EM level (our unpublished results) we raised polyclonal antibodies in chickens against a 12 mer COOH-terminal peptide representing residues 427–438 of rat PV-1 to which a lysine and a glycine were added for coupling purposes (PV-1C peptide). The peptide (KGPPLVNPAVPPSG single letter amino acid code) was coupled to BSA via glutaraldehyde and the resulting conjugate was used as antigen for antibody production in chickens. A total yolk IgY fraction was purified and used for anti– PV-1C pAb affinity purification as described in Materials and Methods. The chicken anti–PV-1C pAb was checked in immunoblotting on PVDF strips containing 10% SDS-PAGE resolved rat lung total membranes proteins. As seen in Fig. 4 a the anti–PV-1C antibody recognizes with high specificity a band of the same molecular weight as the one seen by the 21D5 mAb when reacted with the same material. The specificity of the binding is confirmed by competition with the peptide antigen . Moreover, the anti–PV-1C pAb is highly specific for PV-1; by immunoblotting it detects a single band of either ∼110,000 or ∼60,000 apparent molecular weight in nonreducing or reducing conditions, respectively, and does not detect any protein in either rat plasma or rat lung cytosol . By immunoprecipitation, the chicken anti–PV-1C pAb binds a protein which is recognized by the 21D5 mAb by immunoblotting on the immunoprecipitated material . Silver staining of the immunoprecipitated material detects a single band of ∼120,000 apparent molecular weight in nonreducing conditions. Taken together, these data show that the new chicken anti–PV-1C pAb recognizes PV-1 with high specificity in immunoblotting and immunoprecipitation thus being an appropriate reagent for attempts to localize PV-1 in the rat lung by immunocytochemistry techniques. The anti–PV-1C pAb was used in an immunoblotting assay to check for the presence of PV-1 in different rat tissues . When equal amounts of protein from different rat tissue total membranes were loaded, the antibody detected the protein only in the lung in the linear part of the film, and in the lung and faintly in the spleen and kidney when the exposure time was highly increased. When the protein amount ratio of lung to other tissues was 1:30 the signal was easily detected in spleen, kidney, and liver, thereby matching the data obtained by Northern blotting. No signal was detected in brain, testis, heart, and muscle. Rat lung specimens processed and labeled as described in Materials and Methods were examined by transmission electron microscopy. In the case of preembedding immunocytochemistry, which documents the distribution of PV-1 on the cellular surface, the gold particles were found primarily on the endothelial plasmalemmal vesicles (caveolae) in agreement with the results previously obtained by the immunoisolation procedure of endothelial caveolae ( 37 ). Interestingly, the label was found mostly associated with the neck of the caveolae or their stomatal diaphragms at both fronts of the endothelial cells, although the frequency of the label was considerably higher on the luminal side presumably reflecting higher accessibility from the lumen . In oblique or en face views, the diaphragms occasionally appeared labeled by a cluster of gold particles . Very little label, if any, was found on the endothelial plasmalemma proper, coated pits, or other cellular types to which the label had access in our lung preparations (e.g., epithelial cells lining alveoli and other airways structures). This finding is taken to indicate the specificity of PV-1 localization to endothelial caveolae. Control experiments in which the anti–PV-1 antibody was either omitted or replaced with an irrelevant antibody or preimmune IgY confirmed the specificity of the localization. Moreover, the immunocytochemical findings reinforced the conclusion that PV-1 is a type II membrane protein with the COOH terminus accessible from the microvascular lumina. By immunogold labeling of ultrathin (∼60 nm) rat lung cryosections the label was found at comparable frequencies on the caveolae at both fronts of the endothelial cell as well as vesicles within the cytoplasm (our unpublished results), pointing to the fact that PV-1 is a caveolar resident protein. The present study takes advantage of: (a) the specific procedure we ( 37 ) devised for the purification of caveolae from rat lung endothelium by immunoisolation on anticaveolin antibody–coated magnetic beads; and also (b) the availability of a novel monoclonal antibody (21D5 mAb) ( 10 ) directed against a rat lung endothelial antigen (PV-1). We found that this antigen colocalizes strictly with caveolin on immunoisolated caveolae, thus qualifying for a novel caveolar marker in rat lung endothelium. In light of this fact we decided to isolate this protein in order to obtain sequence information for its identification and further characterization. The PV-1 isolation method presented in this study employs several of its already documented properties: PV-1 is an integral membrane protein resistant to extraction by high salt and high pH, that forms dimers, is N-glycosylated, and is Triton X-100 soluble. Novel findings are the slightly alkaline pI of the dimer and the presence of terminal, nonreduced galactosyl residues in α1-3 linkage (proven by the binding to GS I lectin) on PV-1 glycan antennae. Even though the isolation method yields several protein bands at its final step, as judged by the silver staining of SDS-PAGE resolved proteins from the purified material, it permits a sufficient separation of PV-1 band from other peptides to be useful for band excision and protein sequencing. As the NH 2 -terminal and internal sequences obtained from the PV-1 protein band pointed to a novel protein, we went further for cloning of the PV-1 full length cDNA from a rat lung expression library. The deduced protein sequence encoded by PV-1 full length cDNA contains all three peptides obtained by protein sequencing. Further confirmation that this cDNA encodes for PV-1 came from the expression of PV-1 as a His-tagged protein in E. coli and the detection of an ∼50,000 apparent molecular weight band (which would represent the nonglycosylated form of PV-1 monomer) by the 21D5 mAb only in the transformed bacterial clones. Analysis of the primary structure of the PV-1 confirms its already known properties such as dimer formation, membrane insertion, and glycosylation. Additional information is represented by the possibility of phosphorylation of the cytoplasmic domain, which might be involved in the regulation of the protein, and the presence of the putative protein-protein interaction motif ( 38 ) represented by the proline-rich region at the COOH terminus. Sequence analysis of the PV-1 cDNA and the deduced protein sequence shows that PV-1 is a novel protein, as little homology with other known proteins was found by searches of DNA and protein databases. This is a notable finding when correlated with the results obtained by immunocytochemistry at EM level which demonstrate the strict localization of PV-1 to endothelial caveolae in rat lung: (a) it brings further proof for the special chemistry of the caveolar microdomains (in addition to lipids and caveolins); and (b) it provides another marker for caveolae in rat lung endothelium useful in further studies of these subcellular structures. Moreover, PV-1 strict localization to caveolae in the rat lung endothelium brings additional validation to the procedure of caveolae purification by specific immunoisolation as the results obtained by the two approaches are in agreement. By immunoisolation the label colocalizes strictly with caveolin on immunoisolated caveolae ( 37 ). We assume that the protein is anchored in the membrane and is incorporated within the caveolae upon detachment by sonication. We recognize, however, that by immunocytochemistry the label is found mostly on the necks and the stomatal diaphragms as well as in the vesicles. PV-1 is a single span type II transmembrane protein and its extracellular domain is ∼390 aa long, which would amount to ∼100 nm long if fully extended. Considering the inner diameter of endothelial caveolae of ∼40– 50 nm (average), it might be that the protein is anchored at the level of the caveolar membrane and either: (a) participates in the formation of the diaphragms; or (b) its COOH terminus protrudes through the stomatal diaphragm. The participation of PV-1 in the structure of the diaphragms is further sustained by the possibility of protein-protein interaction via the proline-rich region at the COOH terminus. We have named PV-1 in light of its localization in caveolae in endothelium of rat lung. However, the data by Northern and Western blotting indicate that the messenger and the gene product is present in other organs which contain endothelia in part fenestrated and in part provided with caveolae. The association of PV-1 with the stomatal diaphragms of caveolae in the lung endothelium, and the findings that PV-1 is present in tissues where endothelial caveolae are known to have stomatal diaphragms (4, 24, and references therein) are consistent with a strict caveolar localization. If PV-1 is associated only with the endothelial caveolae in these tissues it might be that this protein has a function in the transport of macromolecules into or across the endothelium. If PV-1 is also associated with other structures (e.g., diaphragms of the transendothelial channels and fenestrae) in these endothelia, its distribution would suggest a sieving function for this protein. The latter hypothesis would be the most interesting as PV-1 would be the first protein of this type to perform such a function. Continuation of this work will find out if PV-1 is restricted only to caveolae in these “mixed” microvascular beds. Precise information on the localization of PV-1 in different microvascular beds is a prerequisite for the formulation of any hypothesis as to its function. | Other | biomedical | en | 0.999998 |
10366593 | S. cerevisiae strains used in this study are listed in Table I . Strains MYY290, MYY291, and MYY298 , MYY403 , MYY535 , and MYY700-MYY721 have been described previously. Strains MYY803 and MYY804 were isolated as pseudorevertants of mdm1-252 as described below. Strains MYY808 and MYY809 were isolated from a backcross of strain MYY803 to strain MYY291. Strains MYY812 and MYY813 were isolated from a backcross of strain MYY803 to MYY291. Strain MYY820, a MAT a strain marked with LEU2 at the RSP5 locus, was created as described below. Strain MYY823 was created by crossing strain MYY809 to strain X21801A (Yeast Genetics Stock Center), sporulating the resulting diploid, and isolating a temperature-sensitive, Ura − , Leu − , haploid spore. Strain MYY825 was created by disrupting one copy of RSP5 with HIS3 in MYY298, as described below. Strains MYY816 and MYY817 in which BUL1 is replaced by LEU2 were generated as described below. Strains MYY826, MYY829, MYY832, MYY833, and MYY834 were generated by transformation of MYY825 with plasmids pRS316-RSP5, pRS316-smm1, pRS316-mdp1-1, pRS316-mdp1-13, pRS316-mdp1-14, respectively, sporulation of transformed strains, and recovery of His + , Ura + , haploid spores. Strains LHY1 (RH448), LHY180 , LHY192 , LHY201 , LHY183 , and LHY21 were obtained from Linda Hicke (Northwestern University) and have been described previously . Media and genetic analyses were as described previously . Second site suppressors were identified by pseudoreversion analysis. Approximately 5 × 10 8 MYY721 ( mdm1-252 ) cells were plated at a density of 0.5–1 × 10 6 cells per plate onto yeast extract/peptone/glucose (YPD) 1 -agar medium at 37°C. Cells were replica-plated every day for 3 d onto prewarmed YPD-agar plates at 37°C. Clones able to grow as serial replica colonies at 37°C were tested for the ability to grow as single isolated colonies at 37°C. Mitochondrial distribution and morphology of apparent revertant colonies were analyzed by staining with the mitochondria-specific vital dye 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) as described previously . To determine if pseudorevertants harbored second site suppressing mutations, candidate strains were backcrossed to strain MYY291 and meiotic progeny were analyzed for temperature-sensitive growth. Backcrossing also revealed whether suppressing mutations caused temperature-sensitive growth defects in the presence of wild-type MDM1. Mitochondrial inheritance in living cells was analyzed by DASPMI staining of cells grown in liquid cultures as described previously . Cellular distribution of Mdm1p structures, mitochondrial outer membranes, and microtubules was examined by indirect immunofluorescence microscopy as described previously . Nuclear and mitochondrial DNAs were visualized by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) . Strain MYY823 was transformed with two different genomic libraries in the centromere-based vectors YCp50 and pSB32 . Four plasmids capable of completely complementing the temperature-sensitive growth of smm1 were isolated: YCp50-3.1 and YCp50-3.10 from the Rose library and pSB-8 and pSB-16 from the pSB32-based library. DNA sequence was determined for each end of the genomic DNA inserts in these plasmids using the pBR322 BamHI cw and BamHI ccw sequencing primers ( Promega ). DNA sequences of plasmid inserts were compared to the Saccharomyces Genome Database using the BLAST program . Strain MYY812 was transformed with a genomic DNA library in plasmid YEp13 . Three plasmids (YEp13-1.1, YEp13-2.2, and YEp13-4.1) containing identical yeast DNA inserts were isolated and analyzed as described above. The smm1 mutation was mapped to the RSP5 locus by integrative transformation and genetic analysis. The integrating vector pRS305-RSP5 was linearized by digestion with MscI and transformed into strain MYY298. The resulting strain was sporulated, and a haploid spore with the LEU2 gene integrated at the RSP5 locus was identified (strain MYY820). This strain was crossed to smm1 strain MYY808, the diploid was sporulated, and haploid progeny were scored for Leu + and growth at 37°C. No recombinants were identified from 28 tetrads, indicating that smm1 mapped to within 1.7 cM of RSP5. The smm2 mutation was mapped to the BUL1 locus by analyzing meiotic progeny resulting from a cross of strain MYY813 ( smm2 ) to strain MYY826, which contained a disrupted copy of bul1 ( bul1 :: LEU2 ). Among 20 tetrads analyzed, all spores were temperature-sensitive, indicating no recombination between smm2 and bul1 and revealing genetic linkage of these two loci within 2.5 cM. Plasmids pRS316-RSP5 and pRS305-RSP5 were created by cloning the 4,031-bp XbaI-XhoI fragment containing the RSP5 gene from plasmid YCp50-3.1 into the XbaI and XhoI sites of pRS316 and into the SalI and XbaI sites of pRS305 , respectively. Plasmid pBS-BUL1 was created by cloning the 5,456-bp NheI-SalI fragment from YEp13-2.2 into the SpeI and SalI sites of pBluescript KS + (Stragene). Plasmid pBS-Δbul1 was created by replacing the region of pBS-BUL1 between the outermost EcoRV sites with LEU2 as follows. After digestion of pBS-BUL1 with EcoRV and treatment with calf intestinal phosphatase (CIP), the 5,834-bp vector backbone was isolated by gel purification. A 2,217-bp fragment containing the LEU2 gene was isolated from plasmid YEp13 after digestion with SalI and XhoI and treatment with the Klenow fragment of DNA polymerase I and ligated into the pBS-BUL1 EcoRV-deleted backbone. Plasmids pTER21, pTER22, pTER23, encoding mutant ubiquitin (K29R, K48R, and K63R mutations, respectively) under control of the CUP1 promoter were described previously . Plasmid pUb, which contains wild-type ubiquitin driven by the CUP1 promoter, was created by replacing the BglII-SalI fragment from pTER22 with the BglII-SalI fragment from plasmid YEp105 . Plasmid pSB-smm1 was isolated by plasmid-mediated gap repair , as described below. Plasmids pRS316-CA, pRS316-mdp1-1, pRS316-mdp1-13, and pRS316-mdp1-14 were generated by PCR-mediated site-directed mutagenesis and fragment-mediated gap repair of pRS316-RSP5, as described below. Plasmid pRS426-smm1 was created by replacing the 3,897-bp region between the BspEI and XhoI sites of pRS426-RSP5 with the corresponding fragment from pSB-smm1. Plasmid pRS316-smm1 was created by replacing the 3,897-bp BspEI-XhoI fragment of pRS316-RSP5 with the corresponding fragment from pRS426-smm1. The smm1 mutation was shown to lie within RSP5 by plasmid-mediated gap repair . Plasmid pSB-H8 was digested with PvuII and ApaI. The resulting vector backbone (17,006 bp) was gel purified, redigested with the same enzyme combination, dephosphorylated with CIP, and transformed into MYY808. 24 independent Leu + isolates were tested for the ability to grow at 37°C. A plasmid, designated pSB-smm1, was isolated from one of the resulting Leu + , temperature-sensitive clones. The DNA region of this plasmid between PvuII and ApaI was sequenced, and the only mutation found was a change of G to T at nucleotide 2258 of RSP5. The sequence of this region of the RSP5 locus was also determined from genomic DNA isolated from MYY808 and MYY823 by asymmetric PCR as described previously . For both strains, the only mutation found was G to T at nucleotide 2258 of RSP5 , resulting in the substitution of valine for glycine at position 753 of Rsp5p (both numbers are relative to +1 of the RSP5 open reading frame). A diploid strain deleted for one copy of RSP5 (strain MYY825) was derived from strain MYY298 by PCR-mediated gene disruption using the HIS3 gene as a selectable marker as previously described . A strain deleted for BUL1 was created by transforming the wild-type diploid strain MYY298 with plasmid pBS-ΔBUL1 which had been digested with BglII and BamHI. A Leu + transformant was then sporulated, and Leu + haploid spores of MAT a (MYY816) and MAT α (MYY817) genotypes were isolated. Gene disruptions were confirmed by PCR analysis. The site-directed mutation C777A was created in RSP5 by PCR in a manner similar to that described by Imhof and McDonnell , and was cloned into pRS316-RSP5 by fragment-mediated gap repair as follows. Two separate PCR reactions were performed using pRS316-RSP5 as a template. The first reaction used the primer pair 5′-CTCACACAgcTTTTAACAGAG-3′ and 5′-GGCGAAGGGGGGATGTG-3′ (binds in the multicloning site of pRS316-RSP5, on the 3′ side of RSP5 ), and the second used the primer pair 5′-GACGAGGTCATTCAATGG-3′ and 5′-CTCTGTTAAAAgcTGTGTGAG-3′. The lowercase letters in these primer sequences indicate mutagenic nucleotides. The products of these two reactions, which overlap by 20 nucleotides, were combined and reamplified in the absence of primers to create a final PCR product containing the C777A mutation and spanning from nucleotide 2134 of RSP5 across the pRS316 multicloning site on the 3′ side of RSP5. Plasmid pRS316-RSP5 was digested with MfeI and SacII and dephosphorylated by CIP treatment. The 7,576-bp vector backbone was gel purified and transformed into MYY823 together with the final PCR product. Sac II cuts pRS316-RSP5 outside of the yeast insert such that one end of the digested vector only has homology with the cotransformed PCR product. Therefore the only event that can repair the gapped plasmid is recombination with the PCR product to generate pRS316-CA. Transformation of MYY823 with digested vector alone yielded nine Ura + clones, whereas cotransformation of digested vector and the C777A-containing PCR product yielded 323 Ura + clones. All Ura + clones tested failed to grow at 37°C. Plasmids were isolated from these clones and digested with NlaIII to verify the presence of C777A, and with BsmAI to verify the absence of smm1. One of these clones was designated pRS316-CA, and sequenced to verify that C777A was the only nucleotide change. The RSP5 mdp1 mutations were created in pRS316-RSP5 by the method described above, using the following mutagenic primers: mdp1-1 , 5′AGTTGATTTGaCACAATACGT-3′, and 5′-ACGTATTGTGtCAAATCAACT-3′; mdp1-13 , 5′-GATTATCGTGaTTACCAAGAG-3′, and 5′-CTCTTGGTAAtCACGATAATC-3′; mdp1-14 , 5′-CCTGTCAACGaGTTTAAAGAT3′, and 5′-ATCTTTAAACtCGTTGACAGG-3′. Plasmids were isolated from yeast and sequenced to verify that the mdp1 mutations were the only changes present. To identify proteins interacting with Mdm1p to mediate mitochondrial distribution and morphology, second site suppressor analysis was performed using the mdm1-252 mutation. This mutation causes temperature-sensitive growth and defects in mitochondrial distribution and morphology, but does not cause abnormal nuclear inheritance . Eight clones capable of growth at 37°C were identified from ∼5 × 10 8 cells. Genetic and microscopic analysis revealed that two of these strains possessed single, distinct mutations which suppressed both temperature-sensitive growth and mitochondrial distribution and morphology defects caused by mdm1-252. These suppressor mutations also conferred temperature-sensitive growth on cells when separated genetically from the original mdm1252 lesion . These new mutations, smm1 and smm2 ( s uppressor of M dm1p-dependent m itochondrial inheritance defects), were found to be unlinked to MDM1. The specificity of suppression by the smm1 and smm2 mutations was determined by crossing cells harboring these lesions to a collection of 10 otherwise isogenic strains containing different mdm1 alleles and analyzing the phenotypes of haploid progeny harboring both the mdm1 allele and the suppressor mutation. Suppression of mdm1 by smm1 was highly allele-specific: smm1 failed to suppress the mutant phenotypes of any allele other than mdm1-252. In contrast, smm2 partially suppressed the mutant phenotypes of several mdm1 alleles including mdm1-202 , mdm1-204 , mdm1-251 , and mdm1-252. Each of these smm2 -suppressed mdm1 mutations is a dominant allele . Additionally, smm2 displayed nonallelic noncomplementation with the recessive mdm1 alleles: heterozygous mdm1 / MDM1 smm2 / SMM2 diploids displayed temperature-sensitive growth. Cells harboring the smm1 or smm2 mutation were analyzed microscopically to assess the possible effect of these lesions on mitochondrial distribution and morphology. At permissive temperature (23°C), mitochondrial distribution and morphology in smm1 cells was indistinguishable from wild-type cells . In contrast, cells harboring the smm1 mutation displayed dramatic aberrations in mitochondrial distribution and morphology, similar to those caused by mdm1-252 , after incubation at 37°C . Indirect immunofluorescence microscopy further revealed that mitochondria in smm1 cells at 37°C formed small round structures of uniform size which failed to enter buds in a large proportion of cells . DAPI staining of smm1 cells indicated that the smm1 mutation was specific for mitochondrial inheritance, as there was no detectable defect in nuclear segregation . Finally, there appeared to be no effect of smm1 on the distribution or stability of Mdm1p cytoplasmic structures . Therefore, smm1 is a mitochondrial distribution and morphology ( mdm ) mutant with a phenotype very similar to that of mdm1-252 cells. In contrast, smm2 mutant cells displayed no defects in mitochondrial distribution or morphology at either permissive or nonpermissive temperatures (data not shown). To understand the mechanism of suppression of mdm1-252 , smm1 was cloned by complementation. Four plasmids which completely restored growth at 37°C to smm1 cells were isolated. These plasmids also corrected defects in mitochondrial distribution and morphology. Restriction enzyme and nucleotide sequence analysis revealed that these plasmids contained overlapping inserts of yeast genomic DNA corresponding to a 5.6-kb region of chromosome V. Transformation of smm1 cells with different DNA fragments derived from the genomic inserts localized complementing activity to RSP5 , an essential gene encoding a ubiquitin-protein ligase containing a HECT domain . Integrative mapping (as described in Materials and Methods) confirmed that the smm1 mutation mapped to RSP5. The smm1 mutation was mapped within RSP5 by plasmid-mediated gap repair, and the molecular identity of the mutation was determined by nucleotide sequencing of the appropriate region of the mutant gene. This analysis revealed a single change in the mutant gene, a transversion of G to T at nucleotide 2258. This mutation in the region of RSP5 corresponding to the conserved HECT domain leads to a change of glycine-753 to valine. To investigate suppression by the smm2 mutation, genomic plasmids that complemented the temperature-sensitive phenotype of smm2 mutant cells were isolated. Three complementing plasmids contained the same genomic DNA insert encoding two genes, RCE1 and BUL1 , located on chromosome XIII. The complementing activity was mapped to BUL1 by subcloning and retesting complementation in smm2 cells. The isolated BUL1 gene was shown to correspond to sequences from the smm2 locus by integrative transformation and mapping. BUL1 was previously shown to encode a 109-kD protein which binds to the ubiquitin-ligase Rsp5p . The smm2 mutation did not cause defects in mitochondrial distribution or morphology (see above). To examine further a possible role for BUL1 in mitochondrial inheritance, a bul1 -null allele was created. Like the smm2 mutant, cells deleted for BUL1 (Δ bul1 ) displayed temperature-sensitive growth but no alteration in mitochondrial distribution or morphology at either permissive or nonpermissive temperature (data not shown). Additionally, the bul1 -null mutation failed to suppress mdm1-252. Previously, several mutations in the HECT domain of Rsp5p were shown to be suppressed by ubiquitin overexpression . To test whether the smm1 mutation could be similarly suppressed, smm1 mutant cells were transformed with plasmids encoding either wild-type or mutant ubiquitin expressed from the copper-inducible CUP1 promoter. These cells were incubated at 37°C in the presence of 100 μM CuSO 4 (to induce high levels of ubiquitin) and examined by microscopy to assess effects on mitochondrial distribution and morphology. Overexpression of wild-type ubiquitin was found to suppress the mitochondrial morphology and distribution defects of smm1 cells . The specificity of this suppression was investigated by overexpression of versions of ubiquitin mutated in one of three critical lysine residues, K29R (UbK29R), K48R (UbK48R), or K63R (UbK63R). Overexpression of UbK29R or UbK48R suppressed the mitochondrial morphology and distribution defects, similar to suppression by wild-type ubiquitin (data not shown). However, the UbK63R mutant failed to suppress smm1 . No effect of ubiquitin overexpression was observed in mdm1-252 cells at either permissive or nonpermissive temperatures (data not shown). In control experiments, the effect of ubiquitin overexpression was examined in wild-type cells. Overexpression of wild-type ubiquitin , UbK29R, or UbK48R had no apparent effect on mitochondrial distribution or morphology. However, expression of UbK63R perturbed mitochondrial inheritance in wild-type cells . A quantitative analysis of this effect revealed that ∼30% of MYY290 cells expressing UbK63R displayed buds devoid of mitochondria, a frequency similar to that seen in MYY823 ( smm1 ) cells . In addition, these cells possessed empty buds of medium to large size and displayed pronounced mitochondrial aggregations . The effect of ubiquitin overexpression was identical whether copper induction was simultaneous with temperature shift or if ubiquitin overexpression was induced for 2 h before temperature shift (data not shown). These results suggest that the formation of polyubiquitin chains linked via lysine-63 is essential for mitochondrial inheritance. Several other temperature-sensitive mutations in RSP 5, the mdp1 mutations, were shown to map to the HECT domain . To determine if these mutations behave like smm1 with respect to mitochondrial inheritance, the effect of three previously described lesions, mdp1-1 , mdp1-13 , and mdp1-14 , on suppression of mdm1-252 and on mitochondrial distribution in the MYY290 genetic background was examined. As expected, each of the mutations conferred a temperature-sensitive growth phenotype on otherwise wild-type cells, but none of the three mdp1 mutations suppressed the mdm1-252 phenotypes (data not shown). To determine whether the mdp1 mutations affected mitochondrial distribution and morphology, cells were incubated at 37°C and examined microscopically after staining with DASPMI. mdp1-1 caused a modest defect in mitochondrial distribution and morphology, although considerably less than that caused by smm1 . Neither mdp1-13 nor mdp1-14 caused any defect in mitochondrial inheritance or morphology relative to wild-type RSP5 . These results indicate that the smm1 mutation confers properties unique from those caused by previously described mutations in RSP5. These findings are largely consistent with a previous study which found that the mdp1 mutation in a different strain background caused no defects in mitochondrial distribution or morphology. The smm1 mutation lies in the HECT domain of Rsp5p, suggesting that the mutation may affect ubiquitin ligase activity or substrate specificity. To test whether the ubiquitin ligase activity of Rsp5p is required for mitochondrial inheritance, a mutation in a key residue of the active site of Rsp5p was created. The conversion of cysteine-777 to alanine (C777A) was shown previously to destroy the ability of Rsp5p to form a covalent intermediate with ubiquitin, thereby destroying its ubiquitin ligase activity . The C777A mutation was generated in a plasmid-borne copy of RSP5 , and the plasmid was transformed into smm1 cells. The C777A mutant rsp5 failed to complement either the smm1 temperature-sensitive growth phenotype or the smm1 defects in mitochondrial distribution and morphology . These results demonstrate that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. To further evaluate the requirement of ubiquitination for mitochondrial inheritance, mitochondrial distribution and morphology were examined in cells defective for ubiquitin-conjugating enzymes (E2-type enzymes). S. cerevisiae possesses genes encoding more than a dozen different E2 proteins, but much of the cytoplasmic ubiquitination activity depends on two proteins with redundant specificity, Ubc4p and Ubc5p . Strains deleted for UBC4 , UBC5 , or UBC1 (or combinations of two of these genes) were examined by fluorescence microscopy after mitochondrial staining. Cells with null mutations in both UBC4 and UBC5 displayed aggregated mitochondria and daughter buds devoid of mitochondria . Both mutant traits were more prevalent after incubation of cells at 37°C, and a quantitation of this effect after 4 h at 37°C revealed 86% of cells with aggregated mitochondria and 52% of budded cells with empty daughter buds. These mutant traits were not apparent in cells with only ubc4 or ubc5 mutations, nor were they found in ubc1 or ubc1 ubc4 mutants (data not shown). In addition, the inheritance and distribution of nuclei were normal in the ubc4 ubc5 mutant cells (data not shown). These results suggest that the ubiquitin-conjugating enzymes Ubc4p and Ubc5p mediate ubiquitination reactions essential for mitochondrial inheritance. We have uncovered a novel role for protein ubiquitination in mitochondrial inheritance. This role was revealed through the characterization of smm1 , a mutation that suppresses the mitochondrial distribution and morphology defects caused by mdm1-252. Six key findings support this new function for ubiquitination. First, smm1 mapped to RSP5 , an essential gene encoding a ubiquitin-protein ligase. Second, the smm1 mutation alone conferred conditional defects in mitochondrial distribution and morphology. Third, the defects caused by smm1 were complemented by wild-type Rsp5p but not by mutant Rsp5p lacking ubiquitin ligase activity. Fourth, smm2 , a second mutation suppressing mdm1-252 , mapped to BUL1 , a gene encoding a protein that binds to Rsp5p and facilitates its activity . Fifth, overexpression of a mutant form of ubiquitin which blocks elongation of certain polyubiquitin chains also caused aberrant mitochondrial distribution and morphology in wild-type cells. Finally, depletion of two ubiquitin-conjugating enzymes, Ubc4p and Ubc5p, caused defective mitochondrial morphology and inheritance. Protein ubiquitination has been found to play a key role in a variety of cellular functions. Prominent among these roles is the ubiquitin-mediated targeting of cytosolic proteins for degradation by the proteosome . Similarly, the proteosome-dependent turnover of several membrane proteins of the endoplasmic reticulum is initiated by their ubiquitination . Ubiquitination of plasma membrane proteins including the yeast Fur4p , Ste2p , and Gap1p has been shown to trigger their endocytosis and transport to the vacuole where they are subsequently degraded in a process independent of the proteosome. Protein ubiquitination has also been found to function in as yet poorly defined roles in the import of certain proteins into peroxisomes and mitochondria . Additionally, recent studies have revealed that the conjugation of protein targets with ubiquitin-like proteins is an important feature of nuclear protein import and cell cycle regulation . Our identification of a role for protein ubiquitination in mitochondrial inheritance represents a novel cellular function for this consequential covalent modification. The smm1 and mdm1-252 mutations display highly specific reciprocal suppression. smm1 did not suppress any other mdm1 allele, nor was mdm1-252 or any other mdm1 allele suppressed by other rsp5 mutations. This specificity and reciprocity suggests that Rsp5p and Mdm1p interact directly to effect normal mitochondrial inheritance. The results suggest further that the smm1 and mdm1-252 mutations interfere individually with this interaction but that the combination of the two lesions restores functionality. Similar reciprocal suppression has been demonstrated for specific mutations in actin and actin-binding protein Sac6p in S. cerevisiae . Future studies will evaluate the possible direct binding or transient interactions of Rsp5p and Mdm1p. The mapping of the smm2 mutation to BUL1 further supports a role for ubiquitination in mitochondrial inheritance. The BUL1 product, Bul1p, was previously identified as a protein that binds to Rsp5p , and it has been proposed to function as a cofactor, modulating the activity or specificity of the ubiquitin ligase. A similar role is played by the E6 protein of human papilloma virus in the ubiquitination of p53 . Although smm2 suppressed mdm1-252 , it also displayed genetic interactions with several other mdm1 alleles, indicating that Bul1p may not interact directly with Mdm1p. Furthermore, neither smm2 nor the bul1 -null mutation caused any apparent defect in mitochondrial inheritance, indicating that Bul1p is not normally involved in this process. One hypothesis consistent with these observations is that one of three Bul1p homologues in S. cerevisiae , Yml111p, Ynr068p, or Ynr069p, may be required for mitochondrial inheritance, and the smm2 mutation might allow Bul1p to supplement or interfere with the activity of its homologue. The target of Rsp5p-mediated ubiquitination associated with mitochondrial inheritance is unknown. Rsp5p was previously shown to ubiquitinate a variety of cellular substrates including the large subunit of RNA polymerase II as well as the plasma membrane proteins Fur4p and Gap1p . Mdm1p appeared to be a reasonable candidate for ubiquitination by Rsp5p, but no evidence of such ubiquitination was obtained. In particular, immunoblot analysis of cellular proteins separated by two-dimensional polyacrylamide gel electrophoresis failed to reveal either ubiquitin associated with Mdm1p or higher molecular weight forms of Mdm1p that might represent ubiquitinated species (data not shown). Other candidates for ubiquitination include proteins integral or bound to the mitochondrial outer membrane. The identification of the relevant Rsp5p substrates may emerge from analysis of proteins ubiquitinated in wild-type cells but not in smm1 or mdm1-252 mutant cells at 37°C. What might be the role of ubiquitination in mitochondrial inheritance? One possibility is that the ubiquitination of one or more key proteins initiates changes in the interaction of mitochondria with Mdm1p structures. In this model, Rsp5p might first bind to Mdm1p and then ubiquitinate a nearby target protein. The consequence of this activity could be either to promote or diminish an interaction between the target protein and Mdm1p. For example, ubiquitination might promote an association of mitochondria with Mdm1p structures as a critical step in the mitochondrial distribution process. Alternatively, ubiquitination of a mitochondrial surface protein might facilitate its dissociation from Mdm1p structures to mobilize mitochondria. The direct effect of ubiquitination could be to target the ubiquitinated substrate for degradation or, alternatively, ubiquitination might function like other types of covalent modifications to alter the activity or structure of the target protein. The dependence of mitochondrial inheritance on lysine-63 (K63) of ubiquitin may provide a clue to the fate of the ubiquitinated target protein. Fur4p modification by Rsp5p-dependent addition of K63-linked polyubiquitin chains leads to the internalization of this protein from the plasma membrane in a proteosome-independent event . In contrast, the addition of polyubiquitin chains linked through lysine-48 leads to the proteosome-dependent degradation of the MAT α2 transcriptional regulator . Because Rsp5p-dependent formation of K63-linked chains appears to be required for mitochondrial inheritance, ubiquitination may play a proteosome-independent role in mitochondrial inheritance. The identification of the relevant substrates of ubiquitination and a characterization of these proteins' molecular interactions should uncover biochemical details of the function of ubiquitination in mitochondrial inheritance. | Study | biomedical | en | 0.999997 |
10366594 | Primary cultures of oligodendrocyte progenitor cells were isolated from P2 neonatal rat brain (Sprague Dawley; Taconic Farms) according to the procedure of Osterhout et al. with slight modifications. In brief, mixed dissociated cultures of neonatal brain were allowed to reach confluence, after which the oligodendrocyte progenitor population was removed by shaking. The progenitor cells were further purified by immunopanning with A2B5 , and plated onto polylysine (PLL)- coated dishes. Progenitors were lysed 24 h after plating and analyzed for Fyn protein expression and kinase activity (see below). Differentiation of progenitor cells was initiated by switching the media to a defined media lacking growth factors . To maintain the cells as progenitors, and prevent differentiation, PDGF and FGF were included in the differentiation media at concentrations that stimulate the maximal proliferation of the cells . The mature oligodendrocytes were analyzed for Fyn expression and activity after 4–6 d in culture. The differentiation state of the cells, either progenitor or mature oligodendrocyte, was determined by immunocytochemical analysis of stage-specific markers such as A2B5 for progenitor cells, and O1, MAG, and MBP for differentiated oligodendrocytes . For the inhibitor studies, PP1, PP2, PP3, and PP4 ( Calbiochem Novabiochem ) were resuspended in DMSO, and included in the culture media at specified concentrations. Cells were grown on PLL-coated 10-mm glass coverslips ( Dynal ab) for all immunochemistry. For oligodendrocyte lineage markers such as A2B5 and O1, the coverslips were rinsed in PBS (10 mM sodium phosphate, pH 7.2, 150 mM NaCl) and incubated in antibody diluted in PBS for 30 min at room temperature (RT). The coverslips were then fixed in 4% paraformaldehyde for 30 min. For Fyn localization, the cells were permeabilized with 0.2% Triton X-100, then incubated with a rabbit antibody to Fyn ( Santa Cruz Biotechnology ) overnight at 4°C. Fluorescein- and rhodamine-conjugated secondary antibodies (Vector Labs) were diluted 1:50 in PBS and left on the coverslips for 30 min. For each immunostain, one coverslip was incubated in the secondary antibody alone as a negative control for background immunofluorescence. The coverslips were rinsed in PBS and mounted on glass slides using Vectashield (Vector Labs). In all staining procedures, labeled cells were visualized using a Nikon Optiphot microscope or a Leica confocal microscope. For analysis of process outgrowth, the distance from the center of the cell body to the tip of the longest process was measured, which in mature oligodendrocytes corresponds to the radius of the extensive process network that surrounds the cell body. For PP1-inhibited cells, this corresponds to the length of the longest process extending from the cell. If a process was not longer than the cell body diameter, it was not measured. For each condition, process outgrowth was measured for 200 individual cells from control and inhibitor-treated cells. Cells were washed twice with cold STE (100 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) and lysed in 1 ml of NP-40 lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 2.5 mM EDTA, 10 mM NaF, 1 mM Na 3 VO 4 , 10 μg/ml aprotonin, 10 μg/ml leupeptin, 1 mM PMSF). Cell lysates were then processed for immunoprecipitation as described . In vitro kinase assays, enolase phosphorylation assays, and Western blotting have been described previously . Quantitation of band intensity in both the Western analysis and kinase assays was accomplished using a Molecular Dynamics PhosphorImager. Antibodies raised against amino acids 1–148 of Fyn for immunoprecipitation have been previously described . Rabbit polyclonal antibodies to Src, Fyn, Lck, and Lyn were purchased from Santa Cruz Biotechnology . Antiphosphotyrosine antibodies 4G10 and PY20 were purchased from UBI and Transduction Labs. MAG antibodies were a generous gift from Dr. Marie Filbin (Hunter College, New York). O1 antibodies were a generous gift from Drs. Steven Pfeiffer and Rashmi Bansal (University of Connecticut, Farmington, CT). Antibodies to neomycin phosphotransferase II (NPTII) were purchased from 5′→ 3′. The K299M Fyn construct contains a point mutation in the ATP binding pocket (lysine 299 was changed to methionine), rendering the kinase inactive . The K299M Fyn cDNA was generated in SP65 using wild-type Fyn as a substrate, and subsequently cloned into the retroviral pLJ vector . The mutation was verified by DNA sequencing. ψ2 cells were transfected with K299M or pLJ plasmids, and cell supernatants containing the retrovirus were collected and used to infect oligodendrocyte progenitor cultures. Primary cultures of oligodendrocyte progenitors typically contain bipolar cells that are characterized by the expression of the surface markers A2B5 (80–90%) and O4 (10– 20%). These progenitors can be induced to differentiate by switching to a serum-free differentiation media 24 h after plating (see Materials and Methods). After several days in culture, mature cells are characterized by marked process outgrowth, with an increase in the number of processes emanating from the cell body and extensive branching of these processes. The result is an intricate lacework of processes that surrounds the oligodendrocyte cell body. The morphological changes are accompanied by the appearance of the cell surface galactocerebroside O1, as well as myelin proteins such as MAG and MBP. At later stages, the extensive process outgrowth gives rise to the formation of myelin-like membrane sheets, which can be visualized by staining for O1 . Fyn is expressed in both the progenitors and differentiated cell populations . In progenitors, Fyn is localized primarily to the cell body and found along the length of the processes. . Likewise in the mature oligodendroycte, Fyn is observed in the cell body and throughout the processes . Staining for O1 and Fyn also confirmed that the majority of Fyn immunoreactivity can be visualized in the cell body and processes of the mature cell . To investigate whether Fyn protein levels changed during differentiation, Western analysis of oligodendrocyte cell populations was performed. The bipolar progenitors were harvested 24 h after plating. Mature oligodendrocytes were harvested after allowing progenitors to differentiate for 5–6 d. Fyn protein is expressed at levels two- to threefold higher in the mature cells compared with progenitors . Moreover, Fyn tyrosine kinase activity in mature oligodendrocytes is 10–30-fold higher than that of the progenitors , as measured by autokinase activity and enolase phosphorylation. This suggests that the specific activity of Fyn is increased 3–15-fold per unit of Fyn protein in the mature cells. Since oligodendrocyte differentiation occurs over 5–6 d in vitro, Fyn activity was examined each day after the plating of oligodendrocyte progenitors to establish the time course of Fyn upregulation during differentiation. Fyn autokinase activity was used as an indication of Fyn activation, as it mimics the results obtained by measuring enolase phosphorylation . The observed increase in Fyn activity occurs within 24 h after switching to differentiation media, which corresponds to 48 h after plating . Remarkably, cells at this time point are indistinguishable from those progenitors harvested at 24 h after plating, both in morphology and in the expression of stage specific markers. Since Fyn activation precedes any morphological changes that accompany differentiation, these observations suggest that the upregulation of Fyn is a very early event in the maturation of oligodendrocyte progenitors. Several members of the Src family of tyrosine kinases are expressed in the neonatal brain, including Src, Fyn, Lyn, and Yes . However, analysis of oligodendrocyte cell lysates revealed that Fyn is the only member of the Src family with significant kinase activity in either cultured oligodendrocytes or progenitor cells. Src is not expressed or catalytically active in these cells; Yes protein is barely detectable in differentiated cells, but is not active . Lyn protein is expressed in both the progenitors and mature oligodendrocytes , but Lyn tyrosine kinase activity was not detected in either population of cells . Analysis of kinase activity was performed using two different substrates, including autokinase as well as enolase phosphorylation for all Src family members . These findings distinguish oligodendrocytes as one of the few cell systems in which Fyn is the major active Src family member. These cells therefore can provide a useful system in which to study Fyn function. Growth factors such as PDGF and FGF stimulate progenitor cell proliferation, regulate cell migration and modulate differentiation in the oligodendrocyte lineage . In vitro, progenitors maintained in the presence of these growth factors fail to differentiate; withdrawal from the cell cycle and terminal maturation is triggered by removal of the growth factors. In the protocol used for this study, progenitors are harvested soon after plating; under these conditions, the progenitors have a low basal level of Fyn kinase activity . Since PDGF and FGF can activate Src and Fyn in other cell types , we examined the possibility that PDGF and FGF are required to activate Fyn in the progenitor cell population. Fyn kinase activity was examined after acute treatment with FGF (20 ng/ml) and PDGF (10 ng/ml) for 5, 10, and 20 min. There was no increase in Fyn activity above basal levels . Since differentiation can be prevented by maintaining progenitors in the presence of growth factors , we also examined the effects of chronic treatment with growth factors. After 3 d in culture with either PDGF (10 ng/ml) or FGF (20 ng/ml), Fyn activity remained at basal levels . Fyn activity was not upregulated until the growth factors were withdrawn, which triggered differentiation. It is also possible that the increase in Fyn activity could be influenced by specific media components. Fyn kinase activity was assayed in cells cultured under several different media formulations, each of which will allow oligodendrocyte differentiation, but vary in their composition . There were no differences in the levels of Fyn kinase activity in the mature oligodendrocytes under these different culture conditions (data not shown). These data indicate that the activation of Fyn is closely associated with the start of progenitor cell differentiation and not stimulated by FGF or PDGF. The lack of growth factor regulation suggests that activation of Fyn in oligodendrocytes is achieved via a novel regulatory mechanism, which is an early event in the terminal differentiation of oligodendrocytes. The upregulation of Fyn kinase activity during differentiation suggests that enhanced tyrosine phosphorylation of potential Fyn substrates should be evident in mature oligodendrocytes. This supposition was verified by the results depicted in Fig. 4 A, in which an increase in tyrosine phosphorylated proteins was observed in both total cell lysates and Fyn immunoprecipitates of differentiated cells. Several proteins were phosphorylated and associated with Fyn as oligodendrocytes differentiate, and these phosphorylated proteins were also observed in Fyn autokinase assays on oligodendrocyte lysates . These proteins were not phosphorylated under the assay conditions required for enolase phosphorylation by Fyn . Interestingly, these proteins were not phosphorylated in autokinase assays using progenitor cell lysates even though there is a low level of Fyn activity, which suggests that phosphorylation of these proteins is part of the maturation program. These phosphorylated bands appear to be novel and specific to oligodendrocytes. They do not correspond to any known Src or Fyn substrates, including Fak, Cbl, p130Cas, annexins, PI3 kinase, or cortactin, and the identification of these potentially novel proteins is currently under investigation. To determine whether activation of Fyn was required for differentiation, we examined the fate of progenitors cultured in the presence of the tyrosine kinase inhibitors PP1 and PP2. These pyrazolopyrimidine derivatives are potent and specific inhibitors of Src family kinases in T cells and compete with ATP to prevent phosphorylation . They have different affinities for each member and do not inhibit other tyrosine kinases at the concentrations effective for the Src kinase family . Since Fyn is the only Src family kinase active in this cell system, Fyn should be the only Src family member affected by the inhibitors. Fyn kinase activity in lysates from mature oligodendrocytes was significantly lower in the presence of PP1 and PP2 , and a corresponding reduction in tyrosine phosphorylation of total cellular protein was observed . Inhibition of Fyn kinase activity by PP1 was concentration dependent, with total inhibition occurring at concentrations of 5 μM and higher. The effects of PP1 are specific since the inactive compound PP3 could not inhibit Fyn kinase . The activity levels of Fyn in oligodendrocytes in the presence of PP3 were comparable to control cells . Tyrosine phosphorylation of proteins in the total cell lysates was reduced but not eliminated in the presence of PP1 . At low concentrations, where PP1 has limited effects on Fyn activity , there was a slight reduction in tyrosine phosphorylation, including Fyn autophosphorylation. As the concentration of PP1 increased, the levels of tyrosine phosphorylation measurably decreased. Fyn autophosphorylation was reduced by 70% at 5 μM PP1 , a concentration at which Fyn kinase activity is totally inhibited . Similar reductions in tyrosine phosphorylation were observed in immunoblots probed with anti-phosphotyrosine antibodies, including proteins at 100–120, 80–85, and 40–45 kD, which may represent Fyn substrates in oligodendrocytes. The observation that tyrosine phosphorylation was not totally eliminated in the presence of PP1 suggests that while Fyn is an important kinase in these cells, other tyrosine kinases may be active and not inhibited by the PP1 inhibitor family. One possibility is Csk, which phosphorylates Fyn at tyrosine 527 and acts as a negative regulator of Fyn . If activation of Fyn tyrosine kinase is indeed responsible for differentiation, then treatment of cells with PP1 should block this process. Progenitors induced to differentiate in the presence of PP1 retracted their processes and clumped together in the dish . This retraction was evident after 1–2 d in differentiation media, just as the control cells started to extend multiple processes. This effect was dose dependent; at low concentrations (1 μM), there was a 10% reduction in process outgrowth after 4 d, as assessed by measuring the radius of the extensive process network that surrounds the oligodendrocyte cell body. At concentrations <1 μM, there were no observable effects on cell morphology (data not shown). In the presence of 5 μM PP1, process outgrowth was significantly inhibited (>95%). All of the cells extended only one or two short, unbranched processes which were generally less that one cell body diameter in length. The treated cells lacked the extensive process network that is characteristic of the differentiated phenotype, and morphologically resembled oligodendrocyte progenitor cells. None of the cells formed the myelin-like membrane sheets observed in cultures of mature oligodendrocytes. The degree of process retraction observed with increasing concentrations of the inhibitors can be correlated with the levels of Fyn kinase inhibition observed in the in vitro kinase assays , which suggests that Fyn kinase activity is required for process extension during oligodendrocyte differentiation. While the inhibition of Fyn activity clearly retarded process outgrowth, it did not affect the expression of the cell surface marker galactocerebroside O1 and myelin proteins MAG and myelin basic protein . The process retraction was reversible, as removal of PP1 resulted in renewed process extension as early as 24 h after the inhibitor removal . To control for any non-specific effects, cells were treated with the related inactive analogues PP3 and PP4, which had no effect on Fyn activity and no effect on differentiating cells in culture . Finally, when progenitors were maintained in FGF and PDGF, mitogens that block differentiation , PP1 had no effect on process outgrowth or cell division . Progenitor cells maintained in 10 μM PP1, a concentration that completely inhibits Fyn kinase activity proliferated normally in the presence of FGF and PDGF, and maintained a bipolar morphology. These data suggest that the PP1 family of inhibitors are not toxic to the cells, but are acting solely to inhibit Fyn kinase activity. Thus, inhibition of Fyn activity appears to block morphological differentiation of progenitors into mature oligodendrocytes, without affecting the synthesis of stage-specific proteins that are characteristically expressed by mature oligodendrocytes. To verify the requirement for Fyn in oligodendrocyte differentiation, a kinase inactive, dominant negative Fyn was introduced into cultures of differentiating oligodendrocyte progenitors. The construct contains a point mutation in the ATP-binding site in which lysine 299 was mutated to methionine . This mutation results in a Fyn protein that cannot bind ATP and is catalytically inactive . The Fyn K299M mutant can still bind to substrate molecules and acts in a dominant negative fashion when expressed with wild-type Fyn in cells . The Fyn K299M cDNA was inserted into the viral pLJ vector containing an IRES sequence linked to the Neo gene, which confers resistance to geneticin, and allows for both selection and immunocytochemical detection of cells infected with the recombinant retrovirus . Oligodendrocyte progenitors were infected with a vector control or Fyn kinase inactive vector using the protocol of Wolven et al. and maintained in differentiation media. Infected cells were detected by immunostaining for neomycin phosphotransferase II (NPTII). Attempts to select for stable cell lines expressing the construct failed, even in low concentrations of G418. However, in transient infections, many cells were positive for NPTII, although the levels of staining varied considerably. Several effects were evident in the cultures infected with the Fyn K299M vector . Cells infected with the pLJ vector alone proceeded to differentiate normally, extending branched processes after culture in differentiation media . Cultures infected with the inactive Fyn kinase construct showed considerable inhibition of process outgrowth after 3 d . During the time course allowed for differentiation, many cells rounded up and detached from the plate, and it is not known whether these cells were expressing the mutant Fyn protein. Others extended processes less than one cell diameter in length and resembled those cells treated with the PP1 inhibitors. A few NPTII positive cells extended processes similar to the vector control. Overall, there was a 50% reduction in the number of cell extending processes longer than one cell body in diameter in the cells expressing the Fyn K299M virus compared with control. Since these data are derived from transient infections in which only 10– 30% of the cells were infected, it was not feasible to assay the level of Fyn kinase activity in the cells expressing the mutant construct. However, the reduction in process outgrowth observed in cultures expressing the dominant negative Fyn supports the hypothesis that Fyn kinase activation is required for the formation of processes that occurs during oligodendrocyte differentiation. The initial observation that Fyn-deficient mice showed reduced myelination in the central nervous system suggested that this tyrosine kinase could be one of the signals involved in regulating myelination during development . However, the underlying mechanism for the failure to myelinate in these mice was unclear. The present study directly examines the potential role of Fyn in myelination using primary cultures of differentiating oligodendrocytes. This is a well-characterized cell system; differentiation proceeds along a defined pathway, marked by discrete stages that can be identified by both morphological changes and the expression of cell surface markers and myelin proteins . Our results indicate that Fyn tyrosine kinase is activated very early after the oligodendrocyte progenitor cells are induced to differentiate . This upregulation occurs at a point before the appearance of MAG in these cells, indicating that another signal triggers Fyn in these cells. Inhibition of the kinase activity during differentiation prevents the extensive process outgrowth and formation of membrane sheets normally observed in cultures of mature oligodendrocytes, suggesting that Fyn may regulate cytoskeletal rearrangements that control process extension. The observed increase in Fyn activity is triggered as progenitor cells are induced to differentiate by maintaining them in a defined differentiation media. Activation of Src family kinases in other cell types is commonly triggered by external signals, such as growth factor–receptor binding, or integrin-substrate interactions . Since cells in the oligodendrocyte lineage are responsive to growth factors, it was surprising that Fyn activation was not affected by growth factor treatment. This finding suggests that another external signal or a novel regulatory mechanism is responsible for Fyn activation. Oligodendrocytes do express integrins on their cell surface that can modulate migration of these cells ; however it is unlikely that integrins are responsible for this response, as there are no abrupt changes in cell adhesion or extracellular substrates when Fyn is activated. Thus, it is likely that a novel regulatory mechanism is responsible for this activation, potentially involving release of a negative regulatory mechanism when the cells are placed in media lacking mitogens. It is possible that Csk, a negative regulator of Fyn, may be highly active in oligodendrocyte progenitors treated with growth factors; thus Fyn activity would be low until the growth factors are removed and Csk is downregulated. Alternatively, the withdrawal of growth factors causes the progenitors to exit the cell cycle and begin to differentiate . Signal transduction pathways that modulate differentiation may be activated once the cells leave the cell cycle, resulting in subsequent activation of Fyn. Elucidation of the upstream signal that triggers Fyn activation may ultimately reveal the specific cellular pathways involved in oligodendrocyte differentiation. Since Fyn is the primary Src family kinase activated during oligodendrocyte differentiation, these cells provide a model system in which to study biological effects of Fyn activation. The inhibition of Fyn, by either a pharmacological inhibitor or by expression of a dominant negative Fyn protein, suggests that Fyn can regulate the cytoskeletal rearrangements that accompany oligodendrocyte differentiation. The localization of Fyn to processes further supports this hypothesis. The precise mechanism underlying this effect is unknown at present; while it is clear that several proteins are phosphorylated when Fyn is activated in these cells, the identity of these proteins is unknown. It is not clear that phosphorylation of any one specific protein is the critical event that modulates the observed process retraction. Examination of tyrosine phosphorylated proteins in the presence of the PP1 inhibitors reveals that there is a general reduction in all phosphorylated proteins, not one protein in particular. This may indicate that Fyn activates another signal transduction pathway that ultimately contributes to the cytoskeletal rearrangement. The dominant negative studies confirm the hypothesis that Fyn is essential for oligodendrocyte process formation. Identification of Fyn substrates in these cells will be necessary to further characterize the intracellular events responsible for the observed biology reported here. The results obtained in this study may in part explain the partial loss of myelin proteins in the Fyn-deficient transgenic mice. If Fyn directs the formation of myelin sheet formation in vivo as it does in vitro, the myelin in these animals may show a reduction in lamellar structure, or oligodendrocyte cell volume. This would lead to a subsequent reduction in the total amount of myelin proteins. It will be interesting to determine if the loss of myelin protein leads to a reduction of the myelin sheath formation in Fyn-deficient mice. The initial phenotypic characterization of the Fyn-deficient mice did not suggest a defect in myelination, as the animals exhibited normal motor function . However, normal nerve conduction and motor function can be realized even if myelin sheath is reduced to 25% of normal . Further examination of the myelin content and the state of oligodendrocyte differentiation from these mice will provide more details on the role of Fyn during myelination. | Study | biomedical | en | 0.999998 |
10366595 | Sf9 cells, the transfer vector pVL1393, and the TA cloning kit were obtained from Invitrogen. Grace's insect cell culture medium was obtained from JRH Biosciences. The transfer vectors BioGreen-His and pAcGHLT-A, as well as baculovirus linearized DNA (BaculoGold), were from PharMingen . Yeastolate was obtained from Becton Dickinson . Lactalbumin hydrolysate, penicillin (10,000 U/ml)/streptomycin (10,000 μg/ ml)/ l -glutamine (29.2 mg/ml) solution, and a human spleen SuperScript cDNA library were from Life Technologies. FBS was purchased from Gemini Bioproducts. Human serum albumin (HSA) was from Intergen Co. [5,6,8,9,11,12,14,15- 3 H]Arachidonic acid (sp act 100 Ci/mmol), 1- O -hexadecyl-2-[ 3 H]arachidonyl-phosphatidylcholine (200 Ci/mmol), and l -1-[1- 14 C]palmitoyl-2-lysophosphatidylcholine (55 mCi/mmol) were from DuPont /NEN. Anti–rabbit horseradish peroxidase–linked F(ab′) 2 IgG fragment, reagents for enhanced chemiluminescence detection on immunoblots, and glutathione–Sepharose beads were purchased from Amersham Pharmacia Biotech . Calcium ionophore A23187 and pluronic acid F-68 (10% solution) were obtained from Sigma Chemical Co. Ionomycin was from Calbiochem-Novabiochem . Okadaic acid (ammonium salt) was from Alexis Biochemicals. Fura-2 acetoxymethyl ester (AM) and the nuclear dye Hoechst 33342 were obtained from Molecular Probes. A donkey anti–rabbit IgG antibody conjugated to Cy3 was obtained from Jackson ImmunoResearch. Reagents for protein determination by the bicinchoninic acid method were from Pierce Chemical Co. Bacillus thuringiensis δ-endotoxin CryIC was generously provided by Dr. Jean-Louis Schwarz (National Research Council, Montreal, Canada) and Dr. Marianne Carey (Case Western Reserve University, Cleveland, OH). The DNA encoding for human cPLA 2 (provided by Dr. James Clark, Genetics Institute, Cambridge, MA) was cloned into the transfer vector pVL1393 as previously described . For fusion-protein construction, the human cPLA 2 coding region was obtained by PCR from a human spleen cDNA library and cloned into the pCR2.1 vector following the TA cloning method. The PCR primers used were 5′-GGATCCTGACTGAAAGCTAGAGGC-3′ and 5′-CAGCCAGTCTCTCATGATCAGTACGAC-3′ . Two in-frame codons present in the 5′ untranslated region were removed by PCR amplification using the primers 5′-GAGAGCGGGTACCCCGGTTTGAAGTGTGAAAACATTTCCTG-3′ (which contains a KpnI restriction site) and 5′-CCTGATTAGGATCCAAAATAAATTCAAAGGTCTC-3′ (which straddles a BamHI site). The PCR product was cut with KpnI and BamHI and cloned into pCR2.1 containing the cPLA 2 gene. The modified cPLA 2 sequence was cloned into the transfer vectors pAcGHLT (for expression of GST-cPLA 2 ) and BioGreen-His (for expression of green fluorescent protein [GFP]-cPLA 2 ) using the SacI and PstI sites. Different mutants of cPLA 2 were obtained by site-directed mutagenesis using a kit from 5Prime→ 3Prime Inc., following the manufacturer's instructions. The mutagenic oligonucleotides used were 5′-CTTGATACTCCAAATCCCTATGTG-3′ (D43N), 5′-CGTTAATGAATGCCAATTATG-3′ (D93N), 5′-GATAGCTCGGACGCTGATGATGAATCAC-3′ (S437A), 5′-GAAGATGCTGGAGCTGACTATCAAAGTG-3′ (S454A), 5′-CTTATCCACTGGCTCCTTTGAG-3′ (S505A), and 5′-CCATCTCGTTGCGCTGTTTCCC-3′ (S727A). A truncated version of cPLA 2 (Δ721–749) was generated by introducing a stop codon after amino acid 720 using the mutagenic oligonucleotide 5′-GAATATAGAAGATAGAATCCATCTCG-3′. A GFP-C2 construct was generated by using the oligonucleotide 5′-GCCCAGACCTATGATTTAGTATGGC-3′ to insert a stop codon after amino acid 144. All mutations were confirmed by sequencing of DNA from several bacterial colonies selected on agar plates. Sf9 cells were routinely cultured in 100-ml vented spinner flasks, at 27°C, in TNM-FH medium (Grace's medium supplemented with 3.3 g/liter yeastolate, 3.3 g/liter lactalbumin hydrolysate, and 1% penicillin/streptomycin/ glutamine solution) containing 10% FBS and 0.1% pluronic acid. Cell density was maintained at 0.2–1.5 × 10 6 cells/ml. Recombinant baculovirus was generated by cotransfection of Sf9 cells with the transfer vector pVL1393 containing the gene for cPLA 2 and linearized baculovirus DNA (BaculoGold; PharMingen ) following the manufacturer's instructions. Viral stocks derived from single virus particles were obtained by plaque purification and amplified (two cycles) following standard procedures. The titer of the working viral stocks was determined by infecting cells with serial dilutions of the stocks, overlaying them with agarose-containing TNM-FH medium, and counting the plaques after 4–6 d of incubation. Sf9 cells were plated on glass coverslips (13 mm diam) in 24-well plates (4 × 10 5 cells/well) and infected with baculovirus containing wild-type cPLA 2 . After infection (50–54 h), Sf9 cells were washed three times in buffer A (10 mM 2-[ N -morpholino]ethanesulfonic acid, 10 mM NaCl, 60 mM KCl, 25 mM MgCl 2 , 4 mM d -glucose, 110 mM sucrose, adjusted to pH 6.2 with Trizma base) and incubated with 2 μM Fura-2 AM (in buffer A) for 60 min at room temperature protected from light. The cells were rinsed twice with buffer A, positioned in a fluorometer cuvette, and maintained at 27°C with continuous stirring. Calcium chloride or EGTA was added to buffer A to reach the final concentrations indicated in the figures. The effect of agonists on the intracellular concentration of calcium was determined by spectrofluorometry as previously described . Sf9 cells were plated in 24-well tissue culture plates (2.5 × 10 5 cells/well) and infected with baculovirus. After infection (30–35 h), they were labeled with 0.2 μCi/well [ 3 H]arachidonic acid and incubated overnight (16–18 h). The cells were washed three times with TNM-FH medium containing 0.1% HSA to remove unincorporated arachidonic acid and then treated with agonists. After stimulation, the medium was centrifuged at 500 g for 10 min, and the amount of radioactivity in the supernatant was determined by scintillation counting. Cells were scraped in 0.5 ml of 0.1% Triton X-100 for determining the total cellular radioactivity. For immunoblot analysis of cPLA 2 , Sf9 cells were scraped in 100 μl of Laemmli buffer and the protein concentration was determined. DTT was added to a final concentration of 25 mM, and samples were boiled for 5 min before loading (0.5 μg/lane) on a SDS-polyacrylamide gel . After electrophoresis, proteins were transferred to nitrocellulose and cPLA 2 was detected by chemiluminescence using a 1:5,000 dilution of polyclonal antiserum to cPLA 2 . Sf9 cells (1.25 × 10 6 cells/35 mm dish) were infected with recombinant baculovirus for 48–50 h. The monolayers were rinsed with 2 ml of PBS and scraped into 100 μl of ice-cold homogenization buffer (10 mM Hepes, pH 7.4, 1 mM EGTA, 0.34 M sucrose, 10% glycerol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF). Cells were lysed with a probe sonicator (3 times for 5 s, then 2 times for 10 s) on ice, and the homogenates centrifuged at 100,000 g for 1 h. The protein concentration in the cytosols was determined and then DTT was added to a final concentration of 1 mM. The cytosols were kept on ice and assayed within 24 h, or stored at −20°C and thawed just before the assay. The PLA 2 and lysophospholipase activities of cPLA 2 were determined by using 1- O -hexadecyl-2-[ 3 H]arachidonylphosphatidylcholine (final concentration 30 μM), or [1- 14 C]palmitoyl-2-lysophosphatidylcholine (50 μM), respectively, as previously described . Reactions were started with the addition of cytosolic protein (10 μg for PLA 2 assays, 5 μg for lysophospholipase assays), and proceeded for 5 min at 37°C. Equal expression of the mutant enzymes was verified by immunoblotting and quantitation of the cPLA 2 bands using a Storm 840 system from Molecular Dynamics. Sf9 cells grown in suspension (6 × 10 8 cells in 600 ml medium) were infected with baculovirus encoding the fusion protein GST-cPLA 2 . Cells were lysed in 20 mM Hepes, pH 7.4, containing 2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Lysates were incubated with 200 μl glutathione–Sepharose beads for 2 h at 4°C. The beads were washed sequentially with 2 ml each of 10 mM Tris (pH 7.5), 10 mM Tris (pH 8.8), 100 mM triethylamine (pH 11.5), and 10 mM Tris (pH 7.5). The beads were mixed with rabbit anti-cPLA 2 antiserum (500 μl, diluted 1:9 in 10 mM Tris, pH 7.5), and then washed with 2 ml each of 10 mM Tris (pH 7.5), 0.5 M NaCl in 10 mM Tris (pH 7.5), and 10 mM Tris (pH 8.8). Antibody was eluted with 2 ml of 100 mM triethylamine (pH 11.5) into tubes containing 1 M Tris (pH 8.0) to adjust the pH. For microscopy experiments, 1.25 × 10 6 cells were plated on 35-mm tissue culture dishes and infected with baculovirus containing the genes for the different GFP-containing fusion constructs at multiplicities of infection ranging from 2 to 10 colony forming units per cell. After infection (40–50 h), cells were washed twice with TNM-FH medium and observed at room temperature using an Olympus Vanox-T Microscope equipped with an Achroplan 63X Zeiss lens and a FITC filter. Digital confocal images were obtained with a SpectraSource Orbis 16 camera, using Slidebook software from Intelligent Imaging Innovations, Inc. Images shown in the figures are representative of at least three independent experiments. For immunofluorescence, Sf9 cells (2.5 × 10 5 ) were plated on 13-mm glass coverslips in 24-well culture plates and infected with baculovirus containing cPLA 2 . After infection (40–50 h), cells were rinsed twice with 0.5 ml PBS, pH 7.5, and then fixed for 15 min with 0.5 ml 3% paraformaldehyde in PBS containing 3% sucrose. After rinsing with PBS, cells were permeabilized with 0.2% Triton X-100 for 15 min, and then incubated for 30 min with HBSS containing 10% FBS (blocking solution). Cells were incubated for 2 h with affinity-purified anti-cPLA 2 antibody diluted 1:20 in blocking solution. After extensive rinsing with PBS, cells were incubated for 1 h with donkey anti–rabbit IgG antibody conjugated to Cy3 (10 μg/ml in blocking solution). Coverslips were mounted on microscope slides in 0.1 M Tris-HCl, pH 8.5, containing 90% glycerol and 2 mg/ml orthophenylenediamine. Cells were observed by digital confocal microscopy using a Cy3 filter. Sf9 cells expressing cPLA 2 release arachidonic acid in response to A23187 and okadaic acid . We have found that CryIC toxin from Bacillus thuringiensis also stimulates arachidonic acid release from Sf9 cells expressing cPLA 2 , but not from cells infected with baculovirus containing the vector alone . The crystal (Cry) protein toxins are inclusions formed by the gram-positive soil bacterium during sporulation. CryIC toxin has been shown to induce an increase in intracellular calcium in Sf9 cells, probably in a receptor-mediated fashion . A 40-kD binding protein for CryIC toxin has been identified recently . Arachidonic acid release in response to CryIC toxin was near maximal at 2 μg/ml, a concentration well below the ED 50 for toxicity (15 μg/ml) for Sf9 cells . Rapid and prolonged release of arachidonic acid was induced by CryIC toxin . An increase in intracellular calcium is an important mechanism for regulating cPLA 2 by promoting its translocation and binding to membrane. However, we demonstrated previously that okadaic acid induces arachidonic acid release in macrophages without increasing intracellular calcium . Experiments were carried out to determine the effect of okadaic acid on intracellular calcium levels in the Sf9 model in comparison to ionomycin and CryIC, which are known to increase intracellular calcium. As shown in Fig. 1 C, okadaic acid did not promote an increase in intracellular calcium in Sf9 cells expressing cPLA 2 , and no effect was seen when the incubation was prolonged for as long as 30 min. In contrast, CryIC toxin induced an increase in intracellular calcium that did not occur as rapidly as with ionomycin, but did reach a similar level. As previously reported, the increase in intracellular calcium induced by CryIC was sustained for at least 15 min (not shown) . GFP was linked to the amino terminus of cPLA 2 and its subcellular localization was visualized in living Sf9 cells. GFP-cPLA 2 , like GFP alone, was uniformly distributed throughout unstimulated cells when observed in different planes of the cell by confocal analysis (data not shown). In Sf9 cells treated with the calcium-mobilizing agonists A23187 and CryIC toxin, as well as okadaic acid, the GFP-cPLA 2 fusion protein exhibited time-dependent translocation to the nuclear envelope . Translocation was evident in at least 80–85% of the cells observed in each experiment. Diffuse fluorescence emanating from the nuclear envelope could also be observed and may represent cPLA 2 bound to endoplasmic reticulum. cPLA 2 that is present both inside the nucleus and in the cytosol in unstimulated cells translocated to the nuclear envelope. Importantly, GFP alone did not translocate in response to agonist treatment and remained uniformly distributed. Translocation of GFP-cPLA 2 to the nuclear envelope was evident by 5–10 min after treatment with the agonists and was nearly complete by 20–30 min . The time course of translocation was not identical from cell to cell in the population and could vary by minutes. As previously reported the agonists induce significant arachidonic acid release by 15 min, which is consistent with the time course of translocation . To further substantiate that GFP-cPLA 2 localizes at the nuclear envelope, Sf9 cells expressing GFP-cPLA 2 were treated with the nuclear dye Hoechst 33342. As shown in Fig. 3 A, GFP-cPLA 2 was uniformly distributed throughout the cell, whereas the Hoechst localized to the nucleus. When GFP-cPLA 2 and Hoechst colocalized, as seen inside the nucleus of the unstimulated cell, the purple color was diminished and the fluorescence appears white. As shown in the cell treated with A23187, GFP-cPLA 2 localized to the nuclear envelope and the perinuclear region, whereas the purple Hoechst stain remained distributed throughout the nucleus. When the GFP-cPLA 2 and Hoechst fluorescence were visualized together a bright ring of GFP-cPLA 2 was seen surrounding the purple nucleus in the stimulated cell. To verify that the translocation of cPLA 2 was not influenced by the GFP moiety, cPLA 2 was expressed alone and as a GFP fusion protein in Sf9 cells and visualized by immunofluorescence using affinity-purified polyclonal anti-cPLA 2 antibody. Localization of cPLA 2 was similar to GFP-cPLA 2 in fixed Sf9 cells . In unstimulated cells, the enzyme was uniformly distributed throughout the cell. In response to A23187, cPLA 2 and GFP-cPLA 2 similarly translocated to the nuclear envelope and perinuclear region. Experiments were also carried out to verify that the GFP-cPLA 2 fusion protein was catalytically active in Sf9 cells and could mediate agonist-induced arachidonic acid release. As shown in Fig. 3 C, cells expressing either cPLA 2 or GFP-cPLA 2 released arachidonic acid to a similar extent in response to A23187 and okadaic acid. GFP-cPLA 2 exhibited a decrease in electrophoretic mobility (gel shift) in response to okadaic acid, as occurs with the native enzyme . We reported previously that treatment of Sf9 cells with okadaic acid increases phosphorylation of cPLA 2 on S727 and to a lesser extent on S437, S454, and S505 . To determine if phosphorylation of these sites was functionally important for okadaic acid– induced arachidonic acid release, the serines were mutated to alanines and the mutant cPLA 2 constructs expressed in Sf9 cells. A double mutant construct, S505A/S727A, was also generated since both sites may act cooperatively in regulating cPLA 2 . In addition, a carboxy-terminal deletion (Δ721–749) was produced to avoid the possibility that serines near S727 (S724, S729, and S731) may become phosphorylated when S727 is converted to an alanine. Sf9 cells expressing the S727A, S437A, or S454A mutant constructs exhibited the same magnitude of arachidonic acid release in response to A23187, CryIC toxin, and okadaic acid as Sf9 cells expressing wild-type cPLA 2 . These results demonstrate that phosphorylation of these sites does not play a functional role in cPLA 2 activation in this model. cPLA 2 containing the S505A substitution was the only phosphorylation site mutant that was less effective (by 50%) at mediating arachidonic acid release. The results with cells expressing cPLA 2 containing either the S505A/S727A double mutation or the S505A single mutation were not significantly different. Arachidonic acid release from Sf9 cells expressing the carboxy-terminal deletion mutant was no different from the response of Sf9 cells expressing wild-type cPLA 2 , confirming that phosphorylation of S727 plays no functional role in this model and that the extreme carboxy terminus of cPLA 2 is not essential. Importantly, the level of expression of wild-type and mutant forms of cPLA 2 was similar . All the mutant cPLA 2 enzymes except S505A were able to gel-shift in response to okadaic acid, demonstrating that the characteristic retardation of the electrophoretic mobility is only due to phosphorylation of S505, and not the other sites. A23187 and CryIC toxin have no effect on cPLA 2 electrophoretic mobility in Sf9 cells . Although okadaic acid induces arachidonic acid release without increasing intracellular calcium, our previous work with macrophages has shown that resting levels of calcium are required . This suggested that the C2 domain of cPLA 2 is functionally important for regulation of the enzyme in response to okadaic acid. The crystal structure of the cPLA 2 C2 domain has revealed several amino acid residues (D40, T41, D43, N65, D93, A94, and N95) that participate in binding calcium, and it has been shown recently that mutation of these residues impairs PLA 2 activity and binding of cPLA 2 to phospholipids . To determine if a functional C2 domain was required for cPLA 2 to mediate arachidonic acid release in response to okadaic acid compared with the calcium-mobilizing agonists, two calcium binding residues (D43 and D93) were mutated to asparagine residues. cPLA 2 containing the D43N, D93N, or D43N/D93N mutations was unable to mediate okadaic acid–induced arachidonic acid release in Sf9 cells . Arachidonic acid release induced by A23187 or CryIC toxin was also dramatically suppressed in Sf9 cells expressing D43N or D43N/D93N cPLA 2 , whereas the D93N mutant was fully functional. In fact, the D93N mutant exhibited a modest but reproducible increased response to A23187, as compared with wild-type cPLA 2 . As shown in the Western blot, the C2 domain mutants of cPLA 2 are expressed at similar levels and gel-shifted in response to okadaic acid. Mutation of D93N to an asparagine resulted in an increased electrophoretic mobility of cPLA 2 . The effect of the C2 domain mutations on translocation of GFP-cPLA 2 in response to okadaic acid and the calcium-mobilizing agonists was evaluated. The D43N or D43N/D93N mutant enzymes were unable to translocate to the nuclear envelope in living Sf9 cells treated with A23187, whereas GFP-cPLA 2 containing the D93N mutation was fully functional and translocated in response to the calcium ionophore . Translocation was evident in at least 85% of the cells observed in each experiment. Similar results were also obtained with CryIC toxin (not shown). In contrast, the D93N mutant did not translocate to the nuclear envelope in response to okadaic acid . The D43N and D43N/D93N constructs were also unable to translocate in response to okadaic acid (not shown). The implication of the above results is that arachidonic acid release induced by okadaic acid in Sf9 cells would show less dependency on extracellular calcium than the response to A23187 or CryIC toxin, but would be dependent on resting levels of intracellular calcium. As shown in Fig. 7 , depleting extracellular calcium dramatically reduced A23187-induced arachidonic acid release from Sf9 cells expressing wild-type cPLA 2 but had less effect on cells treated with okadaic acid. The significant effect of depleting extracellular calcium on okadaic acid–induced arachidonic acid release may be due to the observed decrease in basal levels of intracellular calcium that occurs when extracellular calcium levels are decreased. In normal Sf9 medium containing 9 mM calcium the intracellular calcium concentration was 68 ± 16 nM (mean ± SEM, n = 4), and the level decreased to 17 ± 8 nM when no calcium was added to the medium. With EGTA in the medium, intracellular calcium was below the level of detection (≤5 nM). The agonist-induced level of intracellular calcium can be manipulated by incubating Sf9 cells in medium containing varying levels of extracellular calcium. The effect of extracellular calcium concentration on the ability of CryIC toxin to induce arachidonic acid release in Sf9 cells expressing the C2 mutant constructs of cPLA 2 was evaluated and correlated with the intracellular levels of calcium . Arachidonic acid release induced by CryIC toxin was dependent on extracellular calcium and decreased to near baseline levels when it was chelated with EGTA. Sf9 cells expressing cPLA 2 containing the D43N or D43N/ D93N mutations exhibited little arachidonic acid release above baseline levels in response to CryIC toxin. The ability of cPLA 2 containing the D93N mutation to mediate arachidonic acid release when extracellular calcium was decreased from 9 to 2 mM paralleled the response observed with wild-type cPLA 2 , both exhibiting an ∼30–35% decrease in responsiveness. In contrast, when no calcium was added to the medium, the D93N mutant enzyme was unable to mediate arachidonic acid release, whereas there was still a significant response in cells expressing wild-type cPLA 2 . These results demonstrate that the D43N and D43N/D93N mutations dramatically reduce the ability of the enzyme to mediate arachidonic acid release even at the highest levels of intracellular calcium achieved (630 nM) with CryIC toxin–treated Sf9 cells in medium containing 9 mM calcium. However, cPLA 2 containing the D93N mutation is functional at concentrations of intracellular calcium in the 0.2–0.6 μM range but not at 36 nM intracellular calcium. The results of expressing the C2 domain mutants of cPLA 2 in Sf9 cells suggested that mutating D43 and D93 differentially affected the affinity of the enzyme for calcium. This was verified in vitro by measuring the effect of calcium concentration on cPLA 2 activity of the mutant enzymes in the cytosolic fraction of Sf9 cells in which equal expression of cPLA 2 was confirmed by densitometry . Wild-type cPLA 2 exhibited significant activity at nanomolar calcium that continued to increase substantially up to 1 μM calcium with little additional increase in the millimolar range. Consistent with the results of expressing the C2 mutants in Sf9 cells, mutating D43 had a more profound effect on the calcium sensitivity of cPLA 2 than mutating D93. cPLA 2 containing the D43N or D43N/D93N mutations exhibited very little activity at concentrations of calcium ≤1 μM but exhibited significant activity at millimolar calcium. Unlike wild-type cPLA 2 , the D93N mutant did not exhibit an increase in activity at 50 nM calcium, but did have significant activity in the 0.25–1 μM range. At 1 mM calcium the wild-type and D93N mutant enzymes exhibited similar activities. As previously reported, cPLA 2 exhibits lysophospholipase activity, which is calcium independent . The lysophospholipase activity of cPLA 2 containing the mutations in the C2 domain was not significantly different from wild-type cPLA 2 (data not shown). Experiments were undertaken to determine if the C2 domain of cPLA 2 played a role in the preferential targeting of cPLA 2 to the nuclear envelope. GFP was linked to the amino terminus of the C2 domain and expressed in Sf9 cells. GFP-C2 was uniformly distributed throughout unstimulated cells as observed for GFP-cPLA 2 . A23187 induced translocation of the C2 domain to the nuclear envelope in Sf9 cells with a similar time course as the full-length enzyme. The same results were observed in response to the CryIC toxin (not shown). In contrast to the calcium-mobilizing agonists, okadaic acid did not induce translocation of GFP-C2. Translocation of GFP-C2 to the nuclear envelope in response to A23187 and CryIC toxin required extracellular calcium as observed for full-length cPLA 2 . The calcium-mobilizing agonists did not induce translocation of GFP-C2 in Sf9 cells incubated in medium containing EGTA. However, the subsequent addition of calcium to the medium resulted in translocation of GFP-C2 to the nuclear envelope. The baculovirus expression system in Sf9 insect cells is a useful model to study the regulation of cPLA 2 . This system has been used extensively to study the function of expressed proteins and their roles in signal transduction . The effect of cPLA 2 mutations on arachidonic acid release and translocation can be directly compared in intact, living cells. Importantly, the stimulated arachidonic acid release is due to cPLA 2 since there is little contribution of endogenous PLA 2 enzymes. The responses of Sf9 cells expressing cPLA 2 , such as arachidonic acid release and phosphorylation, are similar to responses that we have observed in murine macrophages and human monocytes . In addition, GFP-cPLA 2 is targeted to the nuclear envelope in response to the calcium-mobilizing agonists and okadaic acid. Using the GFP fusion protein allows translocation to be followed in real time in living cells, and the GFP moiety attached to the amino terminus of cPLA 2 does not appear to influence the function of the enzyme. It should be noted that the mechanisms involved in the regulation of cPLA 2 such as the importance of phosphorylation, calcium, and even the site of subcellular localization, appear cell type– and agonist-dependent. Sf9 cells are ovarian cells and may lack regulatory components present in certain mammalian cells. However, there are also considerable differences in the regulation of cPLA 2 activation among different mammalian cell types. For example, phorbol esters or calcium ionophores alone are poor inducers of cPLA 2 activation (and arachidonic acid release) in many cell types, but can act synergistically. However, in macrophages and certain hemopoietic cells these are potent agonists. Similar to macrophages, Sf9 cells expressing cPLA 2 respond to A23187 and okadaic acid but, unlike macrophages, not to PMA. In unstimulated Sf9 cells, the GFP-cPLA 2 fusion protein was uniformly distributed both inside the nucleus and in the cytosol. This distribution was not influenced by the level of GFP-cPLA 2 expression, which can be varied by decreasing the multiplicity of infection and by decreasing the infection time. In several unstimulated mammalian cells, cPLA 2 has been observed inside the nucleus, in the cytosol, and in both locations depending on the cell type and growth conditions . In many of these cell models cPLA 2 translocates to the nuclear envelope and endoplasmic reticulum in response to cell stimulation, although exceptions have been observed. In confluent endothelial cells treated with histamine, cPLA 2 translocates to both the nuclear envelope and to the plasma membrane at intercellular junctions . A punctate clustering of cPLA 2 has been observed in the cytoplasm of mouse 3T3 fibroblasts overexpressing the epidermal growth factor receptor, and this distribution is not influenced by epidermal growth factor or calcium ionophore . A punctate cytoplasmic localization of cPLA 2 occurs in U937 cells and appears to be due to association with cytoplasmic lipid bodies . Using a variety of agonists that act on cells by diverse mechanisms, it has become clear that there are alternative regulatory pathways that can lead to activation of cPLA 2 and arachidonic acid release . The ability of okadaic acid to activate cPLA 2 and induce arachidonic acid release in macrophages and Sf9 cells without increasing intracellular calcium provides a useful tool to elucidate novel mechanisms involved in cPLA 2 regulation. Previous work in macrophages and other cell models has demonstrated that phosphorylation on S505 is not sufficient for activation of cPLA 2 and arachidonic acid release in the absence of an increase in intracellular calcium . This is also evident in Sf9 cells since a significant portion of cPLA 2 is constitutively phosphorylated on S505 when expressed in Sf9 cells, yet there is no arachidonic acid release unless the cells are treated with okadaic acid or agonists that increase intracellular calcium . This implicates an alternative mechanism in the regulation of cPLA 2 in response to okadaic acid. One possibility was a role for S727 phosphorylation which is preferentially induced in okadaic acid–treated Sf9 cells . S505, S437, and S454 are constitutively phosphorylated on cPLA 2 in unstimulated Sf9 cells, and their phosphorylation state is increased only modestly in response to agonist treatment. These four residues represent the only major phosphorylation sites on cPLA 2 in okadaic acid– treated Sf9 cells. Whether phosphorylation of these novel sites, particularly S727, plays a functional role in cPLA 2 activation has been an important question. The mutagenesis data demonstrate that okadaic acid–induced phosphorylation of cPLA 2 on S727, S437, or S454 plays no functional role in cPLA 2 -mediated arachidonic acid release in this model. Whether phosphorylation of S727 plays a functional role in cPLA 2 regulation in other cells where it has been identified remains to be established . Phosphorylation of S505 does play a role in augmenting arachidonic acid release by both okadaic acid and the calcium-mobilizing agonists. However, it is not absolutely essential since Sf9 cells expressing cPLA 2 containing the S505A mutation still release arachidonic acid to ∼50% of the level of Sf9 cells expressing wild-type cPLA 2 . The role played by S505 phosphorylation in the regulation of cPLA 2 appears to be cell type– and agonist-dependent. In macrophages, S505 phosphorylation is not essential for arachidonic acid release in response to agonists that induce a sustained increase in intracellular calcium but does appear to be required when there is only a transient increase in calcium . In CHO cells overexpressing cPLA 2 , S505 phosphorylation is essential for agonist-induced arachidonic acid release . However, S505 phosphorylation of cPLA 2 is not required for arachidonic acid release in thrombin-stimulated platelets . Our translocation results show a steady accumulation of GFP-cPLA 2 in the perinuclear region of Sf9 cells after addition of A23187 or CryIC toxin. This observation is consistent with recent data showing that a sustained increase in the intracellular calcium concentration is required for the stable association of cPLA 2 -GFP to the nuclear envelope of CHO cells, which correlates with agonist-induced arachidonic acid release . Although okadaic acid does not increase intracellular calcium, our results demonstrate a requirement for a functional C2 domain. Mutagenesis of calcium-binding residues D43 and D93 in the C2 domain quantitatively suppress okadaic acid–induced arachidonic acid release and translocation of cPLA 2 to the nuclear envelope. In addition, lowering the levels of intracellular calcium by including EGTA in the medium diminished okadaic acid– induced arachidonic acid release, suggesting a requirement for the resting levels of calcium. Attempts to completely chelate intracellular calcium by also including BAPTA or Quin-2 were unsuccessful due to adverse effects on Sf9 cells. The differences in the functional responses of Sf9 cells expressing the D43N and D93N mutant enzymes suggest that these mutations differentially affect the affinity of the enzyme for calcium. This is consistent with the structural analysis of the cPLA 2 C2 domain, which binds two calcium ions with different affinities . D43 forms one interaction with each of the two calcium ions, whereas both side chain oxygens of D93 participate in binding only one of the calcium ions at site II. It has been shown recently that purified cPLA 2 containing D43N and D93N mutations exhibits greatly diminished PLA 2 activity and binding to phospholipids as compared with the wild-type enzyme . The calcium requirements are higher for cPLA 2 containing the D43N mutation than D93N, consistent with the functional differences we observe in the Sf9 model. Differential roles for calcium binding residues in the C2 domain of PKCα have also been reported . D246 plays the most critical role for PKCα activation and calcium-dependent binding to lipid vesicles in vitro, and is thought to be due to its ability to coordinate two Ca 2+ ions tightly. Our results demonstrate a pivotal role for D43 in mediating cPLA 2 translocation in intact cells. cPLA 2 containing the D43N mutation is unable to translocate to the nuclear envelope or to mediate arachidonic acid release in response to okadaic acid, which acts at resting levels of calcium (60–70 nM), but also in response to calcium-mobilizing agonists A23187 and CryIC toxin, which increase intracellular calcium to ∼0.6 μM calcium. In contrast, cPLA 2 containing the D93N mutation translocates and releases arachidonic acid at intracellular calcium in the 0.2–0.6 μM range but not at resting calcium. This difference in affinity of the mutant cPLA 2 enzymes is also evident when assayed in vitro. However, the mutations do not adversely affect the conformation of the active site since they all have significant enzymatic activity when assayed in vitro at millimolar calcium, and they exhibit similar lysophospholipase activity as the wild-type enzyme. The mechanisms involved in the preferential targeting of cPLA 2 to the nuclear envelope are not known. However, an important observation from this study is that the C2 domain of cPLA 2 translocates to the nuclear envelope in response to the calcium-mobilizing agonists. This suggests that the sequence determinants for this preferential localization reside in the C2 domain. The C2 domain of cPLA 2 has been shown to preferentially bind to phosphatidylcholine headgroups . However, this would not be expected to be responsible for nuclear targeting since phosphatidylcholine is found in all cellular membranes. The C2 domain of Nedd4 also exhibits preferential binding to phosphatidylcholine but has been shown recently to translocate to the plasma membrane in cells treated with calcium ionophore . Although the C2 domains of Nedd4 and cPLA 2 have similar properties including the same topology (topology II), similar affinities for calcium, and preference for binding amphipathic phospholipid, they exhibit considerable sequence diversity that may be responsible for their differences in subcellular localization. The C2 domain of PKCα, which binds phosphatidylserine, has been shown recently to bind to the plasma membrane when expressed in COS cells . The membrane binding of the PKCα C2 domain occurs at resting levels of calcium, consistent with its higher affinity for calcium for binding to phospholipid vesicles as compared with the cPLA 2 or the Nedd4 C2 domains . Results of several recent in vitro studies have begun to shed light on the mechanisms involved in interaction of the C2 domain and full-length cPLA 2 with phospholipid vesicles. Experiments have shown that the three calcium-binding loops are involved in the binding of the isolated C2 domain to phosphatidylcholine vesicles . These results are consistent with the observation that the two Ca 2+ ions bound to the C2 domain become occluded, and dissociate more slowly upon binding of the C2 domain to phospholipid vesicles . Regions of the cPLA 2 C2 domain that interact with phospholipid micelles have also been identified by 15 N-HSQC spectroscopy revealing a role for the calcium-binding loops, proximal ends of attached β strands, and portions of β strands 2 and 3 . It has been shown recently that calcium induces not only binding but penetration of cPLA 2 and the C2 domain into artificial lipid membranes . An important role for hydrophobic residues in the interaction of the cPLA 2 C2 domain with phospholipid has been implicated from several studies . The residues F35, L39, Y96, and V97 in the calcium binding loops 1 and 3 appear to be particularly important for phospholipid binding and membrane penetration of the C2 domain of cPLA 2 . Two tryptophan residues in the calcium-binding region 3 of PKCα C2 domain also have been shown recently to be essential for binding phospholipid vesicles and membrane penetration in vitro . Mutagenesis studies of the C2 domain of PKCβII have shown that the calcium-induced interaction of PKC with membrane is not due to charge neutralization of the acidic residues . However, unlike cPLA 2 and PKC in which hydrophobic interaction with phospholipid plays a predominant role, the binding of the C2A domain of synaptotagmin I to phospholipid is largely electrostatic . Recent studies have demonstrated that the binding properties of the isolated C2 domain of cPLA 2 do not necessarily predict the binding properties of the full-length enzyme . Binding of the C2 domain to anionic and zwitterionic phospholipid vesicles occurs with similar affinities and requires calcium. However, full-length cPLA 2 binds much tighter to anionic (phosphatidylmethanol) than zwitterionic (phosphatidylcholine) vesicles and, surprisingly, the binding to anionic vesicles does not require calcium. Consequently, this implicates regions downstream of the C2 domain in interacting with membranes containing anionic phospholipids. There is evidence that anionic phospholipids, particularly phosphatidylinositol-4,5-bisphosphate (PIP 2 ), enhance enzymatic activity and promote high affinity binding of cPLA 2 to phospholipid vesicles . Interestingly, PIP 2 enhances both calcium-dependent and -independent binding and activity of cPLA 2 and may represent a physiological equivalent of phosphatidylmethanol . Our results demonstrate that the C2 domain of cPLA 2 can translocate to the nuclear envelope in response to calcium-mobilizing agonists but not in response to okadaic acid. This provides evidence in the intact cell that another region of cPLA 2 may also be involved in membrane targeting induced by agonists that do not increase intracellular calcium. However, mutating calcium-binding residues in the C2 domain of cPLA 2 and depletion of intracellular calcium suppress okadaic acid–induced arachidonic acid release indicating a requirement for the C2 domain and resting levels of calcium for translocation. These data suggest that cPLA 2 translocation to membrane induced by okadaic acid may be coordinately regulated by the C2 domain and another region of the enzyme. The cooperative regulation of membrane association by two domains has been observed in numerous proteins including cytohesin-1, Tiam1, Ras-GRF, and phospholipase C, to name a few . Membrane binding of phospholipase Cδ1 has been studied extensively, and appears to involve a cooperative role for the PH, C2, and catalytic domains . Our results demonstrate that the effects of the C2 domain mutations on the ability of cPLA 2 to mediate arachidonic acid release correlate directly with their ability to translocate to the nuclear envelope. In addition, a differential functional role is shown for specific calcium-binding residues in the C2 domain in cPLA 2 regulation in intact cells. A functional C2 domain is required for agonist-induced translocation of cPLA 2 and arachidonic acid release in the Sf9 model even with okadaic acid, which activates cPLA 2 without increasing intracellular calcium. This suggests that binding of the C2 domain to calcium, even at resting levels, may be required for stable association of the enzyme with the membrane. Interestingly, okadaic acid induces translocation of the full-length cPLA 2 , but not the C2 domain. Our results indicate that the C2 domain is necessary and sufficient for cPLA 2 translocation to the nuclear envelope in response to calcium-mobilizing agonists. However, a functional C2 domain is necessary but not sufficient for translocation in response to okadaic acid, suggesting that additional regions of the enzyme are also involved. | Other | biomedical | en | 0.999996 |
10366596 | The strains used in this study are listed in Table I and were created in this study. Media were prepared as previously described , except that YE medium containing adenine sulfate at the concentration of 75 μg/ml (YEA medium) was used for routine growth of cells. fur1 mutation was tested by the growth on the YEA medium containing 5-fluorouracil ( Sigma Chemical Co. ) at the concentration of 100 μg/ml. Genetic manipulations were carried out as described by Moreno et al. . A portion of the DHC was isolated by PCR using degenerated primers based on regions of highly conserved amino acid sequence that lie in the neighborhood of the P-loop ATP-binding site lying nearest to the NH 2 terminus of the DHC . A 320-bp genomic DNA fragment encoding a partial open reading frame (ORF) was obtained which had ∼52% identity to the DHC sequence of Dictyostelium . A genomic DNA library was screened using the PCR-amplified DNA fragment as a probe, and a 2.7-kb genomic clone (pDHC1-1) was obtained . This cloned fragment was mapped between the mei2 + and leu2 + loci on the right arm of chromosome I from two different cosmid libraries . The complete coding sequence was obtained by recovering plasmids that had been integrated at the dhc1 locus. Plasmids containing different portions of the DHC coding region were integrated at the dhc1 locus of strain CRL152. The genomic DNA of the integrant was isolated, digested with a restriction enzyme, and ligated. The ligated DNA was transformed into Escherichia coli strain STBL2 for subsequent analysis ( GIBCO BRL ). Cloned genomic DNA fragments on the recovered plasmids are schematically shown in Fig. 1 A. Integration plasmids and restriction enzymes used for obtaining dynein clones are as follows: Plasmids of pAY119, pAY123, pAY120, pAY131, and pAY130 were used for the integration to obtain pDHC1-Sac, pDHC1-Sal, pDHC1-Bam, pDHC1-Cla, and pAY142, respectively. pAY-119 was constructed by deleting a 1.2-kb ClaI fragment containing ars1 from pDHC1-1. pAY123 was constructed by inserting an Ecl136II– BamHI fragment of pDHC1-Sac between the SmaI and BamHI sites of pRS405 (Stratagene). pAY120 was constructed by moving an EcoRV fragment of pDHC1-1 into the SmaI site of pRS405. pAY131 was constructed by inserting an XbaI fragment of pDHC1-Sal, of which ends were filled in by Klenow fragments, into the SmaI site of pRS306 . pAY130 was constructed by inserting a BamHI–SmaI fragment of pDHC1-Bam between the Ecl136II and BamHI sites of pRS-306. Restriction enzymes used for obtaining pDHC1-Sac, pDHC1-Sal, pDHC1-Bam, pDHC1-Cla, and pAY142 were SacI, SalI, BamHI, ClaI, and SmaI, respectively. DNA sequences of the cloned fragments were determined by the method recommended by Applied Biosystems Inc., using synthetic DNA primers complementary to the sequence. The dhc1-d1 disruption allele was generated as follows: a 1.8 kb DNA fragment bearing the ura4 + gene was inserted at an EcoRV site located between the BamHI and XhoI sites in the DHC coding region of pDHC1-1. A linear DNA fragment of the heavy chain coding region between the BamHI and EcoRV (located between XhoI and SmaI) sites bearing the ura4 + gene was integrated at the dhc1 + locus of a diploid strain ( h + /h − ade6-210/ade6-216 leu1/leu1 ura4/ura4 ) by one-step gene replacement . The diploid integrants were sporulated and one of the ura + progenies of integrants was further crossed with an h 90 homothallic strain to obtain a dhc1-d1 homothallic strain (DHC102). Homologous integration was confirmed by Southern blot and PCR analyses. To construct strains bearing the dhc1-d2 and the dhc1-d3 mutant alleles, pAY119 and pAY120 were integrated at the dhc1 locus in a haploid strain. Strains bearing dhc1-d2 and dhc1-d3 were used for most of the phenotypic analyses. The dhc1-d4 allele was created as follows: a BamHI–EcoRI DNA fragment of pDHC1-Cla was inserted between EcoRI and BamHI sites of an integration vector, which was constructed by deleting the ClaI fragment containing ars1 from pUR19 . Then, a BamHI–SphI DNA fragment of pDHC1–Bam was inserted between BamHI and SphI sites. The resulting plasmid, pAY144, was linearized by digesting with BamHI and then integrated at dhc1 gene in a diploid strain ( h + /h − ade6-M210/ade6-M216 his2/his2 + lys1/ lys1 + ura4/ura4 ) by one-step gene replacement. The diploid integrants were sporulated and one of the ura + progenies of integrants was further crossed with an h 90 homothallic strain to create a dhc1-d4 homothallic strain (AY160-30A). Homologous integration was confirmed by Southern blot analysis. Cells were grown in YEA medium at 30°C at the cell density of 4 to 6 × 10 6 cells/ml. They were arrested in S phase by incubating for 3 h in the presence of 15 mM hydroxyurea at 30°C. After washing twice with YEA medium, arrested cells were resuspended in fresh YEA medium at the cell density of 2 × 10 6 cells/ml and further incubated at 30°C to synchronously resume mitotic cell cycle. A portion of the culture was taken at intervals and cells were fixed by adding 1/10 vol of 37% formaldehyde. Progression of mitosis was monitored by analyzing chromosomal DNA of fixed cells stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co. ). To examine microtubule morphology, cells were transformed with pDQ105, a multicopy plasmid which carries a green fluorescent protein (GFP)–α-tubulin fusion gene (GFP– atb2 + ) placed under the nmt1 promoter . The transformed cells were grown or sporulated in the medium containing 5 μM thiamine for the repression of the GFP-tubulin expression at low levels. The GFP-labeled microtubules were examined under a microscope as described by Ding et al. . To examine dynamics of the mitotic spindle, cells were used which express the GFP– α-tubulin from the fusion gene integrated at the lys1 + locus. To integrate the fusion gene, PstI–SacI fragment of pDQ105, which contains the GFP– α-tubulin gene placed under the nmt1 promoter, was moved into pYC36, an integration plasmid containing a partial lys1 + gene. The resultant plasmid was introduced into strains CRL152, CRL1521, and AY160-30A to produce strains YY105, GT1521, and GT160-30A, respectively. Integration was confirmed by Southern analysis. Cells of these strains were grown in YEA medium containing 5 μM thiamine at the cell density of 2 to 4 × 10 6 cells/ml and examined for spindle dynamics at 30°C. Live cell analysis of chromosome dynamics was carried out using a computer-operated microscope system, previously described by Chikashige et al. . Wild-type (AY162) and dhc1 (AY163) diploid strains were grown in liquid YEA medium at 33°C to 5 × 10 7 cells/ml. We found that sporulation efficiency was high when meiosis was induced in cells at late log or early stationary phase. The cells were then washed twice and resuspended in EMM medium lacking a source of nitrogen (EMM − N). Meiosis was synchronously induced by incubating the culture at 26°C with shaking. Progression of meiosis was monitored as described in Synchronization of Mitotic Cell Cycle. Chromosome III missegregation was examined using intragenic complementation of two alleles of the ade6 gene on chromosome III, ade6-M210 and ade6-M216, previously described in Molnar et al. . Strains AY1491-1A and AY1491-1B, or AY1491-1C and AY1491-1D, were mated and sporulated on ME plates at 26°C for 3 d. Sporulated cells were suspended in 400 μl of water containing 2 μl of Glusulase ( Sigma Chemical Co. ) and incubated for 16 to 18 h at 30°C to liberate spores and kill vegetative cells. Liberated spores were washed twice with water and plated on YEA and EMM plates supplemented with appropriate amino acids for selecting ade + progenies. More than 100 ade + progenies were tested for disomic chromosome III by streaking on YE plates after growing for several generations on YEA plates. Cells disomic for chromosome III give rise to red colonies on YE plates, since they frequently generate offspring monosomic for chromosome III . Frequency of spores disomic for chromosome III was determined by the frequency of ade + progenies and the ratio of the disomic progenies. The coding sequence of GFP-S65T cloned in pRSET-B vector (Invitrogen Corp.; a gift from Dr. Tsien, University of California, San Diego, CA) was amplified by PCR using primers, CGCGGATCCATGAGTAAAGGAGAAGAACTT and GAAGGCCTATTTGTATAGTTCATCCATGCC. The PCR product was digested with BamHI and StuI and then inserted between the BamHI and SmaI sites of pREP1 vector to form pEG3. A BamHI–SacI fragment of pEG3 containing GFP-S65T with a nmt1 -polyadenylation sequence was cloned into the corresponding polylinker sites of pRS405 to form pAY147. A DNA fragment encoding a COOH-terminal portion of DHC was amplified by PCR using pAY142 as a template and synthetic oligonucleotides, TGGATGCCCTAAGAGATTT and CGGGATCCAATGCACATACTAAAATAAATATC. The PCR product was digested with NsiI and BamHI and inserted between the PstI and BamHI sites of pAY147 to form pAY150, which encodes the COOH-terminal portion of the DHC protein fused with GFP. pAY150 was integrated at the dhc1 locus of CRL152 to make strain CRL1526, replacing the wild-type dhc1 gene with the GFP-fused dhc1 gene. Cells induced into meiosis on solid ME medium were fixed in methanol for 8 min at −30°C. The fixed cells were washed once with 0.1 M potassium phosphate buffer, pH 6.5, and resuspended in the same buffer containing 1 M sorbitol (spheroplast buffer). They were spheroplasted by adding 100 μg/ml of Zymolyase 100T (Seikagaku Kogyo) and 0.1% 2-mercaptoethanol. The spheroplasted cells were washed twice with spheroplast buffer and permeabilized with 1% Triton X-100 in spheroplast buffer. After permeabilization, the cells were washed twice with the phosphate buffer and resuspended in solution F (10 mM potassium phosphate buffer, pH 7.0, 150 mM NaCl, and 1% BSA). Antibody reactions with permeabilized cells were carried out in a microtube at 23°C as described in Hagan and Hyams using solution F for antibody incubations. α-Tubulin and GFP were stained using TAT1 mouse monoclonal anti–α-tubulin antibody and rabbit polyclonal anti-GFP antibody (1:2,000 dilution, CLONTECH Laboratories, Inc.), respectively. FITC-conjugated goat polyclonal anti–mouse antibody (1:100 dilution, Jackson ImmunoResearch Laboratories, Inc.) and rhodamine-conjugated goat polyclonal anti–rabbit antibody (1:100 dilution, Cappel Laboratories/Organon Teknika Corp.) were used for secondary antibodies. DNA was stained with 100 ng/ml DAPI. To examine the frequency of meiotic recombination, two strains bearing appropriate genetic markers were grown on the solid YEA medium at 33°C, mated, and then sporulated on the solid ME or EMM − N medium at 26°C (zygotic meiosis). Alternatively, after mating, diploid cells were selected on the solid EMM medium supplemented with appropriate amino acids. The diploid cells were grown on the solid YEA medium at 33°C and then sporulated on the solid ME medium at 26°C (azygotic meiosis). The frequency of intergenic recombination was examined by tetrad analysis. The frequency of intragenic recombination at the ade6 locus was analyzed, previously described by Ponticelli and Smith . An approximate map of genetic markers used in this study is shown in Fig. 9 . Fluorescence in situ hybridization (FISH) analysis was performed as described with the following modifications. Cells grown on the YEA agar medium were transferred on the ME agar medium to induce meiosis. After incubation at 23°C for 12 to 13 h, they were fixed in 3% paraformaldehyde and 0.2% glutaraldehyde in PEM buffer (100 mM Pipes-KOH, pH 6.9, 1 mM EGTA, 1 mM MgCl 2 ) for 30 min at 23°C. After fixation, the cells were permeabilized and treated with RNase A as described . The RNase A-treated cells were then processed for FISH. For simultaneous staining of the SPB and telomeres, the cells were immunostained for the SPB before being processed for FISH. The cells were reacted with rabbit polyclonal anti–γ-tubulin antibody (1:10,000 dilution; a gift from Dr. Masuda, The Institute of Physical and Chemical Research, Wako, Saitama, Japan) and then with Cy5-conjugated anti-rabbit antibody (1:100 dilution, Nycomed Amersham ). After antibody reactions, the cells were postfixed with 3% paraformaldehyde in PEM buffer for 10 min at room temperature and then washed twice with PEM buffer. For FISH, the cells were suspended in 2× SSC and incubated for 10 min at 23°C. Next, they were resuspended in 2× SSC containing 10% formamide and incubated for another 10 min at 23°C. The concentration of formamide was increased to 20% and then to 40%, step-wise with 10 min incubations at each step. After incubating cells in a 40% formamide solution, they were suspended in hybridization buffer (50% formamide, 5× Denhardt's solution, and 5% dextran sulfate in 2× SSC) containing fluorescent-labeled probes. Fluorescent-labeled probes were prepared as follows: Ribosomal RNA genes, which hybridize to both ends of chromosome III , were used for telomere probes. Alternatively, cosmid cos212 was used; this hybridizes to both ends of chromosome I and II . Plasmid pRS140, which contains centromeric repeats common to all three chromosomes, was used to hybridize to the centromeres . Cosmids, cos256, cos146, cos1228 (from Dr. Yanagida, Kyoto University, Kyoto, Japan) and ICRFc60D0727 (from Resource Center of the German Human Genome Project at the Max-Planck-Institut for Molecular Genetics) were used to hybridize to regions on the chromosome arms . Approximate position of the chromosome regions to which the probes hybridize are shown in Fig. 9 . The DNA used for telomere and chromosome-arm probes was fragmented with RsaI, TaqI, AluI, Sau3AI, and HaeIII, and then labeled with Cy3- or Cy5-dUTP (Nycomed Amersham Inc. ) using an oligonucleotide tailing kit containing a terminal deoxynucleotidyl transferase ( Boehringer Mannheim Co. ). Similarly, the DNA used for centromere probes was digested with RsaI, TaqI, and AluI and labeled with Cy3-dUTP. Concentrations of the fluorescent-labeled DNA probes in the hybridization buffer were ∼15 μg/ml for telomere probes and 30 μg/ml for centromere and chromosome-arm probes. Before the hybridization step the cell suspension containing fluorescent probes was preheated to 70°C for 5 min to denature the DNA. Hybridization was carried out at 37°C for 16 h. After hybridization, the formamide concentration in the hybridization buffer was decreased to 40, 25, 15, and finally 7.5% by step-wise dilution with 2× SSC. For each dilution step, the cell suspension was incubated for 10 min at 23°C. After the concentration was decreased to 7.5%, the cells were resuspended in fresh 2× SSC and incubated for another 10 min at 23°C. Lastly, the cells were washed twice with 2× SSC and then stained for DNA with DAPI. Since the meiotic oscillatory nuclear movement is driven by the dynamic activity of microtubules , microtubule-dependent motor enzymes seemed to be involved. As a first step in examining this possibility, we cloned the dhc gene . A total of 17.3 kb of genomic DNA from the locus containing a candidate gene was examined. Sequence analysis of the cloned fragments revealed that they contained a continuous ORF of 12.6 kb. The ORF encodes a predicted protein of 4,196 amino acids with a deduced molecular weight of 484 kD . A comparison of the amino acid sequence with the protein sequences in the SwissProt database using the BLASTP algorithm revealed a high similarity to the DHC sequences of other organisms. The cytoplasmic dhc gene of Aspergillus nidulans was most similar to the fission yeast sequence (27.4% identity). Dot-plot analysis showed that they are highly similar in their entire length . These results indicate that the cloned ORF encodes the cytoplasmic DHC of fission yeast. Thus, the gene was named dhc1 + . Southern blot and PCR analyses suggested that it is the only dhc gene in this organism (data not shown). The central motor domain of most DHC molecules contains four P-loops, which are putative ATP-binding sites comprising the consensus sequence, GXXXXKS/T . Similarly, the fission yeast DHC contained the consensus sequences at the first three sites corresponding to other DHCs . The fourth site lacked the typical consensus sequence, although sequence around this site was similar to those of other DHCs . The existence of ATP-binding motifs suggests that fission yeast dynein generates motor activity by ATP hydrolysis, like other dynein molecules. To examine the role of the DHC molecule in fission yeast, we disrupted the coding sequence by integrating either the ura4 + gene of fission yeast or the LEU2 gene of budding yeast at the dhc1 + locus by homologous recombination . Cells containing the integration that truncates the putative motor domain ( dhc1-d1 , dhc1-d2 , and dhc1-d3 ) or a deletion of the NH 2 -terminal two-thirds of the heavy chain gene ( dhc1-d4 ) grew in rich and minimal media at temperatures from 20°C to 36°C and their doubling times were similar to those of wild-type cells. Indirect immunofluorescence and living cell analysis using GFP-tagged α-tubulin revealed that nuclear and microtubule morphology of all the mutants were similar to those of wild-type cells. In addition, the number of cytoplasmic microtubules extending along the cell axis in G2 cells were not significantly different, 2.9 ± 0.7 (mean ± SD) for wild-type and 2.7 ± 0.7 for the dhc1-d2 mutant ( n = 40). Cytokinesis took place in the middle of the cell, just as in wild-type cells. In addition, when cells were synchronized in S phase by a DNA synthesis inhibitor, hydroxyurea, the mutant cells underwent nuclear division and cytokinesis with a similar timing to that of wild-type cells . These results indicated that the DHC is not essential for the mitotic growth and the loss of its function does not cause gross defects in mitosis. It has been reported that dynein plays an important role in maintaining proper positioning, orientation, and kinetics of a mitotic spindle through astral microtubules in budding yeast . To seek a possible role of dynein in dynamics of the mitotic spindle, we carried out detailed analysis of spindle behavior in wild-type and the dynein-disrupted cells using GFP-tagged α-tubulin . In wild-type cells, the mitotic spindle was formed at the center of the cell and elongated to ∼1 μm within 2 min . During this early stage of the spindle formation, cytoplasmic microtubules extending along the cell axis disappeared. The spindle continuously elongated to ∼3 μm and its length remained constant until further elongation (anaphase B) begins , as previously reported by Nabeshima et al., . The spindle was always located at the middle of the cell and often changed its orientation . In addition, short astral microtubules radiating from the spindle pole(s) were often observed . The time length of this stage was between 8 and 26 min, varying among cells. After this stage, the spindle elongated along the cell axis at the constant rate to reach its maximum length , and eventually disappeared . Astral microtubules radiating from the spindle poles were observed during elongation . When the dhc1-d2 cells were examined, no significant differences were observed in positioning, orientation, or kinetics of the mitotic spindles or in the behavior of astral-microtubules . Therefore, we conclude that DHC is not required for maintaining proper positioning, orientation, or kinetics of mitotic spindles in fission yeast. We then examined the phenotype of the dynein-disrupted cells during meiotic progression. Haploid cells of opposite mating types are induced to conjugate upon depletion of nitrogen from the growth medium. In conjugating cells, the two haploid nuclei migrate towards each other and fuse at the site of their SPBs (karyogamy). Cells bearing the dhc1-d2 mutation in both mating types underwent conjugation and nuclear fusion to form a zygotic cell containing a single chromosomal DNA mass and one SPB . However, the fraction of conjugated cells with unfused nuclei was significantly larger in the mutant than in the wild-type: of 160 conjugated cells in the stage before meiotic chromosome segregation, unfused nuclei were observed in 21.9% of the mutant cells, whereas only 2.5% of wild-type cells had this phenotype. When unfused nuclei in 10 conjugated cells of the mutant were followed using a portion of DNA-polymerase-α tagged with GFP as a nuclear marker (D.-Q. Ding and Y. Hiraoka manuscript in preparation), all of the nuclei eventually fused to form a single nucleus. These results strongly suggest that nuclear fusion was delayed to some extent in cells lacking a cytoplasmic DHC, but not abolished. The most striking consequence of the dynein disruption was observed after nuclear fusion. In wild-type cells, the fused nucleus immediately started oscillatory movement between the cell poles , as previously reported . In contrast, the fused nucleus of the dynein mutant cells did not show oscillatory movement and remained in the middle of the cell . These observations indicated that the DHC is required for the oscillatory nuclear movement. Despite the lack of oscillatory nuclear movement, many mutant cells underwent two rounds of meiotic chromosome segregation, as observed by the appearance of two and then four nuclei . In wild-type cells, the nucleus performs some hours of oscillatory movement through meiotic prophase and then initiates two rounds of chromosome segregation . Live observations of the nuclear behavior in the dynein-disrupted mutant showed that the nucleus remained in the middle of the cell for ∼3 h after nuclear fusion and then initiated the first chromosome segregation. Unlike wild-type cells, chromosomes sometimes moved separately and failed to form two distinct chromosome masses during the first chromosome segregation . Despite this aberrant behavior, two rounds of chromosome segregation were accomplished within 2 h, as in wild-type cells . Analysis of azygotic diploid cells, which synchronously entered meiosis without undergoing nuclear fusion, suggest that both wild-type and mutant cells accomplished meiotic processes with a similar timing. As shown in Fig. 5 , both wild-type and mutant diploid cells showed similar profiles after nitrogen starvation. Specifically, cells containing a deformed nucleus, which is characteristic of the meiotic stage preceding chromosome segregation, increased at ∼5 h and decreased at ∼8 h. Live observations confirmed that the deformed nucleus of the mutant lacked the oscillatory movement, whereas that of wild-type moved back and forth between cell poles (data not shown). These results suggested that the mutant cells accomplished the meiotic process preceding meiotic chromosome segregation with a timing similar to that of wild-type, despite the lack of nuclear movement. After accomplishing two rounds of chromosome segregation, many mutant cells formed four spores , most of which were viable like those of wild-type cells (Table III ), indicating that chromosomes were properly segregated in those cells. The slight increase in the number of the mutant cells that failed to form four spores, however, suggested increased chromosome missegregation in the mutant . To know if chromosome missegregation is increased in the mutant, we examined the frequency of spores disomic for chromosome III. The frequencies of the disomic spores were 8.0 × 10 −4 for wild-type and 1.1 × 10 −2 for the mutant. The ∼14-fold increase of the disomic spores indicated that chromosome missegregation increased during meiosis I in the mutant. Thus, the dynein function is required for faithful chromosome segregation. Meiotic phenotypes similar to those described were observed in cells bearing each of the different mutations in the dynein gene . In addition, the dynein mutations were recessive for the phenotypes described, since nuclear movement was normal in conjugates of mutant and wild-type cells. In summary, the DHC is required for the oscillatory nuclear movement of meiotic prophase and faithful chromosome segregation during meiosis. Moreover, the successful completion of meiotic processes in the absence of oscillatory nuclear movement in many mutant cells indicates that the movement itself is not essential for the progression of meiosis. Since the oscillatory nuclear movement is dependent upon astral microtubules radiating from the SPB , it is possible that the lack of the movement in the dynein mutants is due to a loss of astral microtubules. We examined microtubule morphology in living meiotic dhc1-d2 cells using GFP-tagged α-tubulin . In a zygote containing a single nucleus, astral microtubules radiated from the SPB , although the oscillatory nuclear movement was not observed. Therefore, the lack of the oscillatory nuclear movement in the mutant is not caused by a loss of astral microtubules. The microtubule morphology of the mutant cells was not significantly different from that observed in wild-type cells during the remainder of meiosis . During karyogamy, astral microtubules radiated from the SPB as each of two nuclei converged to bring the two SPBs together and the meiotic spindles were normal during meiotic chromosome segregation . To further understand the functions of the DHC, we examined its intracellular localization by tagging the COOH terminus of Dhc1p with GFP. The dhc1 + gene was replaced with the tagged gene by chromosomal integration of the DNA encoding GFP, resulting in the expression of tagged Dhc1p under the endogenous promoter on the chromosome. The GFP-tagged Dhc1p (dynein-GFP) was functional, since cells expressing solely the tagged protein showed no aberrant phenotypes in meiosis, particularly in the oscillatory nuclear movement and intragenic recombination at the ade6 locus (data not shown). In mitotically growing cells, the dynein-GFP was not detected (data not shown). Meiotic cells, on the other hand, showed specific intracellular localization. During nuclear fusion through meiotic prophase, the dynein-GFP was localized at the SPB , and astral microtubules . The localization of the dynein-GFP to the SPB was demonstrated by the position of the GFP dot at the protruding edge of the nucleus and its colocalization with the focal point of astral microtubules . This localization was apparent until anaphase of the first meiotic division , but was not detected through the rest of meiosis . The localization to astral microtubules was demonstrated by colocalization of the GFP line with microtubules and further confirmed by the disappearance of the GFP lines by treating the cells with a microtubule-depolymerizing drug, thiabendazole (data not shown). Interestingly, during the oscillatory nuclear movement, a dynein-GFP dot was frequently observed on an astral microtubule(s) at a point where the microtubule(s) contacts the cell cortex in front of the moving nucleus, and the nucleus moved towards it . The dot appeared during the approach of the SPB to the cortex and disappeared after the SPB reached it and reversed the direction of its movement . This observation suggests the presence of a novel structure on the cell cortex that promotes a transient accumulation of the dynein-GFP and is involved in the nuclear movement (see Discussion). If the oscillatory nuclear movement of meiotic prophase facilitates homologous chromosome pairing, as postulated previously , the lack of this movement should affect meiotic recombination. To test this idea, we examined meiotic recombination in the dynein mutant cells. Tetrad analysis investigating the genetic linkage at four regions on three chromosomes revealed that the frequencies of intergenic recombination at the regions were significantly reduced by up to fivefold in the mutant (Table III ). Thus, the intergenic recombination was reduced in the dynein mutant. Interestingly, the recombination frequencies in meioses initiated from the haploid state through karyogamy (zygotic meiosis) and from the diploid state without undergoing karyogamy (azygotic meiosis) were significantly different. In the dynein-disrupted cells, recombination occurs more frequently in azygotic meiosis than in zygotic meiosis at almost every locus. In contrast, in wild-type cells recombination for azygotic meiosis is less frequent. These results indicated that processes of intergenic recombination in azygotic and zygotic meiosis are distinct. We then examined the frequency of intragenic recombination. It has been known that gene conversion occurs at high frequency at the ade6 locus on chromosome III. That is, a strain carrying a mutant allele of the ade6 gene, ade6-M26 or ade6-M375 , spontaneously gives rise to ade + recombinants in high frequency when crossed with a strain carrying another ade6 allele, ade6-469 . As shown in Table IV , the frequency of intragenic recombination of both ade6-M26 and ade6-M375 was reduced by about nine- and fivefold, respectively, through zygotic meiosis in the dynein mutant. Thus, intragenic recombination was also reduced in the dynein mutant and this reduction was not allele-specific. Taken together, these results demonstrated that the dynein function is required for efficient meiotic recombination. In addition, the reduced frequency of the distinct types of recombination suggest that the reduced meiotic recombination results from inefficient pairing of homologous chromosomes, rather than defects in recombination machinery in the dynein mutant. To characterize chromosome pairing in the dynein mutant, we examined the nuclear position of chromosomes by FISH using probes that hybridize to several different chromosomal regions . First, we examined the positions of centromeres and telomeres. It has been shown that centromeres and telomeres have distinct positions during the progression of meiosis . Centromeres locate away from the SPB forming one cluster in each nucleus during nuclear fusion and one or two clusters in most of the elongated zygotic nuclei . In contrast, telomeres locate near the SPB forming a single cluster . It has been inferred that one or two clusters of centromeres in a nucleus reflect the paired or unpaired state of homologous centromeres, respectively . Thus, inefficient pairing of homologous chromosomes should result in a reduced population of cells containing a single centromere cluster. Cells were induced into meiosis and the positions of centromeres and telomeres were determined in zygotic nuclei by FISH analysis. When centromeres were examined the population of cells containing a single centromere cluster was significantly reduced in the dhc1 mutant. The fraction of single-nuclear cells containing a single centromere cluster was about threefold smaller in mutant than in wild-type cells (Table V ). On the other hand, the fraction of those containing dispersed centromeres was significantly larger in the mutant . Dispersed centromeres were not observed during karyogamy in the mutant, as in wild-type. Each of the two nuclei in the fusion process contained a single centromere signal . These results suggest that centromeres fail to form a single cluster and disperse in many dhc1-d2 cells. The reduced population of cells containing a single centromere cluster strongly suggest that the dynein mutant cells failed to achieve efficient pairing of homologous centromeres. When telomeres were examined the fraction of cells containing two telomere signals was larger in the mutant than in wild-type . This mutant phenotype may result from delayed SPB fusion in the mutant, which is likely to be a cause of the delayed nuclear fusion during karyogamy. Otherwise, telomere behavior in dhc1 cells was not significantly different from that of wild-type cells; they form a single cluster near the SPB during karyogamy and during meiotic prophase in most cells. We then examined four different chromosomal regions on the arms of chromosome I and II in the mutant . Greater than 90% of the single-nuclear cells showed one or two nuclear FISH signals in both wild-type and the mutant . However, in the mutant cells the population of cells containing a single FISH signal, which probably indicates the paired state of the regions, was significantly smaller than in wild-type cells (Table VI ). These results strongly suggested that the mutant cells failed to achieve efficient pairing of homologous regions on a pair of homologous chromosomes. Taken together, these results support the idea that homologous chromosomes failed to pair efficiently in the dynein mutant, due to the lack of the oscillatory nuclear movement. We have cloned a fission yeast gene encoding a cytoplasmic DHC and demonstrate that it plays an essential role in the oscillatory nuclear movement of meiotic prophase. In addition, it is probably important for nuclear fusion during karyogamy, since nuclear fusion appeared to be delayed in the dynein mutant cells. Consistent with its role in meiosis-specific nuclear movement, dynein was detected at the SPB and astral microtubules from karyogamy through anaphase of the first meiotic division. It remains to be determined if localization was only detected during meiosis due to a meiosis-specific enhancement of dynein's expression or to its meiosis-specific accumulation at the SPB and microtubules. Involvement of cytoplasmic dynein in microtubule-mediated nuclear migration has been reported in other fungi. In Neurospora crassa , A. nidulans , and Nectria haematococca , dynein is required for the migration of nuclei in the germ tube . The budding yeast, Saccharomyces cerevisiae , requires dynein function for nuclear positioning and migration at the bud neck in mitosis . Specifically, the nucleus penetrates into the bud neck at the onset of anaphase, oscillates several times at the neck, and then divides into mother and daughter cells . In the absence of dynein function, the anaphase nucleus often fails to penetrate into the neck and there are no oscillations . In those cells, the nucleus divides within the mother cell and eventually segregates into mother and bud cells. Thus, cytoplasmic dynein generates forces required for the nuclear penetration and oscillation at the bud neck during anaphase in budding yeast. The dynein-dependent nuclear migration in budding yeast during mitosis and in fission yeast during meiosis share several features. First, both types of nuclear movement are driven by astral microtubules interacting with the cell cortex . Second, both are driven by dynein localized at the SPB and/or astral microtubules . Finally, both are oscillatory, though the amplitude of the oscillation is greater in fission yeast . These similarities strongly suggest that these nuclear movements in two evolutionary divergent organisms are driven by similar mechanisms. Despite many similarities, dynein is not required for mitotic spindle dynamics in fission yeast, as demonstrated by the lack of significant defects in spindle behavior of the mutant. The difference may be due to the different geometry of their cell division. In budding yeast, divided nuclei are segregated into the mother and daughter cells through a narrow path at the bud neck. However, the fission yeast cell is divided by a septum formed at the middle of the cell after the divided nuclei have been segregated to opposite ends of the cell. Thus, the positioning of the nucleus in mitosis may be more important to budding yeast. Alternatively, another DHC, which has not been detected by our analysis, may play a role in mitosis of fission yeast. During the oscillatory nuclear movements, GFP-tagged dynein is localized to the SPB and astral microtubules. It is also concentrated at the point where astral microtubules contact the cell cortex that faces an approaching nucleus. From analogy with other organisms, astral microtubules in fission yeast are probably oriented with their plus ends distal and minus ends proximal to the SPB . Given the localization of dynein and the polarity of the microtubules, we suggest two models for dynein function in the nuclear movement. In the first model, dynein immobilized on the cell cortex generates a pulling force by walking along astral microtubules toward their minus ends. The accumulation of dynein at the point where microtubules contact with the cortex facing an approaching nucleus is consistent with this model. A similar model was proposed for the function of dynein in budding yeast . However, a cortical accumulation of dynein was not observed in this organism. In the second model, dynein motors at the SPB generate a pushing force on the SPB by walking in the minus end direction on microtubules that extend rearward. The localization of dynein at the SPB is consistent with this model. A contribution of pushing forces to create nuclear movement was previously proposed, since the movement was associated with the elongation of microtubules extending rearward . Supporting this model, it has been reported that a pushing by astral microtubules drives preanaphase nuclear migration in budding yeast. However, in this case the pushing force is not generated by dynein . In either model, dynein may also play a role in dynamic changes of microtubule length, which are required for the oscillatory movement. Preliminary results showed that the rate of microtubule shortening, which occurs mostly in front of the moving nucleus in wild-type cells, was significantly altered in the dynein mutant at the meiotic stage preceding chromosome segregation (A. Yamamoto, unpublished results). Future analysis will be required to clarify the functions of DHC in the oscillatory nuclear movement. Most dynein mutant cells completed meiosis and formed four viable spores, despite the lack of oscillatory nuclear movement, and the duration of the meiotic stage preceding chromosome segregation is not obviously different in those cells. These results indicate that the movement is not essential for the progression of meiosis, and that the duration of the stage preceding chromosome segregation is determined independently of nuclear movement. However, the dynein mutant showed significant reduction in meiotic recombination suggesting that nuclear movement is required for efficient meiotic recombination. Our FISH observations of chromosome positioning strongly suggest that the reduced recombination results from inefficient pairing of homologous chromosomes. That is, the formation of a single cluster of centromeres and the colocalization of homologous regions in single-nuclear zygotes, which likely reflects the paired state of homologous chromosomes, were significantly inhibited in the dynein mutant. Preliminary observations of a centromere-linked locus in living cells using lac repressor/operator recognition also support this idea. During oscillatory nuclear movement, the centromere-proximal loci on a pair of homologous chromosomes frequently contact each other in wild-type cells, but rarely in the dynein mutant (A. Yamamoto, K. Nabeshima, M. Yanagida, and Y. Hiraoka, unpublished observation). In addition to this idea, the frequency of both intergenic and intragenic recombination was reduced significantly in the mutant. Given the cytoplasmic role of dynein in nuclear movement, it is likely that the lack of the nuclear movement causes inefficient pairing of homologous chromosomes in the dynein mutant, which brings significant reduction in meiotic recombination. In the mutant, intergenic recombination occurred more frequently in azygotic meiosis than in zygotic meiosis (Table III ). The higher recombination frequencies in azygotic meiosis could be due to the positioning of homologous chromosomes in proximity during mitosis of diploid cells , which may facilitate pairing of homologous chromosomes in subsequent meiosis. However, despite this positioning the recombination frequencies of wild-type cells were lower in azygotic meiosis than in zygotic. Perhaps, a process(es) during karyogamy, which is absent in azygotic meiosis, is required for efficient meiotic recombination in fission yeast. In addition to reduced frequencies of recombination, the dynein mutant showed increased missegregation of chromosomes, probably resulting in the slight increase of cells that failed to form four spores in the mutant. It is most likely that the increased missegregation of homologous chromosomes is caused by reduced crossing over on pairs of homologous chromosomes as a consequence of reduced recombination. The aberrant chromosome behavior that sometimes was observed during the first meiotic chromosome segregation of the dynein mutant may also result from reduced crossing over. Future analysis is required to clarify the relation between crossing over and reductional segregation of homologous chromosomes in the dynein mutant. When the fission yeast nucleus oscillates between the cell poles, its chromosomes form a bouquet structure, i.e., the telomeres remain clustered near the SPB at the leading edge of the moving nucleus . The reduction of the recombination frequency in several mutants of fission yeast in which telomeres failed to form a cluster suggests that formation of the telomere cluster is required for proper pairing of homologous chromosomes . Although the dynein mutants showed a similar phenotype, the defect was not caused by failure to form a telomere cluster, since telomeres were clustered near the SPB in most dhc1 cells . Conversely, mutants defective in telomere clustering still showed nuclear movement indicating that the lack of the nuclear movement is not a cause of the reduced recombination in these mutant cells. These results strongly suggest that telomere clustering and microtubule-mediated nuclear movement are distinct biological events, both of which are required for proper pairing of homologous chromosomes. Requirement of both telomere clustering and nuclear movement for efficient chromosome pairing is consistent with a previously published model . In this model, the nuclear dynamics produce chromosome movement by pulling the clustered telomeres and this chromosome movement aligns homologous chromosomes side by side to promote their efficient pairing . Subsequently, pairing of homologous chromosomes leads to the formation of a single centromere cluster. The nuclear dynamics also cause an elongation of the nucleus and we speculate that the constricted intranuclear space contributes to the chromosome pairing by promoting an efficient encounter of homologous chromosomes. In this scenario, when the nuclear movement is abolished, as in the dynein mutants, homologous chromosomes fail to achieve efficient pairing because they lack the necessary spatial alignment and inefficient pairing results in the reduced meiotic recombination and frequent failure to form a single centromere cluster . The scattering of centromeres observed in dhc1 cells probably corresponds to the chromosomal state, which transiently appears in wild-type cells before the formation of a single centromere cluster , given that a small population of wild-type cells contains dispersed centromeres (Table V ). Similarly, when the telomere clustering is abolished homologous chromosomes fail to pair efficiently and meiotic recombination is reduced, as observed in the mutants defective in telomere clustering . The bouquet configuration of homologous chromosomes in meiotic prophase observed in fission yeast has been described for a wide variety of other organisms, including human, mouse, and maize . It also seems likely that some types of nuclear movement occur during meiotic prophase in organisms other than S . pombe . In addition, it was recently found that chromosomes form the bouquet configuration during meiotic prophase and a microtubule-dependent motor protein is required for proper meiotic recombination and the SC formation in the budding yeast, S. cerevisiae . These observations imply the presence of a similar mechanism to create the spatial alignment of homologous chromosomes in other organisms. Although other organisms seem likely to employ a similar mechanism, the tightly clustered telomeres and the striking nuclear movement, which are characteristic of fission yeast, have not been observed among them. Unlike other organisms, fission yeast does not form the SC during meiotic prophase . This structure is thought to promote alignment and intimate association of homologous chromosomes through their entire length. Supporting the different mode of the chromosome association in fission yeast, FISH analysis of homologous regions in azygotic diploid cells suggested that fission yeast lacks the SC-like stage when all chromosomal regions are engaged in pairing . Due to the lack of the SC, fission yeast may depend more heavily than other organisms on the mechanism consisting of the bouquet arrangement and the nuclear movement for aligning homologous chromosomes. To understand the mechanisms of homologous chromosome pairing, it may be important to examine nuclear dynamics during meiotic prophase from this perspective. | Study | biomedical | en | 0.999997 |
10366597 | Yeast strains are listed in Table I . FY23 and FY86 were provided by Fred Winston (Harvard Medical School, Boston, MA). Y187 and Y190 were provided by Steve Elledge (Baylor College of Medicine, Houston, TX). DDY319, DDY321, DDY760, and DDY496 were constructed as described . Standard methods were employed for growth, sporulation, and tetrad dissection of yeast . Yeast transformations were performed by electroporation or by lithium acetate . The medium for two-hybrid analysis was synthetic medium plus dextrose supplemented with adenine to 10 μg/ml and 3,5-amino-triazole (3-AT) ( Sigma Chemical Co. ) at 25, 50, or 100 mM. Plasmid pRB2247 was constructed by isolating a 1.4-kb product of a BglII partial digest of plasmid pRB2248 and cloning this fragment into the BamHI site of plasmid pGEX-5X-3 ( Pharmacia Biotech, Inc. ) such that the AIP1 gene was in frame with the glutathione- S -transferase (GST) reading frame. Plasmid pRB2251 was constructed by subcloning a 2.2-kb ClaI fragment from AIP1 genomic clone pRB2249 into YCp50 such that AIP1 and bla transcription is divergent. The deletion allele of AIP1 was constructed by double fusion PCR and has been described elsewhere . Plasmids encoding fusions of the GAL4 DNA binding domain (DBD) to SNF1 , the GAL4 DBD to lamin (pAS1-lamin), and the GAL4 activation domain (AD) to SNF4 were provided by Steve Elledge. The construction of the plasmids carrying fusions of the actin–alanine scan alleles to the Gal4 DBD, a fusion of the GAL4 DBD to ACT1 , a fusion of the GAL4 AD to ACT1 (pAIP70), and a fusion of the GAL4 AD to AIP1 previously were described elsewhere . The plasmid encoding a fusion of the GAL4 DBD to AIP1 (pDAb189) was constructed by removing the AIP1 open reading frame from pRB2248 as a BglII partial digest and cloning it into the BamHI site of plasmid pRB1516 (a Cen version of pAS1-CYH2) so that the AIP1 open reading frame is in frame with that of the GAL4 DBD. The construct encoding a fusion of the GAL4 AD to ABP1 (pDAb20) was constructed by excising the ABP1 open reading frame from plasmid pRB1199 as a 1.9-kb XhoI-EcoRI fragment, blunting the EcoRI site with T4 DNA polymerase and cloning into plasmid pACTII (gift of Steve Elledge) that had been cut with XhoI and SalI in which the SalI site had been made blunt with T4 DNA polymerase. The resulting construct expresses all but the first 11 amino acids of Abp1p fused to Gal4p. The constructs encoding fusions of the cofilin mutants to the Gal4p AD (used for the footprinting studies) were constructed by PCR into plasmid pACTII. The cofilin mutant and wild-type alleles were amplified off plasmids using primers DAo-COF1-1 (5′-cgcgccatggaacaaaagatgtctagatct-3′) and DAo-COF1-2 (5′-cggaattcaccttaatgagaaccagcgcc-3′) and vent polymerase ( New England Biolabs Inc. ). Subcloning of cof1-4 required the use of the special primer DAo-COF1-3 (5′-cgcgccatggaacaaaagatgtctagagct-3′) in the place of primer DAo-COF1-1. The PCR products were cut with NcoI and EcoRI and cloned into similarly cut pACTII. Each construct was cloned and tested in duplicates generated from separate PCR reactions. When possible, constructs were confirmed by a BbvI digest. Plasmid pACTII-COF1 was made by PCR amplification of the COF1 open reading frame with oligonucleotides PL70 (5′-gcgcgccatggggtctagatctggtgttgctgttgc-3′) and PL76.2 (5′-gcgcgcggatccttaatgagaaccagcgcctctgc-3′), digestion of the PCR product with NcoI-BamHI, and subcloning of this insert in frame into similarly digested pACTII. To express Aip1p as a GST fusion protein in yeast, primers ARP1 (5′-gcgcgggatccatgtcatctatctctttgaaggaa-3′) and ARP3 (5′-cgcgccggccgctcactcgaggacaacattccacct-3′) were used to amplify the AIP1 open reading frame from genomic DNA. This PCR product was cleaved with BamHI and EagI and cloned into similarly cut pEG(KT) to make plasmid pAR3. To express Aip1p for in vitro translation, primers ARP16 (5′-gcgcgcacatgtatgtcatctatctctttgaaggaa-3′) and ARP4 (5′-gcgcgaagctttcactcgaggacaacattccacct-3′) were used to amplify the AIP1 open reading frame from genomic DNA. This PCR product was cleaved with AflIII and HindIII and cloned into pBAT4 cut with NcoI and HindIII to make plasmid pAR20. Aip1-GST fusion protein for antibody production was purified from bacterial strain UT5600 (Δ [ompT-fepA] leu proC trpE ) (provided by S. Gottesman, National Cancer Institute, Bethesda, MD) carrying plasmid pRB2247 by standard methods . Antibodies were raised in three New Zealand white rabbits by injection of 100 μg GST-Aip1 in 1 ml of adjuvant (RIBI ImmunoChem Research Inc.), three times at 2-wk intervals. 2 wk after the last boost, the animals were exsanguinated. Anti-Aip1 antibodies were affinity-purified on columns to which GST-Aip1p had been conjugated by standard methods . Purified antibodies were concentrated on a Centriplus concentrator (Amicon Inc.). Yeast actin was purified as described previously . However, the formamide eluate from the DNaseI column was dialyzed overnight against three changes of G buffer (5 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl 2 ), and concentrated to 2 ml in Centriprep 10 devices (Amicon, Inc.). The actin was polymerized by adding initiation salts to a final concentration of 100 mM KCl, 2 mM MgCl 2 , and incubating for 2 h at room temperature. Residual actin-binding proteins were stripped from F-actin at this point by slowly adding KCl to 0.6 M and further incubating for 30 min. The polymerized actin was pelleted at 80,000 rpm for 30 min at room temperature in a TLA100.3 rotor ( Beckman Instruments, Inc. ). The pelleted actin was resuspended in G buffer to a final concentration of 50 μM and dialyzed against three changes of 2 liters of G buffer before it was frozen in liquid N 2 and stored at −80°C. Cofilin was purified as a GST-fusion protein from Escherichia coli and subsequently cleaved from GST by thrombin digestion as described previously . Aip1p was purified from the yeast strain DDY130 (carrying the plasmid pAR3) as a GST fusion protein under the control of the GAL promoter. 4 liters of cells were grown at 30°C in synthetic medium with 2% dextrose and without uracil or leucine to an OD of 1.0 at 600 nm, harvested by centrifugation, and resuspended in 4 liters of synthetic medium with 2% glycerol and without uracil or leucine. After an overnight incubation at 30°C to derepress the galactose promoter, cells were again harvested and resuspended in 4 liters of rich medium with 2% galactose. The cultures were induced for 8 h at 30°C before cells were once again harvested, washed twice with 100 ml water, resuspended in 10 ml water, frozen as 50-μl pellets in liquid N 2 , and stored at −80°C. Yeast pellets were lysed in liquid N 2 in a Waring blender and thawed in PBS to a final concentration of 1×, with 1 mM PMSF and 0.5 μg/ml each of antipain, leupeptin, pepstatin A, chymostatin, and aprotin. The lystate was cleared first by spinning at 17,000 g in a Dupont GSA rotor. The supernatant from this spin was cleared by spinning at 50,000 rpm for 50 min in a Beckman 70Ti rotor. This high speed supernatant was dialyzed overnight against PBS and passed twice over a column with a 4-ml bed of glutathione agarose beads ( Sigma Chemical Co. ). The column was washed five times with PBS and incubated with thrombin (5 U/ml; Sigma Chemical Co. ) overnight at room temperature to cleave the Aip1p from the GST. The column was washed with 8 ml 50 mM Hepes, pH 7.2, 50 mM KCl, and the flow-through was concentrated to 2 ml and loaded onto a 1-ml mono-Q anion exchange column ( Pharmacia Biotech Inc. ). A linear salt gradient from 100 to 400 mM KCl was applied to the column and peak fractions containing Aip1p were concentrated to 15 μM, frozen in liquid N 2 , and stored at −80°C. In all cases, two-hybrid analyses were performed by mating strain Y190 carrying constructs encoding fusions to the Gal4 DBD, to strain Y187 carrying constructs encoding fusions to the Gal4 AD. Transformants were lined, spotted, or spread as lawns on selective medium, synthetic complete medium lacking Trp (SC-TRP) or DBD fusions and synthetic complete medium lacking Leu (SC-LEU) for AD fusions. Mating was carried out by replica plating the Y190 and Y187 derivatives together onto yeast extract/peptone/dextrose medium–plates, incubating at 30°C for 24 h, selecting the mated cells on SC-TRP,-LEU. The selected diploids, carrying both DBD and AD fusion constructs, were replica plated to media containing 25, 50, and 100 mM 3-AT ( Sigma Chemical Co. ) and incubated at 25°C. Immunofluorescence was performed by standard protocols using a methanol/acetone fixation . Affinity-purified anti-Aip1p antibody was used at a dilution of 1:100. Affinity-purified rabbit anticofilin was used at 1:100. Guinea pig antiactin antisera (animal 2) was used at 1:2,000. For Aip1p localization, FITC-conjugated goat anti–rabbit IgG (Organon Teknika Corp.) was used at 1:1,000 and rhodamine-conjugated goat anti–guinea pig (Organon Teknika Corp.) was used at 1:1,000. For Aip1p localization in wild-type and act1-111 strains, rhodamine-conjugated goat anti–rabbit IgG (Cappel; ICN Biomedicals) was used at 1:1,000. For cofilin localization in DAY30 and DDY1264 FITC-conjugated goat anti–rabbit IgG (Cappel; ICN Biochemicals) was used at 1:1,000 and rhodamine-conjugated goat anti–guinea pig IgG (Cappel; ICN Biochemicals) was used at 1:1,000. To evaluate actin filament sedimentation in the presence of Aip1p and cofilin, 3.75 μM actin was polymerized at room temperature in F-buffer (5 mM Tris, pH 7.5, 0.7 mM ATP, 0.2 mM CaCl 2 , 2 mM MgCl 2 , 100 mM KCl, 0.2 mM DTT). After 45 min, polymerized actin (final concentration, 2.5 μM) was added to variable concentrations of Aip1p (final concentration, 0.012–0.5 μM) and/or cofilin (final concentration, 0.125–0.5 μM) in F-buffer. The reactions were incubated at room temperature for 20 min and centrifuged at 90,000 rpm for 20 min at 23°C in a TLA100 rotor ( Beckman Instruments ) to pellet the actin filaments. Equal proportions of the pellets and supernatants were fractionated on 12% SDS-PAGE gels and proteins were visualized by Coomassie blue staining. Protein levels were quantified using NIH Image software. To test the cofilin dependence of Aip1p binding to F-actin, variable concentrations of yeast F-actin were incubated for 20 min at 25°C with equimolar amounts of cofilin or control buffer. [ 35 S]Methionine-labeled in vitro–translated (TNT quick coupled transcription/translation; Promega Corp. ) Aip1p from plasmid pAR20 was incubated for 20 min with the cofilin–F-actin and pelleted for 20 min at 90,000 rpm in a TLA100 rotor ( Beckman Instruments ). Equal amounts of supernatants and pellets were fractionated on 13% SDS-PAGE gels and visualized by autoradiography. Results were quantified on a PhosphorImager using ImageQuant software (STORM 860; Molecular Dynamics, Inc.) and on an IS2000 densitometer using AlphaImager software. Images of actin and cofilin molecular models were generated on a Silicon Graphics Indigo™ workstation running Sybil software (Tripos Inc.). Coordinates for actin (file 1ATN) and yeast cofilin (file 1CFY) were retrieved from the Brookhaven database. Aip1p was first identified as a 67-kD yeast protein that interacts with actin in the two-hybrid system and is the first discovered member of a family of conserved proteins . Homologues of Aip1p have been identified in Schizosaccharomyces pombe , Physarum polycephalum , Dictyostelium discoideum , Caenorhabditis elegans (where there are two homologues), Mus musculus , and humans. Additionally, members of the Aip1p family show weak homology to proteins that contain reiterated motifs called WD repeats. These repeats were first identified in β subunits of trimeric G-proteins and have since been found in proteins with highly diverse functions. Members of the Aip1p family contain from four to eight WD repeats. To identify additional Aip1p ligands we used a two-hybrid construct of Aip1p fused to the GAL4 DBD (plasmid pDAb189) to screen a large set of yeast actin interacting proteins and cell polarity proteins fused to the GAL4 activation domain. Included in this set were clones encoding AIP1 , AIP2 , AIP3 , OYE2 , SRV2 , PFY1 , COF1 , BNR1 , LAS17 , MNN10 , ABP1 , RVS167 , BEM1 , FUS1 , and SAC6 . As can be seen in Fig. 2 , we found that in addition to actin, Aip1p also interacted with Cof1p (yeast cofilin). These interactions are specific since no activation was observed between Aip1p and the transcription factor Snf4p or between Aip1p and the actin cortical patch protein Abp1p (data not shown). Furthermore, the Aip1p–actin and Aip1p–cofilin two-hybrid interactions are reciprocal. An apparent cofilin–cofilin interaction also was detected but is likely the result of bridging through actin. The focus of this study is the functional significance of the Aip1p– cofilin interaction. To obtain a structural framework for understanding the functions of the Aip1p–actin and cofilin–actin interactions, we used actin mutations in conjunction with the two-hybrid system to identify likely sites of interaction for these proteins on actin. This approach was described previously for the identification of the binding footprint of Aip1p on actin and was repeated here to identify the binding footprint for cofilin on actin . Yeast strain Y187, carrying a fusion of the Gal4p AD to cofilin, was mated to strain Y190 containing plasmids encoding fusions of 35 actin mutants to the Gal4p DBD . Diploids were replica plated on 3-AT medium to assess activation of the His3p two-hybrid reporter . Failure to grow on this medium indicates that the actin mutant contained in that strain is defective for the cofilin–actin interaction. This result suggests that the mutation may lie in or near the cofilin binding site on actin. We discovered that eight actin mutants failed to interact with cofilin . Of these eight, five have thus far failed to interact with any actin-binding protein tested ( act1-107 , act1-130 , act1-127 , act1-128 , and act1-108 ) and probably encode either unfolded or unstable proteins. Therefore, nothing can be concluded from these mutants. However, the remaining three mutants ( act1-103 , act1-106 , and act1-126 ) display specific effects on the cofilin–actin interaction . The three mutations that specifically disrupt the cofilin– actin interaction are located in a small region of subdomain III on actin. Interestingly, these three mutations form one-half of the Aip1p binding footprint that includes act1-109 , act1-111 , and act1-112 , as well as act1-103 , act1-106 , and act1-126 . These data are consistent with the model that Aip1p binding to actin is facilitated by cofilin. A large set of mutant alleles of cofilin has been constructed and the yeast cofilin structure has been determined . This presented us with the opportunity to use our two-hybrid methodology to identify surfaces on cofilin required for its interactions with Aip1p and actin. Toward this end, we cloned the cofilin mutants into the two-hybrid activation domain vector pACTII (gift of S. Elledge) and scored the ability of these mutants to interact with Aip1p and actin. Four cofilin mutants failed to interact with both actin and Aip1p: cof1-9 , cof1-16 , cof1-17 , and cof1-20 . Two cofilin mutants specifically failed to interact with actin: cof1-6 and cof1-14 . Three mutants, cof1-4 , cof1-13 , and cof1-22 , failed to interact with Aip1p but interacted well with actin. We displayed the two-hybrid data on the molecular model of cofilin . In agreement with the in vitro binding data of cofilin mutants to actin , the two-hybrid data identified a ridge that is involved in the actin interaction , on the edge of the disc-shaped cofilin protein. A subset of the mutations that disrupted the cofilin–actin interaction constitute part of the Aip1p footprint on cofilin . A different set of mutations specifically affected cofilin interactions with Aip1p and not actin (shown in blue). These data are consistent with the model that Aip1p binding to cofilin is facilitated by actin because, according to this model, disruption of the cofilin–actin interaction would be predicted to also disrupt the Aip1p–cofilin interaction. Overall, the footprinting data suggest that there is a ternary complex between Aip1p, cofilin, and actin, and that the members of this complex make distinct contacts with each other. The actin cytoskeleton consists of a large number of interacting components. Often the deletion of a gene encoding one of these components does not in itself cause a readily detectable phenotype. However, combinations of mutations can produce informative synthetic phenotypes that suggest a shared or parallel function for the proteins involved . Deletion of the AIP1 gene had no effect on cell growth on a variety of media at a variety of temperatures (data not shown). Therefore, we investigated its genetic interactions with mutations in genes that encode other actin-binding proteins. First we crossed the aip1Δ strain to the collection of clustered-charged-to-alanine mutants of COF1 ( cof1-4 , cof1-5 , cof1-6 , cof1-7 , cof1-10 , cof1-11 , cof1-12 , cof1-13 , cof1-15 , cof1-18 , cof1-19 , cof1-21 , and cof1-22 ). The results of these crosses are shown in Table II . The aip1Δ mutation is synthetically lethal with cof1-5 and cof1-22 , which are both temperature sensitive for growth on their own and show defects in actin turnover rates in vivo at the permissive temperature . In addition, the aip1Δ mutant is synthetically lethal with cof1-4 , which has no growth phenotype of its own but has actin organization defects as visualized by rhodamine-phalloidin staining . The aip1Δ mutant is also synthetically temperature sensitive at 37°C with cof1-6 , which has no actin organization or growth phenotype on its own . We examined actin and cofilin localization in the aip1Δ cof1-6 double mutant. At the permissive temperature, the double mutant grows slowly and has actin clumps in the mother cell. These clumps stain with rhodamine-phalloidin, which binds specifically to F-actin and not G-actin. At the restrictive temperature, actin is depolarized, actin clumps are apparent, and unbudded cells accumulate. Cofilin colocalizes with the actin structures at both the permissive and restrictive temperatures (data not shown). We also determined if the aip1Δ mutation displayed synthetic lethality with any of the viable actin–alanine scan alleles of actin ( act1-1 , -101 , 102 , 104 , 105 , 108 , 111 , 113 , 115 , 116 , 117 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 129 , 132 , 133 , 136 ) and act1-159 , which decreases rates of actin filament turnover in vivo and in vitro . When a yeast strain carrying the act1-159 mutation was crossed to the aip1Δ strain, double mutant spores failed to grow at 25°C. We also observed subtle synthetic growth defects in double mutants containing the aip1Δ allele and three other actin alleles: act1-133 , act1-119 , and act1-125 (data not shown). These three mutations are not located near the Aip1p interaction interface on actin , suggesting that the weak synthetic interactions are not a function of compromised Aip1p–actin interactions but of cumulative defects in cytoskeletal function. Finally, we determined if the aip1Δ allele was synthetically lethal with deletion alleles of six other components of the yeast cortical cytoskeleton: ABP1 , SLA1 , SLA2 , TWF1 , CAP2 , and SAC6 . No clear synthetic lethality was found. However, the sac6 Δ, cap2 Δ -1 , and sla1 Δ mutations had slight synthetic growth defects in combination with the aip1Δ allele (data not shown). As a further test of the importance of the Aip1p–actin and Aip1p–cofilin interactions in vivo, we sought to determine if Aip1p colocalizes with actin and cofilin. Toward this end, we generated antibodies to a GST-Aip1p fusion protein. These antibodies specifically recognize a 67-kD protein on Western blots of wild-type (FY23) yeast extract that is of the expected size based on the primary sequence of AIP1 . This band is absent from AIP1 deletion strain DAY53 , but is restored when a low copy number vector carrying a 2.2-kb AIP1 genomic fragment is introduced into the deletion strain . Wild-type strain FY23×86 was stained with the anti-Aip1p antibodies and guinea pig antiactin antibodies . Aip1p localized only to cortical actin patches and was not detected along the actin cables. This localization pattern is identical to that observed for cofilin , but simultaneous colocalization of Aip1p and cofilin was not possible, since both antibodies were raised in rabbits. Since both Aip1p and cofilin appear to be found in all actin cortical patches recognizable by antiactin antibodies, we presume that Aip1p and cofilin colocalize. Aip1p also localized diffusely throughout the cytoplasm and this cytoplasmic staining is not seen in the aip1Δ strain (data not shown). To assess the importance and relevance of the Aip1p– actin two-hybrid interaction, we localized Aip1p in a strain bearing actin mutations that disrupt the Aip1p–actin two-hybrid interaction. Fig. 5 A shows Aip1p localization in the act1-111 strain TDS143. Aip1p was not detected in cortical patches in the mutant strain and there is an apparent increase in the cytosolic Aip1p signal. The failure of Aip1p to localize in this strain is not due to a loss in the ability of cofilin to bind to actin . Similarly, Aip1p localization to cortical patches in an act1-112 strain is severely compromised , whereas cofilin localization to patches in this strain is not affected . Aip1p was well localized to cortical patches in act1-119 , act1-132 , act1-124 , and act1-125 strains (data not shown) indicating that Aip1p mislocalization is not caused by generalized defects in the actin cytoskeleton. These results suggest that Aip1p must bind to F-actin for stable association with cortical actin patches and is consistent with the two-hybrid data suggesting that Aip1p contacts actin in the vicinity of the act1-111 and act1-112 mutations. To test the importance of the Aip1p–cofilin interaction on Aip1p localization, we examined Aip1p localization in viable cofilin mutants . Both Aip1p and cofilin were localized by indirect immunofluorescence in strains bearing 14 different cof1 alleles: cof1-4 , cof1-5 , cof1-6 , cof1-7 , cof1-8 , cof1-10 , cof1-11 , cof1-12 , cof1-13 , cof1-15 , cof1-18 , cof1-19 , cof1-21 , cof1-22 , and a wild-type congenic strain (Table II ). The cells were grown at 25°C, a permissive temperature for all the strains, before fixation. Aip1p localized to patches in all of the cofilin mutants except the strain carrying the cof1-19 allele. Fig. 5 E shows Aip1p localization in cof1-19 strain DDY1264. As was seen with the act1-111 strain, Aip1p is lost completely from the cortical patches in cof1-19 cells and there is an apparent increase in the cytoplasmic pool of Aip1p. Double labeling of this strain with antiactin and anticofilin antibodies showed that Cof1-19p is associated with cortical actin patches . The cof1-19 strain, like the aip1Δ strain, is viable and has a wild-type growth phenotype. We have examined the actin cytoskeleton in these strains and found no obvious defects. However, both strains do appear to have slightly aberrant cortical actin patches: they appear by rhodamine-phalloidin staining to be slightly larger or perhaps to contain more F-actin (data not shown). In addition, the cof1-19 cells have slightly depolarized actin patches and misoriented actin cables. To examine the role of Aip1p in cofilin localization, we immunolocalized cofilin in the aip1Δ strain. Surprisingly, cofilin localized not only to cortical patches but also to actin cables . We confirmed colocalization of cofilin with actin cables by double staining with the guinea pig antiactin antibody, as shown in Fig. 6 C. This result suggests that Aip1p is required to restrict cofilin to cortical actin patches in the yeast actin cytoskeleton. We asked if exclusive localization of cofilin to cortical actin patches depends on localization of Aip1p to these patches. To address this question, we examined cofilin localization in the cof1-19 mutant strain DDY1264, in which Aip1p is localized in the cytoplasm. As can be seen in Fig. 6 F, Cof1-19p is localized to both the patches and the cables. Fig. 6 E shows the same cells stained with the antiactin antibody confirming association of Cof1-19p with the actin cables. Though act1-111 and act1-112 mutants also fail to localize Aip1p to actin patches , we were unable to confirm that cofilin also localizes to actin cables in these strains because their actin cytoskeletons are more generally disrupted and cables are undetectable by antibody staining (data not shown). We next asked if the localization of other proteins normally localized to the cortical actin patches was affected in the aip1Δ strain. We found that immunolocalization of Abp1p, fimbrin/Sac6p, and coronin/Crn1p was unaffected by the absence of Aip1p (data not shown). This indicates that the role of Aip1p in cofilin localization is specific and not reflective of gross structural defects in the cortical patches. To evaluate directly the functional and physical interaction between cofilin, actin, and Aip1p, we investigated the effects that these proteins might have together in vitro. Aip1p was expressed in yeast as a GST fusion protein under the control of the GAL promoter. GST–Aip1p was affinity-purified from extracts on glutathione-agarose beads, cleaved from GST by thrombin digestion, and purified by cation exchange chromatography. This protein has two additional amino acids NH 2 -terminal to the primary sequence of Aip1p. No contaminants are apparent in the preparation on overloaded (5 μg) Coomassie stained gels (data not shown). To evaluate the interaction of Aip1p with F-actin at steady state, we carried out cosedimentation assays with 2.5 μM prepolymerized yeast F-actin. For all of the assays described here, after 20 min of coincubation the reaction is at steady state as evaluated by light-scattering at 400 nm (data not shown). Although a small proportion (5–10%) of purified Aip1p sedimented in the absence of actin in these assays, this amount did not perceptibly increase upon addition of actin (data not shown). Given the two-hybrid interaction of Aip1p and cofilin, it seemed possible that an actin–Aip1p interaction might be mediated by cofilin. To test this hypothesis, we performed the cosedimentation assay in the presence of recombinant cofilin, which binds to F-actin and accelerates disassembly rates (increasing subunit turnover), but does not significantly change actin polymer levels at steady state . Strikingly, in the presence of both Aip1p and cofilin, we observed a dramatic shift of actin and cofilin from the pellet to the supernatant . This shift might be explained by invoking a monomer sequestering model as applies for twinfilin , a protein that binds stoichiometrically to actin monomer, preventing nucleotide exchange and polymerization. We examined the effects of stoichiometry on the Aip1p-dependent shift by varying the concentration of Aip1p or cofilin in cosedimentation assays with constant concentrations of F-actin (2.5 μM). Intriguingly, though the shift into the supernatant showed a linear dependence on cofilin, it did not require Aip1p at similar stoichiometry. In fact, a significant shift occurred at molar ratios of Aip1p/cofilin/actin as low as 1:50:50 , and can even be seen at molar ratios of Aip1p/cofilin/actin as low as 1:200:200 . We used radiolabeled in vitro–translated Aip1p to evaluate binding to F-actin at a low concentration of Aip1p, which would not promote net disassembly of the filaments. The in vitro–translated Aip1p product sedimented with actin filaments. This cosedimentation was abolished by addition of excess nonradiolabeled Aip1p or by dilution of the sample, suggesting that the binding is specific (data not shown). To establish the dependence of this binding on cofilin, we pelleted increasing concentrations of actin filaments with or without stoichiometric cofilin with in vitro– translated Aip1p. Aip1p cosedimented with the F-actin in the absence of added cofilin, but addition of 1:1 cofilin increased the amount that cosedimented . The binding of Aip1p to cofilin–F-actin is saturatable with a K d of ∼4 μM. Similar results were obtained by Western blot analysis of identical experiments with purified Aip1p (data not shown). We next examined how much added cofilin was required to get the increased binding. The amount of Aip1p cosedimenting with 7.5 μM F-actin falls off linearly with cofilin concentration . Aip1p cosedimentation with F-actin could be increased in the presence of cofilin either because cofilin creates more binding sites for Aip1p, or because cofilin increases the affinity of Aip1p for actin. To distinguish between these possibilities, we added 15 and 45 nM purified Aip1p to 5 μM cofilin-saturated actin filaments (which is below the concentration that saturates Aip1p binding) and ran the sedimentation reaction. A threefold higher concentration of Aip1p increases the fraction of Aip1p cosedimenting with actin, suggesting that Aip1p binding sites are not saturated, and that the cofilin-dependent increase in binding reflects an increased affinity of Aip1p for F-actin in the presence of cofilin (data not shown). To determine stoichiometries of these proteins in yeast cells, we estimated the ratio of Aip1p/cofilin/actin in the cell by comparing immunoblots of whole-cell extracts and purified proteins of known concentration (data not shown). Aip1p, cofilin, and actin are each present in whole-cell extracts at a ratio of about 1:1:5 or 1:1:10. Aip1p was originally identified by its two-hybrid interaction with actin . Further two-hybrid analysis revealed an interaction between Aip1p and cofilin raising the possibility that actin, Aip1p, and cofilin might form a ternary complex. The existence of such a ternary complex is supported by evidence that Aip1p and cofilin are dependent on each other and on actin for their correct localization in vivo . Binding experiments with purified proteins also support an Aip1p–cofilin–actin complex . Binding of purified or in vitro–translated Aip1p to F-actin is concentration-dependent and increases at high ratios of cofilin/actin. Since cofilin is a contaminant in yeast actin preparations, despite efforts to deplete it (∼1 μM/100 μM actin), we cannot rule out the possibility that contaminating native cofilin is responsible for the baseline binding to F-actin in the absence of added recombinant cofilin. Thus, it is possible that Aip1p binding to F-actin is strictly cofilin-dependent. We were unable to show a direct interaction between Aip1p and cofilin and/ or G-actin by native gel shift or by cosedimentation with GST–Aip1p, further suggesting that Aip1p interacts with cofilin on F-actin. A large set of mutations in both actin and cofilin was used in conjunction with the two-hybrid system to identify regions of actin and cofilin involved in the Aip1p interaction . Those data that describe the cofilin–actin interface appear sound since they agree with similar studies using biochemical , modeling , and structural (Amy McGough, personal communication) approaches. The binding footprint (as obtained by two-hybrid analysis) for Aip1p on the surface of actin partially overlaps with the cofilin binding site (red). Similarly, the footprints of Aip1p and actin on cofilin overlap as four cofilin mutants were specifically defective for both Aip1p and actin binding. Though overlapping interaction interfaces are consistent with both competitive interactions and a ternary complex, we favor the latter model because it is consistent with the localization and in vitro binding data discussed above. While most of our structural data can be incorporated into a coherent model for interaction in a ternary complex, there were several discrepancies between the biochemical and two-hybrid data. First, only by two-hybrid assay was cof1-9 defective for binding to actin. Additionally, although cof1-6 has a wild-type growth phenotype, by two-hybrid analysis it appears to be completely defective for actin binding. This interaction has not been tested biochemically. These discrepancies may be an artifact resulting from the fusion to Gal4p in the two-hybrid system. Alternatively, the cofilin-actin two-hybrid interaction might be subtly different than that observed in vitro with purified components. Note that cof1-19 is the only cofilin mutant that is defective for Aip1p localization, but it appears to interact well with both Aip1p and actin by two-hybrid analysis. This cofilin mutant may interact well with actin and Aip1p in the two-hybrid complex but have subtly altered binding properties in vivo. Though the AIP1 deletion mutant has no obvious phenotype on its own, allele-specific synthetic lethality was observed between aip1Δ and act1-159 , cof1-4 , cof1-5 , cof1-22 , and cof1-6 (at 37°C). These results suggest that in the double mutants, a common function is compromised enough that cell viability is lost. Because act1-159 , cof1-5 , and cof1-22 have all been shown to decrease the rate of F-actin disassembly in vivo , we postulate that Aip1p also promotes actin filament turnover. This conclusion is supported by our biochemical studies of Aip1p that demonstrate that it causes cofilin-dependent actin filament disassembly . Note that Cof1-22p has defects in actin binding in vitro, but that Cof1-5p does not , suggesting that the synthetic interaction is not simply a function of compromised actin binding by cofilin. The cofilin–actin interaction has not been tested biochemically for cof1-4 or for cof1-6 . Specific sorting of cofilin to cortical patches but not cytoplasmic cables is lost in aip1Δ and cof1-19 strains. One model that could explain these results in terms of the in vitro effects of Aip1p on actin filaments assumes that two populations of actin cables, one cofilin-bound and one tropomyosin-bound, form in yeast cells. Cofilin-bound cables would undergo net depolymerization in the presence of Aip1p, as occurs for purified actin filaments in vitro. Tropomyosin, which is localized to actin cables , can compete for cofilin binding sites on actin . Thus, tropomyosin would stabilize a subset of cables against Aip1p–cofilin depolymerization and these would go on to be the normal cables visualized in cells. In the aip1Δ strain or in a cofilin mutant that mislocalizes Aip1p ( cof1-19 ), Aip1p would not be able to function synergistically with cofilin to destabilize the filaments, and both cofilin-bound and tropomyosin-bound populations of filaments would be maintained. In support of this model, cofilin also localizes to rare actin cables in act1-159 tpm1 Δ and act1-159 mdm20 Δ double mutant strains , which would be predicted to have hyperstable F-actin structures that would not be readily disassembled by cofilin–Aip1p. The aip1Δ allele was also found to enhance the defects observed in specific actin mutants ( act1-119 , act1-125 , and act1-133 ) and in null alleles of genes encoding several components of the cortical actin cytoskeleton (Sac6p, Sla1p, and Cap2p). We believe that these double mutants most likely suffer from a general, cumulative derangement of the actin cytoskeleton, a conclusion that further supports that the actin cytoskeleton is affected in the aip1Δ strain. We demonstrated that Aip1p causes cofilin-mediated actin filament depolymerization in vitro . Interestingly, we discovered that Aip1p can induce cofilin-mediated actin filament depolymerization at stoichiometries as low as 1:50:50 Aip1/actin/cofilin. On the other hand, cofilin must be present at a 1:1 ratio with actin for optimal Aip1p-mediated activation of depolymerization. Though we were able to detect concentration-dependent F-actin cosedimentation of Aip1p at a low molar ratio with actin (by Western blot analysis or using in vitro–translated Aip1p), we were unable to detect cosedimentation at higher Aip1p/actin ratios. One hypothesis that explains these results is that Aip1p saturates binding at a low stoichiometry with F-actin. A second hypothesis is that high ratios of cofilin/actin are required for Aip1p binding, but that at high concentrations of Aip1p, net depolymerization prevents cosedimentation of Aip1p with actin. A model that is consistent with substoichiometric or cofilin-dependent filament binding, net filament depolymerization, and the two-hybrid footprinting data is that Aip1p enhances the weak severing activity of cofilin . Aip1p is the first protein aside from actin and LIM-kinase to show a physical interaction with cofilin. The fact that Aip1p is highly conserved in eukaryotes suggests that it may be a cofactor for cofilin activity in all eukaryotic cells. Though the mechanistic details of these interactions remain to be elucidated fully, our biochemical data demonstrate clearly that Aip1p stimulates cofilin-mediated actin filament disassembly and our genetic and cell biological data provides powerful evidence for the relevance of this activity in vivo. | Study | biomedical | en | 0.999996 |
10366598 | Grasshopper ( Schistocerca americana ) eggs at the 50% stage were sterilized and dissected in grasshopper saline. Thoracic and abdominal ganglia from ventral nerve cords were transferred to an Eppendorf tube containing an enzyme solution (2 mg/ml collagenase dispase; 0.5% ficin). After a 1-h incubation at 31°C, the ganglia were rinsed three times in L-15 medium ( GIBCO-BRL ), and then triturated ∼30 times with a BSA-coated Gel saver II pipette tip (USA/Scientific Plastics). The dissociated cells were placed on a dish with a glass coverslip bottom coated with poly- l -lysine (1 mg/ml for 24 h; Sigma Chemical Co. ) and goat anti–horseradish peroxidase antibody (1:50 dilution, incubated for 2 h at 31°C immediately before use; Jackson ImmunoResearch). Cells were allowed to adhere to the coverslip for 10 min, and then were transferred to an incubator (31°C) for 15–20 h before use. Eggs at the 32–33% stage were sterilized and dissected in grasshopper saline, and the embryos were transferred to a dish with a poly- l -lysine (5 mg/ml) coated glass coverslip bottom. Limb buds were placed ventral side down, opened with a glass needle, and spread flat on the coverslip . Mesodermal cells were removed with a suction pipette, exposing the Ti1 pioneer afferent neurons. Fillets were maintained in supplemented RPMI culture medium at 32°C in a heated stage chamber (Medical Instruments) on a Nikon inverted microscope during the experiments. Calcium was elevated by photolysis of caged calcium compounds (DM-nitrophen from Cal-Biochem ; nitrophenyl [NP] 1 -EGTA from Molecular Probes; Nitr-5 from Cal-Biochem ). It was not possible to measure calcium levels with Fura-based ratio-imaging in these experiments, because the excitation wavelengths for Fura compounds overlap extensively with the caged calcium compound photolysis wavelengths. Consequently, resting calcium levels, and levels after photolysis, were measured by fluorescence of Calcium-Green-1 (CG-1; Molecular Probes). Initially, the relationship between CG-1 fluorescence and calcium ion concentration in the presence of caged calcium was examined. In vitro calibration of the Ca 2+ -sensitive dye CG-1 fluorescence was carried out under conditions matching, as much as possible, the subsequent photolysis experiments on cultured neurons (see below). Five solutions with different Ca 2+ concentrations (Ca 2+ -free, three intermediate Ca 2+ levels, and saturated Ca 2+ ) were prepared by mixing different amounts of Ca-EGTA and K 2 -EGTA, each with a total EGTA concentration of 20 mM. These solutions all contained 1.2 mM NP-EGTA and 0.06 mM CG-1 and all were adjusted to pH 7.2 and ionic strength 175 mM by addition of K-MOPS and K-gluconate. Free [Ca 2+ ] in each solution was calculated using a K d for EGTA adjusted for an ionic strength of 175 mM, obtained by linear interpolation of the hydrogen- and calcium-binding constants of EGTA at 100 and 250 mM, and calculating the resultant K d at pH 7.2 using the equations of Tsien and Pozzan . Free [Ca 2+ ] was calculated from numerical solution of the equilibrium buffer equations for a mixture of 20 mM EGTA, 1.2 mM NP-EGTA, and 0.06 mM CG-1, using K d s of 179, 90, and 190 nM, respectively. These calculations were checked by measuring [Ca 2+ ] in solutions without NP-EGTA and CG-1, using Ca 2+ -selective electrodes (Microelectrodes Inc.), and confirming that the measured [Ca 2+ ] levels were close to those calculated for such solutions. The volumes of our final calibration solutions were not large enough to permit the use of Ca 2+ -sensitive electrodes. For calibration measurements, solutions were placed in 20-μm path-length microcuvettes (Vitro Dynamics). CG-1 fluorescence was converted to Ca 2+ concentration using the following equation: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[Ca^{2+}]={\mathit{K}}_{d}({\mathit{F}}-{\mathit{F}}_{min})/({\mathit{F}}_{max}-{\mathit{F}})\end{equation*}\end{document} where K d is the dissociation constant for CG-1, and F max is the fluorescence level for saturating Ca 2+ and F min for zero Ca 2+ . K d and ratio of F max / F min were obtained from the calibration curve . We also tried to produce a calibration curve for the CG-1/NP-EGTA mixture after photolysis of NP-EGTA by amounts similar to those in our experiments. To do this, we exposed the microcuvettes to large-field illumination using the same optical arrangements as for photolysis in cells, calculating the amount of NP-EGTA photolysis and the expected effect on the free [Ca 2+ ] in each calibration solution, and measuring the CG-1 fluorescence in each Ca 2+ -buffer solution. Wide-field illumination was used to minimize the rapid reduction in [Ca 2+ ] that would occur after photolysis due to rapid diffusion of uncaged Ca 2+ out of the photolysis spot into the large volume of the cuvette. Although [Ca 2+ ] should have increased after photolysis, fluorescence dropped in all solutions, apparently due to bleaching of CG-1 by the photolysis illumination. This invalidated our attempt to obtain a CG-1 Ca 2+ -calibration curve in the presence of partially photolyzed NP-EGTA. In contrast to these observations, in cells filled with NP-EGTA and CG-1, localized photolysis always led to an increase in CG-1 fluorescence. This is probably because, in nerve axons, unbleached CG-1 would quickly replace locally bleached CG-1 by diffusion, whereas Ca 2+ itself would diffuse much more slowly because it is bound to nondiffusible native buffers in cytoplasm. Therefore, the local CG-1 fluorescence would quickly recover to the level appropriate for nonbleached CG-1 and Ca 2+ released from NP-EGTA photolysis. There was no easy way to replicate this situation of rapid CG-1 diffusion but restricted Ca 2+ diffusion in our calibration experiments. However, the effects of NP-EGTA photolysis on the Ca 2+ sensitivity of CG-1 are likely to be modest , so we may use the calibration curve without photolysis to get a reasonable idea of the approximate levels of [Ca 2+ ] i reached in our experiments. To examine [Ca 2+ ] i elevation induced by photolysis, we next loaded cultured embryonic CNS neurons with CG-1 and caged calcium compounds. The cells were first exposed to membrane-permeable NP-EGTA (2 μM), CG-1 (2 μM), and 0.02% Pluronic (Molecular Probes) for 40 min at room temperature. Then, after washing three times, cells were rested for ∼1 h before experiments. For photolysis, UV light from a 100-W mercury lamp was passed through a 505 dichroic mirror and a 60×, NA 1.4 Nikon objective lens, or through a Nikon UV-2A filter block and a Nikon 40×, NA 1.3 objective lens (UV light passage through these combinations was similar). Flash duration, ranging from 100 to 400 ms, was controlled by an electronic shutter (Ludl Electronic Products Ltd.). After photolysis, Ludl filter wheels were repositioned to place in the light path a ND filter (1–4) to reduce photo damage to cells, and an appropriate excitation filter set . Fluorescent images were collected by a CCD camera (SenSys; Photometrics), and transferred to imaging software (Metamorph; Universal Imaging ). CG-1 fluorescence initially was measured 2–3 s after photolysis, and at 2–3 s or longer intervals thereafter. The CG-1 concentration loaded into axons of cultured neurons was estimated to be ∼60 μM by comparing the resting fluorescence of filled axons to that of a micropipette shank of similar diameter filled with various test concentrations of CG-1 and with [Ca 2+ ] buffered to ∼100 nM with 20 mM of a Ca-EGTA mixture. Acetoxymethyl ester compounds load into cells at rates highly dependent on their molecular weights, which are 1,147 and 654 D for esterified CG-1 and NP-EGTA, respectively. Cytoplasmic NP-EGTA should accumulate ∼10–20 times faster than CG-1 , to a level of ∼1.2 mM. To estimate how high [Ca 2+ ] i might rise under our experimental conditions, we measured the photolysis efficiency of our illumination spot by opening the field stop iris and exposing a 20-μm path length microcuvette (VitroCom) containing 5 mM DM-nitrophen and 2.5 mM Ca 2+ plus 0.1 mM Fluo-3 (Molecular Probes) in 100 mM KCl and 10 mM MOPS, pH 7.2. Exposure duration was varied until a duration was found that just caused a large increase in Fluo-3 fluorescence, measured shortly after photolysis. This provides an estimate of the time needed to photolyze half the DM-nitrophen , which we found to be 80 ms when a 10% neutral density filter was used to attenuate photolysis light intensity. Assuming equal quantum efficiencies for Ca-free and Ca-bound DM-nitrophen , we converted the calculated photolysis rate for DM-nitrophen (87/s without the neutral density filter) to a photolysis rate for NP-EGTA (21.6/s) from their relative quantum efficiencies and ultraviolet absorbances . The effect of a 200-ms exposure to this light on the [Ca 2+ ] i in a cell was simulated computationally by solving differential equations representing competing buffer reactions, NP-EGTA, and a Ca 2+ extrusion process . We included 1.2 mM NP-EGTA and 0.06 mM CG-1 plus 3 mM endogenous Ca 2+ buffer (buffer ratio 150:1, K d = 20 μM, k on = 2 × 10 8 M −1 s −1 ) and a first-order calcium removal process with pump rate 10 −4 s −1 . Total [Ca 2+ ] in the mixture was set to produce a resting [Ca 2+ ] i of 60 nM. Association and dissociation rates for NP-EGTA were 1.91 × 10 7 M −1 s −1 and 1.36 s −1 . CG-1 had a Ca 2+ association rate of 6 × 10 8 M −1 s −1 and K d of 119 nM . Fig. 1 b shows the expected elevation in [Ca 2+ ] i in a neuronal process exposed to our Hg lamp through our microscope optics for 200 ms. [Ca 2+ ] i should rise to ∼4 μM at the end of the light pulse, then drop within 3 s to 750 nM, and then gradually back to baseline within 15 s. Since the exact loading of NP-EGTA is uncertain, we also performed calculations assuming a cytoplasmic concentration of 0.6 mM NP-EGTA. In that case, [Ca 2+ ] i should rise to nearly 2 μM, and drop within 3 s to 400 nM. We also estimated the [Ca 2+ ] i in cultured CNS neurons using the values of K d and ratio of F max / F min from the CG-1 calibration curve and the measurements of CG-1 fluorescence inside the cultured neurons. The peak Ca 2+ level after a 200-ms photolysis was obtained using the following equation: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[Ca^{2+}]_{p}={\mathit{K}}_{d}({\mathit{F}}_{p}/{\mathit{F}}_{r}-{\mathit{F}}_{min}/{\mathit{F}}_{r})/({\mathit{F}}_{max}/{\mathit{F}}_{r}-{\mathit{F}}_{p}/{\mathit{F}}_{r})\end{equation*}\end{document} where F p is the CG-1 fluorescent intensity of the axon at peak [Ca 2+ ] i after photolysis, and F r is the CG-1 fluorescence at resting [Ca 2+ ] i before photolysis. From our measurements of CG-1 fluorescent intensity inside axons in culture after photolysis, the average F p / F r was ∼1.743 ± 0.075 (± SEM, n = 3). Minimal CG-1 fluorescence was measured from cells bathed in Ca 2+ -free grasshopper saline with addition of 5 mM EGTA and 5 mM ionomycin (Molecular Probes). The average F min / F r was ∼0.423 ± 0.066 (± SEM, n = 3). F max / F r can then be calculated using the ratio of F max / F min from the in vitro calibration. Using Eq. 2 and all the values we obtained for each parameter, [Ca 2+ ] p inside cultured axons was ∼360 nM. Similarly, the resting [Ca 2+ ] i was estimated using the following equation: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[Ca^{2+}]_{r}={\mathit{K}}_{d}({\mathit{F}}_{r}/{\mathit{F}}_{min}-1)/(\ddot {{\mathit{F}}}_{max}/{\mathit{F}}_{min}-{\mathit{F}}_{r}/{\mathit{F}}_{min}).\end{equation*}\end{document} Again, using the values of K d and F max / F min from our in vitro calibration and the measurements of minimal CG-1 fluorescence inside actual axons in zero calcium solution, [Ca 2+ ] r was ∼60 nM from Eq. 3 . Our procedure for estimating F min may overestimate this parameter, so our estimates for [Ca 2+ ] r and [Ca 2+ ] p may be somewhat lower than actual values. Ti1 afferent neurons on limb fillets were pressure injected (Narishige USA Inc.) with caged Ca 2+ injection solution (50 mM Nitr-5 with 20 mM CaCl 2 or 20 mM NP-EGTA with 10 mM CaCl 2 , 140 mM K Hepes, pH 7.3). To estimate the amount that was injected, solutions also contained 2 mM rhodamine-dextran (Molecular Probes). Rhodamine fluorescence in injected neurons was compared with that of micropipettes of similar diameter containing various known concentrations of rhodamine-dextran. We found that injection with 3–5 pulses (1.0 PSI; 300 ms duration) gave a dilution of ∼1/40 in the cell regions selected for photolysis, with an expected Nitr-5 concentration of ∼1.25 mM. From the relative absorbances and quantum efficiencies of DM-nitrophen and Nitr-5, we calculated a photolysis rate of 21.8 s −1 for Ca 2+ -loaded Nitr-5, and 7.9 s −1 for Ca 2+ -free Nitr-5. Assuming a Nitr-5 concentration of 1.25 mM and a Ca 2+ loading that leaves the resting [Ca 2+ ] i at 60 nM, UV exposures of 100 and 400 ms are predicted to elevate [Ca 2+ ] i to 300 and 900 nM, respectively . Successfully injected neurons were labeled with 1,1′-dihexadecyl-3,3, 3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) by gently touching the cell body membrane with DiI crystals on an electrode tip . Within ∼10–15 min, the lipophilic dye diffused to the growth cone and labeled all filopodia. For control experiments, Ti1 neurons were either labeled with DiI alone or injected with BAPTA injection solution (50 mM Nitr-5, 20 mM CaCl 2 , 140 mM K Hepes, 50 mM K 2 BAPTA, pH 7.3) and labeled with DiI. Photolysis for 100, 150, and 200 ms was calculated to elevate [Ca 2+ ] i to 98, 111, and 120 nM, respectively . The distribution of flash durations for control experiments, between 100 and 200 ms, was matched to the distribution of flash durations that had induced filopodial extension. Using a diaphragm to select a small area (20–30 μm in diameter) on the growth cone, nascent axon, or on filopodia, injected cells were illuminated either through the 505 dichroic/60× objective, or the UV-2A filter block/ 40× objective. Starting with a 100-ms flash, photolysis time was increased in 50-ms increments until a morphological response was observed or until a maximum flash length of 400 ms was reached. Using the CCD camera with a ND1 or ND2 neutral density filter and a Chroma phycoerythrin filter block , DiI labeled processes were imaged for 50–200 ms. For growth cones and nascent axons, sets of 3–5 focal planes were taken: the axons are 1–2 μm in diameter; for 3-focal plane sets, one plane was taken through the middle of the axons, and one 1–1.5 μm above and below the axons; for 5-focal plane sets, the additional planes were at least 2 μm away from the axon. Measurement of filopodial length was done using the MetaMorph Imaging System or NIH Image. To confirm that similar results would be observed in culture, plated neurons were loaded with NP-EGTA (as above), rinsed, and then incubated in 6 μg/ml DiI solution for 15 min. Cells were rested for 1 h before experiments. Since the cytoskeletal scaffold of filopodia primarily comprises F-actin bundles, we examined the disposition of F-actin in these cells with respect to the sites of filopodial protrusion after photolysis of caged calcium. Initially, F-actin disposition was examined in fixed cells. Cells were fixed in glutaraldehyde (0.25, 0.1, 0.5, and 1%), formaldehyde (3.7%), or paraformaldehyde in an actin stabilizing buffer. Similar results were obtained with all fixes, although the following paraformaldehyde fixation resulted in the most consistent, low background, preparations for light microscope: cells were fixed in 4% paraformaldehyde in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl 2 , pH 6.9) for 20 min, washed in PHEM three times and then permeabilized with 0.1% Triton X-100 or 0.02% saponin in PBS (150 mM NaCl, 20 mM Na 2 HPO 4 , pH 7.4) for 5 min. After being washed three times in PBS, cells were incubated in rhodamine-phalloidin (1 μg/ml; Molecular Probes) for 20 min. Cells were then rinsed in PBS, cleared in glycerol and mounted with Vectashield mounting medium (Vector Labs), and either photographed directly in a Nikon microscope, or imaged in a Bio-Rad 1024 confocal microscope. For live labeling, Ti1 afferent neurons in limb fillets were injected with caged Ca 2+ solution containing 0.1–0.25 mM rhodamine-phalloidin, and photolyzed (as above). For in situ CCD imaging of rhodamine-phalloidin, 200–400-ms exposures with a Chroma phycoerythrin filter set were generally used with ND 0.6. Photolysis sites were imaged for at least 20 min preceding the photolysis flash. For examination of the locations of filopodia with respect to F-actin patches, multiple image planes (3–6 per set) were taken. We also examined actin dynamics by injecting rhodamine-actin into Ti1 neurons. Rhodamine-actin solution (Cytoskeleton) was dialyzed into Hepes injection buffer (1 mM Hepes, pH 7.2, 0.2 mM MgCl 2 , 0.2 mM ATP) and concentrated (2 mg/ml) with microconcentrators (Amicon). Using the calibrations described above (Materials and Methods), we measured resting [Ca 2+ ] i and [Ca 2+ ] i after photolysis in 13 cultured CNS neurons loaded with NP-EGTA from 6 different cell culture experiments. Loaded neurons were viewed with differential interference contrast (DIC) optics, and imaged with the CCD camera before and after photolysis of a small (20–30 μm) region at or near the growth cone. Qualitatively, CG-1 fluorescence rose sharply after photolysis and returned gradually to the resting level within ∼10–15 s . To quantify the calcium concentration changes, minimal CG-1 fluorescence ( F min ) was measured by placing neurons in calcium-free medium with a calcium chelator (EGTA) and ionomycin (to admit calcium). Peak CG-1 fluorescence ( F p ) was measured after a 200-ms photolysis. K d and F max / F min values from the calibration curve then were used with Eq. 2 , to calculate peak calcium ion concentration and, using Eq. 3 , resting [Ca 2+ ] i . Intracellular [Ca 2+ ] before and after photolysis for a cultured cell is shown in Fig. 2 b2. Resting Ca 2+ concentration was estimated to be ∼60 nM. The observed calcium ion concentration of 360 nM 3 s after a 200-ms photolysis compares well with the computed value of 410 nM for an [NP-EGTA] of 0.6 mM. The extrapolated [Ca 2+ ] i immediately after photolysis is ∼1 μM. Based upon the computed and observed photolysis-induced calcium elevation in neurons in culture, neurons next were examined in situ. Ti1 neurons extending axons on embryonic limb bud epithelium were injected with NP-EGTA or Nitr-5. Comparisons with known concentrations of a fluorescent marker (rhodamine-dextran) were used to determine the intracellular injection regimen to achieve a desired concentration of caged calcium compound. The photolysis rate for this concentration and compound were used to compute Ca 2+ concentrations resulting from flashes of 100–400 ms . The neurons were double-labeled with DiI for CCD imaging. Segments of nascent axon 20–30 μm in length and just behind the growth cone were selected for photolysis. These regions, while capable of extending filopodia, normally did so at a lower rate than the leading edge of the growth cone (we did not use the leading edge of the growth cone for our experiments because the high frequency of filopodial protrusion made it difficult to isolate the effect of Ca 2+ elevation). Using injection regimens selected to load the neurons with ∼1.25 mM Nitr-5, we started with 100-ms flashes, and increased flash length in 50-ms increments. We found that flashes in the 100–200-ms range usually became effective in effecting filopodial protrusion. These flashes should result in intracellular peak Ca 2+ concentrations of ∼300–800 nM . A single photolysis flash typically was followed by elongation of filopodia within the flash zone, and also by extension of new filopodia . We measured morphological changes of filopodia after photolysis at 32 photolysis sites on 22 Ti1 neurons from 22 limb fillets . New filopodia could appear within 1 min, and were several micrometers in length by 5 min. The number of filopodia in the target region was significantly increased after the flash . It peaked ∼11–15 min after the flash , and was substantially reduced by 21– 30 min after the flash. The total length of all filopodia in the flash zone increased and decreased with a similar time course . Although multiple flashes appeared to sustain filopodial protrusion , we did not systematically study their effects. We also performed 13 photolysis experiments on 8 different cultured cells, and found that the filopodial responses after photolysis in all 13 experiments were similar to those occurring in Ti1 neurons in situ (data not shown). These results suggest that calcium ion elevation can promote elongation of existing filopodia, and can initiate protrusion of new filopodia. To determine whether the observed effects on filopodia were due to Ca 2+ elevation per se, we used three control conditions . First, to control for normal protrusive activity, the number and length of filopodia after a flash were compared with the number and length of filopodia in the same time interval preceding the flash. Second, to control for possible photo-effects of DiI fluorescence, the number and length of filopodia were measured in neurons labeled with DiI, but not injected with caged calcium. Third, to control for possible effects of the caging molecule, or the photolysis thereof, the number and length of filopodia were measured in neurons coinjected with Nitr-5 and a calcium buffer (BAPTA). In none of these control conditions were significant effects on filopodia observed. These results indicate that [Ca 2+ ] i elevation after photolysis specifically was required to induce filopodial protrusion. In addition to nascent axons, individual filopodia also were targeted. Filopodia of these axons have a consistent uniform diameter . We selected target zones that contained 10–20-μm regions along the length of one or more filopodia, but did not contain nascent axon, lamellae, or a region of a filopodium that was enlarging into a branch. Photolysis of these regions typically resulted in one or more new filopodia arising along the length of the targeted filopodium . In a controlled set of 21 filopodia exposed to regional photolysis, no new filopodia arose in neurons labeled with DiI but not injected with caged calcium (7 filopodia tested), or in neurons injected with caged calcium and with BAPTA (7 filopodia tested); by contrast, filopodial branching events occurred after photolysis in all neurons injected with caged calcium alone (7 filopodia tested). This result suggests that all components necessary to generate a filopodium are present in filopodia and can be activated by local calcium elevation. Since generation of new filopodia requires polymerization of new F-actin, we examined the disposition of F-actin in these cells and its response to calcium elevation. We started by examining F-actin in fixed, cultured CNS neurons, using a variety of actin stabilization and fixation procedures (Materials and Methods). We imaged F-actin disposition in 76 fixed grasshopper embryonic CNS cells from 18 cell culture experiments. We observed conventional F-actin distribution including (a) relatively little F-actin in the central domain of the growth cone and in the interior of the nascent axon; (b) concentrated F-actin at the leading edge of the growth cone; (c) concentrated F-actin at the bases of some (presumably extending) branches, and some filopodia; and (d) cortical F-actin along the nascent axon . We looked at live labeling of actin in Ti1 pioneer neurons in situ by injecting rhodamine-actin into cell bodies. Injected rhodamine-actin diffuses rapidly into these growth cones and filopodia, and predominantly appears to reveal F-actin structures, presumably because of the high density of labeled monomers . We injected four Ti1 neurons with rhodamine-actin; the actin labeling was similar in all cells. The observed labeling was similar to that in fixed, rhodamine-phalloidin labeled neurons . As in fixed cells, the cortical labeling was not uniform, but usually was relatively dense in some areas, and relatively sparse in others. Some of the dense labeling occurred in fairly distinct patches . Patches could be localized to the bases of filopodia, or to other cortical regions. To confirm that these patches were F-actin, and to monitor their activity, we injected 22 live Ti1 neurons with low concentrations of rhodamine-phalloidin . These axons and growth cones appeared very similar to rhodamine-actin injected growth cones. When axons were observed for several minutes , cortical F-actin was seen to change, with patches forming or disappearing, increasing or decreasing in density, and sometimes moving distally or proximally along the axon. We then coinjected caged calcium with rhodamine-phalloidin, targeted regions of the nascent axon with photolysis flashes, and imaged subsequent changes in filopodia (with rhodamine filters) and in F-actin. New filopodia arose from the target regions, and we examined the relationship between the sites of F-actin accumulation, and the sites of filopodial protrusion. In some cases, new filopodia arose from sites where no preexisting F-actin patch had been observed; some prominent F-actin patches within target zones were not the sites from which filopodia emerged; and, often, filopodia emerged at the sites of preexisting F-actin patches. A case where several filopodia emerged from F-actin patches in the target zone is shown in Fig. 5 e. To quantify the relationship between filopodial protrusion and F-actin accumulation along the axons, we counted the total number of new filopodia after photolysis experiments and determined whether those filopodia were associated with F-actin patches or not. The data from 24 experiments are summarized in Table I . For each experiment, three to six images at different focal planes were collected every 5 min in the photolysis area, and each cell was followed for ∼30 min after photolysis. In this sample set, ∼84% of new filopodia were initiated from preexisting F-actin patches, ∼10% were from F-actin patches newly formed after photolysis, and ∼6% from cortical sites without detected F-actin accumulation. Thus, new filopodia were disproportionately, but not exclusively, associated with F-actin patches. Using intracellular caged calcium compounds, we investigated the effect of direct elevation of calcium ion concentration on filopodia extended from target regions of nascent axons, and on the relationship between lateral filopodia and cortical F-actin. The selected target regions were just proximal to the growth cones of Ti1 afferent neurons undergoing axonogenesis on an epithelial substrate in situ. After a brief (50–200 ms) photolysis flash, intracellular Ca 2+ concentration probably rose within a second to a peak of ∼1 μM, and then declined rapidly back to the resting level within 10–15 s. The increase in [Ca 2+ ] i induced elongation of existing filopodia, and protrusion of new filopodia. Elongation or protrusion could begin within less than a minute, and peaked ∼10–15 min after the flash. Photolysis of distal regions of individual filopodia induced new filopodia (branching) in the flash zone. In neurons coinjected with rhodamine-phalloidin, F-actin was observed to be nonuniformly distributed in the submembrane cortex of the nascent axon; F-actin accumulations formed, moved, coalesced, fragmented, and dispersed. Photolysis-induced filopodia emerged disproportionately from F-actin patches. We used caged calcium compounds to elevate [Ca 2+ ] i because we wanted to localize the calcium change spatially and to restrict the initial signaling event to calcium (by-passing upstream signaling elements). A disadvantage of this approach is that overlap in activation wavelengths precludes ratio imaging with Fura-based compounds, since the measurement procedure would itself photolyze caged calcium and elevate [Ca 2+ ] i . Consequently, we estimated [Ca 2+ ] i changes using a single-wavelength indicator, CG-1. In vitro, we measured, and plotted, CG-1 fluorescence at different calcium concentrations . In models and in AM-loaded cultured neurons , respectively, we computed, and measured, resting [Ca 2+ ] i and [Ca 2+ ] i after photolysis. Computed and measured values were in reasonable agreement. We then used photolysis flashes of the intensity and length evaluated in models and in cultured neurons to photolyze caged calcium in injected neurons in situ. We were not successful in estimating [Ca 2+ ] i with CG-1 in Ti1 neurons in situ, perhaps because they have more rapid Ca 2+ extrusion than cultured neurons so that the induced [Ca 2+ ] i elevation was largely dissipated by the time [Ca 2+ ] i measurements could begin. Elevation of intracellular calcium concentration throughout a growth cone by application of serotonin, by depolarization, or by calcium ionophore can cause a reduction in the number of filopodia . This result is consistent with the loss of F-actin (and microtubules) seen after calcium elevation , with calcium-mediated growth cone collapse that can be induced by repellent guidance molecules , and with the reduction in outgrowth mediated by spontaneous calcium spikes , or caged calcium–induced calcium transients . In contrast, an increase in the number of filopodia can be initiated by calcium elevation induced by a large-scale electric field , depolarizing electrode , or local [K + ] elevation . These different outcomes may be due to the localization of the calcium elevation or to the level of [Ca 2+ ] i reached . In addition, intracellular events after depolarization are not restricted to elevation of calcium concentration, and other intracellular effects might influence the calcium influx– dependent outcomes seen with different methods of depolarization. Our direct elevation of calcium concentration with photolysis of caged calcium bypasses these other possible upstream events, and makes a strong case that local calcium elevation can, by itself, directly initiate protrusion of filopodia. In contrast to protrusion of new filopodia, lengthening of already existing filopodia has been a consistently observed result of elevation of intracellular calcium ion concentration by several methods , including extracellular K + , current passing electrodes, calcium ionophore and, now, photolysis of caged calcium ions. The calcium levels, onset, and time course of this response observed with caged calcium are comparable to those features observed with other methods. Growth cones can project many filopodia that can extend several tens of micrometers, potentially greatly increasing exploration of the embryonic environment. This potential is realized by the surprising capabilities of filopodia. Single filopodial contacts can steer the growth cone in situ or initiate growth cone retraction . Recent work has examined the sensory signaling and motor capabilities of single filopodia. In response to depolarizing extracellular current, calcium ionophore, soluble neurotransmitters , or contact with laminin-coated beads , isolated filopodia can generate a calcium-elevation response followed by restoration of calcium concentration to the resting level. In isolated filopodia, however, stimuli that elevated calcium resulted in shortening rather than lengthening . This raises the issue of whether calcium signaling that is restricted to a filopodium can activate an effector response , particularly since organelles that may mediate calcium signal amplification may not be present within filopodia . We tested this issue by using photolysis of caged calcium to restrict calcium elevation to filopodia . We found that filopodia can generate an effector response to this calcium signal by initiating multiple new filopodia within the illuminated region . Thus, single filopodia apparently contain all of the receptor, signaling, and effector molecular machinery necessary to generate an outgrowth response. Furthermore, transient calcium elevation that is confined to the filopodium itself is sufficient to trigger the signal cascades to initiate filopodial protrusion. Interestingly, one of the most consistent early responses seen in situ when a filopodium contacts a powerful attractive substrate, the surface of a guidepost cell, is initiation of multiple new filopodia at the contact site . Although considerable progress has been made in understanding actin assembly and mechanics , many features of the signaling pathways controlling cell motility and generation of filopodia remain unresolved. Filopodial assembly can be driven by small GTPases , particularly cdc42, and associated proteins . There is substantial evidence that calcium-sensitive proteins affect outgrowth, filopodia, actin, and actin assembly controllers . Using coinjection of caged calcium and rhodamine-phalloidin, we examined the relationship between disposition of F-actin along nascent axon shafts, local elevation of calcium concentration, and the protrusion sites of filopodia . We observed that F-actin is not uniformly distributed in the cortex of nascent axons, but occurs in accumulations of varying density that can be motile and transient. After photolysis, new filopodia emerge most frequently from preexisting F-actin accumulations, although new accumulations sometimes form before a filopodium emerges, and in a few cases no accumulation is observed at a filopodial protrusion site (Table I ). We did not observe a consistent increase in number of F-actin patches after photolysis; transient [Ca 2+ ] i elevation may induce filopodial protrusion not by initiating more de novo F-actin patches, but by activating the actin regulatory proteins located near or inside the preexisting F-actin patches to reorganize actin filaments and initiate filopodial formation. In budding yeast, F-actin accumulations, termed patches , contain actin-binding proteins and also move laterally in the cortex . In growth cones and nascent axons, there also may be dynamic, transient aggregations of actin assembly–associated proteins that can either generate a new filopodium, or disassemble. Such aggregates may be preferentially activated to generate a filopodium by local elevation of calcium concentration. | Study | biomedical | en | 0.999998 |
10366599 | B3′: 5′GCTGAGTACG CTCGAG GTAGGGGAGCTGGAGGGC3′; B5′: 5′CGCTTCTG GAATTC CCCAAAGGGCCTCTGAG3′; C3′: 5′GCTAGCATCG CTCGAG CCACACCATGGCAGATG3′; D5′: 5′CGCTTCTG GAATT\\C CCCACGGTTGGCATG3′; E3′: 5′TATCG CTTAAG TCGACGATGTAGAGCTGATGGG3′; E5′: 5′CGGTACGT GAATTC AAGAAGGCGCTGCC3′; F3′: 5′GCTAGCATCG CTCGAG ATACTTGGAATTTTTGG3′; F5′: 5′CGTCATCAGC GAATTC CCGAAAGGTCCACTTCG3′; G3′: 5′CTCGCCTAGC CTCGAG CCACACCATTGTTGAGG3′; L3′: 5′TCGTGAGGATC CTCGAG CTACTGGAGCCGCGACAGGC3′; L5′: 5′CTAGACCT GAATTC CCAATGGCGACTGCGACCCC3′; M3′: 5′CGAGTAGCAT GTCGAC CAGGACAATCTTAAAGGA3′; M5′: 5′GCTACACTAG CCGCGG GAATTCGGCACGAGGCG3′; N3′: 5′CGAGTAGCAT GTCGAC TCACAGGACAATCTTAAA3′; N5′: 5′GCTACACTAG CCGCGG CCACCATGGAGCAAAAGCTCATTTC TGAAGAGGAC TT GAAT CGCGGCGGGGCGATGGG3′. Restriction enzymes sites incorporated into the primers to aid in cloning are underlined. The yeast two-hybrid screen for PS2 interacting proteins was performed essentially as described by Golemis et al. , with the necessary plasmids and cDNA library obtained from Dr. Roger Brent (Harvard Medical School, Cambridge, MA). The PS2-loop bait was constructed by PCR amplification of the region from human PS2 using primers B5′ and B3′. The resulting 150-bp PCR fragment was double digested with EcoRI and XhoI and ligated into the corresponding sites of the pEG202 LexA fusion plasmid. This bait construct and the LacZ reporter plasmid pSH18-34 were cotransformed into yeast strain EGY48 which was then transformed with ∼5 μg of human fetal brain cDNA library in pJG4-5. 1.5 × 10 7 of the resulting transformants were plated on Gal/Raf/CM-ura-his-trp-leu plates to screen for transcriptional activation of the chromosomally integrated LEU2 reporter gene. 100 Leu+ yeast colonies were picked to a Glu/CM-ura-his-trp master plate, then replica-plated to Glu/CM-ura-his-trp-leu, Gal/Raf/CM-ura-his-trp-leu, Glu/Xgal/CM-ura-his-trp and Gal/Raf/Xgal/ CM-ura-his-trp plates to test for galactose-dependent leu2 and lacZ expression. Dual expression of the reporters was displayed by 15 colonies, from which the library plasmids were recovered in Escherichia coli strain JBe15 and subsequently transformed back into EGY48 containing pSH18-34 and the pEG202LexA/PS2-loop B bait or one of several negative controls to test the specificity of the interaction with PS2-loop B. The library plasmids that produced a strong and specific interaction with PS2-loop B were recovered in E . coli strain DH1 and the DNA sequence of their inserts was determined. DNA and protein sequences were analyzed using MacVector 6.5 (Oxford Molecular). Homology searches of the NCBI databases were performed using the BLAST program. Three of seven putative interactors were independent clonings of the same cDNA which we named calmyrin. For further experiments, clone 7, a library plasmid which contained full-length calmyrin cDNA was digested with EcoRI and XhoI and subcloned into pBluescript KS(−) or pGST/His T1 vector ( Pharmacia Biotech, Inc. ). The specificity of the calmyrin interaction was tested against three PS2-loop region constructs, two different PS1-loop constructs of which one was further mutated to the corresponding PS2 sequence, one PS2 COOH-terminal construct, and a lamin control. A conserved 31–amino acid loop region (designated PS2-loop C) was obtained by using primers B5′ and C3′ to PCR generate a 93-bp fragment. The more divergent region of the loop (designated PS2-loop D) was generated using primers D5′ and B3′. A construct encoding the final 40 amino acids of PS2 (designated PS2-Cterm) was created using primers E5′ and E3′. The corresponding loop B and loop C regions in PS1 were PCR amplified using primers F5′ with F3′ or G3′, respectively, from a full-length PS1 clone obtained from Dr. S.S. Sisodia (University of Chicago, IL). The PS1-loop C region, which differs by only three amino acids from the corresponding PS2-loop (containing a threonine instead of a proline at position 281 , a leucine in place of an isoleucine at position 282, and a threonine for an alanine at position 291), was mutated at each of the three divergent residues, singly, and in every possible combination to the corresponding PS2 sequence using appropriate PCR primers and the QuikChange site-directed mutagenesis method (Stratagene). A control bait construct which contained the first 31 amino acids of lamin B was obtained by PCR with primers L5′ and L3′ from lamin B cloned in pBluescript KS(−) . All PCR-amplified regions were digested with EcoRI and XhoI, cloned into pEG202, and confirmed by DNA sequencing. These various baits were transformed into EGY48 and found by immunoblotting of yeast extracts to express appropriately sized lexA-PS fusion polypeptides. Three isolates from yeast transformed with the calmyrin in pJG4-5 (clone 7) plus each PS bait or the control lamin bait were assayed for β-galactosidase enzyme activity in liquid cultures using ONPG ( O -nitrophenyl β- d -galactopyranoside) as a substrate . 32 P-labeled DNA probes were prepared via standard random primer labeling of 100 ng of full-length calmyrin cDNA or human β-actin cDNA control. A human multiple tissue Northern (MTN) blot and a human brain multiple tissue Northern blot II ( CLONTECH Laboratories, Inc.) were hybridized with the calmyrin probe at 68°C overnight, washed in 0.1× SSC at 50°C, and exposed to film. The blots were then stripped of the calmyrin probe and reprobed with the β-actin control ( CLONTECH Laboratories, Inc.). The original pGST construct or the pGST construct containing the complete calmyrin sequence fused COOH-terminally and in-frame with GST was transformed into CAG456 bacteria. Unfused GST and GST/calmyrin fusion protein induction with IPTG, incubation with glutathione agarose, and elution with reduced glutathione were as described in Janicki and Monteiro . The pGEM-CMV vector, a CMV-driven expression plasmid containing a COOH-terminal myc-tag , was used for protein expression in HeLa cells. A calmyrin construct containing an in frame COOH-terminal myc epitope was created by PCR amplifying the calmyrin fragment from pBS-calmyrin with primers M5′ and M3′ resulting in a ∼600-bp PCR product that was digested with SacII and SalI and ligated into pGEM-CMV. An NH 2 -terminal myc-tagged calmyrin construct was also created by PCR using primer N5′ with primer N3′ to introduce eleven residues of the myc epitope followed by four residues encoded by 5′ untranslated calmyrin sequence linked to the complete calmyrin coding sequence. The resulting ∼600-bp PCR product was digested with SacII and SalI and ligated into pGEM-CMV. An untagged full-length calmyrin expression construct was created by digesting pBS-calmyrin with SacII and XhoI, gel isolating the ∼650-bp fragment, and ligating it to SacII/SalI linearized pGEM-CMV. The cloning and expression of both full-length PS2 and the PS2 construct deleted of loop and COOH-terminal sequence [pPS2(268aa) + Myc] were described previously . Expression of full-length wild-type neurofilament light (NF-L) subunit was achieved using the CMV-NF-L expression construct . Purified GST/calmyrin protein and GST/PS2(NH 2 -terminal) fusion protein were sent to Covance Research Products for inoculation into rabbits. The specificity of these rabbit antibodies was determined by immunoblotting and immunofluorescent staining of HeLa cell transfected with calmyrin or PS2. For immunoblotting, the anti-calmyrin and anti-PS2 antibodies were used at a 1/500–1/700 dilution and detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies and SuperSignal Substrate ( Pierce Chemical Co. ). HeLa cells were grown in DME supplemented with 10% FBS and transiently transfected with appropriate plasmid DNAs as calcium phosphate precipitates . Alternatively, 20 μg of DNA and 2 × 10 6 HeLa cells were electroporated at 960 μF and 0.3 kV. HeLa cells were transfected directly on glass coverslips, fixed, and antibody stained as described in Janicki and Monteiro . Antibodies used were rabbit anti-calmyrin serum (diluted 1:250), goat anti-PS2(NH 2 -terminal) antibody (diluted 1:150; Santa Cruz Biotechnology, Inc. ), rabbit anti-lamin serum , rabbit anti-NFL serum (diluted 1:250; generated in this lab using recombinant-purified, bacterially expressed, mouse neurofilament light chain), M30 CytoDEATH mouse anti-cytokeratin 18 antibody (diluted 1:50; Boehringer Mannheim ), fluorescein- and rhodamine-conjugated donkey anti–rabbit, anti–goat, and anti–mouse antibodies (Jackson ImmunoResearch Laboratories, Inc.). Fluorescence staining of cells was visualized on an inverted Leica DM IRB microscope and images were captured using a Photometrics SenSys camera and manipulated with IPLab Spectrum and Multiprobe software ( Scanalytics ) on a Power Macintosh. Confocal microscopy and image processing was performed using the ×100 objective of a Leica confocal and imaging system (Leica Inc.) with the kind help of Dr. Timothy Mical and Dr. Joseph Gall (Carnegie Institution, Baltimore, MD). Spleen, brain, kidney, liver, heart, and skeletal muscle tissues were dissected from an adult mouse, chopped with a razor blade in 1–2 ml lysis buffer , homogenized on ice, briefly sonicated on ice, and centrifuged at 2,000 rpm for 5 min. Tissue lysate supernatants were collected, their protein concentration was determined by the BCA Protein Assay (Pierce), and 100 μg of each sample was separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with the rabbit anti-calmyrin antibody. Kidney and heart tissues from 8–12 2-d-old mice were chopped with a razor blade, resuspended in 2 ml 0.25% collagenase in PBS, vortexed, incubated at 37°C for 15 min, centrifuged, and washed 3× with PBS. Cells were cultured in EGM medium supplemented with BBE (Clonetics) and 10% FBS for 2–7 d. For immunofluorescence, cells were cultured directly onto coverslips and fixed and stained as described above. Nondetergent soluble and insoluble fractions of HeLa cells were prepared essentially as described by Gerace and Globel . HeLa cells (∼1 × 10 6 ) were collected 24 h after transfection by scraping the cells in ice-cold PBS and centrifugation at 10,000 g . The cells were resuspended in 0.25 ml 10 mM triethanolamine-HCL (pH 7.4), 10 mM KCl, 1.5 mM MgCl 2 , 5 mM iodoacetamide, and 1 mM Pefabloc ( Boehringer Mannheim ). After 10 min incubation on ice the cells were disrupted with 10 gentle strokes in a 0.5-ml Potter-Elvehjem homogenizer. Next, 0.25 ml 10 mM triethanolamine-HCL (pH 7.4), 270 mM KCl, 1.5 mM MgCl 2 , 5 mM iodoacetamide, and 1 mM Pefabloc were added and after mixing, the homogenates were centrifuged at 100,000 g for 15 min in a Beckman TLX ultracentrifuge. The supernatants were removed and the pellets resuspended in lysis buffer to a volume equal that of their respective supernatants. Triton X-100–treated HeLa cell fractions were prepared by lysing the transfected cells in 0.5 ml ice-cold 1% Triton X-100, 10 mM triethanolamine-HCL (pH 6.9), 140 mM KCl, 1.5 mM MgCl 2 , 5 mM iodoacetamide, and 1 mM Pefabloc. After 10 min incubation the lysates were centrifuged at 140,000 g for 15 min. Supernatants were collected and the pellets resuspended in lysis buffer. Equivalent volumes of the supernatant and pellet fractions of the detergent-treated and untreated cells were separated by SDS-PAGE and immunoblotted using the rabbit anti-calmyrin antibody or the rabbit anti-lamin antibody. The same cell fractionation procedure was used on primary cell cultures prepared from mouse kidney. After transfection, sodium pyruvate to a final concentration of 1 mM and 0.1–0.2 mCi 3 H-myristic acid ( Amersham Life Science Inc.) were added to the fresh media in each cell culture dish. At ∼24 h after transfection, cells were scraped off the bottom of the dish and the media was collected and centrifuged 5 min at 3,000 rpm. The cell pellet was washed with PBS, centrifuged, resuspended in 400 μl lysis buffer (50 mM Hepes, 100 mM KCl, 0.3% NP40, 1 mM EDTA, 1 mM EGTA, pH 7.5, + protease inhibitor cocktail with aprotonin, leupeptin, and PMSF; Boehringer Mannheim ), and homogenized on ice. Insoluble material was pelleted and the supernatant was collected and diluted with an equal volume of dilution buffer (50 mM Hepes, 1 mM EDTA, and 1 mM EGTA, pH 7.5). 150 μl of the lysates were incubated with 5 μl of antibody (rabbit anti-calmyrin, rabbit anti-PS2, or rabbit control preimmune serum) for 2 h at 4°C. 45 μl of a slurry of protein A–Sepharose beads ( Pharmacia Biotech, Inc. ) was then added to the lysates and incubated with rotamixing for another 1 h. The beads were pelleted by centrifugation, and after removal of the supernatant, the beads were washed four times with lysis/dilution buffer. All of the immunoprecipitate and one-sixth of the supernatant sample were separated by SDS-PAGE. After Coomassie blue staining and destaining, the gel was soaked for three 15 min changes in DMSO, immersed in 22% PPO (2,5-diphenyloxazole) for 90 min, washed in water, dried, and exposed to film by fluorography for 1 wk to 2 mo. After ∼24 h, mock or PS2-transfected HeLa cells (∼1 × 10 6 cells) were washed in ice-cold PBS, scraped into PBS, and pelleted by centrifugation for 5 min at 3,000 rpm. The cells were resuspended in 400 μl of lysis buffer (see myristoylation section), sonicated, and homogenized on ice. Insoluble material was pelleted and the supernatant was collected and diluted with an equal volume of dilution buffer. All of the soluble lysate was incubated overnight at 4°C with CNBr-activated Sepharose beads ( Pharmacia Biotech, Inc. ) coupled with an equivalent amount (240 μg) of either purified GST or GST/calmyrin. The Sepharose beads were then pelleted by centrifugation and supernatant containing unbound protein was removed. The beads were washed with 2.5 M KCl, resuspended in Laemmli buffer, and 1/2 of the sample was separated by SDS-PAGE and immunoblotted for the presence of PS2 using a goat anti-PS2(NH 2 -terminal) antibody. Duplicate dishes of HeLa cells (plated at ∼3 × 10 5 /100 mM dishes) were transfected with various combinations of pGEM-CMV-calmyrin, pGEM-CMV-PS2, and control vector (a CAT basic expression vector; Promega ). After ∼48 h, floating cells from each dish were harvested by collecting all of the media, centrifuging 5 min at 3,000 rpm, and removing all but ∼0.5 ml of the supernatant. After vortexing, the exact volume of each cell suspension was measured. Cell numbers were counted twice for each sample on a hemacytometer. These cell counts were adjusted according to the initial resuspension volume to give the total number of floating cells per dish. The counts for the two independent dishes of each transfection construct combination were averaged and graphed. There was a direct correspondence between floating cells and apoptotic cells, with ∼85% of floating cells showing positive CytoDEATH staining. Using the yeast two-hybrid system, a human fetal brain cDNA library was screened for proteins that bind the loop region of PS2. Full-length PS2 was unsuitable as bait presumably because it could not be transported into the nucleus due to the presence of hydrophobic transmembrane domains. In addition, as it is one of the most divergent regions between the presenilin proteins, we believed our chances of finding PS2-specific interactors would be increased. After finding that our initial PS2-loop construct (residues 270–361) self-activated transcription, we truncated the bait reducing it to the first 50 amino acids in order to eliminate several acidic residues and designated it PS2-loop B . The PS2-loop B bait construct, the lacZ reporter plasmid, and human fetal brain cDNA library plasmids were transformed into yeast, and out of 1.5 × 10 7 primary transformants screened, 15 putative interactors were isolated. Isolated library prey plasmids were tested for their ability to reproduce the specific interaction phenotype when coexpressed with the loop bait but not with unrelated baits (such as human lamin B). Clones that produced the specific interaction phenotype were sequenced and identified via BLAST homology database search. Interestingly, three of the interactors were independent cDNAs all containing the full coding sequence of a recently identified calcium-binding protein, but with varying NH 2 -terminal untranslated extensions. Two other groups have recovered this calcium-binding protein in yeast two-hybrid screens and have named it CIB, for its calcium- and integrin αIIb–binding ability , and KIP, due to its interaction with eukaryotic DNA-dependent protein kinase, DNA-PKcs . Rather than pick between these two names we have chosen to refer to this protein as calmyrin (for calcium-binding myristoylated protein with homology to calcineurin) because it describes its inherent properties without bias towards its multiple binding partners. To quantify the binding specificity of calmyrin to the PS2-loop and to determine if this protein also interacts with the PS1 loop which is 45% identical in amino acid sequence, we measured the β-galactosidase activity in yeast liquid assays. When cotransformed with calmyrin, the PS2-loop B bait produced an 8.5-fold increase in β-galactosidase activity over the lamin B negative control, while the corresponding region of PS1 (PS1-loop B) produced only a 1.9-fold increase in activity . A PS2–COOH-terminal construct, containing the COOH-terminal 39– amino acid sequence downstream of the eighth TMD also did not appear to interact with calmyrin. To further map the binding site of calmyrin within the PS2-loop, two new baits were constructed which divided the loop into a conserved portion, loop C (28 out of the 31 amino acids are identical to PS1), and a divergent region, loop D . Since calmyrin did not interact preferentially with the comparable loop region of PS1 (PS1-loop B) we expected the calmyrin binding site to be within the divergent region of the PS2-loop sequence (PS2-loop D). To our surprise, the PS2-loop D bait interacted very weakly with calmyrin, a 2.2-fold increase over control . However, the highly conserved region of the PS2 loop, PS2-loop C, produced a 74-fold increase in activity . In comparison, the corresponding PS1-loop C construct increased activity only 5.8-fold. Although the two PS-loop C baits are highly conserved in sequence, they differ by three amino acids, with PS1 containing threonine residues at positions 281 and 291 instead of proline and alanine, respectively , and a leucine instead of an isoleucine at position 282. We investigated how these three divergent residues influenced calmyrin interaction with the PS-loop C region in yeast two-hybrid assays by introducing the PS2 amino acids into the PS1 bait, so that each of the three divergent residues were mutated singly, and in every possible combination, to the corresponding PS2 sequence. These data indicated that all three residues contributed in different and complex ways towards the interaction . Interestingly, calmyrin interaction was restored to approximately half the PS2 level by single mutation of residue 281 to a proline, a residue which would be predicted to introduce a kink in the loop. In comparison, single mutation of residue 282 to an isoleucine did not increase binding to any significant extent, whereas mutation of residue 291 to an alanine increased binding to a third that of the PS2 level. Double mutants confirmed the importance of residues 281 and 291. When both proline and alanine were present together (T281P, T291A) they increased binding substantially, producing an approximately twofold higher level of binding compared with the wild-type PS2-loop bait. This mutant suggests that isoleucine at residue 282 in PS2 may actually compromise binding, as this would be equivalent to the triple substitution (T281P, L282I, T291A; i.e., turning it back to the PS2 sequence). Consistent with this expectation, isoleucine 282, when present together with alanine 291, did not increase binding above that of the latter alone, whereas paradoxically when it was substituted together with proline 281 it increased binding 1.7-fold higher than when proline was substituted alone. The 191–amino acid sequence of calmyrin has a number of notable features . Sequence comparison indicates that calmyrin is most closely related to human calcineurin B, the regulatory subunit of protein phosphatase 2B, sharing 25% identity and 44% overall similarity. The protein contains two complete EF hands, a conserved motif involved in calcium binding, and in fact, was shown to bind radiolabeled calcium in blot overlay assays . The protein also contains an NH 2 consensus myristoylation site, a cotranslational modification involved in targeting proteins to membranes. To verify the size and expression pattern of calmyrin transcripts, Northern blot analysis of poly(A) + RNA isolated from multiple adult human tissues was performed . The calmyrin probe hybridized to an ∼1.2-kb transcript that was ubiquitously expressed in the tissues examined, extending the evidence that it is widely expressed and implying that it plays a common function in most if not all cells. Although mRNA expression was relatively low in brain, a Northern blot of specific brain regions showed that the expression of calmyrin transcripts was easily detectable and fairly uniform . To study further the calmyrin protein, rabbit polyclonal anti-calmyrin antibodies were generated to affinity-purified GST/calmyrin fusion protein . By immunoblotting, these antibodies appear to be highly specific for calmyrin as they reacted only with the appropriately sized polypeptides (∼22-25 kD) in HeLa cells overexpressing calmyrin cDNAs . Lane 1 of Fig. 3 B shows that at the depicted exposure time the antibodies failed to detect any endogenous calmyrin in untransfected lysate. Only after prolonged exposure did a faint calmyrin band appear (data not shown), indicating that endogenous levels of this calcium-binding protein are relatively low in HeLa cells. However, consistent with our Northern blot analysis, an endogenous immunoreactive band at ∼22 kD was detected in human adult brain lysate . The anti-calmyrin antibodies also successfully detected the mouse form of this protein in several mouse tissue lysates due to the high conservation between the human and mouse calmyrin proteins . The significance of the faster migrating immunoreactive band in mouse skeletal muscle has not been determined. Since the subcellular localization of calmyrin was unknown, we used the anti-calmyrin antibody to determine its distribution in mammalian cells by indirect immunofluorescence microscopy. In primary cultures from mouse heart tissue endogenous calmyrin localized to the nucleus and in a reticular-like pattern throughout the cytoplasm . This staining was clearly distinguishable from the nonspecific background produced when probing with rabbit preinnoculation serum (data not shown), and moreover, this staining pattern was reproduced by overexpression of calmyrin upon transfection (see below). As PS2 is a transmembrane protein and our yeast two-hybrid findings indicated that calmyrin interacts with PS2, the membrane targeting potential of the consensus myristoylation site in calmyrin especially intrigued us. To determine whether calmyrin is myristoylated in vivo, we added 3 H-myristic acid to the media of HeLa cells transfected with untagged calmyrin. For comparison, HeLa cells were also transfected with calmyrin constructs that had myc tags fused at either the NH 2 - or COOH-terminal ends of the protein. The prediction was that the myc tag (MEQKLISEEDLN) fused at the NH 2 -terminal end would disrupt myristoylation since it moved the glycine residue that is essential for myristoylation more downstream . After 24 h, the cells were lysed and calmyrin was immunoprecipitated with the anti-calmyrin antibody. Myristoylated proteins were visualized by fluorography after SDS-PAGE . The fluorograph of labeled HeLa cell lysates indicated immunoprecipitated C-myc– tagged calmyrin and untagged wild-type calmyrin were myristoylated as evident by incorporation of the radioactive 3 H-myristic acid label (band in lanes 4 and 6 indicated by an arrows) while, as expected, the N-myc tagging of the protein prevented myristoylation (absence of band in lane 2). The lower panel of this figure contains an immunoblot of these same HeLa cell lysates to show that both NH 2 - and COOH-terminally tagged calmyrin proteins were expressed efficiently and to equivalent levels, whereas untagged calmyrin accumulated at lower protein levels, explaining the fainter myristoylated calmyrin band seen in lane 6 as compared with lane 4. In fact, when the ratio of calmyrin protein to radioactive 3 H-myristic acid labeling is compared for C-myc–tagged and wild-type calmyrin proteins they are similar, which is expected since myristoylation is thought to occur cotranslationally . The reason for higher expression of C- and N-myc– tagged calmyrin proteins is not known but perhaps fusion of the myc epitope affects protein stability or toxicity, allowing the proteins to accumulate to higher levels. Similar attempts to demonstrate myristoylation of endogenous calmyrin in mouse and human cells were unsuccessful, presumably because of low protein expression or the slow turnover of the protein. Once we established that calmyrin was indeed myristoylated, next we determined whether this protein was associated with the membranes of fractionated cells. Transfected HeLa cells were fractionated in the absence of any detergents into a soluble (cytosolic) supernatant and an insoluble (membrane and cytoskeletal) pellet. Equivalent amounts of supernatant and pellet cell fractions were separated by SDS-PAGE and immunoblotted for the presence of lamins and calmyrin . Lamins A and C, cytoskeletal components used as a control of the fractionation process, were detected as 68- and 66-kD polypeptides in the insoluble pellet as expected . The majority (>85%) of the calmyrin was found in the insoluble fraction. Since this manner of cell fractionation does not distinguish membrane components from other insoluble structures, the cells were also fractionated in the presence of 1% Triton X-100 which solubilizes membranes. After this procedure, the calmyrin protein shifted to the soluble (membrane) fraction whereas the lamins, as expected, remained insoluble . The same fractionation was performed on primary cultures of mouse kidney and showed an analogous pattern of membrane localization for endogenous calmyrin . Interpreted together, these cell fractionation results provide strong biochemical evidence that calmyrin is associated with cell membranes. On account of the faint staining of endogenous calmyrin in primary and established cell cultures, calmyrin was forcibly expressed in HeLa cells by transient transfection of untagged and myc-tagged calmyrin constructs for further immunofluorescent localization studies. As seen in Fig. 5 A, cells expressing untagged calmyrin had strong staining in the nucleus and cytoplasm, a pattern very similar to the subcellular localization of endogenous calmyrin detected in mouse cells. At higher magnification, many of these transfected cells showed clear calmyrin staining of thin projections from the cell surface as well as a reticular staining in the cytoplasm consistent with membrane targeting to the plasma membrane and ER . Cells expressing C-myc calmyrin had greater variation in staining with many showing prominent localization to the ER and plasma membrane and often less staining in the nucleus . Double immunofluorescence staining for calreticulin, an ER marker protein, and calmyrin showed that within the cytoplasm a notable portion of calmyrin colocalized with calreticulin (data not shown), corroborating the impression that in these transfected cells calmyrin localization includes, but is not limited to, ER membranes. In contrast, cells expressing N-myc calmyrin showed predominant nuclear staining, more diffuse cytoplasmic staining, and less staining at the plasma membrane which was especially evident in low expressing cells . This observed reduction in membrane association was not surprising considering our previous finding that this NH 2 -terminally tagged construct failed to be myristoylated. To address whether the bright nuclear staining was due to calmyrin localization within the nuclear envelope or throughout the nucleoplasm, we double stained wild-type calmyrin transfected HeLa cells for calmyrin and lamins A/C. According to confocal microscopy, lamins A and C had rim fluorescence consistent with their known localization as a caged meshwork of filaments tethered to the inner nuclear envelope . In the same confocal Z-section (1.0-μm section) where lamins had rim fluorescence, calmyrin immunoreactivity was present throughout the cell and clearly within the nucleoplasm . Overall, these results indicated calmyrin localizes to many different cellular compartments, consistent with the protein having dynamic targeting properties. Of particular interest to this study was the comparison of calmyrin and PS2 staining patterns when overexpressed individually in HeLa cells. Although the two staining patterns overlapped in part, especially the ER reticular staining of untagged and C-myc calmyrin, PS2 staining was readily distinguishable by its exclusive ER and nuclear envelope staining pattern . When calmyrin was coexpressed with PS2, its staining pattern was dramatically altered such that it colocalized almost completely with PS2 . As exemplified by the two cells shown in panels A and B, the calmyrin protein was less apparent in the nucleus in coexpressing cells than in cells transfected solely with calmyrin . Another indication that these two proteins bind each other was seen in a small subset of cells where calmyrin and PS2 colocalized distinctively in unusual intranuclear spots . The intranuclear spots did not colocalize with anti-centromere staining by double immunofluorescent microscopy (data not shown) suggesting that they are distinct from the PS-immunoreactive structures observed by Li et al. . The shift in calmyrin localization and the nearly identical staining patterns between PS2 and calmyrin (see merged images) in these coexpressing cells provide persuasive evidence that these two proteins interact in vivo. Furthermore, when calmyrin was cotransfected with a PS2 construct deleted of the loop and all sequence COOH-terminal of it, the staining patterns displayed significantly less overlap; as seen by patchy aggregates of PS2 which excluded calmyrin . The failure of this PS2 deletion construct to completely colocalize with calmyrin in aggregates, which contrasts with the colocalization of the wild-type PS2 protein and calmyrin in nuclear inclusions, enhances our view that the PS2-loop region facilitates binding of calmyrin. Despite results from yeast two-hybrid assays, cell fractionation experiments, and histological colocalization, which all consistently argue for an interaction between calmyrin and PS2, our initial attempts at demonstrate binding of the two proteins in vitro proved difficult. After trying various combinations of affinity chromatography and immunoprecipitation with GST fusion proteins , in vitro translated proteins, and HeLa cell–expressed proteins under several different buffer conditions, two of these experiments provided further evidence for the binding of calmyrin and PS2. First, HeLa cell lysates of overexpressed PS2 were incubated with purified GST-calmyrin, or GST alone, that had been covalently coupled to Sepharose. The two Sepharose columns were then washed, and retention of PS2 was determined by immunoblotting with anti-PS2 antibody. Fig. 7 A shows that GST-calmyrin Sepharose bound PS2 with approximately threefold greater affinity (lane 4, see arrow) than control GST-coupled Sepharose (lane 3). The second verification of binding was the coimmunoprecipitation of myristoylated calmyrin protein from cotransfected HeLa cell lysates with anti-PS2 antibodies . The myristoylated calmyrin protein did not immunoprecipitate when the preimmune anti-PS2 serum was used but, as expected, could be immunoprecipitated with the anti-calmyrin antibody . Since we had previously shown that overexpression of PS2 in HeLa cells causes apoptosis , we wished to determine what effect overexpression of calmyrin would have on cell viability. To detect apoptosis we used the M30 CytoDEATH antibody. This mouse monoclonal binds an epitope of cytokeratin 18 which is exposed only after caspase cleavage, an early event in apoptosis . Consistent with our previous findings, Fig. 8 A shows that a subset (two out of three) of cells overexpressing PS2 appeared apoptotic (notably only those that had rounded up) according to both CytoDEATH positive staining and condensed nuclei. Similarly, when calmyrin was overexpressed, analogous apoptosis was observed . Cotransfection of PS2 and calmyrin induced even higher apoptosis. To convey more clearly the high levels of apoptosis that resulted from overexpressing these two proteins, example images captured at low magnification are provided. Fig. 8 C shows that at 16 h after cotransfection almost 13% of PS2-expressing cells (which presumably also expressed calmyrin since PS2 and calmyrin staining showed a near 1:1 correspondence [data not shown]) on coverslips were positive for CytoDEATH staining. By 40 h the proportion of apoptotic cells had increased to ∼50% of the PS2-stained cells. The high level of apoptosis seen on coverslips was striking, especially since this method only captured a brief “window” of the cells progression into apoptosis as during programmed cell death HeLa cells lose their adherence on coverslips and float away into the media. This phenomenon explains the reduction in total cells, and most notably PS-expressing cells (only 10 cells), remaining on the coverslip at 40 h after cotransfection . In the examples shown in Fig. 8 only a subset of PS overexpressing cells were apoptotic, indicating a time-dependent process, whereas the corollary that all apoptotic cells were also overexpressors always held true. In contrast when a control protein, the neurofilament light (NF-L) subunit, was overexpressed in HeLa cells , minimal apoptosis (< 1%) was detected and total cell counts and expression levels remained high even at 40 h. It is remarkable that the single apoptotic cell in this field did not stain for NF-L and that, conversely, there are several rounded-up and highly expressing cells (presumably those in mitosis), none of which appeared apoptotic. Fields of cells overexpressing calmyrin or PS2 individually showed levels of apoptosis above the neurofilament background, but less than coexpressors. Unfortunately, due to variation between (and even within) coverslips, this method did not prove suitable for statistically significant quantification. Because CytoDEATH labeling on coverslips only captured a narrow “window” of cells undergoing apoptosis, we decided to quantify the total amount of cell death accumulated over time by counting the total number of floating cells in the media after transfection with various amounts of plasmid DNAs encoding calmyrin and PS2. This simple method was more reliable in quantifying cell death. As graphed in Fig. 9 , transient overexpression of PS2 increased cell death in a dose-dependent manner, whereas cell death induced by calmyrin overexpression reached a plateau at 10 μg of transfected DNA. More interestingly, when both proteins were coexpressed in the linear cell death range of their respective DNAs, cell death increased 5.9-fold over the control, compared with 2.7- and 2.4-fold for the same respective transfection amounts of calmyrin and PS2 individually, suggesting that these two proteins have additive effects in promoting cell death. When these floating cells were collected and stained with the CytoDEATH antibody, ∼85% of the cells stained positive for this marker of apoptosis, bolstering our belief that counting floating cells is a reliable measure of cell death. In this study, we demonstrate by several criteria that human PS2 protein interacts with a recently discovered calcium-binding protein which we refer to as calmyrin. First, calmyrin interacts with PS2-loop sequence in yeast two-hybrid assays. Second, the two proteins bind to each other by affinity chromatography and can be coimmunoprecipitated. Third, the two full-length proteins colocalized when coexpressed in vivo. The interaction of calmyrin with PS2 is also noteworthy since it is the first protein, to our knowledge, that interacts preferentially with PS2 (at least by yeast two-hybrid analysis) suggesting distinct functions for the highly homologous presenilin proteins. Two lines of evidence favor the PS2-loop region as the critical site of calmyrin interaction: reduced in vivo colocalization when calmyrin was coexpressed with a loop-deficient PS2 construct and increased yeast liquid culture binding of calmyrin to the PS2-loop rather than the PS2– COOH-terminal domain. Deletion analysis indicated that calmyrin binding was mediated primarily by the NH 2 -terminal 31 amino acids of the PS2-loop. Remarkably, despite only a three–amino acid difference, the comparable loop region of PS1 interacted with less than one-tenth the strength in similar yeast two-hybrid assays. Site-directed mutagenesis in which the three divergent PS2 residues were introduced singly and in double combinations into PS1 indicated that, in fact, all three amino acids produce variable affects on the specificity of calmyrin for PS2. Particularly interesting was the pronounced increased in binding conferred by the conversion of a threonine at positions 281 and 291 in PS1 to proline and alanine, respectively. In contrast, the leucine at position 282 in PS1 when converted to isoleucine as in PS2 caused pleotrophic effects, increasing, decreasing, and inducing no change in interaction depending on its context with the other two residues. These data suggest that minor alterations in the sequence of the PS loop induce conformational changes in this region with dramatic consequences to protein–protein interactions. Furthermore, the loop region is a site associated with several PS-processing phenomena, including proteolytic cleavage, caspase cleavage, as well as abnormal splicing . Apart from calmyrin, several other proteins have been found to interact with the PS-loop, namely, γ-catenin, filamin, calselinin, mu-calpain, and armadillo protein p007 . Our data showing that minor (single amino acid) alterations in the loop sequence can produce dramatic changes in protein binding not only has implications in terms of calmyrin function, but may also have important consequences for the other processing events and binding partners associated with this region. Therefore, it is not surprising that many FAD mutations map to the PS1 loop. In addition to the importance of protein–protein interactions for localization, our immunofluorescence microscopy and biochemical fractionation studies indicate that the myristoylation of calmyrin is important for the dynamic targeting of this protein to several subcellular compartments including: the cytoplasm, long projections of the plasma membrane, and the nucleoplasm. Elegant studies of recoverin, a myristoylated calcium-binding protein involved in signaling in the retina, have established that this protein alternates between conformations in which the myristoyl group is exposed or sequestered, conformations that are dependent on calcium binding . These calcium-myristoyl switches are a known mechanism for protein targeting and signal transduction. Radiolabeling and biochemical studies show that calmyrin is myristoylated and associated with membranes. At present, we cannot clearly tease apart the roles that myristoylation and protein–protein interactions play in the in vivo targeting of calmyrin to membranes. In fact, our evidence suggests that both are important. Yeast two-hybrid assays with loop constructs containing site-directed mutations clearly show the importance of protein–protein interactions in mediating the association between calmyrin and the integral membrane protein, PS2. Additionally, fusion of the Gal4-acidic blob sequence at the NH 2 -terminal end of calmyrin in the yeast two-hybrid clones would be expected to prevent this fatty acid modification suggesting that myristoylation is not essential for the interaction. Paradoxically, however, it is the myristoylated form of calmyrin that we were able to show coimmunoprecipitated with PS2. Perhaps insertion of the myristoyl group into the lipid bilayer initiates a conformational change that enhances the affinity of calmyrin for PS2. Analogously, the Gal4-acidic blob may have maintained calmyrin in the conformation that was more prone to binding PS2. Cells overexpressing calmyrin proteins capable of being myristoylated showed greater variation in staining patterns often with increased targeting of calmyrin to the cytoplasm and plasma membrane suggesting that myristoylation may be involved in this dynamic behavior. The calmyrin that localized to the cytoplasm had a reticular-like staining pattern which colocalized with PS2 staining when the two proteins were coexpressed. We believe that the reticular staining represents targeting of calmyrin protein to the ER since we and others have shown that overexpressed PS2 was localized to the nuclear envelope and ER . Interestingly, in PS2-cotransfected cells relatively little calmyrin was present in the nucleus, and instead, the entire population almost completely colocalized with PS2 at the ER. This redistribution to the ER is consistent with the stoichiometric change of binding sites available for calmyrin once PS2 was overexpressed. However, the possibility that PS2 expression may alter processing or intracellular targeting of calmyrin can not be ruled out. As myristoylation is known to be important for membrane targeting, it is curious that a significant pool of calmyrin is present within the nucleoplasm despite the lack of a classical nuclear localization signal. Calmyrin is small enough to passively diffuse through the nuclear pores (proteins ∼65 kD and larger must be actively transported) and may be sequestered within the nucleoplasm by binding to nuclear resident proteins such as DNA-PKcs which has also been shown to bind calmyrin in yeast two-hybrid assays . The localization of calmyrin to the long projections of the plasma membrane may similarly represent binding to calmyrin's other known interactor, αIIb-subunit of integrin. These results indicate that calmyrin may traffic between several proteins and factors suggesting a role for calmyrin in complex signaling processes. How these processes relate to AD, and/or apoptosis is not known but it is interesting that Volado, a novel integrin which dynamically mediate cell adhesion and signal transduction, was recently identified as a new memory mutant in Drosophila . Also DNA-PKcs is the only known eukaryotic protein kinase activated by DNA double-strand breaks which is a lesion induced during apoptosis . The possibility that calmyrin, integrins, DNA-PKcs, and PS2 are in any way connected or involved in human diseases is intriguing, yet speculative. In hypothesizing a physiologic role for the interaction between PS2 and calmyrin, we are especially interested in exploring the involvement of calcium and apoptosis. It is noteworthy that calsenilin, another Ca 2+ -binding protein with sequence similarity to recoverin, binds to the COOH-terminal region of presenilin proteins and like calmyrin redistributes with presenilin proteins in cotransfected cells . Calmyrin does not share a high degree of amino acid similarity to calsenilin, instead the protein sequence of calmyrin is most homologous to human calcineurin B, the regulatory subunit of the Ca 2+ -calmodulin–dependent protein phosphatase 2B, which plays important roles in stress, apoptosis, cell calcium signaling, and signal transduction . The greatest homology is found in the regions surrounding calcineurin B's four calcium-binding EF hand motifs and its NH 2 -terminal myristoylation site. Although these regions are relatively well conserved, sharing 44% overall similarity, calmyrin appears to have only two functional EF hands as the two NH 2 -terminal motifs contain several insertions that are predicted to disrupt calcium binding. Naik et al. have demonstrated that calmyrin can indeed bind calcium, but it is unknown whether this property regulates phosphatase activity. If calmyrin behaves similarly to calcineurin B in phosphatase regulation, it may have some relevance to AD where there is speculation that PHF formation and tau hyperphosphorylation occurs due to misregulation of protein phosphatases or kinases . Our cell death findings imply that the binding of calmyrin to PS2 may be related to PS2 function in apoptosis. In a previous study we found that overexpression of PS2 in HeLa cells induced apoptosis. The current finding that coexpression of calmyrin with presenilins in HeLa cells increased apoptosis suggests that the two act in concert in a pathway or pathways regulating cell death. Although we have not determined the pathway through which the two proteins function during programmed cell death, the fact that calmyrin is a calcium-binding protein raises some obvious possibilities. First, calmyrin may “sense” Ca 2+ changes and subsequently regulate PS2 function. Alternatively, PS2 proteins (including FAD mutants) may alter calcium homeostasis resulting in a change in calcium binding by calmyrin which could then trigger a signal transduction cascade. This latter possibility is attractive since overexpression of presenilins has been shown to cause perturbations in calcium homeostasis . Interestingly, the apoptosis rescue screen in which the PS2 ALG3 fragment was isolated (see introduction) yielded another cDNA named ALG2, which caused antisense inhibition of a calcium-binding protein . However, when ALG2 was expressed in the sense orientation, this calcium-binding protein induced apoptosis . It could be argued that coexpression of any calcium-binding protein with presenilins would cause increased cell death. This is clearly not the case as overexpression of another calcium-binding protein, calbindin D28k, suppressed the proapoptotic functions of PS . An imbalance in calcium regulation could be catastrophic to the cell due to the central role calcium plays in cellular processes including its participation in the induction phase of apoptosis . Although there is some disagreement as to whether AD involves a perturbation of calcium regulation , the consensus of research (opinion) is indicative of such a defect. The uncertainty is in part complicated by lack of reliable and easy methods to measure intracellular calcium, let alone compare them in different individuals. Nevertheless, numerous studies have shown that calcium levels are altered in cells cultured from AD patients, especially those harboring (or transfected with) presenilin genes containing FAD-linked mutations . In summary, our results suggest that calmyrin, a calcium-binding myristoylated protein, may play dynamic and diverse roles in intracellular signaling, and we propose that it is important in the modulation of presenilin function. Understanding the complex interplay between calcium regulation, apoptotic signaling, and protein–protein interactions will no doubt aid in deciphering the mechanisms through which the presenilins function which in turn could provide insight into the pathogenesis of Alzheimer's disease. | Other | biomedical | en | 0.999999 |
10366600 | Reagents were obtained from the following sources: medium 199 modified Earle's salt solution ( Gibco Life Technologies); Clonetics EGM-2 medium (TCS Biologicals Ltd.); Nutridoma NS ( Boehringer Mannheim Ltd.); human fibronectin, heparin, endothelial cell growth supplement, bromodeoxyuridine (BrdU), cytochalasin D, 2,3-butanedione 2-monoxime, TRITC-phalloidin, 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid), and mouse monoclonal anti–human HLA class I antigen antibody ( Sigma Chemical Co. ); TNF-α (Insight Biotechnology); mouse monoclonal anti-CD45 and anti-CD14 antibodies (Leucogate; Becton Dickinson ); mouse monoclonal anti-CD58 antibody and FITC-labeled goat anti–rabbit IgG (Southern Biotechnology Associates); mouse monoclonal anti-CD14 (SH-M1) antibody (Autogen Bioclear UK Ltd.); tetramethylrhodamine dextran with a molecular weight of 10,000 (Molecular Probes); mouse monoclonal anti–E/P-selectin (clone BBIG-E6), mouse monoclonal anti–ICAM-1 (function blocking, clone BBIG-I1), mouse monoclonal anti–VCAM-1 (function blocking, clone BBIG V1), mouse monoclonal anti–human E-selectin (function blocking, clone BBIG-E4), mouse monoclonal anti–human P-selectin (function blocking, clone 9E1), goat anti–human VCAM-1 polyclonal antibody, and goat polyclonal anti– ICAM-1 antibody (R&D Systems); mouse monoclonal anti-myc (9E10) antibody ( Santa Cruz Biotechnology ); FITC- and TRITC-labeled goat anti–mouse and donkey anti–goat antibodies (Jackson ImmunoResearch Laboratories); ML-7 ( Calbiochem ); Limulus Amoebocyte Lysate test (Biowhittaker Inc.); protein assay kit (Bio-Rad); enhanced chemiluminescence kit ( Amersham International plc ); and polystyrene plates (Costar Corp.). Rabbit polyclonal antiezrin, antimoesin, and antiradixin antibodies were generously provided by Paul Mangeat (Montpellier, France); pcDNA3 N19RhoA (myc epitope–tagged) was a kind gift from Alan Hall (London, UK). HUVECs were a kind gift from Ruggero Pardi (Milano, Italy). The cells were cultured in TC Nunclon flasks coated with 10 μg/ml human fibronectin in medium 199 modified Earle's salt solution containing 1.25 g/liter NaHCO 3 and Glutamax and supplemented with 20% FCS, 100 μg/ml endothelial cell growth supplement, 1% Nutridoma NS, and 100 μg/ml heparin. Cells were cultured at 37°C in humidified air containing 5% CO 2 . For experiments cells were used between 3 and 7 passages. MMVE-tsLT cells (kindly provided by Catherine Clarke, London, UK) were human mammary microvascular endothelial cells expressing a temperature-sensitive SV-40 large T antigen to extend their life span in culture. MMVE-tsLT cells were cultured in EGM-2 medium and used for experiments up to 15 passages. For microinjection experiments cells were grown on glass coverslips coated with 10 μg/ml human fibronectin until confluent. Glass coverslips were marked with a cross using a diamond pen to facilitate the localization of microinjected cells. To obtain quiescent cells, the culture medium was replaced by medium containing 10% FCS but no heparin or other growth factors. Cells were incubated in this medium for 72 h and tested for DNA synthesis by incubation with 10 μM BrdU for 24 h. The cells that had incorporated BrdU were visualized as described previously . Over 97% of cells were quiescent and did not incorporate BrdU. Single donor platetephoresis residues were purchased from the North London Blood Transfusion Service. Mononuclear cells were isolated by Ficoll-Hypaque centrifugation (specific density, 1.077 g/ml) preceding monocyte separation in a Beckman JE6 elutriator. Monocyte purity was assessed by flow cytometry using directly conjugated anti-CD45 and anti-CD14 antibodies and was routinely >85%. All media and sera were routinely tested for endotoxin using the Limulus Amoebocyte Lysate test and rejected if the endotoxin concentration exceeded 0.1 U/ml. To activate endothelial cells, TNF-α was added at 100 ng/ml for 4 or 24 h. Cell viability was tested after treatment with TNF-α by a trypan blue exclusion test. After 4 h endothelial cells were washed four times in culture medium to remove TNF-α and then purified human monocytes were added at 5 × 10 5 cells/ml and incubated for a further 2 h before fixation. The monocyte/endothelial cell ratio in cocultures was 3.2 ± 0.2. Where indicated, cytochalasin D at 0.05 μg/ml was added to cell cultures 3 h after addition of TNF-α and incubated for 1 h. C3 transferase was added to culture medium at 15 μg/ml 1 h after the addition of TNF-α and incubated together with TNF-α for a further 3 h. To determine the contribution of each monocyte-binding receptor to monocyte adhesion, endothelial cells were incubated for 1 h with function-blocking antibodies against E-selectin, P-selectin, ICAM-1, and VCAM-1 at 10 μg/ml before the addition of monocytes. The number of adherent monocytes and monocyte spread area was determined using a confocal laser scanning microscope (MRC500; Bio-Rad). To determine changes in the spread area of monocytes, images of the basal aspect of the cells were collected and the area was measured using the software integrated to the MRC500 (SOM, version 6.42 a) against a Geller MRS-2 magnification reference standard 131 R3 (Agar Scientific Ltd.). To estimate the extent of monocyte adhesion, the number of monocytes bound per endothelial cell was calculated. Cell cultures were stained for F-actin with TRITC-phalloidin and incubated with mouse monoclonal anti-CD14 (SH-M1) antibody diluted 1:100 and FITC-labeled anti–mouse IgG diluted 1:100 in order to facilitate identification of the adherent monocytes. In each experiment >500 endothelial cells were scored to calculate monocyte adhesion, and experiments were performed in triplicate. In some experiments endothelial cells were stained for E- and P-selectin, ICAM-1, and VCAM-1 as described below. The recombinant proteins, V14RhoA, N17Rac1, N17Cdc42, and C3 transferase, were expressed in Escherichia coli from the pGEX-2T vector as glutathione S-transferase fusion proteins and purified as described previously . Protein concentrations were estimated using a protein assay kit (Bio-Rad). Proteins were microinjected into the cytoplasm of quiescent HUVECs 3.5 h after stimulation with TNF-α. After a 15-min incubation, the cells were washed four times in culture medium and monocytes were added to endothelial cell cultures. To identify injected cells, tetramethylrhodamine dextran (molecular weight of 10,000) at 5 mg/ml was microinjected together with recombinant proteins. C3 transferase was microinjected at a concentration of 4 μg/ml, V14RhoA was microinjected at ∼100 μg/ml, N17Rac1 at 7 mg/ml, and N17Cdc42 at 2 mg/ml. In experiments involving receptor clustering C3 transferase was added to the culture medium at 15 μg/ml, 1 h after the addition of TNF-α, and incubated together with TNF-α for a further 3 h. To express N19RhoA, an expression vector containing myc epitope– tagged N19RhoA cDNA (pcDNA3-N19RhoA) was microinjected at 0.05 mg/ml together with tetramethylrhodamine dextran into cell nuclei at the same time as the addition of TNF-α, and cells were incubated for a further 3 h before adding antibodies to induce receptor clustering or for 4 h before assaying monocyte adhesion. Cells expressing N19RhoA were identified with the mouse monoclonal anti–myc epitope antibody 9E10 and FITC-labeled anti–mouse antibody: 84% ± 10% of microinjected cells expressed detectable levels of N19RhoA. To induce receptor clustering, TNF-α was added to endothelial cells and then after 3 h mouse monoclonal antibodies to E-selectin, ICAM-1, VCAM-1, HLA class I antigen, or CD58/LFA-3 were added to cells at a final concentration of 10 μg/ml and incubated for 1 h at 37°C. The mouse monoclonal anti–human E/P-selectin antibody used here recognizes both E- and P-selectin on the surface of endothelial cells. Using mouse monoclonal antibodies that specifically recognized only E- or P-selectin, we determined that TNF-α–activated HUVECs expressed predominantly E-selectin and only very low levels of P-selectin, and therefore the results obtained with the anti–E/P-selectin antibody relate to E-selectin. After incubation with primary antibodies, TNF-α and the primary antibodies were removed from the cell medium and 10 μg/ml of FITC-labeled goat anti–mouse antibody was added to the cells for 30 min. Cells were then washed three times in PBS, fixed with 4% formaldehyde dissolved in PBS for 10 min at room temperature, permeabilized for 6 min with 0.2% Triton X-100, and then incubated with 1 μg/ml TRITC-phalloidin for 45 min to stain actin filaments, or for 1 h with rabbit polyclonal antiezrin, antimoesin, or antiradixin antibodies diluted 1:200, followed by 5 μg/ml TRITC-labeled goat anti–rabbit antibody for 1 h. The specimens were mounted in moviol. To examine the extent of spontaneous receptor clustering upon the addition of the primary antibodies only, TNF-α–stimulated HUVECs were incubated for 1 h with the primary antibodies as described above, and then fixed. Fixed cells were then incubated with the secondary antibody for 30 min, washed, permeabilized, and stained for actin filaments. For controls, nonstimulated HUVECs or HUVECs that were stimulated with TNF-α for 4 h were used. The cells were then fixed, incubated with primary and secondary antibodies, and then permeabilized and stained for actin as described above. In experiments with MLCK inhibitors, ML-7 (40 μM) and 2,3-butanedione 2-monoxime (BDM) (5 mM) were added to cell cultures together with the primary antibody, 3 h after stimulation with TNF-α. After a 1-h incubation, the cells were washed as described before, and the inhibitors were added again together with the secondary antibody and incubated for 30 min. The cells were then washed, fixed, and stained for actin filaments as described above. Confocal laser scanning microscopy was carried out with an LSM 310 or 510 ( Zeiss ) mounted over an infinity corrected Axioplan microscope ( Zeiss ) fitted with a ×10 eyepiece, using either a ×40 NA 1.3 or a ×63 NA 1.4 oil immersion objective. Image files were collected as a matrix of 1024 × 1024 pixels describing the average of 8 frames scanned at 0.062 Hz where FITC and TRITC were excited at 488 nm and 543 nm and visualized with a 540 ± 25 and a 608 ± 32 nm bandpass filters, respectively, where the levels of interchannel cross-talk were insignificant. To measure the cell surface expression of E-selectin, ICAM-1, and VCAM-1 we used an ELISA assay as described by Zund et al. with some modifications. In brief, HUVECs were plated onto fibronectin-coated 96-well polystyrene plates at a density of 2 × 10 4 cells/well and grown for 48 h. The cells were then incubated in starvation medium (10% FCS) for 24 h. The cells were stimulated with TNF-α for 4 or 24 h and, where indicated, cytochalasin D at 0.05 μg/ml was added to cell cultures 3 or 23 h after addition of TNF-α and incubated for 1 h. C3 transferase was added to the culture medium at 15 μg/ml either 1 or 21 h after the addition of TNF-α and incubated together with TNF-α for a further 3 h. In controls, C3 transferase and cytochalasin D were added to nonstimulated cells. Cells were washed three times in PBS and then fixed in 1% paraformaldehyde at 4°C for 15 min. The cells were washed three times in PBS and then incubated overnight with 1% BSA solution in PBS at 4°C. All wells were then washed three times in PBS and incubated for 2 h at room temperature with 200 μl/well of 0.5% BSA solution containing 5 μg/ml of mouse monoclonal anti–human E/P-selectin antibody, mouse monoclonal anti–human ICAM-1 antibody or goat anti–human VCAM-1 antibody. Subsequently, cells were washed three times in PBS and incubated with 200 μl of HRP-conjugated rabbit anti–mouse IgG or donkey HRP-conjugated anti–goat IgG solution at 1:1,000 in 0.5% BSA for 1 h at room temperature. After washing, plates were developed by addition of peroxidase substrate, 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) and OD at 650 nm was determined on a microtiter plate spectrophotometer. Data are presented as mean ± SD (background subtracted). HUVECs were grown to confluence in 40-mm plastic petri dishes precoated with human fibronectin and starved for 24 h in 10% FCS as described above. TNF-α was added as indicated and incubated with cells for 4 or 24 h. Cytochalasin D and C3 transferase were added as described above (cell surface immunoassay). Cells were washed, lysed, and debris was removed by centrifugation at 14,000 rpm. The protein concentration was measured using a Bio-Rad protein assay. Equal amounts of protein were separated by SDS-PAGE on 7.5% polyacrylamide gels under nonreducing conditions, and transferred to nitrocellulose membranes, which then were blocked overnight with 5% nonfat dry milk powder in TBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl) containing 0.05% Tween 20. This was followed by incubation with anti–E/P-selectin, mouse monoclonal anti–ICAM-1, or goat anti–VCAM-1 antibodies diluted 1:500 in TBS-Tween containing 1% dried milk powder for 2 h. Blots were then washed three times in TBS-Tween and incubated for 1 h at room temperature with HRP-conjugated sheep anti–mouse antibodies diluted 1:2,000. Membranes were developed using an enhanced chemiluminescence kit ( Amersham International ). Human monocytes showed low levels of adhesion to unstimulated, quiescent HUVECs . The few adherent cells retained a regular, rounded morphology similar to that observed in a suspension of freshly isolated monocytes . Monocyte adhesion increased sevenfold after stimulation of endothelial cells with TNF-α . To determine whether Rho in endothelial cells plays a role in monocyte adhesion, we treated cells with C3 transferase, an exoenzyme produced by Clostridium botulinum which inhibits Rho by ADP ribosylation . The TNF-α–induced increase in monocyte adhesion was reduced by 55% in HUVECs incubated with C3 transferase . Monocyte adhesion was also inhibited in TNF-α–stimulated HUVECs which were microinjected with C3 transferase 30 min before the addition of monocytes . Under the conditions used here, C3 transferase induced loss of stress fibers but did not induce cell rounding . To provide further evidence for the involvement of Rho in monocyte adhesion, HUVECs were microinjected with a plasmid encoding N19RhoA, a dominant negative RhoA protein. In cells expressing N19RhoA, monocyte adhesion was reduced by 75% compared with control-injected cells . As Rho is known to induce actin reorganization, we specifically investigated the involvement of the endothelial cell actin cytoskeleton in monocyte adhesion by pretreating endothelial cells with cytochalasin D before addition of monocytes. Cytochalasin D inhibits addition of actin monomers to the barbed ends of actin filaments , and as expected it induced a decrease in actin cables in HUVECs . It was necessary to wash out cytochalasin D from the medium before addition of monocytes, to prevent it acting on the monocytes. Control experiments showed that although the effects of cytochalasin D on the actin cytoskeleton of HUVECs were reversible, few actin cables reappeared during the 2-h incubation with monocytes. By 4 h after cytochalasin D removal, however, the actin cytoskeleton was essentially indistinguishable from that of untreated cells (data not shown). Monocyte adhesion was substantially inhibited in HUVECs pretreated with cytochalasin D , indicating that the actin cytoskeleton in endothelial cells is important for promoting monocyte adhesion. Monocytes attached to TNF-α–treated HUVECs showed an approximately twofold increase in their spread area relative to monocytes on unstimulated endothelial cells . Many monocytes become elongated and extended lamellipodia, characteristic of a migratory phenotype . The few monocytes that did attach to C3 transferase– or cytochalasin D–treated activated HUVECs did not spread significantly . As with HUVECs, adhesion of monocytes to nonactivated MMVE-tsLT cells was low and adherent monocytes remained rounded and unspread . The addition of TNF-α to MMVE-tsLT cells increased the number of adherent monocytes fivefold . This effect was almost completely inhibited by the addition of C3 transferase or cytochalasin D to endothelial cells stimulated with TNF-α . Similar to HUVECs, the treatment of MMVE-tsLT cells with C3 transferase or cytochalasin D inhibited monocyte spreading on endothelial cells . As these results indicate that endothelial cell Rho is involved in regulating monocyte adhesion, we investigated whether the related proteins, Rac and Cdc42, also affect this process. Microinjection of TNF-α–activated HUVECs with dominant inhibitory Rac or Cdc42 protein, N17Rac1 or N17Cdc42, did not significantly change monocyte adhesion or spreading . The N17Cdc42 and N17Rac1 protein preparations were active as they were able to inhibit the formation of stress fibers in TNF-α–activated HUVECs . We conclude that Rho but not Rac or Cdc42 in endothelial cells is involved in the formation and maintenance of intercellular adhesions between monocytes and endothelial cells. As our results indicate that Rho is required in endothelial cells for monocyte adhesion, we investigated the effects of introducing activated Rho into endothelial cells. Microinjection of constitutively activated Rho protein, V14RhoA, into quiescent, unstimulated cells did not enhance monocyte adhesion , whereas in TNF-α–treated HUVECs V14RhoA induced a small but significant increase in monocyte adhesion . Monocytes tended to form clusters around the cells microinjected with V14RhoA . This was not a nonspecific consequence of microinjection because injection of fluorescent dextran into TNF-α–activated endothelial cells did not have a significant effect on monocyte adhesion . These results provide further evidence for a role of Rho in promoting monocyte adhesion. To investigate how Rho regulates monocyte adhesion and spreading, we first determined the involvement of different receptors for monocytes in mediating monocyte adhesion to TNF-α–activated monocytes. We focused specifically on E-selectin, ICAM-1, and VCAM-1, as these are the three major monocyte-binding receptors on HUVECs known to be upregulated by TNF-α . As expected from previous studies , quiescent endothelial cells showed very low surface expression levels of E-selectin, ICAM-1, and VCAM-1 . TNF-α increased the surface expression of these three proteins by 4 h after stimulation . The levels of spontaneous receptor clustering were relatively low. ICAM-1 in TNF-α–treated cells tended to accumulate in the region of intercellular junctions . The first changes in the expression of E-selectin were detectable by immunofluorescence 1 h after cell activation (data not shown). An increase in intracellular levels of E-selectin indicating de novo synthesis of the protein was also observed 2 h after stimulation with TNF-α . Addition of function-blocking antibodies against E-selectin, ICAM-1, and VCAM-1 showed that each of these receptors contributed to monocyte adhesion to TNF-α–activated HUVECs. Antibodies against E-selectin, ICAM-1, and VCAM-1 inhibited monocyte adhesion by 17% ± 5%, 34% ± 9%, and 24% ± 8%, respectively. Anti–P-selectin antibodies did not significantly reduce monocyte adhesion, reflecting the low level of P-selectin expression in HUVECs (see Materials and Methods). Inclusion of all four antibodies against E-selectin, P-selectin, ICAM-1, and VCAM-1 inhibited monocyte adhesion by 61% ± 12%. Similar results with adhesion-blocking antibodies carried out in stationary (nonflow) conditions have been reported previously . These results indicate that E-selectin, ICAM-1, and VCAM-1 are major monocyte-binding receptors on TNF-α–activated HUVECs, although this does not rule out a contribution of other receptors such as ICAM-2, CD31/PECAM-1, or αvβ3 in monocyte–endothelial cell interactions . One possible explanation for the decreased binding of monocytes to endothelial cells treated with C3 transferase or cytochalasin D is that there are lower levels of monocyte-binding receptors on the cell surface. To investigate this possibility, a cell surface immunoassay was performed on fixed monolayers of TNF-α–activated HUVECs . Endothelial cells were stimulated with TNF-α for 4 or 24 h either with or without addition of C3 transferase for the last 3 h or cytochalasin D for 1 h before fixation. Analysis of receptor levels by ELISA showed that neither C3 transferase nor cytochalasin D induced any significant changes in the surface expression of E-selectin , ICAM-1 , or VCAM-1 . Western blot analysis of TNF-α–treated HUVECs showed that the overall level of expression of E-selectin, ICAM-1, or VCAM-1 in HUVECs was also not altered by treatment with C3 transferase or cytochalasin D (data not shown). Clustering of E-selectin in endothelial cells has been postulated to be an initial step leading to the linkage of receptors to the cytoskeleton and stabilization of cell adhesion . We observed an accumulation of E-selectin on the surface of activated HUVECs around the margin of adhering monocytes and an overall increase in receptor clustering often unrelated to the position of a monocyte . The position of adhering monocytes was determined by F-actin staining . ICAM-1 also accumulated along the margin of attached monocytes outlining fine protrusions formed by the monocyte membrane . In contrast, VCAM-1 did not significantly change its distribution upon the adhesion of monocytes. Clustering of receptors was seen only in a few places where monocytes were attached and the positions of clusters were unrelated to monocyte margins . In endothelial cells that were treated with C3 transferase, E-selectin accumulation and clustering of ICAM-1 at the margin of the few adhering monocytes were much reduced. This lack of clustering could be a consequence of weaker monocyte adhesion which is unable to signal sufficiently to induce clustering, or represent a requirement for Rho in the process of clustering, which in turn is required for stable adhesion. We have shown that although C3 transferase and cytochalasin D did not change the expression levels of monocyte-binding receptors, they inhibited the binding and spreading of monocytes on endothelial cells and inhibited receptor clustering around the adherent monocytes. To investigate further the role of Rho in regulating receptor clustering we mimicked clustering induced by adhering monocytes by incubating HUVECs with primary antibodies against E-selectin, ICAM-1, and VCAM-1 and then cross-linking them with fluorescently labeled secondary antibody. This technique of clustering membrane receptors with specific antibodies has been described previously . Treatment of TNF-α–activated HUVECs with antibodies against E/P-selectin induced spontaneous clustering of E-selectin which was enhanced by the addition of secondary, cross-linking antibodies . Clustering of E-selectin with primary antibodies alone or with primary antibodies followed by secondary antibodies was significantly reduced by the treatment of endothelial cells with C3 transferase or cytochalasin D . Both C3 transferase and cytochalasin D also inhibited stress fiber formation, but under the conditions used did not induce cell rounding or detachment . ICAM-1 in TNF-α–treated HUVECs incubated with only the primary antibody was localized mainly in the intercellular junctions . Addition of secondary (cross-linking) antibodies caused a disappearance of ICAM-1 from the junctions and clustering of the receptors on the cell surface . This was not a consequence of loss of intercellular junctions, as VE-cadherin localization to junctions was not altered by ICAM-1 cross-linking (data not shown). C3 transferase significantly inhibited antibody-induced clustering of ICAM-1 on the cell surface . It also reduced the localization of ICAM-1 to cell junctions, although again VE-cadherin localization was not altered (data not shown). Expression of dominant negative N19RhoA protein in endothelial cells also inhibited antibody-induced ICAM-1 clustering and E-selectin clustering (data not shown), providing further evidence that Rho plays a specific role in regulating receptor clustering. Some clusters of VCAM-1 were present on the surface of TNF-α–activated HUVECs treated with anti–VCAM-1 antibody , but addition of secondary antibodies caused a marked increase in VCAM-1 clustering . Pretreatment of the cells with C3 transferase or expression of N19RhoA (data not shown) inhibited clustering of VCAM-1 induced by the secondary antibodies. Clustering induced by the primary anti–VCAM-1 or anti– ICAM-1 antibodies alone was also inhibited by C3 transferase, and ICAM-1 and VCAM-1 clustering was similarly inhibited by cytochalasin D treatment (data not shown). Cross-linking of E-selectin, ICAM-1, and VCAM-1 with antibodies was accompanied by increased stress fiber accumulation and appearance of intercellular gaps, indicative of increased contractility . Adhesion of monocytes to TNF-α–activated endothelial cells also induced stress fiber formation , consistent with previous observations . Antibody-induced clustering of HLA class I antigen or CD58/LFA-3, a receptor of the Ig superfamily , did not induce stress fiber formation, showing that this is not a general response to receptor clustering but is restricted to specific receptors (data not shown). C3 transferase inhibited the formation of stress fibers induced by the use of cross-linking antibodies or by monocyte adhesion , indicating that Rho is required for this response. To investigate whether the mechanism of receptor clustering on HUVECs is linked to stress fiber formation and/or is dependent on the activity of MLCK, as was suggested for the Rho-mediated assembly of integrins into focal contacts , we used two MLCK inhibitors ML-7 and BDM. ML-7 is a potent and selective inhibitor of both Ca 2+ -dependent and -independent MLCKs . BDM acts as an inhibitor of muscle myosin ATPase activity . Both inhibitors caused a loss of stress fibers in HUVECs, as previously reported , but did not inhibit the antibody-induced clustering of E-selectin , ICAM-1 , or VCAM-1 (data not shown). These results show that neither Rho-mediated stress fiber formation nor myosin-dependent contractility is necessary for receptor clustering, implying that Rho independently induces the clustering of monocyte-binding receptors and stress fiber formation. Therefore, we sought to determine whether F-actin or associated proteins were detectably linked with clusters of monocyte-binding receptors. The localization of E-selectin , ICAM-1, and VCAM-1 (data not shown) after antibody-induced receptor clustering was not related to stress fibers in HUVECs . Some large clusters of E-selectin , ICAM-1, and VCAM-1 (data not shown) colocalized with F-actin, and in these cases the F-actin often appeared to form a ring around the cluster . However, F-actin did not detectably colocalize with most clusters of receptors, suggesting that if they are linked with the actin cytoskeleton this does not require the presence of large actin-containing structures. Ezrin/radixin/moesin (ERM) proteins can provide a link between some membrane receptors and the actin cytoskeleton, and in particular ICAM-1 has been reported to interact with ezrin in vitro . Antibody-induced clusters of ICAM-1 , VCAM-1 , and E-selectin (data not shown) often colocalized with moesin , ezrin, and radixin (data not shown). This was observed with both small and large clusters of receptors . This colocalization of ERM proteins with monocyte-binding receptors was not a nonspecific consequence of antibody-induced receptor clustering, as antibody-induced clusters of HLA class I antigen did not colocalize with moesin , ezrin, or radixin (data not shown). Similarly, clusters of CD58/LFA-3 did not colocalize with ERM proteins (data not shown). In vivo, leukocyte adhesion to endothelial cells is a prerequisite for subsequent transmigration across the endothelium into underlying tissues. In this paper we have demonstrated that Rho in endothelial cells is required for the adhesion and spreading of monocytes, and modulates the clustering of the monocyte-binding receptors, E-selectin, ICAM-1, and VCAM-1, induced by monocyte adhesion or by cross-linking antibodies. This clustering is dependent on the actin cytoskeleton, as it is prevented by cytochalasin D, an inhibitor of actin polymerization, which also inhibits monocyte adhesion. These results demonstrate that responses in endothelial cells activated by receptor engagement are crucial for stable monocyte adhesion, and suggest that Rho may regulate the linkage between monocyte-binding receptors and the actin cytoskeleton to allow the formation of adhesion foci between monocytes and endothelial cells. The precise links between leukocyte-binding receptors and the actin cytoskeleton have not been identified, but ICAM-1, ICAM-2, and E- and L-selectins have all been reported to associate with actin-binding proteins . Clustering of adhesion receptors has been suggested to play a mechanical role in strengthening cell-cell or cell-extracellular matrix adhesion. In fibroblasts, tension transmitted via extracellular matrix proteins to integrins can strengthen their linkage to the cytoskeleton, and lead to further clustering of integrins . Similar responses may occur after leukocyte binding to endothelial cells. E-Selectin clusters and associates with the actin cytoskeleton during leukocyte adhesion; this linkage increases the stress resistance of the ligand-receptor binding and can be inhibited by cytochalasin D . We have observed clustering of both E-selectin and ICAM-1 at the margin of adhering monocytes, and in addition found that F-actin colocalized with large clusters of antibody cross-linked receptors, suggesting association of these clusters with the actin cytoskeleton. Clusters of E-selectin, ICAM-1, and VCAM-1 also colocalized with ERM proteins, which are known to interact with F-actin . Association of ERM proteins with the clustered receptors is receptor specific as we did not observe any colocalization of ERM proteins with clusters of HLA class I antigen or with CD58/LFA-1, a receptor for CD2 . The involvement of F-actin in the cross-linking of monocyte-binding receptors and strengthening of monocyte-endothelial adhesion is further supported by the observation that clustering is inhibited by C3 transferase and cytochalasin D. VCAM-1 did not localize at the margins of adherent monocytes, although its clustering by cross-linking antibodies was also dependent on Rho activity. Therefore, it is likely that monocyte adhesion and spreading on endothelial cells depends initially on Rho-regulated clustering of E-selectin and ICAM-1, and that VCAM-1 plays a role at later stages of monocyte migration. Recent evidence suggests that clustering of leukocyte-binding receptors plays a signaling as well as a mechanical role in endothelial cells. For example, adhesion of monocytes has been reported to induce a transient increase in the cytosolic free calcium concentration and also stress fiber assembly in HUVECs, and these responses are mimicked by incubation with antibodies against E-selectin, VCAM-1, or platelet/endothelial cell adhesion molecule (PECAM-1) . In addition, cross-linking of ICAM-1 on brain endothelial cells was reported to activate Rho and to induce Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 cas . Our observation that stress fiber formation is stimulated in HUVECs after monocyte adhesion or antibody-induced clustering of E-selectin, ICAM-1, and VCAM-1 suggests that these events also activate Rho. Although we have reported previously that TNF-α itself induces stress fiber formation in quiescent HUVECs , this is a transient response and by 4 h after TNF-α addition, when monocytes or cross-linking antibodies were added, the background level of stress fibers was low. As the cross-linking of HLA class I antigen and CD58/LFA-3 did not result in increased stress fiber formation, this response appears to be limited to receptors involved in leukocyte interaction. The mechanisms whereby monocyte-binding receptors transduce signals in endothelial cells have not been established, but interestingly E-selectin can interact via its cytoplasmic domain with paxillin and focal adhesion kinase , which are known to be involved in integrin-mediated signaling and are activated via a Rho-regulated pathway . E-Selectin itself is a target for protein phosphorylation: its cytoplasmic domain contains several potential phosphorylation sites, at least one of which becomes phosphorylated in cytokine-activated HUVECs and may therefore act to recruit signaling proteins . The cross-linking of E-selectin, ICAM-1, and VCAM-1 induces stress fiber formation, but MLCK inhibitors which are known to prevent the formation of stress fibers do not inhibit receptor clustering. This suggests that occupancy of these receptors leads to Rho activation, which then activates at least two separate signaling pathways, one leading to stress fiber assembly and another to receptor clustering. As stress fiber formation is not required for receptor clustering, receptor clustering represents a new response mediated by Rho signaling. Interestingly, introduction of activated V14RhoA into endothelial cells only slightly increased monocyte adhesion, suggesting that endogenous Rho is strongly activated during monocyte binding and that this is sufficient to induce near-maximal monocyte adhesion. Some of the downstream signaling partners involved in Rho-induced stress fiber formation have been identified , and it will be interesting to determine which of these are involved in receptor clustering on endothelial cells. In some circumstances, Rho can be activated indirectly via Cdc42 and Rac , but as the binding of monocytes to endothelial cells was not inhibited by dominant negative inhibitors of Cdc42 and Rac, clustering of receptors is likely to be an effect of direct activation of Rho by receptor engagement. The effect of inhibiting Rho on monocyte binding and receptor clustering closely resembled that caused by cytochalasin D, suggesting that Rho regulates the linkage of the actin cytoskeleton to the surface receptors. Incubation of cells with cytochalasin D leads to gradual loss of actin filaments, as cytochalasin D acts by binding to barbed ends of actin filaments and preventing them from further polymerization or shortening . Interestingly, C3 transferase and cytochalasin D have also been reported to inhibit the clustering of Fcγ receptors on macrophages induced by opsonized particles, and concomitantly inhibit Fcγ receptor–induced protein tyrosine phosphorylation, calcium release, and actin cup formation . Together with our results, this suggests that Rho may be more generally involved in mediating the linkage of receptors to the actin cytoskeleton, and that this linkage and resultant receptor clustering is important for cellular signaling. Precisely how Rho alters receptor clustering is not known. It could be required for the formation of links between receptors and the actin cytoskeleton, once receptors have diffused in the plasma membrane, and thereby stabilize transient clusters of receptors. Alternatively, it could be actively involved in regulating the diffusion of receptors in the plasma membrane. The mechanism by which Rho regulates links between receptors and the actin cytoskeleton could well involve ERM proteins, which interact with both F-actin and several adhesion receptors, including ICAM-1 , and which we have found colocalize with clusters of ICAM-1, VCAM-1, and E-selectin. ERM proteins also interact with RhoGDI, which is normally found in the cytoplasm in complex with Rho proteins, and may thereby facilitate Rho targeting to cell adhesion receptors and subsequent activation . In conclusion, our data indicate that Rho regulates the clustering of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 on the surface of endothelial cells, probably by enhancing their association with the actin cytoskeleton. The assembly of cytoskeletal connections with the clusters of membrane receptors could then provide “footholds” for attached monocytes and create the tension required for their spreading and migration, thereby mimicking the more static nature of extracellular matrix. A similar requirement for association with the actin cytoskeleton has been reported for integrins in focal adhesion complexes and for L-selectin– and LFA-1–mediated adhesion in leukocytes . Clustering of monocyte-binding receptors is not dependent on stress fibers, in contrast to their involvement in focal adhesion assembly . The formation of stress fibers that accompanies cross-linking of membrane receptors may instead serve to provide a more rigid cell structure to facilitate monocyte migration. | Other | biomedical | en | 0.999997 |
10366601 | Restriction enzymes, TaqI polymerase, and RadPrime DNA labeling system was purchased from GIBCO BRL . The Biotechnology Core Lab (Fred Hutchinson Cancer Research Center, Seattle, WA) prepared oligonucleotide primers used for PCR. The multiple tissue Northern blot and hybridization buffer were purchased from CLONTECH Laboratories, Inc. FBS was from Summit. Other cell culture reagents included BSA, trypsin, and hydrocortisone ( Sigma Chemical Co. ); cholera toxin ( Calbiochem-Novabiochem Corp. ); aminoguanidine ( Aldrich Chemical Co. ); EGF (Collaborative Biomedical Products); and keratinocyte growth medium (KGM; Clonetics). A 600-bp cDNA clone (Ep-1) corresponding to the helical region of the human α3 laminin chain was used to screen a murine fetal kidney library. Based on sequence homology, the resulting cDNAs were confirmed to be the murine equivalent of the α3-laminin chain. The clones were later found to be compatible with published sequence for the murine laminin α3 chain . The α3 laminin cDNAs were then used to screen a 129Sv genomic library (kindly provided by Dr. Phil Soriano, Fred Hutchinson Cancer Center, Seattle, WA) and multiple clones corresponding to the LAMA3 gene were identified. A 1.2-kb NsiI/SacI genomic fragment that contained the murine equivalent of exon A3 from the LAMA3 gene was replaced with a neo cassette driven by the PGK promoter. The construct was flanked 5′ and 3′ by a 0.8-kb BglII/NsiI fragment and a 5-kb SacI/SacI fragment, respectively. Both flanking sequences were derived from genomic fragments corresponding to the LAMA3 gene. A PGK-driven diphtheria toxin expression cassette was placed 5′ to the 0.8-Kb BglII/NsiI fragment. The construct was linearized with XhoI and electroporated into embryonic stem (ES) cells. Colonies were selected for G418 resistance and screened for homologous recombination using PCR as described . The PCR strategy included an oligonucleotide from the neo gene (5′ TCGCAGCGCATCGCCTTCTA 3′) and an oligonucleotide specific for the LAMA3 gene (5′ AACCCTGGCTAGTCTGGAAC 3′) upstream of the 0.8-kb NsiI/BglII fragment used in the targeting construct. PCR was performed in a DNA Thermal Cycler ( Perkin-Elmer Corp. ) for 40 cycles as follows: 93°C for 30 s; 55°C for 30 s; 65°C for 3 min. Positive clones were further characterized by Southern blot analysis using genomic DNA digested with NsiI and hybridized with an XbaI fragment that is 5′ to the PGKneo insert. Tissue culture and blastocyst injections were performed as previously described . For histology, samples were fixed using 10% formalin, rinsed in PBS, dehydrated through graded alcohol, and embedded in paraffin. Sections were stained with hematoxylin and eosin. For immunohistochemistry, frozen tissues were embedded directly in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc.). Cryostat sections (8–10 microns) were extracted with 1% Triton in PBS and fixed with 2% formaldehyde in PBS (20 min). Tissue sections were either stained using a Vectastain ABC kit (Vector Labs) or processed for immunofluorescence. For the ABC kit, sections were blocked with 10% goat serum, incubated with primary antibody for 2 h, washed, incubated with a biotinylated secondary antibody, washed, incubated with peroxidase-conjugated avidin, washed, and developed using diaminobenzidine plus nickel chloride. For immunofluorescence, tissue sections were blocked, incubated with primary antibody for 2 h, washed, incubated with FITC or rhodamine-conjugated secondary antibodies, and washed. Sections were mounted in a solution containing 25 mg/ml of 1,4-diazobicyclo-(2,2,2)octane in glycerol and visualized for immunofluorescence using a Zeiss Microscope. Tissue was fixed with half strength Karnovsky's fixative plus 0.1% tannic acid, rinsed in 0.1 M cacodylate buffer, and post-fixed in 2% osmium tetroxide . Samples were dehydrated in graded ethanols and propyleneoxide and then embedded in Polybed 812 resin. Thin sections (80–90 nm) were cut and stained with uranyl acetate. A JEOL 100-SX electron microscope was used for examining and photographing the samples. Neonatal mouse pups were killed by decapitation, rinsed in 70% ethanol and PBS, and skinned. The skin was digested in 0.25% trypsin overnight at 4°C for 14 h. The epidermis was separated from the dermis and placed in N-medium according to the protocol of Hager et al. . N-medium is MEM (0.06 mM Ca 2+ ) plus 7.3% chelexed FBS supplemented with culture supernatant from freshly isolated fibroblasts. N-medium also contains hydrocortisone, cholera toxin, aminoguanidine, and EGF. Cells are released from the epidermis into N-medium by shaking. The cells were directly seeded onto dishes that were untreated or coated with 10 μg/ml of type IV collagen (Collaborative Biochemical Products; Becton Dickinson Labware ). Immortalized mouse epidermal keratinocytes (MEKs) were cultured in KGM containing 0.06 mM calcium chloride (Clonetics Corp.). Adhesion assays on tissue sections were as follows: cryostat sections of split skin from wild-type and mutant animals were attached to the lid of a petri dish. Tissue sections were washed with PBS and blocked with 0.5% BSA. Trypsinized human foreskin keratinocytes that had been resuspended in KGM (Clonetics Corp.) were labeled with calcein-AM (Molecular Probes, Inc.) for 15 min at room temperature . Cells were washed with PBS and incubated with tissue sections for 1 h in the presence or absence of inhibitory antibodies. After incubation, cells were gently washed once using PBS, fixed with 2% formaldehyde in 0.1 M sucrose cacodylate buffer for 20 min, washed three times in PBS, and rinsed with distilled water. Sections were mounted with a solution containing 25 mg/ml of 1,4-diazobicyclo-(2,2,2)octane in glycerol and visualized for fluorescence using a Zeiss microscope. Rat mAbs against mouse β1 and γ1 laminin were purchased from Chemicon International, Inc. A rat mAb against β4 integrin was purchased from PharMingen . Dr. Takashi Hashimoto (Kurume University, Kurume, Fukuoka, Japan) kindly supplied human mAb 5E against bullous pemphigoid antigen 230 . Dr. Eva Engvall (Burnham Institute, La Jolla, CA) provided polyclonal antibodies for α1 and α2 laminin chains, which were made against the E3 fragment of EHS laminin and recombinant domain VI from the α2 chain, respectively. A polyclonal antibody prepared against recombinant α5 laminin was prepared by Dr. Jeffery Miner (Washington University, St. Louis, MO) as previously described . A polyclonal antiserum that was prepared against laminin 5 isolated from rat 804G cells was supplied by Dr. Jonathon Jones . A mouse mAb, D3-4, that cross-reacts with mouse laminin 5 was prepared in this lab by Susana Gil as previously described . Secondary antibodies were purchased from Vector Labs, Southern Biotechnology Associates Inc., and Jackson ImmunoResearch Laboratories, Inc. Northern blot analysis showed that two major transcripts for the laminin α3 chain are expressed in multiple mouse tissues . The schematic illustration in Fig. 1 indicates that multiple laminin trimers can be derived from these gene products. Therefore, to introduce a mutation into the LAMA3 gene, we developed a strategy that would ablate all possible α3-laminin trimers. We removed the murine equivalent of exon A3 of the LAMA3 gene and flanking sequence from the 5′ NsiI site (Ns*) to the 3′ SacI site (S*) replacing it with a PGK-driven neomycin ( neo ) cassette . The coding sequence of exon A3 is common to both the α3a and α3b transcripts . Consequently, removal of exon A3 will cause a frame shift mutation followed by a premature stop codon that will disrupt the α3a and α3b transcripts produced by the LAMA3 gene . The targeting construct was introduced into 129Sv ES cells by electroporation and homologous recombination occurred at a frequency of 12%. Southern blot analysis on genomic DNA digested with NsiI confirmed the presence of the mutant allele . Five clones that contained the mutant allele were used for blastocyst injection resulting in the generation of multiple chimeric animals. Chimeric animals generated from ES clones 5-2, 15-4, and 19-1 were crossed with C57BL/6J females. ES-agouti coat color identified pups with germline transmission of the mutant gene. Mice heterozygous for the LAMA3 mutation were crossed to obtain homozygous null offspring. Homozygous null animals derived from ES clones 5-2, 15-4, and 19-1 displayed similar phenotypes, so we continued our studies with the animals generated from ES clone 5-2. PCR analysis was used to determine the genotype of the offspring by using primers designed to detect the wild-type and mutant alleles . Homozygous α3−/− null animals appeared indistinguishable from α3+/− and α3+/+ wild-type littermates at birth . After birth, homozygous α3−/− null animals, referred to as mutant animals, develop progressive blistering of the forepaws, limbs, and oral mucosa . 25% of the newborn pups were homozygous null, suggesting that failure to express α3 did not cause embryonic lethality in the C57BL/6J genetic background. However, the animal in Fig. 3 C showed more extensive blistering than most of the mutant littermates, suggesting that the skin phenotype may be more severe in some of the affected animals. In most cases, the limbs and paws of the mutant animals were visibly red and bleeding , even when blistering lesions were not detected. It was noted that the content of the mutant stomach was substantially smaller and the mutant animals weighed 40–50% less than wild-type littermates. The presence of milk in the intestine of mutant animals indicates that there was no gastric obstruction, such as pyloric atresia, which can occur in JEB patients with mutations in integrin α6β4 . The mutant animals died 2–3 d after birth from a failure to thrive, possibly caused by dehydration and malnutrition. Paraffin-embedded sections of skin from wild-type and mutant animals were analyzed using hematoxylin and eosin staining . The lesions present in mutant skin were confirmed to be junctional blisters caused by a separation at the dermal–epidermal junction . The epidermis of mutant skin showed distinct organizational changes in lesional areas when compared with nonlesional areas. In lesional areas, the epidermis contained clusters of cells in the superbasal layer that maintained an undifferentiated morphology similar to the pearl cells described in β4 null animals . It was noted that the basal cells present in lesional regions were sparse and contained flattened nuclei . These alterations contrasted with the organization of the epidermis in nonlesional regions. In nonlesional regions of mutant skin , the morphology of the basal cells and the organization of the epidermis were similar to that of the wild-type skin . Immunostaining confirmed the absence of laminin 5 from the epidermal BM of mutant skin , consistent with the blistering phenotype. Using mAb D3-4, we sought to examine the expression of all α3-laminin heterotrimers in skin. mAb D3-4 immunoprecipitates multiple α3-containing heterotrimers from human keratinocytes , including laminin 5 (α3β3γ2) and laminin 6 (α3β1γ1), suggesting that it interacts with α3 chain. Immunostaining showed that mAb D3-4 was positive in wild-type skin, but absent from the epidermal BM of mutant skin . The absence of staining in the mutant BM with mAb D3-4 shows that we have removed all laminin trimers containing the α3 chain from the BM of mutant skin. We also found that α3-laminin was absent from multiple tissues in late stage mutant embryos including the tissues that were shown by Northern blot analysis to express the α3a and α3b transcripts (data not shown). In contrast, immunostaining showed that the expression of the β1 and γ1 laminin chains were maintained in the wild-type and mutant tissues (Table I ). The β1 and γ1 subunits of laminin form trimers with different α chains to generate tissue-specific laminin isoforms . Therefore, we used antibodies against the α1-, α2-, and α5-laminin chains to identify which laminin isoforms are present in the epidermal BM of wild-type and mutant skin. Strong staining for α5 laminin is maintained in wild-type and mutant skin, indicating that laminin 10 (α5β1γ1) or 11 (α5β2γ1) may be present in the epidermal BM. The results, summarized in Table I , indicate that multiple laminin isoforms remain in the BM of mutant skin. However, none of the other laminins are sufficient to stabilize the adhesion of epithelium to the BM. The skin phenotype of mutant animals is consistent with an autosomal recessive disorder in humans known as junctional epidermolysis bullosa-gravis . The loss of anchorage function in mutant epidermis led us to examine the organization and composition of HDs in mutant skin. HDs link the laminin 5-rich BM to the keratin cytoskeletal through integrin α6β4 . BP230 stabilizes the adhesion plaque on the cytoplasmic side . Immunohistochemical staining showed that integrin α6β4 and BP230 are expressed and are polarized in the basal cells of wild-type and mutant skin. Integrin α6β4 and BP230 both displayed an unexpected discontinuous organization in blistered and nonblistered regions of mutant skin when compared with the wild-type . This discontinuity suggests that the absence of laminin 5 effects organization of HD components in basal cells, even in nonlesional areas. Furthermore, these results identify a novel phenotypic alteration in HDs that may be diagnostic in cell adhesion defects stemming from abnormalities in the basement membrane zone (BMZ). We used transmission electron microscopy to examine the ultrastructure of HDs in tissue. HDs appear normal in wild-type animals, containing a basal and subbasal electron dense plaque . Anchoring filaments extend from the basal lamina toward the HD. In the cytoplasm, keratin filaments accumulate near the subbasal plaque . This fine structural organization was lacking in mutant animals. The epidermis of mutant skin contained electron dense plaques at the plasma membrane, but lacked discernible subbasal plaques . Intermittently, rudimentary HDs formed at the plasma membrane in the mutant animals . Consistent with our immunostaining results, there was discontinuity in the formation of electron dense plaques resulting in a complete absence of HDs in some regions of the basal cells in mutant skin . Taken together, these findings indicate that the formation and stability of continuous HDs at the basal surface requires laminin 5, whereas basal polarization of integrin α6β4 and BP230 proceed in the absence of laminin 5. To evaluate the adhesive properties of the BM in vivo, short term adhesion assays were done on cryostat sections of split skin from wild-type and mutant animals (Gil, S.G., T.A. Brown, and W.G. Carter, manuscript in preparation). Previous studies have indicated that the function of β4 integrin can be more effectively evaluated when the function of β1 integrins are suppressed . Therefore, we used cytochalasin D to suppress the function of β1 integrins by inhibiting the organization of the actin cytoskeleton, which allowed the function of β1 and β4 integrins to be distinguished. Tissue adhesion assays were done using human foreskin keratinocytes (HFKs) instead of mouse keratinocytes because some of the inhibitory antibodies do not cross-react with mouse cells. Fig. 7 A shows that HFKs adhere well to the BM of wild-type tissue regardless of whether they are untreated or pretreated with cytochalasin D . The cytochalasin D-resistant adhesion could be blocked using an inhibitory antibody against α6 integrin indicating that this interaction is dependent on integrin α6β4. In contrast, pretreatment of HFKs with cytochalasin D resulted in complete inhibition of adhesion to the BM of mutant tissue demonstrating the loss of a functional interaction between integrin α6β4 and laminin 5. However, in untreated HFKs, we were able to detect adhesion to the mutant BM , suggesting that a ligand for β1 integrin is present in the BM of mutant tissue. This led us to investigate which β1-integrin receptor was required for adhesion to the mutant BM. The results showed that mAb P1B5, an inhibitory antibody against integrin α3, significantly reduced adhesion to the BM of mutant tissue when compared with the control . A comparison of several representative areas indicated that the inhibition observed in the presence of P1B5 was ∼80%. A combination of anti-α3 and anti-α6 inhibitory antibodies allowed for complete inhibition of adhesion to the BM of mutant skin . The nature of any contribution from integrin α6, if significant, awaits further investigation. It is sufficient that in the presence of mAb P1B5 adhesion of HFKs to the mutant BM is reduced by >80% demonstrating that a ligand for integrin α3β1 is detectable in laminin 5 deficient tissue. In contrast, mAb P1B5 did not effect adhesion of HFKs to the wild-type BM due to the interaction of laminin 5 with integrin α6β4. This was confirmed by using a combination of GoH3 and P1B5, which completely blocked adhesion of HFKs to the wild-type tissue . Therefore, in mutant tissue, we were able to detect a ligand for integrin α3β1 that could not be detected in wild-type tissue because of the dominant interaction of laminin 5 with integrin α3β1 and integrin α6β4. These results provide evidence that other endogenous BM proteins can serve as a ligand for α3β1, but not for the anchorage function mediated by integrin α6β4 in the epidermis. MEKs were isolated from wild-type and mutant skin. The yield of cells and seeding density was comparable for normal and mutant MEKs. However, in contrast to normal MEKs, mutant MEKs failed to survive when plated on an untreated culture dish . Survival of mutant MEKs was restored when cells were plated on an exogenous ligand such as collagen . These results show that ablation of laminin 5 is sufficient to prevent the survival of mutant MEKs in the absence of an exogenous ligand. This concept was further reinforced with the generation of a laminin 5 deficient cell line. Using the E6/E7 transforming genes from papilloma virus we immortalized wild-type and mutant epithelial cells. The mutant epithelial cells remained dependent on exogenous ligand for survival and this dependence was confirmed in a growth curve . Using flow cytometry, there was no accumulation of a pre-G1 peak , suggesting that extensive apoptosis was not occurring in the mutant cells (data not shown). Studies were initiated to identify receptor/ligand interactions that could rescue the survival defect in mutant MEKs. Laminin 5-enriched ECM or immobilized anti-integrin β4 antibody were able to rescue mutant MEKs, suggesting that interactions of laminin 5 with integrin α6β4 contribute to the survival of epithelial cells. Because keratinocytes use integrin α3β1 and α6β4 to interact with laminin 5, both receptors may be contributing to the enhanced survival observed on laminin 5 . We could not evaluate the contribution from integrin α3β1 separately because antibodies that react with mouse are not available. However, rescue of mutant MEKs on collagen or immobilized anti-α2 antibody (data not shown) indicate that ligation of β1 integrins is also sufficient to rescue survival of mutant MEKs. In contrast to mutant cells, the MEKs derived from normal animals retained a high survival rate and characteristic epithelial morphology regardless of whether they were plated on untreated culture dishes, exogenous ligand, or immobilized antibody . Therefore, within the limits of this assay, we found that ligation of β1 or β4 integrins are sufficient to rescue the survival defect resulting from ablation of laminin 5. These results confirm the proposal that laminin 5 secreted into the ECM by cultured keratinocytes is the primary adhesive ligand produced by these cells, even though an additional ligand for integrin α3β1 resides in the BM. Laminin 5 is expressed in a variety of epithelial BMs. In skin, we observed severe defects in basal keratinocytes resulting in abnormal HDs and loss of anchorage function in the BM of the mutant epidermis. We predicted that if laminin 5 were involved in late stage differentiation we would be able to detect abnormalities in other target organs that develop late in gestation. Examination of cross-sections from neonatal mice revealed gross abnormalities in the developing incisors of mutant animals. Therefore, developing incisors of wild-type and mutant animals were selected for further investigation. Cross-sections from medial to lateral regions of the head were taken to evaluate histogenesis of the incisors at different stages of maturation . Progressive differentiation is identifiable from the base of the tooth (region I) to the tip (region III and VI). The stages of differentiation are outlined for ameloblasts , which are the specialized epithelial population that deposit enamel on one side of the developing rodent incisor . Mitotic preameloblasts of the inner dental epithelium located at the base of the tooth in region I develop into post-mitotic, secretory ameloblasts in region II and produce the enamel layer of the tooth. A sharp boundary is visible at the junction between the ameloblasts and the stratum intermedium . This boundary corresponds to cytoplasmic filaments, not a BM. The BM is located between the developing tooth and the ameloblast . As a result, the nuclei of the ameloblast become polarized away from the BM . Consistently, immunostaining with mAb D3-4 showed intense deposition of the laminin α3 chain between the secretory ameloblasts and the enamel boundary in the wild-type incisor . Positive staining with mAb D3-4 was absent from a comparable region of the mutant tooth confirming the removal of laminin α3 chain trimers from the developing incisor. Cross-sections from comparable regions of the wild-type teeth and mutant teeth were evaluated. In wild-type incisors, secretory ameloblasts of region II were discernible as elongated epithelial cells with Tomes' processes extending toward the deposited enamel . Differentiation continued normally with the appearance of ruffled edge mature ameloblasts in region III followed by the formation of the reduced enamel epithelium in region IV . A discrete morphological change occurs in region IV: the stratum intermedium no longer marks a discrete boundary because the ameloblasts, stratum intermedium, stellate reticulum, and outer dental epithelium become incorporated into the stratified epithelium referred to as the reduced enamel epithelium. In mutant animals, incisor development appeared normal until the onset of enamel secretion. Presecretory ameloblasts in region I of wild-type and mutant animals are indistinguishable. At the onset of enamel secretion in region II, the ameloblasts of mutant incisors were shorter with visible undulations at the edges when compared with wild-type incisors . As differentiation proceeded, the ameloblasts of the mutant incisor continued to be reduced in size relative to the wild-type teeth and enamel deposition did not appear normal. The enamel edge of mutant incisors appeared frayed in comparison to the enamel deposited in wild-type incisors . The abnormal appearance and size of the mutant ameloblasts made it difficult to distinguish the transition from secretory ameloblasts of region II to mature ameloblasts of region III. In region IV, where the ameloblasts and the adjacent stratum intermedium form the reduced enamel epithelium, tissue organization was completely disrupted . This disorganization corresponded precisely with the point where the stratum intermedium no longer formed a discrete boundary . At this junction between region III and IV the reduced enamel epithelium should form a stratified epithelium. This does not occur in the mutant tissue . These results define a role for the α3 subunit of laminin 5 in murine tooth development and provide a biological basis for the hypoplastic enamel that has been described in human JEB-G . Our results show that loss of α6β4-laminin 5 anchorage functions have profound effects on HD organization. Discontinuities in localization of integrin α6β4 and BP230 were so prominent in mutant tissue that they could easily be detected at the light microscope level by immunohistochemical staining with anti-β4, -α6, or -BP230 antibodies. Analysis with other cellular markers, such as keratin-1 or keratin-14, did not display alterations that would allow us to distinguish between wild-type and mutant skin (data not shown). In contrast, the discontinuous staining of α6β4 and BP230 was reproducible in 100% of the homozygous null pups allowing us to readily identify wild-type and mutant skin. Because the discontinuity of β4-integrin and BP230 staining occurred in both lesional and nonlesional regions, our data suggests that HD alterations are a primary consequence of laminin 5 deficiency rather than a secondary effect caused by the lesion. Consistently, a subpopulation of patients lacking either laminin 5 or bullous pemphigoid antigen 180 (BP180) display a similar disorganization of HD proteins (Brown, T.A., and W.G. Carter, unpublished observation), suggesting that this phenotypic alteration may have diagnostic value in patients with structural abnormalities in the BMZ. It was noteworthy that the discontinuity of α6β4 and BP230 appeared to occur at cell–cell boundaries, suggesting that stability of cell–cell junctions may be reduced in these regions. The notion that cell-substrate adhesion can regulate interactions at cell– cell junctions has been established in epithelial cells. Tiam1/Rac signaling in epithelial cells can promote either cell–cell adhesion or cell migration depending on the type of matrix used for cell adhesion . Similarly, laminin 5 interactions with integrin α3β1 selectively promote intercellular communication of basal keratinocytes through gap junctions . Ablation of the LAMA3 gene causes a phenotype similar to a lethal variant of human epidermolysis bullosa, JEB-G. Clinical features of JEB-G include mechanical fragility of the skin, growth retardation, oral erosions, gastrointestinal and genitourinary tract involvement, dental abnormalities, hypoplastic HDs, and high morbidity . JEB-G is an autosomal recessive disorder that has been associated with mutations in the LAMA3 , LAMB3 , and LAMC2 genes of laminin 5. Mutations in the LAMA3 gene have been documented in only a small fraction of the JEB-G cases, which led us to wonder if null mutations in the LAMA3 gene would result in embryonic lethality, particularly since we removed all trimers containing the α3 chain from the BM, including laminins 5–7. On the contrary, we found that mice homozygous for the null mutation were born at the expected frequency of 25%, suggesting that embryonic lethality did not occur in the C57/BL6 genetic background. The reduced number of patients with mutations in the LAMA3 gene relative to the other genes that encode laminin 5 may be due in part to a reported hotspot in the LAMB3 gene . In vitro studies on primary and immortalized keratinocytes have indicated that laminin 5 contributes to keratinocyte survival, which may have relevance to pathology of human JEB-G. Jonkman et al. described an individual who is mosaic for mutations in the COL17A1 gene encoding BP180, a component of HDs. Surprisingly, the subpopulation of keratinocytes from this mosaic individual that express BP180 display a survival advantage in culture and in the skin of the individual . Thus, both laminin 5 and BP180 provide a survival advantage for keratinocytes. Curiously, keratinocytes from individuals with JEB-pyloric atresia with inherited defects in β4 appear to survive in culture as well as or better than wild-type keratinocytes (Gil and Carter, unpublished observation). Additional experiments will be necessary to determine if the survival advantage observed in wild-type cells is due to a direct effect of laminin 5 on cell cycle regulation through integrins. It has been shown that adhesion of primary keratinocytes to laminin 5 promotes entry into the cell cycle through signaling pathways that are generated by ligation of integrin α6β4 . Laminin 5 may also promote cell proliferation through a second signaling pathway involving integrin α3β1 . Consistently, our results indicate that exogenous ligands are not adequate for longterm survival of mutant MEKs (Ryan, M.C., unpublished observation), suggesting that the survival contributions from laminin 5 may not simply be due to adhesion. Whether or not exogenous laminin 5 is sufficient to rescue defective cellular functions caused by the absence of endogenous laminin 5 remains to be determined, particularly since endogenous and exogenous laminin 5 may have different biological functions. Exogenous laminin 5 is a scatter factor for carcinoma cells that do not make endogenous laminin 5, but not for carcinomas that deposit endogenous laminin 5 . Similarly, we have observed that keratinocytes from JEB-G patients with defective laminin 5 expression and MEKs from LAMA3 null animals will both scatter in response to exogenous laminin 5 while normal cells do not (Gil, S.G., M.C. Ryan, and W.G. Carter, unpublished observation). Future studies will determine if exogenous laminin 5 or transfection with α3-laminin cDNAs can rescue cellular defects stemming from the removal of laminin 5. The integrity of the epidermis of LAMA3 null animals remained intact during development and birth indicating that adhesion independent of laminin 5 may provide sufficient developmental instruction and stability for survival before birth. Junctional blisters developed in the affected pups several hours after birth and were usually restricted to the forepaws, limbs, and oral mucosa. A similar blistering phenotype was found in mice carrying a disruption in the β3 subunit of laminin 5 that was caused by insertion of an intracisternal-A particle into the LAMB3 gene . The relatively restricted blistering phenotype of the LAMA3 null animals contrasts with the phenotype of pups lacking integrin α6β4, the anchorage receptor for laminin 5. Pups lacking either integrin α6 or β4 displayed extensive blisters over the entire body surface and died within hours of birth . The extensive skin fragility may have been caused by weakening in the cytoplasm and at the BMZ that resulted in the formation of both simplex and junctional blisters in these animals . The epithelium in β4 null animals was also susceptible to apoptotic cell death. Using a tunnel staining assay (data not shown), we did not detect apoptosis in LAMA3 null animals. The accelerated and heightened severity of the blistering in the β4 null animals may also have contributed to the onset of apoptosis in these animals. We noted organization changes in the epidermis of LAMA3 null animals that were similar to the β4 null animals. In particular, we identified cell clusters that appeared undifferentiated in the superbasal cell layer similar to the pearl cells described in the β4 null animals . Immunostaining of skin with anti-β4 antibodies identified positive staining in the superbasal cell layer (data not shown), suggesting that cellular differentiation in lesional regions of the epidermis may be abnormal in LAMA3 null animals. Laminin 5 can regulate both anchorage and motility of epithelial cells through integrin α6β4 and α3β1, respectively . Consistently, analysis of skin from integrin α3 null animals has revealed alterations in the epidermis stemming from the absence of integrin α3β1 function . In particular, the integrin α3 null animals showed a disorganized BM and blister formation at the dermal–epidermal junction . Similar to LAMA3 null animals, bleeding was identifiable in the paws of integrin α3 null pups . Whether these two observations are connected remains to be determined. The bleeding that occurred in the paws of the LAMA3 null pups was detectable even before a blister had formed, suggesting that it was a primary defect. The bleeding may be an indication of an abnormality stemming from the role of α3-laminin in an alternative trimer such as laminin 6 or 7. Using a novel adhesion assay to directly assay BM function in vivo we found that the mutant BM could no longer induce adhesion by integrin α6β4. These results confirm that functional interactions between integrin α6β4 and laminin 5 have been eliminated in homozygous null animals. This data has implications for the organism as a whole because it indicates that integrin α6β4 may be unligated or no longer functional in multiple tissues, which may contribute to the neonatal lethality in homozygous null animals. In contrast to loss of α6β4 function, cell adhesion via integrin α3β1 was retained in the mutant BM. Because we have demonstrated that laminin 5 is absent from the mutant BM, the most logical conclusion is that we are detecting an alternative ligand for integrin α3β1 that is present in the BM of mutant skin. Our immunostaining experiments have identified several laminin isoforms which may be candidate ligands for integrin α3β1 in the epidermal BM (Table I ). In a recent study that compared the ligand binding activities of different laminin isoforms, laminin 10/11 was identified as a potent substrate for adhesion of lung carcinoma cells via integrin α3β1 . The α3β1-mediated adhesion to laminin 10/11 was comparable to laminin 5 and found to be greater than adhesion to laminin 1 or laminin 2/4 . Likewise, we have shown that adhesion of HFKs via integrin α3β1 is better on laminin 5 than on laminin 1, suggesting that laminin 1 does not significantly contribute to adhesion of keratinocytes in vivo . Accordingly, laminin 10/11 or a new laminin isoform may contribute to α3β1-mediated adhesion and will be investigated as a possible second ligand for integrin α3β1 in epidermis. We have established a role for laminin 5 in late stage differentiation of ameloblasts in developing incisors of mutant animals. The phenotypic alterations found in mutant incisors are consistent with the dental abnormalities and enamel hypoplasia described for JEB-G patients . Enamel hypoplasia has also been reported for an epidermolysis bullosa patient with a confirmed mutation in the ITGB4 gene , implicating a role for integrin α6β4 in amelogenesis. In our studies, ameloblast abnormalities were first detected at the onset of enamel secretion and continued throughout ameloblast differentiation, culminating in the disorganization of the reduced enamel epithelium. The phenotypic alterations coincided nicely with the deposition of α3-laminin trimers in the wild-type incisor where we identified positive staining for the laminin α3 chain along the edge of the differentiating ameloblasts. No staining was detected in a comparable region of the mutant incisor, confirming the absence of laminin 5 in the mutant tooth. Positive staining for integrin β4 (data not shown) suggests that the ameloblast differentiation may be dependent on laminin 5 interactions with integrin α6β4. The deposition of laminin α3 chain in the wild-type tooth is consistent with recent studies that have shown that the subunits of laminin 5 are expressed in differentiating ameloblasts, even during enamel secretion when laminin 1 expression has disappeared . It is interesting that abnormalities were detected in the mutant tooth at the onset of enamel secretion because ultrastructural analysis of developing teeth have shown that the basal lamina disappears during the secretory stage of amelogenesis and then reforms during ameloblast maturation . Our results suggest that laminin 5 has a unique role in regulation of ameloblast differentiation and that the requirement for laminin 5 may begin at the onset of enamel secretion. Furthermore, the disorganization that occurred in the reduced enamel epithelium emphasizes a role for laminin 5 in the maintenance of stratified epithelium. | Other | biomedical | en | 0.999997 |
10366602 | LN was purified from Engelbreth-Holm-Swarm (EHS) tumor by the method of Timpl et al. . SDS-PAGE of purified preparations shows bands at ∼215 kD (β and γ chains) and ∼400 kD (α chain). The latter was not recognized by an antiserum to LN α2 chain (a gift from Dr. Peter Yurchenco) but did react with an anti-LN antiserum produced by immunizing rabbits with purified EHS tumor LN. This antiserum recognizes all three subunits of LN 1 (α1, β1, and γ1), but does not cross react with agrin or LN α2 chain. Anti-LN IgG was purified by chromatography on Affigel Blue (BioRad Laboratories) according to the manufacturer's instructions. An antiserum to LN α2 chain was raised against the recombinant G domain of this chain and does not cross-react with LN α1 chain. mAb 5D3 to LN (Life Technologies) has been previously characterized . Antiserum to the integrin β1 subunit was generated by immunizing rabbits with purified rat integrin β1 subunit . mAb IIH6 recognizes a unique carbohydrate epitope on α-DG while the antiserum to fusion protein B recognizes a portion of the core protein of both α- and β-DG. mAb NCL–β-DG is directed against the last 15 amino acids of the COOH terminus of β-DG (Novocastra Laboratories Ltd.). The antiserum to α-SG, raised against a peptide corresponding to the last 19 amino acids of the COOH-terminal domain of α-SG, specifically recognizes a single protein of ∼50 kD in purified DGC from rabbit muscle and in crude protein extracts from skeletal muscle and C2 cells. mAb HUC1-1 to muscle actin was purchased from ICN Pharmaceuticals. A 1.8-kb fragment of the mouse DG cDNA extending from approximately −100 bp (5′ to the translation start site) to the HindIII site situated at +1,725 bp was removed by digestion of a mouse DG cDNA subclone in bluescript SK(−) with NotI-HindIII. This Not1-HindIII fragment was then subcloned in the antisense orientation into the pRcCMV expression vector (Invitrogen) using standard subcloning techniques . Before transfection, the plasmid was linearized by digestion with BglII. C2 cells were plated on 10-cm tissue culture plastic dishes (Falcon) maintained at 37°C, 8% CO 2 atmosphere, in growth medium consisting of DMEM (low glucose; Life Technologies) supplemented with 20% FBS (heat inactivated; Life Technologies), 0.5% chick embryo extract (ultrafiltered; Life Technologies), and penicillin/streptomycin (Life Technologies). Stable transfections were carried out by calcium phosphate coprecipitation after methods of Yoshihara and Hall . In brief, when C2 myoblasts reached 70% confluence, they were harvested by treatment with trypsin/EDTA, replated at a 1:20 dilution on fresh 10-cm dishes and allowed to attach overnight. On day 2, the medium was changed 3 h before transfection and DNA/Ca 2 PO 4 coprecipitate, prepared according to published procedures , and was added directly to the culture medium (5 mg of linearized plasmid per dish). Cells were returned to the incubator for 16 h, and on the following day, the medium and precipitate were removed. The cells were washed briefly with Dulbecco's PBS plus 0.5 mM EDTA to remove excess precipitate. Fresh medium was added and 24–36 h after the beginning of transfection it was replaced with selection medium consisting of growth medium supplemented with G418 (750 μg/ml active concentration; Life Technologies). Selection was carried out for up to 10 d, or until all cells of an equivalent, untransfected culture were killed. At that point, drug-resistant C2 cell colonies could easily be seen on transfected plates. Colonies were then picked and expanded for further characterization. Stable clones were maintained in growth medium supplemented with 70 μg/ml active G418. Low (6–20) passage 11F, 11E, 9B, 10C, 11A, and control C2 cells were cultured on tissue culture plastic dishes (Falcon) coated with 0.15% gelatin and maintained in growth medium until confluent. Cultures were switched to fusion medium (DMEM high glucose, 1% horse serum) and allowed to differentiate for an additional four days. Some cultures were treated with 12 nM LN on the third day of fusion. Control and transfected C2 clones differentiated into myotubes for 3–5 d were washed three times with ice-cold Ca/Mg-free PBS, then extracted into 0.2-ml/10-cm dish of 1× SDS sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) preheated to 85°C. The remaining extract was scraped and transferred to a 1.5-ml microcentrifuge tube, and heated at 95°C for 5 min. It was subsequently passed five times through a 30-gauge syringe needle and centrifuged at 16,000 g for 10 min to remove insoluble debris. A portion of the lysate was precipitated with trichloroacetic acid/deoxycholate for protein determination . For some experiments, cultured myotubes were subjected to subcellular fractionation generating soluble fractions and KCl-washed light and heavy microsomes . To assess expression of the integrin β1 subunit by Western blotting, cells were scraped in ice-cold PBS and membrane proteins were detergent extracted in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100, 20 mM N -ethylmaleimide, Complete protease inhibitor cocktail ( Boehringer Mannheim ) for 10 min on ice. Protein concentration was determined and extracts were diluted in 5× SDS sample buffer without dithiothreitol. Cellular proteins released into the culture medium were assayed by collecting medium from two 10-cm dishes of differentiated cells. The medium was pooled, concentrated ∼100-fold and partially purified using Centricon Plus-20 centrifugal filter devices ( Millipore ). All samples were assayed for protein content before electrophoresis on 7.5 or 10% SDS–polyacrylamide mini gels (0.75-mm thickness; BioRad Laboratories) at 20-mA constant current for 1 h. Fractionated proteins were electroblotted onto nitrocellulose membranes (BA-S 75; Schleicher and Schuell) under standard conditions (100-V constant voltage for 1 h). Blots were stained with Ponceau-S red to assess transfer, then incubated in 10 ml of Blotto (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Tween 20, 5% dried skim milk powder) at room temperature for 1 h and subsequently in an appropriate dilution of primary antibody in 4–5 ml Blotto for 1 h at room temperature with constant agitation. Primary antibodies used in Western blotting included: mAb IIH6 culture supernatant (1:10); antiserum to fusion protein B (1:50); antiserum to α-SG (1:1,000); mAb to β-DG (1:350); antiserum to LN (1:100); antiserum to the integrin β1 subunit (1:5,000); mAb to sarcomeric actin (1:1,000). After incubation with the primary antibody, blots were washed 4× 15 min in TBS-Tween (10 mm Tris, pH 7.5, 150 mM NaCl, 1% Tween 20), then incubated with the appropriate HRP-labeled secondary antibody diluted in Blotto for 1 h at room temperature with constant agitation. Blots were washed in TBS-Tween (4× 15 min) and labeled bands were visualized by enhanced chemiluminescence (Mandel/ NEN Life Science Products) after exposure to x-ray film (Hyperfilm-ECL; Amersham Life Sciences ). Blots were often stripped in 0.2 M glycine, 0.1% (vol/vol) Tween 20, pH 2.5, for 30 min, rinsed in PBS and reprobed with mAb HUC1-1 to muscle actin to take into account differences in the levels of DGC and integrin expression due to differences in the proportion of differentiated myotubes among cultures (see text). Cultures of confluent myoblasts (day 0), or of cells allowed to differentiate for 2 or 4 d, were washed twice with PBS then fixed and stained with Coomassie blue (25% propanol, 10% acetic acid, 0.1% Coomassie brilliant blue) for 10 min at room temperature. Cultures were washed twice in PBS, air dried and visualized in brightfield on a Zeiss Axioskop. For immunocytochemistry, myotube cultures were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for 20 min, rinsed three times with PBS, blocked for 1 h in PBS + 1% horse serum, then incubated overnight at 4°C with the primary antibody. For immunostaining of live cells, the primary antibody was added directly to the culture medium and cells were incubated for 30 min at 37°C in 8% CO 2 , then fixed as described above. Cells were then washed and incubated with the appropriate biotinylated secondary antibody for 1 h, followed by fluorescein-conjugated streptavidin and rhodamine-conjugated α-bungarotoxin (α-BTX; Molecular Probes) for 20 min at room temperature. Staining was visualized with epifluorescence illumination on a Zeiss Axioskop. Primary antibodies used include: mAb IIH6 ascites, 1:100 dilution; antiserum to LN 1, 1:50 dilution; mAb 5D3, 1:100 dilution; antiserum to LN α2 chain, 1:150 dilution; antiserum to the integrin β1 subunit, 1:100 dilution. To assess the level of apoptosis in myotube cultures, cells were differentiated for 3 d, then fixed sequentially in 2% formaldehyde and 4% neutral-buffered formalin for 10 min each at room temperature and washed twice in PBS, pH 7.4. Cultures were permeabilized by incubation in either 0.1% Triton X-100 for 15 min at room temperature or in ethanol/acetic alcohol (1:4) for 5 min at –20°C, then processed for TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; Apoptag Plus In Situ Apoptosis Detection Kit; Oncor). Negative controls were run for each experiment by omitting the anti-digoxigenin antibody or the terminal deoxynucleotidyl transferase enzyme. Positive control slides were provided with the kit. Cultures were counterstained with 0.1 μg/ml DAPI to visualize the total number of nuclei present and to confirm by morphological criteria that TUNEL stained nuclei were not mitotic or necrotic. The number of apoptotic nuclei and the total number of nuclei were counted with a 63× objective on a Zeiss Axioskop. 20 fields were quantified per coverslip and three coverslips were analyzed per cell type and per experiment. Collapsed myotubes and masses of dead cells were not included in the quantification since the number of labeled nuclei could not be accurately determined. C2, 11F, 11E, 9B, and 10C cells were cultured in 96-well plates (Falcon) coated with 0.15% gelatin and assayed at day 0 (confluent myoblasts), and days 2 to 4 in fusion medium. To determine cell number, plates were thoroughly washed with PBS, frozen at −80°C and labeled with the Blue DNA assay kit (Molecular Probes). Hoechst fluorescence was measured with a Cytofluor 2300 fluorometer using a 360 nm excitation/460 nm emission filter set. Empty wells coated with gelatin were used as controls for background fluorescence. Proliferating cells were labeled using the colorimetric cell proliferation ELISA kit ( Boehringer Mannheim ). In brief, cells were incubated for 2 h in medium containing 10 μM bromodeoxyuridine (BrdU), fixed for 30 min at room temperature, incubated for 2 h with the anti-BrdU antibody, washed, and incubated for 5 min in the substrate solution. The reaction was stopped with 1 M sulfuric acid and the plates were immediately read with an ELISA plate reader at 450 nm using a reference wavelength of 690 nm. Wells where the BrdU was omitted were used as control for nonspecific labeling of the antibody. Plates for the cell loss and proliferation assays were prepared on the same day and from the same aliquot of cells. Four replicate wells were prepared per cell type and per plate. Proliferation is expressed as a ratio of the average number of cells per well as determined by Hoechst staining on an age-matched plate. To assay membrane integrity cells differentiated for 4 d on tissue culture dishes coated with gelatin were washed in serum-free DMEM and incubated for 1 min in 0.2% Trypan blue in DMEM. Cells were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at room temperature, washed in PBS and then dehydrated in 70% and 95% ethanol for 1 min each. Cultures were counterstained with eosin (0.1% eosin in 95% ethanol, 2.8 μl/ml concentrated acetic acid) for 1 min then washed three times 1 min each in 100% ethanol. Cultures were visualized on a Zeiss Axioskop. Blue nuclei were counted with a 40× objective for three sets of 10-cm dishes (10–15 fields/dish) for C2 and 11E cells. Large aggregates of dead cells and cell debris were not included in the quantification. Statistical significance for cell loss, proliferation, and membrane integrity assays was determined for three to four independent experiments using a simple ANOVA test followed by the Fisher's t -test. The n represents the individual experiments. For statistical analysis of the percentage of apoptotic nuclei, each experiment was evaluated separately since the permeabilization procedure used was not always the same. A simple ANOVA test was also used followed by the Fisher's t -test. The n represents the number of dishes used (three for each experiment). The correlation between cell number and α-DG expression for C2, 11F, and 11E cells was determined using a simple regression analysis followed by Fisher's z-test (null hypothesis: correlation coefficient = 0). The relative cell number was obtained from quantification of Hoechst fluorescence and the average levels of α-DG expression for each cell type were assessed by densitometry from seven individual Western blots. In each Western blot, extracts from all three cell types had been assayed side by side. To assess the role of α-DG in the deposition and organization of muscle ECM, the C2 muscle cell line was transfected with a DG cDNA fragment in the antisense orientation under the control of the cytomegalovirus promoter . G418-resistant clones were selected and screened by examining the levels of α-DG expression relative to untransfected controls or cells transfected with the vector alone. Five clones, 11E, 11F, 9B, 10C, and 11A were chosen for further characterization. Control C2 cells, and antisense clones were cultured to confluence then induced to fuse into myotubes as described in Materials and Methods. After 4–5 d in culture, the myotubes were extracted directly in SDS sample buffer and equal protein loads were fractionated by SDS PAGE and transferred onto nitrocellulose membranes. Fig. 1 B shows a representative blot probed with mAb IIH6, which recognizes a carbohydrate epitope on α-DG . Control C2 cells show a typically broad band of muscle α-DG extending from 130–160 kD. By contrast, only low levels of α-DG expression were detected in 11F cells and even lower levels were detected in 11E cells (this blot is greatly overexposed to allow for visualization of α-DG expression in the 11E line). Nearly normal levels of α-DG expression were detected in the 9B, 10C, and 11A clones possibly as a result of low expression of the antisense construct, and these clones were used as additional controls for any nonspecific effects resulting from the transfection or selection procedures. Densitometric analysis of films from Western blots exposed over a range of times to insure that the emulsion was not overexposed, revealed a 50–60% decrease in α-DG in 11F cultures and an 80–90% decrease in 11E cultures. The blot in Fig. 1 B was also probed with an antiserum raised against α-SG, one of the four transmembrane components of the SG complex which is associated with, but distinct from, the DG complex . No reduction in the expression of this 50-kD protein was seen in either α-DG antisense clone compared with C2 cells . A similar result was obtained for β-SG (not shown). Since mutations affecting the expression of any one member of the SG complex can lead to the loss of all other members from the sarcolemma , we deduced that the SG complex is unaffected in the antisense clones. This result is also in agreement with the reported relative independence of the DG and SG complexes . In addition, Western blots of extracts probed for LN , collagen IV and perlecan (Montanaro, F., and S. Carbonetto, unpublished data), integrin β1 AChR α, δ, and γ subunits, or for the MuSK receptor tyrosine kinase showed no reduction in expression of these proteins in 11F and 11E cells, nor in 9B, 10C, and 11A cells, confirming the specificity of the antisense construct. Since mAb IIH6 is known to recognize a carbohydrate epitope on α-DG involved in binding to LN , it was important to demonstrate that the defect in the antisense-expressing clones was not simply due to aberrant glycosylation of α-DG. Therefore, similar blots were probed with an antiserum to an α-DG fusion protein (anti-fusion protein B) which recognizes the DG core protein . For C2 cells as well as 11F and 11E antisense clones, the relative intensity of labeling by this antiserum is very similar, albeit generally weaker , to that observed with mAb IIH6 suggesting that the decreased expression of α-DG in the 11E and 11F clones is due to decreased levels of α-DG polypeptide and not merely altered glycosylation. To confirm further that the expression of membrane associated α-DG was similarly altered, C2, 11F, and 11E myotubes were fractionated into soluble and KCl-washed light microsome fractions, then analyzed by SDS-PAGE and Western blotting with mAb IIH6. The decrease in membrane associated α-DG seen in the antisense clones relative to control C2 cells closely parallels that seen for the levels of total α-DG . Little, if any, expression of α-DG was detected in the soluble fraction or heavy microsome fraction (data not shown) of either control C2 cells or antisense clones, suggesting that the latter are not defective in the localization of α-DG to the cell membrane. Consistent with this β-DG, like its cotranscript α-DG, is also reduced by 70 and 88% in 11F and 11E cells, respectively, but not significantly in 9B, 10C, and 11A cells, when compared with C2 cells . This confirms that DG synthesis is reduced in antisense-expressing cell lines and that the reduced levels of α-DG do not result simply from shedding into the medium. α-DG is a high affinity LN binding protein in vitro and colocalizes with LN in muscle cells . Since LN isoforms have been reported to play an important role in myoblast fusion , we compared the ability of C2, 11F, 11E, 9B, 10C, and 11A myoblasts to differentiate into myotubes. In low density cultures 11F and 11E myoblasts appeared somewhat flatter and more spread than control C2 myoblasts. However, the proliferation rate and survival of 11F and 11E myoblasts in these cultures were indistinguishable from C2 myoblasts. In cultures approaching confluence little morphological difference could be seen between C2, 11F, and 11E myoblasts . One day after transfer to fusion medium, myoblasts assumed a more elongate shape and on the second day of differentiation numerous thin myotubes could be seen in C2 as well as in 11F and 11E cultures . After 4 d of differentiation, many large myotubes were found in all cultures indicating that 11F and 11E myoblasts can differentiate into myotubes with the same time course as C2 cells although 11F and 11E cultures had lower densities of myotubes (discussed below). 9B, 10C, and 11A cells also differentiated normally within the same time frame as C2 cells but did not show any appreciable decrease in cell density with differentiation. 11A myoblasts seemed to replicate more slowly compared with the other clones and usually required 3 d to reach confluence rather than 2 d like C2 cells and all the other clones, although, once confluence was reached, differentiation into myotubes proceeded at the same rate as C2 cells. It should be noted that after 4 d in fusion medium, cultures of 11F, 9B, 10C, and 11A cells had a very low proportion of myoblasts compared with C2 cells. This may be a reflection of heterogeneity in the parental C2 line relative to the transfected myoblast clones. Thus, substantial depletion of α-DG in 11F and 11E cells does not obviously affect these aspects of muscle cell differentiation in agreement with studies implicating integrins as the major receptors mediating the effects of ECM on myoblast alignment and fusion . However, even in the 11E cell line α-DG expression is not completely abolished and it remains possible that the residual α-DG is sufficient to play a role in the differentiation of myoblasts into myotubes. In addition to α-DG, several LN receptors of the integrin superfamily such as the α7β1, α9β1, and α3β1 integrins are expressed in skeletal muscle raising questions as to the relative contributions of integrins and α-DG in LN deposition on myotubes. As a first step in exploring this issue, C2 cells were immunostained with mAb IIH6 to α-DG or with antibodies recognizing several LN isoforms and the distribution of these proteins was compared in DG-deficient clones. In C2, 11F, and 11E cells, α-DG immunoreactivity is punctate and uniformly distributed over the myotube surface . However, the intensity of staining is greatly decreased on the surfaces of 11F and even more so on 11E myotubes relative to C2 myotubes . A similar pattern was obtained with live cultures labeled with this antibody (data not shown) confirming that the residual α-DG is properly targeted to the cell surface in the antisense cell lines. In all cell types, dense patches of α-DG immunoreactivity were found to be associated with spontaneous AChR clusters . Thus, the antisense cDNA construct affects the amount of α-DG on the surface of myotubes but not its distribution. In adult skeletal muscle, LN α2 and γ1 chains are expressed both synaptically and extrasynaptically, LN β1 chain is excluded from synaptic regions, while LN α4, α5, and β2 chains are only found at the NMJ . To map the distribution of most LN heterotrimers expressed in C2 cells, we used an anti-LN antiserum to LN α1, β1, and γ1 chains as well as the rat mAb 5D3 which recognizes mouse LN α1, α2, β1, and γ1 chains. Both antibodies gave similar results, showing a patchy distribution of LN on the surface of C2 myotubes with some larger aggregates . Since α-DG is diffusely distributed on the surface of C2 myotubes and there appears to be a pool of α-DG unbound to LN, it is difficult to determine from their distributions alone whether LN and α-DG may be interacting with one another. Nevertheless, decreased expression of α-DG in 11F and 11E cells leads to a dramatic reduction in LN immunoreactivity on the myotube surface suggesting that LN is bound to α-DG. Occasionally, dense accumulations of LN immunoreactivity were seen on the surfaces of 11F and, much less frequently, on 11E myotubes and these often coincided with densities of AChRs . 11A, 9B, and 10C myotubes expressed LN on their surface in a pattern and amount similar to C2 myotubes . Western blots of total extracts of C2, 11A, 9B, 10C, 11F, and 11E myotubes probed with the anti-LN antiserum showed similar levels of LN expression indicating that there is no defect in the biosynthesis of LN per se in the DG-deficient clones. Furthermore, very little LN is released into the culture medium , and no difference could be seen in the pattern of LN staining between cultures immunolabeled live or after fixation indicating that 11F and 11E myotubes do not accumulate LN intracellularly. Rather, most of the LN in 11F and especially in 11E cells is deposited on the surface of the dish in large, irregularly shaped deposits found between live cells (not shown). This paucity of surface LN and the extensive extracellular deposition of LN observed in cultures of antisense-expressing myotubes suggest a deficit in the ability of LN to bind to myotubes deficient in α-DG. Because of the high expression of LN α2 chain in skeletal muscle and its involvement in some types of muscular dystrophy in both humans and mice, we looked specifically at the distribution of this LN chain in C2 cells and DG antisense clones. In C2, 11F, and 11E cells LN α2 chain immunoreactivity was detected only on the surface of myotubes and never on the culture dish. In contrast with its abundance in mature skeletal muscle, LN α2 is rather sparse in C2 cells and could not be detected by Western blot on crude protein extracts (data not shown) but has been detected after enrichment by immunoprecipitation . LN α2 immunoreactivity on the cell surface was associated with AChR clusters in C2, 11F, and 11E myotubes. Outside of these clusters, LN α2 immunoreactivity was diffuse and faint. Integrin localization in C2 cells was determined with an antiserum to the β1 subunit which is common to all integrin heterodimers expressed in muscle that recognize LN . In all cultures, myotubes were intensely labeled, whereas myoblasts showed a much fainter punctate staining pattern. For C2 as well as 11F myotubes the integrin β1 subunit either had a diffuse punctate pattern or was found in large, intensely immunoreactive, oval patches . In 11E cells, a strip of intense immunoreactivity occasionally ran the length of the myotube. In cultures double labeled with α-BTX, immunoreactivity for the integrin β1 subunit only rarely overlapped with AChR clusters , indicating that this receptor is not likely to be responsible for the presence of LN at these sites. To further investigate any differences in the levels of expression of this integrin subunit, we used Western blotting to simultaneously probe the same blot for expression of the integrin β1 subunit and sarcomeric actin . Expression of sarcomeric actin has been shown to increase with muscle differentiation and is therefore an indicator of the relative proportion of myotubes versus myoblasts in our cultures. Under these conditions, Western blotting confirmed that all clones express similar levels of the integrin β1 subunit, indicating that β1 integrins are not upregulated to compensate for decreased DG expression in 11F and 11E cells. Conversely, the integrin β1 subunit is not downregulated in 11F and 11E cells indicating that the decrease in LN immunoreactivity on the surface of 11F and 11E myotubes is not due to a lack of integrin expression and that integrins are unable to compensate for decreased α-DG expression. In summary, the level of surface LN correlates well with α-DG levels in C2, 11F, and 11E myotubes, suggesting that α-DG is responsible for the assembly of an LN-rich ECM on the surface of myotubes. However, the amount of α-DG staining appeared more diffuse and extensive than that of LN, in C2 and, to a lesser extent, 11F myotubes, possibly because a significant proportion of α-DG was either free or else bound to another ligand, such as agrin . As a further test of the ability of α-DG to function as a LN receptor, we assessed whether excess surface α-DG was capable of binding exogenously added LN and whether such binding could redistribute α-DG on the myotube surface as shown previously for Xenopus myocytes . C2 and antisense-expressing myotubes were incubated overnight in the presence of 12 nM EHS purified LN 1 (α1β1γ1), then fixed and immunostained for α-DG or LN. A frequent feature of the LN treated C2 myotubes was the presence of large plaques of α-DG immunoreactivity as well as similar plaques of LN immunoreactivity suggesting that exogenous LN can bind to and reorganize the unbound pool of α-DG on the myotube surface . Plaques of α-DG and LN immunoreactivity were also seen on the surface of LN-treated 11F myotubes but these were of smaller size and much less frequent . No such plaques were found on LN-treated 11E myotubes, nor did exogenous LN bind well to 11E myotubes, consistent with the relative abundance of α-DG in these clones. This correlation of LN binding with α-DG expression and the redistribution of α-DG by exogenous LN suggest strongly that α-DG is a functional LN receptor. As noted previously , the aggregation of α-DG by LN may be an early step in ECM assembly, which, from our data, appears to be dependent on the level of α-DG expression. Although antisense clones were able to differentiate normally , a consistently higher degree of cell loss was observed after differentiation in 11F and 11E cultures. DAPI staining of cultures after 4 d in fusion medium confirmed a decreased number of nuclei in 11F and 11E cultures compared with C2 cultures . The degree and time of onset of cell loss in the antisense clones was quantified spectrofluorometrically after staining DNA with Hoechst dye. For the first 24 hours in fusion medium, C2, 11F, and 11E cultures have comparable numbers of cells. Subsequently, significant drops in cell number compared with C2 cultures are first detected at day 2 in 11E cultures and at day 3 in 11F cultures and by day 4, cultures of 11F and 11E cells emit 45 and 65% less Hoechst fluorescence, respectively, when compared with C2 cultures . Statistical analysis revealed a linear correlation between cell number in C2, 11F, and 11E cultures at day 4 and the amount of α-DG expressed . In contrast, 9B and 10C clones behaved essentially like C2 cells. 11A cells also showed an increase in cell number similar to C2 cells but were not included here because the slightly slower proliferation rate of 11A myoblasts led to an asynchronous differentiation of these cultures compared with the other clones rendering any comparison difficult. Thus the level of α-DG expression correlates with the amount as well as the time of onset of cell loss during differentiation of myoblasts into myotubes. Since the number of cells increases with differentiation in control cultures of C2, 9B, and 10C cells, it was important to establish whether the decreased cell number in DG-deficient clones was due to actual cell loss or a deficiency in proliferation. BrdU incorporation, as measured by a colorimetric assay, was used to determine proliferative activity. When relative proliferation rates were expressed as a ratio of BrdU incorporation over total cell number as determined by Hoechst fluorescence, 11F and 11E cells did not have lower proliferation rates than C2, 9B, or 10C cells . In fact the loss of cells at day 2 in 11E cultures seems to cause a drop in cell density sufficient to stimulate proliferation of the remaining myoblasts as indicated by the increase in BrdU incorporation in 11E cultures at day 3 . This increased proliferation translates into a small increase in cell number at day 4 . The reduction in cell number observed in both α-DG–deficient clones is therefore unlikely to be due to decreased proliferation rates but reflects a genuine loss of cells in these cultures due to increased apoptosis or necrosis. Visual inspection of DAPI-labeled cultures revealed the presence of a greater number of nuclei with condensed chromatin in cultures of antisense clones compared with control cells. Since apoptotic cell death leads to chromatin condensation, we used the TUNEL method to assay for apoptosis . To verify the specificity of this method in our assay, we counterstained the cultures with the nuclear dye DAPI allowing us to also identify apoptotic cells by morphological criteria. We found that cells with marked condensation of chromatin and cytoplasm (apoptotic cells) as well as cytoplasmic fragments with condensed chromatin (apoptotic bodies) were intensely labeled by the TUNEL method. Generally, labeled cells were in the latest stages of apoptotic cell death and had often assumed a round morphology. This method labeled both individual, poorly attached cells and nuclei within aggregates. It was therefore difficult to determine whether TUNEL-positive nuclei belonged to former myoblasts or myotubes. More rarely, nuclei of adherent cells were labeled, albeit less intensely. These nuclei were often found within myoblasts but were occasionally also seen in myotubes. Since in most cases TUNEL-positive nuclei could not be definitely assigned to myoblasts or myotubes, we quantified the number of apoptotic nuclei without attributing them to a particular cell type . A small proportion of nuclei was TUNEL-positive in differentiating C2, 11A, 10C, and 9B cultures, while a larger number of nuclei, often within rounded, poorly attached cells, were labeled in day 2 and older cultures of 11F and 11E cells . At day 3, 11F, and 11E cultures have 3 and 11 times more TUNEL-positive nuclei compared with cultures of C2 cells . These data indicate that reduced α-DG expression results in a persistent loss of myoblasts after transfer to fusion medium and a correspondingly significant increase in apoptotic cell death in both myoblasts and myotubes. Studies of dy/dy and mdx mice suggest that loss of membrane integrity is linked to a loss of interaction between dystrophin and the actin cytoskeleton, rather than a decreased cell surface expression of the DGC . However, recent studies have provided evidence that mutations affecting the expression of SGs can result in a loss of membrane integrity . Since we perturbed the expression of DG without affecting the SGs, we set out to determine the role, if any, of DG in the maintenance of membrane integrity. We used the vital dye Trypan blue to visualize myoblasts or portions of myotubes where membrane integrity was compromised. In our hands, Trypan blue stained nuclei much more intensely than the cell cytoplasm and allowed a direct visualization of the extent of membrane damage on each multinucleated myotube. In cultures of C2, 11F, and 11E cells maintained 3 d in fusion medium cell death has peaked and Trypan blue labeled the nuclei of some myoblasts . At this time myotubes often had one or two blue nuclei, indicating a localized loss of membrane integrity and only rarely were all nuclei in a myotube labeled. Notably, there were no significant differences between C2 and 11E cultures in either the number of myotubes with one or more nuclei stained with Trypan blue or in the proportion of myoblasts versus myotubes with blue nuclei . Therefore, a substantial reduction of α-DG expression at the surface of myotubes does not lead to a loss of membrane integrity. In muscle, the DGC is thought to link two proteinaceous matrices, the ECM and the subplasmalemmal cytoskeleton, providing structural support for the interposed plasma membrane. According to this widely held model α- and β-DG associate to form the core of the DGC with α-DG bound to LN and β-DG to dystrophin . To investigate functional aspects of this model, we have perturbed the expression of α- and β-DG by generating muscle cell lines stably transfected with an antisense DG cDNA expression construct. After several transfections and screening of many stable lines we identified two clones of C2 cells in which expression of DG was reduced significantly i.e., 40–50% and 80–90%, respectively. The two DG-deficient cell lines retain the ability to fuse and form myotubes, and express near normal levels of α- and β-SG, two other DGC proteins. Similarly, expression of other membrane proteins such as the AChR, and the MuSK receptor tyrosine kinase and β1 integrin are indistinguishable from parental C2 cells. Three other clones, 9B, 10C, and 11A, which were subjected to the same transfection and antibiotic selection and have wild-type levels of α- and β-DG, fuse normally and have no obvious cell loss indicating that the altered phenotype of 11E and 11F cells is not a trivial outcome of transfection and antibiotic selection. These observations argue that 11E and 11F cells differ only in their abnormally low expression of DG. An alternative possibility, especially in light of the relatively small number of clones we were able to isolate, is that an unidentified mutation in the antisense-expressing cell lines affects the glycosylation of α-DG so that it is poorly detected by mAb IIH6 . In fact, we do see a slightly faster migration during SDS-PAGE of α-DG from 11F and 11E myotubes . This may well be due to altered glycosylation since most of the apparent mass of α-DG on SDS-PAGE is due to carbohydrate moieties . However, an antibody directed against the core protein (anti-fusion protein B antiserum) also reveals a decrease of at least 50–80% in α-DG levels, which is equivalent to that seen with mAb IIH6 . Thus, α-DG appears to be expressed at low levels in 11F and 11E cells. That the residual α-DG is recognized about equally by a polyclonal antiserum to DG and by mAb IIH6 which is to a binding site on α-DG suggests that the small shift in electrophoretic mobility of α-DG in the antisense clones does not affect the interaction of α-DG with LN. The concomitant decrease of β-DG by 70–90% in 11F and 11E cells compared with control C2 cells, further suggests that any possible defects in glycosylation do not lead to excessive shedding of α-DG into the medium of 11F and 11E cells. To date there have been no reports of any human or animal myopathy linked to mutations in the DG gene. Mice null for the DG gene die very early in development suggesting that mutations which compromise its expression in tissues other than skeletal muscle may lead to embryonic lethality in humans. Previous studies indicate that α-DG functions as a receptor for two ECM proteins agrin and LN. For LN, this includes observations on: (a) binding of LN to α-DG isolated from muscle and nervous system ; (b) colocalization of LN with α-DG in muscle and other tissues ; (c) inhibition of LN-dependent differentiation in kidney by a mAb IIH6 to α-DG ; (d) coprecipitation of LN with dystrophin in cultured muscle cells ; (e) disruption of Reichert's basement membrane in mice rendered null for the DG gene . More recently, Henry and Campbell have implicated DG in basement membrane assembly in cultured embryoid bodies, though it is unclear whether this is mediated uniquely by α-DG binding to LN or also to other ECM molecules such as agrin and perlecan . In support of the hypothesis that α-DG is a LN receptor in muscle, we find that lower α-DG levels in 11E and 11F cells result in a corresponding reduction in the level of exogenous LN bound to the surfaces of these lines when compared with parental C2 cells. Moreover, there is a clear decrease in the deposition of endogenous LN on the surface of myotubes. This is not obviously a result of reduced synthesis and secretion of LN since DG-deficient cells have normal levels of LN and the ECM which forms on the culture substratum between the myotubes appears equivalently LN-rich in DG-deficient and parental C2 cells. The loss of surface LN in 11F and 11E myotubes does not appear to be a secondary consequence of a general disruption of the ECM since the distribution of collagen IV is not significantly affected (Montanaro, F., and S. Carbonetto, unpublished observations). Instead, DG deficiency leads to a rather selective loss of LN from the myotube ECM implicating α-DG as a functional LN receptor necessary for proper ECM assembly in skeletal muscle. Vachon et al. have suggested recently that the “de facto receptor” for LN 2 (α2β1γ1) in skeletal muscle is the α7β1 integrin. They report a decrease of the α7β1 integrin in human and mouse muscular dystrophies where mutations in the LN α2 chain lead to loss of LN 2 or expression of a truncated form. They further note that α-DG expression is unaffected in these instances concluding that it is not necessary for LN assembly in vivo. However in the dy/dy and dy 2J mutant mice there is a compensatory upregulation of LN 8 (α4β1γ1) which replaces LN 2 and could be responsible for the maintained expression of the DGC at the cell surface. Alternatively, expression of the DGC might be more dependent on its association with dystrophin than with its extracellular ligands. In fact, muscle cells in culture have a pool of surface α-DG that is not bound to LN . Similarly, in the dystrophin mutant mdx mouse, the observation that LN is present in the muscle ECM in spite of the dramatic decrease in the DGC at the cell surface, does not eliminate α-DG as a LN receptor in skeletal muscle. For example, although the α7β1 integrin is expressed at wild-type levels in mdx mice , a large fraction of LN in skeletal muscle in these mice and DMD patients is unusually soluble indicative of a weak binding to the muscle cell surface . Indeed, an early sign of pathology in DMD is the separation of the basement membrane from the muscle cell surface . Furthermore, no obvious colocalization of LN and the integrin β1 subunit is observed in cultures of C2 cells or of primary myotubes from mouse or human and in C2 myotubes the distribution of the LN α2 chain does not match that reported for the α7 integrin subunit . More importantly, in mice null for the integrin α7 gene the distribution of LN in skeletal muscle appears normal and the progressive muscle degeneration seems to be predominantly due to defects at the myotendinous junction . Thus, as Mayer et al. , we also propose integrins and α-DG may function as independent receptor complexes which together provide a link between LN and the muscle membrane that is necessary for muscle homeostasis. The DGC has been postulated to act as a superstructure for the muscle cell surface, and its loss has been correlated with disruption of the plasma membrane, necrosis, and in some cases apoptosis . In vivo, apoptosis could be a secondary consequence of the inflammation caused by the constant degeneration and regeneration of muscle fibers that occurs in the absence of dystrophin. Indeed, apoptotic cell death in skeletal muscle of the mdx mouse appears to be mainly caused by activated inflammatory cells that infiltrate the muscle mass and subsequent release of the cytotoxic protein perforin . However, our studies and those of Vachon et al. suggest that other apoptotic pathways might also be activated. Vachon et al. have studied the effect of LN 2 (α2β1γ1; merosin) on myotube stability in human and mouse muscle cell lines in culture. Several spontaneous variants deficient in LN 2 expression were cloned and found to have increased myotube degeneration after fusion. Addition of exogenous LN 2 increased myotube numbers in all clones and transfection with a human LN α2 chain cDNA decreased myotube degeneration and the abnormally high level of apoptosis in these cell lines. In our studies, decreased expression of α-DG disrupts LN expression on the surface of myotubes, and we expected to see a similar pattern of myotube degeneration. While there are some apoptotic nuclei within adherent myotubes, most TUNEL-positive nuclei were found within amorphous, loosely adherent masses, which could have been detached or “collapsed” myotubes, or clumps of dead myoblasts. Possibly, deficiency of α-DG could cause myotubes to detach more readily than a reduction in the expression of a single LN isoform. Indeed, α-DG–deficient cells would have impaired binding to most if not all LN heterotrimers expressed by muscle cells, as well as agrin and possibly perlecan . However, Henry and Campbell reported that absence of DG in embryonic stem cells affects basal lamina assembly but not adhesion to a LN-coated substratum. Similarly, preliminary results show no detectable difference in the ability of 11F and 11E myoblasts to adhere to LN-coated dishes compared with control myoblasts. Vachon et al. also reported that myoblasts from LN-deficient clonal variants have a reduced ability to fuse. In our studies, 11E and 11F cells fuse and differentiate normally indicating that α-DG may not mediate the effects of LN on myoblast fusion and that these are likely integrin mediated . However myoblast survival was compromised in both clones after serum withdrawal from the onset of differentiation . Serum withdrawal is typically associated with cell death, and we observed a decrease in the number of cells in all cultures within 24 h of switching to fusion medium. While C2 cells and all control clones had completely recovered after 48 h, 11F and 11E cells continued to be lost. Although death of DG-deficient myoblasts could be due to loss of adhesion, we located some adherent myoblasts that were TUNEL-positive indicating that apoptotic cell death can precede cell detachment. It is interesting to note that during differentiation of C2 myoblasts, the level of α-DG expression increases dramatically and its glycosylation changes (Leschziner, A., and S. Carbonetto, unpublished observations) suggesting that it might play a particular function at this developmental stage. Our results suggest that DG is important for myoblast survival during differentiation in culture and we speculate that loss of DG in vivo might affect muscle regeneration by satellite cells. Straub et al. have studied the permeability of muscle fibers to the vital dye Evans blue in dystrophic mice. In mdx mice skeletal myofibers have increased permeability to this hydrophilic dye most likely through disruptions in their plasma membranes which allow serum proteins to enter these presumably necrotic cells . In contrast, dy/dy mice with mutations in the LN α2 chain develop a more severe muscular dystrophy with a minor loss in membrane integrity as reflected by a relative impermeability to Evans blue and by normal levels of creatine kinase in the serum. In both dy/dy mice and patients with merosin-deficient congenital muscular dystrophy skeletal myofibers are apoptotic . Furthermore, recent studies have shown a strong correlation between loss of membrane integrity and lack of SGs at the muscle cell surface . In our studies, decreased expression of DG does not appear to affect the expression of α- and β-SG. We are currently investigating whether these SGs are correctly expressed at the sarcolemma of DG-deficient myotubes as would be expected from observations that membrane integrity is not compromised in myotubes from both 11F and 11E cells . Our results support the notion that the DG and SG complexes are expressed independently of one another in muscle cells and that they perform distinct functions vis à vis membrane integrity. The data presented here raise questions about how α-DG, a peripheral membrane protein, may transduce an extracellular signal to suppress apoptosis in either myoblasts or myotubes. α-DG is bound tightly to its transmembrane partner β-DG which, in turn, can interact with the SH2/SH3 domain containing adapter protein Grb 2 . An additional, second messenger, nitric oxide synthase, associates with syntrophins , equipping the DGC further for intracellular signaling. Thus, one can envision a scenario wherein these signaling intermediates are activated by LN binding to α-DG transmitting a signal to the cell interior via β-DG. To our knowledge, however, there is no precedent for an extracellular peripheral membrane protein like α-DG transmitting signals in this manner. Although no evidence is presently available for the involvement of the DG complex in the initiation of signaling, DG has been shown to be involved in the aggregation of acetylcholine receptors and to act downstream of the muscle specific tyrosine kinase receptor MuSK . Apoptosis or its prevention is often associated with activation of receptors to growth factors or ECM molecules. DG could be part of a cell surface signaling complex and/or act downstream of other tyrosine kinase receptors. LN/α-DG complexes on the myotube surface are involved in the assembly of a heterogeneous basal lamina that would include constituents of the ECM (e.g., glial growth factor, agrin, collagen) which directly activate receptor tyrosine kinases or act as coreceptors for them . Their inclusion in a two dimensional array of ECM in close proximity to the plasma membrane may facilitate activation of these or similar receptors inhibiting cell death. Also, the assembly of an ECM of proteins occurs coincident with assembly of an intracellular network which may “trap” and concentrate receptor tyrosine kinases activating them in a ligand-independent manner. Integrins may offer another route for intracellular signaling after LN binding to α-DG. Integrin expression changes dramatically during muscle differentiation , affecting the passage from a proliferative to a quiescent state in myoblasts , and promoting fusion . In mature myotubes, Vachon et al. speculate that the LN receptors responsible for regulating apoptosis in their studies are members of the integrin superfamily viz. α 7 β 1 . Integrins are well-known to be involved in apoptosis in nonmuscle cells where loss of integrin-mediated adhesion results in the downregulation of Bcl 2 and upregulation of Bax expression , as well as activation of the ICE protease cascade , all of which are intermediates in apoptotic cell death. Interestingly, α-DG accumulates at focal adhesions and colocalizes with the integrin β1 subunit when fibroblasts are grown on a LN substrate . Furthermore, Yoshida et al. recently reported evidence for cross-talk between integrins and the DGC. In their studies the α5β1 fibronectin receptor in L6 myoblasts can associate with dystrophin and the DGC. Fibronectin, or amino acid mimetics of fibronectin, stimulate phosphorylation of α- and γ-SG. Perhaps, similar cross-talk occurs between α7β1 heterodimer, and the DGC. α-DG binds to the last 2 globular domains in the LN α1 and α2 chains, a region distal to that of any known integrin-binding site so that LN bound to an integrin should be able to bind α-DG or vice versa. Binding of LN to α-DG stimulates aggregation of α/β-DG on muscle cells which may increase the chance of interacting with an integrin. Thus, α-DG, in addition to its function as a structural element of the cell surface may, by stimulating ECM assembly, enhance the interaction of ligands embedded in the ECM with integrins and other transmembrane receptors that suppress apoptosis. In conclusion, our data provide strong evidence that α-DG functions in muscle cells as a LN receptor which mediates ECM assembly. Furthermore, they indicate that the DGC regulates apoptosis in culture, which may have important implications for novel functions of the DGC as a signaling complex. | Study | biomedical | en | 0.999996 |
10377181 | Indirect immunofluorescence with primary mAb plus secondary fluorochrome-conjugated goat anti–mouse Ig antibodies (Southern Biotechnology Associates) and flow cytometry (FACSCalibur™; Becton Dickinson ) determined NK cell phenotypes. NK cell clones were identified using an anti-CD16 mAb (Immunotech). Expression of KIRs recognizing group 1 HLA-C alleles (KIR2DL2), group 2 HLA-C alleles (KIR2DL1), and HLA-Bw4 alleles (KIR3DL1) was determined with mAb EB6, GL183, and Z27, respectively ( 29 ) (provided by L. Moretta, University of Genova, Italy). Anti-HLA class I mAb and ME1 and B27M2 anti–HLA-B27 mAb were obtained from American Type Culture Collection (ATCC). NK cell–enriched preparations were obtained from PBMC by negative immunomagnetic selection ( Dynal ) of T cells, B cells, and monocytes with anti-CD3, -CD20, and -CD14 mAb (Immunotech) and were cultured with 100 U/ml rIL-2 for 4–6 d before cell-killing assays. For NK cell cloning, PBMC depleted of T cells by negative immunomagnetic selection with anti-CD3 mAb (OKT3; obtained from ATCC) were plated at the concentration of 10 cells/well in 96-well microtiter plates, activated with PHA, and cultured with IL-2 and irradiated feeder cells as described elsewhere ( 30 ). Bulk-cultured and -cloned NK cells were used as effectors in standard ( 30 ) 51 Cr-release cytotoxicity assays using autologous gene–transferred T cells as targets. E/T ratio was 10:1. Standard 51 Cr-release cytotoxicity assays against cell lines transfected with class I genes (provided by L. Moretta) determined NK clone allospecificity. Specificity for group 1 (Cw4-related) and group 2 (Cw3-related) HLA-C allotypes was analyzed using the HLA class I–negative cell line 721.221 and 721.221 cells transfected with Cw*0401 or Cw*0302 genes, respectively ( 31 ). Specificity for HLA-Bw4 allotypes (such as HLA-B27) was analyzed using untransfected C1R cells and C1R cells transfected with B*2705 gene ( 29 ). Specificity for the nonclassical MHC class Ib molecule HLA-E was analyzed using C1R cells expressing HLA-B7 ( 29 ) (cell surface expression of HLA-E is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules, such as HLA-B7) ( 3 ). E/T ratio was 10:1. Results are presented as percent inhibition compared with lysis of the untransfected 721.221 or C1R cells. Clones lysed the untransfected cells at levels exceeding 60%, whereas lysis of autologous cells (PHA lymphoblasts) never exceeded 5%. NK clones were considered specific for a given allotype when cytotoxicity was <50% of control lysis obtained against untransfected cells. The PA317 amphotropic packaging cell line was transfected with the pLXSN retroviral plasmid ( 32 ), G418-selected, and subcloned. A high retroviral titer clone was used to infect cells. To construct a retroviral vector unable to produce the neo protein, the SV40 neo transcriptional unit was deleted from the LXSN plasmid by BamH1–Nae1 restriction digestion, Klenow blunting, and religation. The modified plasmid was transiently transfected in Phoenix amphotropic packaging cells ( 33 ) to produce retroviral particles. To construct a retroviral vector carrying a neo gene encoding a mutated protein that cannot produce potentially nonprotective peptides, the 209 cystine TGT codon of the neo gene was mutated to TGA to truncate the reading frame. The LXSN plasmid was consequently used as a template in a PCR reaction with the following mutagenizing oligonucleotides: 5′-TGCAAAAAGCTTGGGCTGCAGGTC, 3′-CCCAGCCGGCCTCAGTCGATGAATC. The PCR product was cloned and replaced in the original LXSN plasmid at the Hind III–NgoIV fragment. The final plasmid was sequenced to ensure that the open reading frame of the neo gene was maintained throughout the cDNA to the new stop codon. The plasmid was used to transiently transfect Phoenix cells and produce retroviral particles. PHA-activated, 48-h IL-2–cultured T cells (PBMC forming rosettes with SRBC) were infected by repeated cycles of centrifugation in the presence of viral supernatants. For cloning, T cells were plated at the concentration of 0.5 cells/well in 96-well microtiter plates and cultured with 100 U/ml IL-2 and irradiated feeder cells. The gene-transferred T cell clones were identified by PCR on DNA. For PCR on DNA, 10 5 lymphocytes were lysed in 100 ml of a buffer containing 10 mM Tris/HCl, 50 mM KCl, 2.5 mM MgCl2, 0.1% gelatin, 0.45% NP40, 0.45% Tween 20, and 100 mg/ml proteinase K. 10 ml of extract was used for a PCR reaction with the following ψ region primers: 5′-TGGTTCTGGTAGGAGACGAG, 3′-GCTTCCCAGGTCACGATGTA. Reverse transcriptase (RT)-PCR was performed with published neo primers ( 34 ). Peptides were synthesized by solid-phase method on an automated multiple peptide synthesizer (AMS 422; Aimed) using F-moc chemistry ( 35 ). Purity was confirmed by reverse-phase HPLC. TAP2-deficient RMA-S cells transfected with the human β 2 m alone (referred to in this report as RMA-S) or in combination with the HLA-B*2705 class I gene (RMA–S–B27) were cultured for 24 h at 26°C ( 8 ). Peptides were added in two separate doses: 100 μM at the onset of the experiment and an additional 100 μM 12 h later. Peptide loading was documented by surface stabilization of HLA-B27 as measured by immunofluorescence and flow cytometry. RMA-S and RMA-S–B27 cells were 51 Cr-labeled overnight during the peptide pulsing. After labeling, cells were used as targets in standard cell-killing assays with KIR3DL1 + NK clones as effectors ( 30 ). Some cytolytic assays were performed in the presence of Z27 anti-KIR3DL1 mAb (500 ng/ml) ( 29 ). Donor 1: A2/A33, B52/B35, Cw4; donor 2: A2/A11, B51/B27, Cw2; donor 3: A2/A28, B44/B18, Cw5/Cw7; donor 4: A11/A28, B8/B35, Cw4/Cw7. Peripheral blood T lymphocytes were infected with LXSN, and the transduced cells were selected in G418. The selected cells as well as nontransduced control cells were used as targets in cytotoxicity assays with autologous IL-2–cultured NK cells as effectors. Transfer of LXSN conferred susceptibility to lysis by autologous NK cells in three consecutive, independent experiments . Immunofluorescence analysis of MHC class I expression showed that killing was not the consequence of a downregulation of MHC class I molecules at the surface of the transduced cells (not shown). The next step was to identify whether expression of the heterologous selectable marker gene, neo , or the defective retroviral mRNA itself (through translation of short open reading frames) was responsible for susceptibility of LXSN-infected cells to lysis. To this end, we measured NK killing of autologous T lymphocytes transduced with the same retroviral vector that did not contain the neo cDNA. T cells underwent the gene transfer procedure and were cloned by limiting dilution. Cells infected with LXSN were also cloned by limiting dilution without antibiotic selection. PCR on DNA identified neo -positive, “empty” vector–positive, and nontransduced control targets for autologous NK cell–mediated killing . neo expression was confirmed by RT-PCR. Remarkably, all of the neo -transduced clones and none of the empty vector–transduced clones (and none of the noninfected control clones) were killed by autologous NK cells. Therefore, within the LXSN retroviral vector, the neo gene was responsible for conferring susceptibility to NK lysis. The neo cDNA might include sequences that could be translated into heterologous peptides, replacing the autologous peptides at given MHC class I alleles, so that NK cells with the appropriate MHC receptors recognize the neo peptide– loaded alleles. To test this hypothesis, several NK clones were obtained from four random donors and used for cytotoxicity experiments with autologous LXSN-infected cells as targets. Interestingly, as illustrated in Fig. 2 A, some NK clones from donors 1, 2, and 3, but not from donor 4, killed the neo gene–expressing autologous targets. The three well-defined KIR specificities ( 1 , 2 ) were presumably present in these donors, because donor 1 and donor 2 expressed group 1 HLA-C and Bw4 HLA-B alleles; donor 3 expressed group 1 HLA-C, group 2 HLA-C, and Bw4 HLA-B alleles; donor 4 expressed group 1 and group 2 HLA-C but not Bw4 alleles (this donor, in contrast to the other donors, was homozygous for HLA-B alleles of the Bw6 group, which is not recognized by KIR) (see Materials and Methods for HLA typing). Clones were, therefore, analyzed for these KIR specificities by the use of target cells expressing the HLA-Bw4 allele B27, the group 2 HLA-C allele Cw3, and the group 1 HLA-C allele Cw4 and for expression of the corresponding KIR . Control targets, not recognized by KIRs, were cells expressing B7 (the binding of signal sequence peptides from certain class I alleles, such as B7, regulates the expression of HLA-E, a nonclassical MHC class Ib molecule recognized by CD94-NKG2A) ( 3 ). All clones from donors 1, 2, and 3 that lysed the neo -expressing autologous targets, but none of their nonlytic clones, exhibited Bw4 specificity and expressed KIR3DL1, a Bw4 receptor. Fig. 2 B illustrates data obtained from one lytic and one nonlytic clone, and Table I summarizes data from all clones of donors 1, 2, and 3. These three donors expressed alleles of the Bw4 group. In contrast, donor 4, whose LXSN-infected cells were resistant to autologous NK lysis, did not express Bw4 alleles. Thus, without exception, Bw4 specificity and the Bw4 receptor KIR3DL1 were the distinctive features of NK clones triggered to lyse genetically modified autologous targets. Moreover, and again without exception, lack of Bw4 specificity and failure to express KIR3DL1 defined clones that did not recognize the genetically modified autologous targets. As expected, several clones coexpressed multiple specificities and the corresponding KIRs. It was therefore remarkable that, in spite of the fact that a number of specificities might be involved, expression of the Bw4 receptor KIR3DL1 appeared to be both necessary and sufficient for recognition of the autologous gene-modified cells. Immunofluorescence analysis of HLA-B27 on the LXSN- infected cells from donor 2 revealed that susceptibility to lysis was not a consequence of selective downregulation of Bw4 molecules at the surface of the transduced cells (data not shown). The coexpression of other KIRs besides KIR3DL1 by lytic clones suggests that neo gene expression might also lead to the production of peptides interfering with MHC recognition of other receptors. However, the pivotal role of KIR3DL1 in detecting expression of the neo gene is emphasized by the presence of one neo peptide with anchor residues for Cw4 and a residue at position 8 that prevents recognition of HLA-Cw4 by KIR2DL2 (i.e., QYDDAVY F L) ( 11 ). As shown in Table I , clones expressing KIR2DL2 did not kill the gene-transferred cells unless they coexpressed KIR3DL1. Recognition of the Bw4 allele HLA-B27 by KIR3DL1 is the most extensively studied model of peptide-specific recognition of MHC class I by NK cells (8– 10). A comparison of the amino acid sequence of the neo protein with sequences of peptides reported to block recognition of HLA-B27 by KIR3DL1 (Table II ) showed that several nonamers within the neo protein possess (i) anchor residues at positions 2 and 9 that allow binding to HLA-B27 ( 36 ) and (ii) residues at positions 7 and/or 8 that are known to prevent KIR3DL1-mediated recognition of HLA-B27 ( 8 – 10 ). In addition, other P7 and/or P8 residues that prevent recognition of HLA-B27 were present within neo peptides anchoring to Bw4 alleles of the other donors (Table II ). We therefore asked whether neo peptides actually bind Bw4 alleles and if this complex specifically affects interaction with KIR3DL1. To this end, we synthesized one of the HLA-B27–binding neo peptides shown in Table II , GRLGVADRY, and the analogue peptide GRLGVAIHY, in which aspartic acid and arginine at positions 7 and 8 had been replaced by isoleucine and histidine, respectively. As isoleucine and histidine at positions 7 and 8 are critical for the interaction between HLA-B27 and KIR3DL1 ( 8 – 10 ), the latter peptide was expected to be a protective version of the original neo peptide. Peptides were loaded onto HLA-B27 molecules of RMA-S cells, as indicated by the surface stabilization of HLA-B27 molecules shown in Fig. 3 A. Peptide-pulsed RMA-S–B27 and control RMA-S cells were used as targets for lysis by three randomly chosen KIR3DL1 + (Bw4-specific) NK clones . Binding of the neo peptide was unable to protect RMA-S–B27 cells from NK lysis. In contrast, the analogue peptide conferred protection from lysis. Protection was mediated by HLA-B27 on target cells because no protection was conferred to control RMA-S cells and by KIR3DL1 on the NK clones because it was abrogated by the addition of anti-KIR3DL1 mAb. Similar results were obtained with the other HLA-B27–binding neo peptides shown in Table II . Therefore, exogenously loaded neo peptides prevent recognition of HLA-B27 by KIR3DL1. To document whether the endogenous production of these neo peptides is indeed capable of triggering autologous NK lysis, a stop codon preventing translation of the last 56/265 amino acids of the protein was created in the neo cDNA . The resulting truncated neo protein does not contain two of the four HLA-B27–binding, nonprotective peptides shown in Table II and used for the experiments in Fig. 3 (GRLGVADRY and QRIAFYRLL). T lymphocytes from an HLA-B27 + donor were transduced with the truncated neo protein–expressing gene and used as targets for autologous NK lysis by KIR3DL1 + clones . This deletion of ∼1/5 of the protein reduced lysis by over 60%. Importantly, the deleted fragment did not contain other HLA-B27–binding peptides with P7 and/or P8 residues that could have triggered autologous NK lysis. Therefore, the reduction of NK-mediated lysis of autologous T lymphocytes infected with the mutated vector must be attributed to the deletion of the two nonprotective peptides. Taken together, the experiments in Figs. 3 and 4 indicate that neo gene expression by LXSN-infected cells from HLA-B27 + individuals can generate peptides that prevent KIR3DL1-mediated recognition of HLA-B27 and trigger autologous NK lysis. The fact that 721.221 or C1R cells commonly transfected with class I genes for NK recognition employ neo selection may seem perplexing and opens the question as to why these cells, when they express Bw4 alleles, are not rendered susceptible to lysis by neo peptides. Our data show that the susceptibility to lysis conferred by the neo gene is limited. On the other hand, the degree of protection conferred to 721.221 or C1R cells by class I transfection seldom reaches the 100% level of unmanipulated autologous cells, even when NK clones with single class I allele specificity are used. It is therefore plausible that the neo gene also partially antagonizes the protective effect of Bw4 allele expression in 721.221 or C1R cells. The fact that clones with specificity for Bw4 allotypes were responsible for killing autologous LXSN-infected cells implies that it should be possible to predict that HLA-Bw4 + individuals may possess an NK cell repertoire with potential to clear gene therapy–modified cells. Individuals (like donor 4) homozygous for the reciprocal group of HLA-B allotypes, Bw6, should tolerate the engineered cells. Assessment of the impact of the present findings on survival of gene-transferred cells will require monitoring of gene expression at serial times after the reintroduction of engineered cells in vivo. We had the opportunity to evaluate two patients with adenosine deaminase deficiency undergoing adenosine deaminase gene therapy with the LXSN vector (carrying neo ) ( 26 ). One patient expressed alleles of the Bw4 and Bw6 groups, and the other was homozygous for Bw6 alleles. Both patients had undergone gene therapy simultaneously 4 mo before our assessment. The HLA-Bw4 + patient, compared with the HLA-Bw6 + patient, exhibited 100-fold lower levels of the transduced gene. Finally, our observations, by demonstrating that NK cells can selectively detect the expression of heterologous genes, provide a general model of the NK cell–mediated control of viral infections. | Study | biomedical | en | 0.999996 |
10377182 | For cell culture, healthy, PPD skin test– negative donors from the laboratory staff at Case Western Reserve University were bled and genotyped as below. In the pilot case-control analysis, a different population of 89 unselected patients and 114 control subjects who were Hindu, residents of London, and identified as being of Gujarati origin were recruited from Northwick Park Hospital, Harrow, England. The peak migration of Gujaratis to west London followed political change in East Africa in the decade 1970–1980. There is a high incidence of tuberculosis amongst Gujaratis in Harrow of ∼128/100,000 ( 25 ), with an unusual excess of extrapulmonary disease in females. Within this community, 35–65% of marriages are prearranged, marriage to non-Gujarati Hindus is rare, and marriage to non-Hindus is exceptional (Patel, P., and R.J. Wilkinson, unpublished observations). 62% of subjects in this study were bacille Calmette-Guérin vaccinated. All 89 patients (average age 42.3 ± 1.7 yr; 56 females and 33 males) had culture- or biopsy-proven tuberculosis. All patients had free access to optimal medical care. The median duration of symptoms at diagnosis was 31 d (21 and 90 d being the 25th and 75th quartile values), thereby minimizing the effect of chronicity on clinical presentation. The definition of clinical phenotype was based on the International Classification of Disease 9 classification, and the overwhelming majority of patients were judged to have delayed postprimary (reactivation) disease. Patients known to be immunosuppressed (e.g., by HIV infection or corticosteroid therapy) were excluded. Mantoux testing was performed by the intradermal injection of one tuberculin unit of PPD (Evans Medical). The resultant diameter of transverse induration was recorded after 48 h. This low dose of tuberculin is routinely used in the United Kingdom to avoid necrotic reactions. All 114 nonconsanguineous (spouses of patients where possible) healthy controls (average age 42.9 ± 1.2 yr; 54 females and 60 males) were recruited from the tuberculosis contact clinic at the same hospital and had documented contact with tuberculosis (often multiple). All were PPD skin test–positive, asymptomatic, and had normal chest radiographs. 10/114 (8.7%) received chemoprophylaxis. These subjects were recruited between June 1995 and May 1998; in June 1998, all remained disease free. Ethical permission for this case-control analysis was obtained from the Harrow local research ethical committee . The genotypes were determined as previously described ( 20 , 22 ). DNA was isolated by phenol-chloroform extraction, and 5 ng was used in the PCR amplification of the IL-1Ra VNTR region, using 0.05 μM of the following primers: 5′-TCC TGG TCT GCA GGT AA-3′ and 5′-CTC AGC AAC ACT CCT AT-3′. The mixture was heated to 96°C for 1 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, 70°C for 1 min, and then a final 7 min at 70°C. Products were run on an ethidium bromide–stained, 1.5% agarose MR gel ( Boehringer Mannheim ) and visualized directly. A 304-bp fragment of the IL-1β gene from −702 to −398 was amplified using the following primers: 5′-TGG CAT TGA TCT GGT TCA TC-3′ and 5′-GTT TAG GAA TCT TCC CAC TT-3′, using the same cycling conditions as above. The products were digested overnight at 37°C with 5 U Ava 1 and run on a 2.5% gel as above, generating the following patterns: single band of 304 bp, A2/A2 homozygote; two bands at 190 and 114 bp, A1/A1 homozygote; all three bands, heterozygote. A 249-bp fragment of the IL-1β exon 5 was amplified using the following primers: 5′-GTT GTC ATC AGA CTT TGA CC-3′ and 5′-TTC AGT TCA TAT GGA CCA GA-3′. The mixture was heated for three cycles of 94°C for 2 min, 55°C for 2 min, 74°C for 1 min, then 32 cycles of 94°C for 1 min, 55°C for 1 min, 74°C for 1 min, and then a final 10 min at 70°C. The products were digested overnight at 65°C with 2.5 U Taq 1 and run on a 3% gel, generating the following patterns: single band of 249 bp, A2/A2 homozygote; two bands at 135 and 114 bp, A1/A1 homozygote; all three bands, heterozygote. PBMCs were separated over a Ficoll ( Pharmacia Biotech ) gradient. Preliminary experiments established that conventional separation of monocytes by adherence to plastic, harvesting, and replating led to spontaneous release of IL-1Ra. To reduce such activation, freshly isolated PBMCs were cultured at 2.5 × 10 6 /ml in 24-well plates in RPMI 1640 (Biowhittaker) without antibiotics in the presence of 2% autologous serum. In each experiment, the number of monocytes present in PBMCs was determined by washing off nonadherent cells (×3) in a duplicate well and then detaching the adherent cells using ice cold PBS and a cell scraper. Monocyte counts were generally ∼10% of the total PBMC numbers. Preliminary experiments showed that IL-1Ra production under these conditions was detectable by 4 h and reached a plateau by 10–12 h, with no further significant increase during the next 12 h. There was no significant difference in production between experiments in which the nonadherent cells had been removed by washing and wells containing unseparated PBMC, indicating that the adherent cells were responsible for the IL-1Ra secretion. We therefore collected, and froze at −70°C, PBMC supernates after 10 h of culture. In some cases, cell lysates were prepared by adding an equal volume of PBS and then freeze-thawing once. In this way we established that the ratio of IL-1Ra secreted into the supernate to that remaining in cell lysates was consistently >10:1, irrespective of time point, stimulus, and genotype. M . tuberculosis H37Ra and H37Rv was prepared and aliquotted as previously described ( 26 ). Aliquots were vortexed for 15–20 min before use at an infection ratio of 0.1 or 1 M . tuberculosis bacilli/1 PBMC (corresponding to ∼1:1 and 10:1 per monocyte). PPD of M . tuberculosis was the gift of Lederle Labs. (American Cyanamid Co.) and used at 0.1–100 μg/ml. Recombinant TGF-β, IL-4, and IFN-γ, and the neutralizing antibodies to IL-1β (mouse IgG 1 ), IL-6 (polyclonal goat IgG), TGF-β (polyclonal chicken IgY), and TNF-α (mouse IgG 1 ), and appropriate isotype control antibodies were purchased from R & D Systems, Inc. All recombinant cytokines, PPD, M . tuberculosis , and neutralizing antibodies used were tested for endotoxin contamination by the Limulus amebocyte assay (Biowhittaker) and were either free or contained very small levels (always <2ng/mg) of endotoxin. Maxisorp (Nunc, Inc.) plates were coated overnight at 4°C with 100 μl of the following coating antibodies in PBS: 2 μg/ml anti–human IL-1β mAb or 5 μg/ml of anti– human IL-1Ra mAb (both from R & D Systems, Inc.). After washing in PBS/0.05% Tween 20 (×3), the plates were blocked for 1 h at room temperature (rt) using 300 μl 1% BSA/5% sucrose/ 0.05% NaN 3 in PBS. After three further washes, duplicate 100-μl samples and dilutions of standard cytokines were then incubated for 2 h at rt. After washing (×4), 100 μl of the following biotinylated detection antibodies were added in diluent (0.1% BSA, 0.05% Tween 20 in TBS, pH 7.3): 100 ng/ml anti–human IL-1β antibody or 20 ng/ml anti–human IL-1Ra antibody (both from R & D Systems, Inc.). After 2 h at rt, the plates were washed (×5) and 100 μl streptavidin horseradish peroxidase (Jackson Immunoresearch) at 1:5000 in diluent was added. After 20 min, six final washes were followed by the addition of 100 μl of 3,3′,5,5′-tetramethylbenzidine hydrochloride solution in perborate ( Sigma Chemical Co. ) to each well. The reaction was stopped by adding 50 μl/well 0.5 N H 2 SO 4 , and the plates were read at 450 nm in an ELISA reader. The sensitivity of each cytokine ELISA was as follows: IL-1β, <1 pg/ml and IL-1Ra, 0.05 ng/ml. 5 × 10 7 freshly isolated PBMCs were used to obtain ∼5 × 10 6 adherent cells. This population of cells is up to 90% monocytes by cytostaining and is 99% viable ( 27 ). After resting overnight, the adherent cells were infected as above with M . tuberculosis at 1:1. After 4 h, the cells were harvested, and total RNA was extracted using guanidinium isothiocyanate, CsCl 2 density gradient centrifugation, and ethanol precipitation. 2 μg of the resultant RNA was hybridized overnight according to the manufacturer's instructions to a cocktail of [ 32 P]UTP (Du Pont)-labeled complimentary RNA probes ( PharMingen ) for IL-1α, IL-1β, IL-1Ra, IL-6, IL-10, IL-12 p40 and p35, TNF-α and -β, TGF-β 1–3 , LT-β, and the housekeeping genes L32 and GAPDH at 56°C. Single-stranded RNA was digested by incubation with RNase for 45 min at 37°C and the protected fragments reextracted by ethanol precipitation. The products were electrophoresed on a 5% denaturing polyacrylamide gel; a negative control RNA and the unhybridized radioactive probe were run in each experiment. The gel was exposed overnight using a Biorad Geldoc 1000. The identity of the protected bands was confirmed by reference to the unhybridized probes and quantitated by reference to bands for the housekeeping genes L32 and GAPDH. This assay was performed as previously described with minor modifications ( 26 ). In brief, adherent cells were plated in triplicate wells in 96-U microtiter plates (Corning Glass Works) and readhered for 2 h. Cells were infected with M . tuberculosis H37Ra at 1:1, 10:1, and 100:1 (bacillus/cell) in 30% autologous serum. After 2 h, noningested bacteria were removed by washing gently (×3) with prewarmed RPMI 1640. Each well then received RPMI 1640 containing 2% autologous serum, and the plates were cultured in a humidified incubator at 37°C in the presence of 5% CO 2 for as little as 1 h (time 0 sample) up to 10 d. Duplicate wells contained 2 μg/ml of neutralizing anti–IL-1Ra (goat IgG; R & D Systems, Inc.) or the same amount of isotype control antibody. At the end of the culture period, supernates were aspirated, and the plates containing the infected adherent cells were frozen at −70°C. To determine the number of intracellular bacteria in the CFU assay, the plates were thawed and cells lysed with 0.25% SDS in PBS for 12 min and then neutralized using 20% BSA. The lysates were then 10-fold serially diluted with 7H9 broth (Difco Labs., Inc.), and three 10-μl aliquots of each dilution were plated on Middlebrook 7H10 agar (Difco Labs., Inc.). The plates were then incubated for 19 d at 37°C in humidified air with 5% CO 2 . At the end of this culture period, the number of CFUs in each of the three replicate spots was enumerated for at least two consecutive dilutions using a stereomicroscope and averaged. Using this technique, extracellular growth of mycobacteria as assessed by culture of the supernates is consistently >1 log lower than intracellular growth ( 26 ). The rate of intracellular growth expressed as doubling time was determined by reference to the logarithmic growth from the cultures. Values throughout are quoted or shown as the mean ± SE. Normally distributed variables were analyzed by paired or unpaired t test. P values reflect two-tailed values of t . Unpaired nonparametric variables were analyzed by the Mann-Whitney U test. Contingency analysis was performed using Fisher's exact test of probability. First, we examined the M . tuberculosis –stimulated production of IL-1Ra by culture of 2.5 × 10 6 PBMCs for 10 h. Culture supernates were assayed for IL-1Ra content, and the results were normalized to the number of monocytes in culture. The relationship between polymorphism in IL-1RN in 17 donors homozygous for the A1 allele (IL-1Ra A2 − ) and 16 donors at least heterozygous for A2 (3 A2/A2, 13 A1/A2; IL-1Ra A2 + ) and the M . tuberculosis– induced secretion of IL-1Ra was determined. The other alleles of IL-1RN were very rare and therefore could not be assessed. The M . tuberculosis– stimulated IL-1Ra response of A2/A2 homozygotes did not differ from A1/A2 (data not shown), confirming the previous finding that IL-1Ra A2 is codominant ( 21 ). The unstimulated production of IL-1Ra was slightly, but not significantly, higher in the IL-1Ra A2 + group . Stimulation by M . tuberculosis (0.1 and 1:1 bacillus/cell) caused a dose-dependent increase in IL-1Ra secretion irrespective of genotype. However, the median response of the IL-1Ra A2 + group was 1.9 times greater at both doses of M . tuberculosis tested ( P = 0.02 at 1:1). In a subset of 16 healthy subjects, the dose response of IL-1Ra induction to PPD was also determined . Although IL-1Ra A2 − individuals showed a dose-dependent increase in IL-1Ra secretion, this did not become statistically significant until the dose of PPD was 100 μg/ml. The response of IL-1Ra A2 + individuals was 2.1–3.6 times higher, depending on the dose. In contrast, induction of IL-1Ra in IL-1Ra A2 + donors was significant at 1 μg/ml. Thus, IL-1Ra A2 + donors appeared more sensitive to PPD stimulation. The median production of IL-1Ra in response to LPS (10 μg/ml) was also 1.82 times greater in the IL-1Ra A2 + donors (6.6 ± 1.3 vs. 3.6 ± 0.5 ng/ml/10 5 monocytes, P = 0.012). We next determined the level of IL-1β in the same culture supernates used for the analysis of IL-1Ra. In contrast to the IL-1Ra polymorphism, the two polymorphisms in the IL-1β gene did not correlate with the M . tuberculosis –stimulated production of IL-1β to the same extent. The median M . tuberculosis (at 1:1)-stimulated production of IL-1β in subjects positive for the −511 A2 ( n = 20) was 635 ± 119 pg/ml and 404 ± 261 pg/ml in A1/A1 homozygotes ( n = 8). The corresponding figures for the +3953 polymorphism were 404 ± 84 pg/ml (A2 + , n = 12) and 643 ± 171 pg/ml (A1/A1 homozygotes, n = 16). IL-1β production did tend to be higher in IL-1Ra A2 − subjects, but only significantly so in response to M . tuberculosis at 0.1:1 ( P = 0.01) (data not shown). As a pure antagonist of IL-1, IL-1Ra competes for occupancy of IL-1RI, and it has been estimated that IL-1Ra needs to be present in a large molar excess (25–50×) to antagonize IL-1β significantly ( 28 ). Therefore, the ratio of IL-1Ra/ IL-1β is likely to be more relevant to regulation of the inflammatory response than the absolute value of either cytokine. The molar ratio of IL-1Ra/IL-1β was therefore calculated for each supernate and was significantly higher in IL-1Ra A2 + individuals ( P ≤ 0.05) in response to doses of both M . tuberculosis and PPD at 1, 10, and 100 μg/ml , in some cases with only minor overlap between the groups. By contrast, the response to LPS did not differ significantly between the groups. Fig. 2 B shows that the highest ratios likely to result in antagonism of the IL-1β response to PPD and M . tuberculosis stimulation (especially at lower doses likely to be relevant to M . tuberculosis –infected foci) were observed in the majority of IL-1Ra A2 + individuals but only in a minority of IL-1Ra A2 − subjects. The bulk of the experiments were performed using attenuated M . tuberculosis H37Ra. Therefore, parallel determination of IL-1Ra and IL-1β secretion using the same doses of M . tuberculosis H37Rv in three donors (one A1/A1 and two A1/A2) was also performed. The level of each cytokine was very similar, such that at an infection multiplicity of 1:1 the IL-1Ra/IL-1β ratio when stimulated by H37Rv was 4.1, 16.8, and 12.6 for the three donors and 6.2, 17.3, and 8.0, respectively when stimulated by H37Ra. We thus have no reason to suspect that the findings using M . tuberculosis H37Ra would not apply to virulent clinical isolates. We next sought to investigate association between the polymorphisms and the expression of mRNA. Ribonuclease protection assay was performed on RNA from 13 donors, all of different genotypes. The spontaneous expression of IL-1Ra and IL-1β transcript was low. There was no constitutive expression of any other monocyte cytokine, indicating that this low expression was unlikely to have been due to a nonspecific effect of cellular activation during isolation. Within 1 h, M . tuberculosis induced IL-1Ra gene expression in all individuals, irrespective of genotype, together with the mRNAs for IL-1β and TNF-α and followed slightly later (2 h) by IL-1α and IL-6. At hour 4, there was higher induction of IL-1Ra in the IL-1Ra A2 + subjects consistent with the protein data, although the difference was not statistically significant (Table I ). The IL-1β +3953 allele A2 was associated with significantly lower production of IL-1β transcript ( P = 0.04). Taken together, we interpret these observations to indicate that the alleles are associated with differences in transcription, but the dissociation between induction and secretion, particularly in the case of IL-1β, indicates that posttranscriptional mechanisms also influence cytokine secretion. The results so far showed that in response to M . tuberculosis or its PPD, IL-1Ra gene expression is induced within 1 h, large quantities of protein are secreted within 10 h, and differences between individuals could be related to their genotypes. However, an indirect modulating influence of M . tuberculosis via increased translation of preexisting IL-1Ra mRNA or an effect of other cytokines (such as TGF-β, TNF-α, IL-1β, and IL-6) produced by monocytes early in response to infection is also possible. We investigated this possibility by assessing the ability of antibodies known to neutralize the biological effects of TGF-β, TNF-α, IL-1β, and IL-6 on M . tuberculosis– stimulated production of IL-1Ra. Control wells received isotype-matched antibodies. No consistent effect on constitutive or stimulated IL-1Ra secretion was seen, irrespective of genotype, cytokine, or dose of antibody used (up to 1,000-fold the ED 50 concentrations). TGF-β modulates the human response to tuberculosis ( 29 , 30 ) and has also been reported to increase IL-1Ra in some ( 31 ) but not all studies ( 32 ). We therefore also evaluated the effect of rTGF-β (0.1–10 ng/ml) on both M . tuberculosis –stimulated and –unstimulated IL-1Ra production in 12 individuals (6 IL-1Ra A2 − and 6 IL-1Ra A2 + ). No significant enhancement of the early secretion of IL-1Ra was seen (data not shown). However, rIL-10 (0.1–10 ng/ml) caused a significant dose-dependent increase in the M . tuberculosis – stimulated IL-1Ra/IL-1β ratio in IL-1Ra A2 + and IL-1Ra A2 − donors at all doses tested ( P < 0.02), an effect largely due to the suppression of IL-1β production . The addition of rhIL-6, however, caused no significant change in the IL-1Ra/IL-1β ratio in either group. It has also been shown that the lymphocyte production of IFN-γ and IL-4 can differentially modulate IL-1β and IL-1Ra production ( 33 ). Our coculture system excluded the possibility of an obscuring effect of T cell cytokines by the sole use of PBMCs from PPD − individuals and a short culture duration. In fact, the production of IFN-γ was negligible in the M . tuberculosis– stimulated cultures (20 pg/ml) from these subjects. To investigate the possibility that T cell cytokines modulate M . tuberculosis– induced IL-1Ra and IL-1β secretion, rhIFN-γ or rhIL-4 were added (0.1–10 ng/ml) to cultures. IL-4 caused a dose-dependent increase in both unstimulated and M . tuberculosis –stimulated IL-1Ra production, which was most significant in the M . tuberculosis –stimulated IL-1Ra A2 + group ( P = 0.002 at 10 ng/ml). Furthermore, IL-4 also significantly decreased IL-1β production in M . tuberculosis –stimulated cells from both genotypes ( P < 0.05 at 10 ng/ml). By comparison, IFN-γ led to a dose-dependent increase in M . tuberculosis –stimulated IL-1β production that was most marked in the IL-1Ra A2 + group ( P = 0.052 at 10 ng/ml). Thus, IFN-γ tended to increase IL-1β production in M . tuberculosis –stimulated cells without affecting IL-1Ra production, whereas IL-4 increased IL-1Ra production irrespective of genotype and also depressed IL-1β secretion. This differential effect was reflected in the mean M . tuberculosis –stimulated IL-1Ra/IL-1β ratio, which increased in response to IL-4 even at the lowest dose of 0.1 ng/ml ( P < 0.01, both groups combined). By comparison, higher doses of IFN-γ (1–10 ng/ml) were required to reduce the IL-1Ra/IL-1β ratio significantly . We next investigated the effect of IL-1Ra polymorphism on the rate of intracellular replication of M . tuberculosis . Monocytes from 22 donors (12 IL-1Ra A2 − and 10 IL-1Ra A2 + ) were infected with M . tuberculosis at various multiplicities (1:1, 10:1, and 100:1 bacillus/cell) and then cultured in vitro for up to 240 h. Cell lysates were set up for M . tuberculosis CFU assay at 0, 24, 96, 168, and 240 h. Although there was interindividual variation in the establishment of initial infection, there was no significant difference between the IL-1Ra A2 − and IL-1Ra A2 + groups. Logarithmic growth was established in 8 donors. The remainder showed either minimal or linear intracellular growth of mycobacteria only, with no difference between IL-1Ra A2 − and IL-1Ra A2 + donors. In those donors in whom logarithmic growth did occur (5 IL-1Ra A2 − and 3 IL-1Ra A2 + ), the doubling time of M . tuberculosis was estimated from the growth curve. Data from these individuals is shown in Table II . Intra- and interindividual differences did not appear to be related to the presence or absence of the IL-1Ra A2 allele. These data therefore contrast with the readily demonstrable increase in IL-1Ra secretion conferred by the A2 allele in the same donors (shown in parentheses in Table II ). In each experiment, triplicate wells were also included to assess the effect of 2 μg/ml neutralizing antibody to IL-1Ra (and goat IgG isotype control). No consistent effect of these antibodies on intracellular growth was seen (data not shown). We next sought in vivo correlates by determination of the frequency of the IL-1β and IL-1Ra polymorphisms in patients with tuberculosis and healthy PPD-reactive control subjects in a pilot case-control analysis of Gujarati asians in west London. This population is distinct and has a high incidence of tuberculosis with an excess of extrapulmonary forms. Individual alleles at each locus were in Hardy-Weinberg equilibrium. The IL-1β (−511) allele 1 was in linkage disequilibrium with IL-1β allele 2 and vice-versa ( P < 0.03). In addition, there was weaker linkage between IL-1Ra A2 and IL-1β A2. No allele or genotype, singly or in combination, was associated with an increased risk of tuberculosis (Table III ). We concluded that, in this population, these polymorphisms have little effect on susceptibility to tuberculosis per se. The in vitro data indicated that the IL-1Ra A2 − /IL-1β A1 + haplotype was associated with low IL-1Ra protein and gene expression and higher corresponding IL-1β values, implying a proinflammatory phenotype. We therefore examined association between the gene polymorphisms and the presenting form of post-primary tuberculosis (Table IV ). This proinflammatory haplotype was more common in patients with pleural disease ( P = 0.028 by comparison with control subjects). Pleural tuberculosis represents a contained disease phenotype associated with a high DTH response and marked proinflammatory cytokine responses at the site of disease ( 34 , 35 ). By comparison, the IL-1Ra A2 was more common in patients with extrapulmonary disease ( P = 0.009, by comparison with pleural disease). A similar reduction in DTH as manifested by cutaneous reactivity to PPD was also associated with the presence of the IL-1Ra A2 allele: the proportion of IL-1Ra A2 + individuals progressively decreased at higher grades of Mantoux . We have investigated the effect of polymorphisms in the IL-1β and IL-1Ra genes on M . tuberculosis –stimulated cytokine production in vitro and their relevance in patients with tuberculosis. When compared with healthy IL-1Ra A2 − subjects, A2 + subjects as a group secreted nearly twice as much IL-1Ra in response to both laboratory-adapted and virulent M . tuberculosis , PPD, or LPS. The mean fold induction of IL-1Ra mRNA was also nearly twice that of IL-1Ra A2 − subjects. The two polymorphisms in the IL-1β gene were not clearly associated with the level of M . tuberculosis –stimulated IL-1β production in vitro, although the IL-1β A1 + haplotype was associated with significantly increased M . tuberculosis– induced expression of the IL-1β gene. The individual molar ratios of IL-1Ra/IL-1β, which determine the net effect of these cytokines in response to PPD and M . tuberculosis, were clearly higher in IL-1Ra A2 + subjects. Furthermore, the IL-1Ra/IL-1β ratios were affected by cytokines, as IL-4 upregulated IL-1Ra production and downregulated IL-1β production. IL-10 greatly suppressed and IFN-γ moderately enhanced the production of IL-1β. In patients with tuberculosis, the proinflammatory IL-1Ra A2 − /IL-1β A1 + haplotype was unevenly distributed, being more common in patients with pleural disease and less common in those with extrapulmonary disease. A further finding, consistent with the in vitro observations, was that the IL-1Ra A2 + haplotype was associated with a reduced Mantoux response to PPD of M . tuberculosis : 60% of tuberculin-nonreactive patients were of this type. Our study of IL-1RN gene expression indicates the early induction by M . tuberculosis of its mRNA together with IL-1β, IL-1α, TNF-α, and IL-6. Although IL-1Ra A2 was associated with an increased induction of the IL-1RN gene, the exact mechanism of increased IL-1Ra production requires further elucidation. Whereas the fold induction of IL-1β mRNA was higher than that of IL-1Ra and could also be related to both IL-1β polymorphisms, the amount of secreted IL-1β protein was much less. In addition, the IL-1β polymorphisms could not so readily be related to protein secretion. This observation is consistent with other data ( 36 ) and indicates a dominant influence of both posttranscriptional and posttranslational events on the secretion of IL-1β. Many cytokines can upregulate IL-1Ra expression in vitro ( 17 ). The production of IL-1Ra, however, was unaffected by antibody neutralization of IL-1β, IL-6, TGF-β, and TNF-α, suggesting that M . tuberculosis or its products induce the early production of large quantities of IL-1Ra by a direct mechanism. IL-1β is involved in the early recruitment of inflammatory cells to M . tuberculosis– or PPD-induced granulomas ( 37 – 41 ). Submaximal occupancy of IL-1RIs can mediate the full biological effects of IL-1β, and as a consequence, it has been postulated that IL-1Ra needs to be present in a large molar excess in order to exert its antagonism ( 28 ). In tuberculosis, this condition would be best fulfilled in IL-1Ra A2 + subjects ; the IL-1Ra A2 allele was associated with reduced DTH and was lower in frequency in patients with pleural tuberculosis, consistent with the in vitro data and suggestive of biological significance. Antigen-specific lymphocytes are also necessary for the DTH reaction to proceed. In our experiments, IL-4 increased IL-1Ra secretion, particularly in stimulated cultures from IL-1Ra A2 + subjects . The production of IL-4 in tuberculosis has been best demonstrated in T cell clones ( 42 ), but one study has also documented small amounts of antigen-specific secretion of IL-4 by PBMCs ( 43 ). As cell-associated IL-4 is a stimulus for IL-1Ra, there is the possibility that relatively small amounts of IL-4 may greatly affect the IL-1Ra response ( 33 ). IFN-γ decreased and IL-10 increased the IL-1Ra/IL-1β ratio mainly through an effect on IL-1β secretion. Both IFN-γ and IL-10 are produced by PBMCs and at disease sites in patients with tuberculosis ( 29 , 44 , 45 ). Our data therefore suggests that the polymorphism in the IL-1Ra gene may exert regulatory influence on cytokine circuits beyond its direct effect on IL-1Ra production. There is both epidemiological and experimental evidence of a dissociation between DTH and protection from tuberculosis ( 46 , 47 ). Our finding that IL-1Ra appears to influence DTH with minimal effect on either the intracellular growth of M . tuberculosis in vitro or disease susceptibility in the case-control study further suggests a basis for the dissociation between DTH and susceptibility. In addition to disease susceptibility, the degree of cutaneous reactivity to PPD after bacille Calmette-Guérin vaccination in both mono- and dizygotic twins and in siblings is also heritable ( 48 , 49 ). Our in vitro data clearly suggest a functional basis for the observed association between reduced DTH and A2 of the IL-1RN gene. Although our case-control analysis was modest in size, there was a distinct difference in IL-1Ra A2 + frequency between patients with pleural and extrapulmonary tuberculosis, and this preliminary data encourages us to determine in larger studies whether this association is generalizable to other populations. As our data also support a heritable component in the quantitative skin response to PPD, another appropriate strategy would be to perform a genome-wide search, which may not only confirm the involvement of the IL-1 locus but also potentially identify loci of relevance to other infectious processes as well ( 50 ). As the frequency of the IL-1Ra A2 allele is approximately six times lower in Gambia ( 51 ) and also in Kenya (Wilkinson, R.J., and P.A. Zimmerman, unpublished observations), perhaps this gene has been subject to natural selection by different major infectious diseases in India or Africa. It would also be interesting to determine whether a high IL-1Ra allele A2 frequency is present in populations with a high degree of PPD “anergy” ( 3 ). The association between the IL-1Ra genotype and disease expression supports the hitherto unproven concept that host genes can influence disease phenotype in tuberculosis ( 52 ). We propose that the early recruitment and activation of inflammatory cells by IL-1β to foci of tuberculous infection is in turn downregulated by IL-1Ra that, under polymorphic host control, acts to limit the resultant DTH. This hypothesis could be readily tested in IL-1 and IL-1Ra gene knockout mice ( 53 , 54 ). Reduction of DTH by targeted immunotherapy with either IL-1Ra or other engineered antagonists of IL-1RI ( 55 ) may also be a possible approach to modulation of immunopathologic cytokine circuits in tuberculosis. | Study | biomedical | en | 0.999998 |
10377183 | Mice deficient for the expression of CD40 ( 22 ), MHC class II ( 23 ), β2-microglobulin ( 24 ), and RAG-2 ( 25 ) have been described previously. Mice exhibiting a mutation in their H-2D b molecule (B6.C–H-2 bm13 ) were provided by Dr. P. Ohashi, Ontario Cancer Institute, Toronto, Canada. Transgenic mice expressing a TCR specific for peptide p33 derived from LCMV presented in association with H-2D b and transgenic mice expressing a TCR receptor specific for the HY antigen have been described previously ( 26 , 27 ). Mice were bred under specific pathogen-free conditions according to Swiss federal law. Class II −/− mice were back-crossed on a C57BL/6 background, and C57BL/6 mice were used as controls. CD40 +/− mice were used as controls for CD40 −/− mice. The LCMV isolate WE was provided by Dr. P. Ohashi, Toronto, Canada and grown on L cells at a low multiplicity of infection. VSV serotype Indiana was originally provided by R.M. Zinkernagel (Institute of Experimental Immunology, Zürich, Switzerland) and grown on BHK cells at a low multiplicity of infection. Mice were immunized intravenously with LCMV (200 PFU) or VSV (2 × 10 6 PFU), and 8 or 6 d later, respectively, spleen cell suspensions were prepared and tested directly in a 51 Cr-release assay, using LCMV-derived peptide p33 (KAVYNFATM)- or VSV-derived peptide (SDLRGYVYQGLKSG)-pulsed EL-4 cells as target cells ( 28 ). Lytic units were calculated for a 50% level of cell lysis. As different control mice were used for CD40 −/− versus class II −/− mice, results are expressed as percent lytic units of controls. Spleen cells from TCR-transgenic control mice (10 6 cells) were adoptively transferred into normal C57BL/6 recipient mice. 2 h later, mice were challenged with LCMV. After 8 d, spleen cells were harvested and stained for CD8 (PE; PharMingen ) and transgenic Vα2 expression (FITC; PharMingen ), and the presence of TCR-transgenic cells was assessed by flow cytometric analysis. To assess cytolytic effector function, spleen cells were tested in 51 Cr-release assays using peptide p33 (KAVYNFATM)-pulsed EL-4 target cells. To distinguish endogenous CTL activity of the C57BL/6 recipient mice from CTL activity of the transferred TCR-transgenic T cells, EL-4 cells pulsed with peptide MB6 (KAVVNIATM) were also used as target cells. MB6 is recognized by the TCR-transgenic T cells but not by the polyclonal C57BL/6 T cells ( 29 ). Mice were immunized intravenously with LCMV (200 PFU) or VSV (2 × 10 6 PFU). 6 or 5 d later, respectively, spleens of uninfected and virus-infected mice were dissected, cut into pieces, and digested twice with collagenase D ( Boehringer Mannheim ) for 30 min at 37°C in a shaking water bath. Cells were recovered by centrifugation and resuspended in an Optiprep™ (Nycomed Pharma) gradient as previously described ( 30 ). Low density cells were then incubated for 30 min on ice with FITC-labeled anti-CD11c (1:400; PharMingen ) and with PE-labeled anti–B7-1, anti–B7-2, anti-CD40, and isotype control, in the presence of 5% normal mouse serum. Cells were washed and analyzed by a FACSCalibur™ flow cytometer ( Becton Dickinson ), excluding propidium iodide– positive cells. Percent upregulation of median fluorescence intensity (MFI) was calculated as follows: % upregulation = ([MFI induced − MFI control]/MFI control − 1) × 100. Transgenic mice expressing a TCR specific for LCMV were intravenously injected with 10 μg LCMV-derived peptide p33 (KAVYNFATM), A4Y (KAVANFATM), V4Y (KAVVNFATM), or S4Y (KAVSNFATM). After 24 h, DCs were isolated from spleens of treated and, as control, untreated mice and monitored for expression of B7-1, B7-2, and CD40 as described above. Activation of T cells was assessed by measuring CD69 (PE) expression on CD8 + T cells (FITC). For the T cell stimulation assay, DCs were sorted using a FACStar PLUS™ , obtaining a purity >97%. Different numbers of sorted DCs (H-2 b ) were added to 2 × 10 4 purified T cells obtained from spleens of H-2 d BALB/c (MLR) or female HY-transgenic mice (H-2 b on a RAG-2 background) and cultured in 96-well plates (Falcon; Becton Dickinson ) for 3 d. T cell proliferation was assessed by [ 3 H]thymidine (1 μCi/well) uptake in a 16-h pulse after 72 h. CD8 + T cells were positively selected from mesenteric LNs of a RAG-2 −/− LCMV-specific, TCR-transgenic mouse by anti-CD8–coated magnetic beads (GmbH; Miltenyi Biotec). DCs (10 5 cells/well) obtained from control B6, CD40 −/− , and MHC class II −/− mice were pulsed for 1 h with 10 μg/ml peptide p33, A4Y, V4Y, or S4Y, washed, and cocultured with 3 × 10 5 naive CD8 + T cells at 37°C. After 20 h, DCs were double-stained with FITC-labeled anti-CD11c and PE-labeled anti–B7-1, anti–B7-2, and anti-CD40 and analyzed by a FACSCalibur™ flow cytometer, excluding propidium iodide–positive cells. RAG-2 −/− mice were irradiated (3 Gy) and reconstituted with 5 × 10 6 bone marrow cells from CD45.1 congenic C57BL/6 mice and 5 × 10 6 bone marrow cells derived from CD45.2 TCR-transgenic B6.C–H-2 bm13 mice. 10 wk later, recipient mice exhibited large numbers of TCR-transgenic T cells and a mixed DC population of both CD45.1 and CD45.2 allotypes. DCs were isolated 24 h after injection of peptide or saline as described above, triple-stained with APC-labeled anti-CD11c, FITC-labeled anti-CD45.2, and PE-labeled B7-2, anti-CD40, or negative control, and analyzed by using a FACSCalibur™ flow cytometer. To compare the role of CD4 + T cells versus the presence of CD40 for the induction of LCMV-specific CTL responses, control, CD40 −/− , and MHC class II −/− mice that lacked Th were immunized with LCMV. At the peak of the response, i.e., 8 d after infection, spleen cells were harvested and tested in a 51 Cr- release assay. To be able to quantify the relative numbers of CTL effector cells in the different mice, lytic units were calculated . Absence of MHC class II or CD40 did not affect the frequency of CTL precursor after infection with LCMV. The Th dependence of the VSV-specific CTL response was analyzed next. Control, CD40 −/− , or MHC class II −/− mice were injected intravenously with VSV, and the presence of CTLs was assessed 6 d later in a 51 Cr-release assay. In contrast to LCMV-specific CTL responses, VSV-specific CTL responses were reduced in the absence of Th in MHC class II −/− mice by 75–80%. Similar results were obtained in mice depleted of CD4 + Th (data not shown). However, CTL responses were completely normal in CD40 −/− mice. Thus, surprisingly, CD40 did not mediate the Th-dependent component of the VSV-specific CTL response . CD40 and MHC class II have been implicated in thymic selection ( 23 , 31 ). We therefore wanted to exclude the possibility that alterations in the CTL populations in CD40 −/− or MHC class II −/− mice were responsible for the Th independence of the CTL response. Thus, T cells from transgenic mice expressing a TCR specific for LCMV ( 26 ) were adoptively transferred into the different mice before viral infection. It has been shown previously that this leads to a dramatic expansion of transgenic T cells ( 32 ). 8 d after infection, at the peak of the antiviral response, presence of T cells expressing the transgene-encoded TCR (Vα2) was assessed in the spleens of the mice. The absolute number and the frequency of transgenic T cells was comparable in control, CD40 −/− , and MHC class II −/− mice . Moreover, a comparable lytic activity of the transgenic cells was observed in a 51 Cr-release assay on peptide p33–pulsed target cells . Similar results were obtained with peptide MB6, which is selectively recognized by the transgene-encoded TCR (data not shown). These results demonstrate that a defined population of CTLs expressing a single TCR can be stimulated by LCMV in the absence of both CD40 molecules and CD4 + Th. DCs have been shown to be activated in vitro upon stimulation with various inflammatory cytokines and, in particular, after stimulation with CD40L ( 33 , 34 ). This activation step leads to a maturation of DCs that is thought to be essential for the generation of immune responses ( 35 ). The upregulation of costimulatory molecules on DCs is a hallmark of this maturation step. To analyze whether viral infection may induce maturation of DCs in vivo, mice were infected with LCMV, and DCs were isolated 6 d later from the spleen and directly analyzed by flow cytometry . LCMV infection induced the upregulation of B7-1, B7-2, and CD40 on splenic DCs. Surprisingly, LCMV infection induced generalized activation of DCs, as the great majority displayed upregulated costimulatory molecules. To analyze the role of Th and CD40 in the activation of DCs, mice deficient for the expression of MHC class II (and therefore lacking Th) and CD40 −/− mice were infected with LCMV, and DCs were isolated 6 d later . Maturation of DCs was induced irrespective of the presence of CD40 or Th, indicating that DC maturation was triggered by CD8 + T cells. However, DCs also matured in class I −/− mice , indicating that Th are sufficient but not necessary for activation of DCs. Thus, virus-specific CD4 + and CD8 + T cells are able to trigger an activation program in DCs. Taken together, these data demonstrate that (a) both virus-specific CD8 + and CD4 + T cells are able to trigger maturation of DCs in vivo and (b) CD40 is not involved in this process. LCMV exhibits a high virulence in vivo and is able to activate T cell responses in the absence of a variety of accessory molecules. In contrast, VSV exhibits a low virulence in vivo, and T cell responses depend to a higher degree on costimulation ( 11 ). To analyze whether the low virulence of VSV may be reflected in inefficient activation of DCs in vivo after infection, mice were inoculated with VSV, and splenic DCs were isolated 5 d later (Table I ). Indeed, upregulation of costimulatory molecules on DCs occurred less efficiently after infection with VSV compared with LCMV. Thus, the low virulence of VSV was reflected in inefficient activation of DCs. The majority of splenic DCs exhibited an activated phenotype after infection with LCMV. As, by the time of the analysis, little LCMV was left in the spleen (data not shown), it was unlikely that only virally infected DCs underwent maturation. However, it remained possible that few virus-infected DCs nucleated the CTL response that subsequently activated splenic DCs. To analyze whether viral infection was required for activation of DCs, transgenic mice expressing an MHC class I–restricted TCR specific for LCMV ( 26 ) were injected with the LCMV- derived peptide p33, and DCs were isolated 1 d later. The naive CD8 + TCR-transgenic T cells were able to induce maturation of DCs within 24 h after activation in the complete absence of viral infection . We have recently developed a panel of altered peptide ligands that trigger transgenic T cells with various efficiencies ( 36 ). Peptide A4Y behaves as a weak agonist, peptide V4Y behaves as a partial agonist, and peptide S4Y is a strict antagonist. If these altered peptide ligands were injected, the efficiency of the peptides in activating T cells, as assessed by upregulation of CD69 expression , was directly reflected in their ability to trigger maturation of DCs . Interestingly, although peptide A4Y is able to induce proliferation of specific T cells in vitro ( 36 ), it fails to efficiently trigger maturation of DCs in vivo and induced only low level expression of CD69. This finding is compatible with the observation that weak antigens generally require the presence of Th to induce CTL responses in vivo, because only CD8 + T cells interacting with strong agonist peptides are apparently able to stimulate maturation of DCs, thereby replacing T help. Whether a direct interaction of CD8 + T cells with DCs was required for maturation was analyzed next. To this end, chimeric mice were generated, using bone marrow from H-2 bm13 mice. The TCR of the transgenic mouse line used in our experiments recognizes peptide p33 presented by H-2D b . H-2 bm13 mice exhibit a mutation in the D b molecule that does not allow presentation of peptide p33 ( 37 , 38 ). Thus, DCs from H-2 bm13 mice fail to specifically stimulate TCR-transgenic T cells, although the TCR-transgenic cells are efficiently positively selected by H-2 bm13 and are reactive to peptide p33 ( 37 ). RAG-2 −/− mice were reconstituted with a mixture of bone marrow derived from CD45.2 + H-2 bm13 TCR-transgenic mice and bone marrow from CD45.1 + C57BL/6 mice. These chimeric mice therefore exhibit a population of CD45.2 + H-2 bm13 DCs and a population of CD45.1 + H-2 b DCs and express a transgenic TCR specific for peptide p33 in association with H-2D b . 10 wk after reconstitution, chimeric mice were injected with peptide p33 or saline, and activation of CD45.1 + and CD45.2 + DCs was analyzed. Both CD45.1 + DCs (H-2 b ) that present peptide p33 and CD45.2 + DCs (H-2 bm13 ) that fail to present peptide p33 exhibited an activated and matured phenotype after injection of specific peptide (Table II ). In contrast, DCs derived from TCR-transgenic H-2 bm13 mice injected with peptide p33 did not exhibit activated DCs, confirming that peptide p33 was not presented by H-2 bm13 (data not shown). To analyze CD8 + T cell–induced maturation of DCs in a two-cell coculture system, purified CD8 + T cells derived from TCR-transgenic mice on a RAG-2 −/− background were mixed with freshly isolated, peptide-pulsed splenic DCs and incubated at 37°C for 20 h. Analysis of the expression of B7-1, B7-2, and CD40 demonstrated efficient maturation of DCs triggered by peptide-specific CD8 + T cells . Moreover, as seen in vivo, weak, altered peptide ligands were inefficient at inducing the maturation program in DCs. As expected, MHC class II −/− and CD40 −/− DCs matured comparably to control DCs, indicating that the CD8 + T cell–mediated maturation of DCs occurred in the absence of CD40 and Th. We next analyzed the immunogenicity of DCs after activation by CTLs. Male transgenic mice (H-2 b ) expressing a TCR specific for peptide p33 derived from LCMV were injected with peptide p33 or saline, and splenic DCs were isolated 24 h later and purified by cell sorting (purity >97%). Titrated numbers of DCs were used to stimulate purified allospecific T cells (H-2 d ) derived from BALB/c mice, and proliferation was assessed 3 d later by means of [ 3 H]thymidine incorporation. DCs derived from p33-immunized mice were able to induce efficient proliferation of allospecific T cells, whereas DCs derived from control animals were barely able to stimulate T cells . Alternatively, DCs were used to stimulate T cells derived from transgenic mice expressing a TCR specific for HY in association with class I ( 27 ). To avoid the contribution of T cells with endogenous TCR α chains, T cells derived from HY–TCR-transgenic mice on a RAG-2 −/− background were used. As expected for a Th-dependent CTL response, control DCs stimulated only low-level proliferation of specific T cells. In contrast, CTL-activated DCs derived from p33-primed animals triggered much stronger proliferation of specific T cells . Thus, CTL-mediated activation of DCs rendered Th-dependent CD8 + T cell responses largely Th independent. This study demonstrates that maturation of DCs occurs in vivo after viral infection in the absence of Th and CD40. Surprisingly, this Th-independent maturation was triggered by virus-specific CD8 + T cells and not by the viral infection per se. Moreover, the matured DCs were able to induce a classically Th-dependent CTL response in the absence of Th, indicating that antiviral CD8 + T cells can replace Th in vivo. These results help explain why viruses are able to trigger Th-independent CTL responses, and highlight important differences between virus- and tumor-specific CTL responses. It has been known for some time that viruses are able to stimulate protective CTL responses in the absence of CD4 + T cells (12– 20). However, CTL responses are impaired in the absence of Th after infection with poorly replicating viruses such as VSV ( 17 ). To date, it has not been analyzed whether CD4 + T cells assist CTLs via a CD40L-dependent mechanism under these circumstances. This study demonstrates that this is not the case. Thus, even the Th-dependent part of a virus-specific CTL response is not dependent upon the CD40–CD40L interaction. Compared with tumor-specific CTL responses, where Th assist CTL induction by activating DCs via CD40 triggering, the Th-dependent production of cytokines, such as IL-2, seems to be more important during viral infections. This interpretation is consistent with the previous observation that cytokine secretion by Th after viral infection is not impaired in the absence of CD40 ( 39 ). A large proportion of DCs isolated from virally infected mice exhibited a mature phenotype. This maturation of DCs occurred in the absence of Th and CD40. Various stimuli are known to activate DCs. Specifically, microbial components, such as LPS, bacterial DNA, or cell walls; inflammatory cytokines; and CD40 triggering are able to stimulate expression of costimulatory molecules on DCs and induce their migration to central lymphoid organs ( 6 , 7 ). Moreover, it has been shown that LPS is able to activate splenic DCs in vivo and trigger the migration of DCs from the red pulp to T cell areas ( 40 ). Interestingly, infection of splenic APCs by influenza virus directly induces the expression of costimulatory molecules and enhances the immunogenicity of DCs ( 8 , 41 ). Thus, it was possible that the observed Th-independent maturation of DCs was (a) directly mediated by viral components, (b) mediated by non-T cells such as NK cells, or (c) mediated by CD8 + T cells. To address this question, various gene-deficient mice were infected with LCMV, and the maturation of DCs was assessed. Maturation of DCs occurred in class II −/− as well as class I −/− mice. These results demonstrated that DC maturation could be mediated by either CD4 + or CD8 + T cells. DCs isolated from transgenic mice expressing a class I–restricted TCR 24 h after injection of specific peptide also exhibited an activated phenotype. This demonstrated that DC maturation triggered by CD8 + T cells could occur in complete absence of viral infection. Interestingly, the maturation stimulus delivered by CD8 + T cells was not dependent upon a direct interaction of CD8 + T cells with DCs. In contrast, CD8 + T cells could activate DCs in trans, and DCs that failed to present the relevant peptide due to a mutation in the class I molecule also underwent maturation in TCR-transgenic mice injected with peptide. Thus, freshly activated CD8 + T cells are able to induce generalized activation of DCs, even in the absence of direct T cell–DC contact. There are parallels between the activation of DCs by CD8 + T cells described here and the IFN system. Type I IFNs induce an antiviral state in many different cell types. This antiviral state is induced in trans, i.e., virally infected cells are able to stimulate neighboring cells via secretion of type I IFNs. Similarly, CD8 + T cells induce an immunostimulatory state, not only in virally infected DCs that present the relevant peptides but also in neighboring APCs. However, the two systems are apparently not identical, because (a) type I IFNs induce an antiviral rather than an immunostimulatory state in DCs and (b) DC activation by CD8 + T cells occurs in the absence of functional type IFN receptors (data not shown). Using the in vitro system of CD8 + T cell–mediated DC activation and T cells or DCs derived from various gene-deficient mice together with neutralizing antibodies and other blocking molecules, we tried to define the factor responsible for the observed maturation of DCs. So far, we can exclude maturation mediated by CD28, CD40L, TRANCE, TNF, IL-1, IL-4, IL-6, IL-17, IFN-α/β, and IFN-γ, but have not yet identified the critical molecule. Future studies will therefore address the cloning of the as yet elusive factor. It has been observed that viruses are able to generate CTL responses in the absence of Th ( 12 – 20 ). Interestingly, those viruses that induced strong CTL responses in the presence of Th also did so in the absence of Th, suggesting that the Th dependence of the response may simply be determined by the overall strength of the response. Along a similar line, recombinant viral proteins injected in association with insect cell debris as an adjuvant were able to induce very strong CTL responses by cross-priming ( 42 ), and this cross-priming occurred in the absence of Th ( 42 ). Thus, the failure of model antigens to induce CTL responses in the absence of Th is not absolute but rather seems to be determined by the strength of the response. The results presented here may provide an explanation for these findings by suggesting that generation of CTL responses may occur as a threshold phenomenon due to a positive feedback mechanism. Accordingly, virus-infected DCs are able to activate a few naive CTLs. These activated T cells in turn secrete inflammatory cytokines/chemokines that may lead to the activation of neighboring APCs. The number of CD8 + T cells that can be activated in such a way determines the efficiency of DC activation. Thus, if only few CD8 + T cells are activated initially, the response may be abortive, as too few DCs undergo maturation. In contrast, if a sufficient number of CTLs is triggered, widespread activation of DCs may occur, and an immunostimulatory program is initiated in lymphoid organs that renders the response independent of Th. Thus, during tumor-specific responses or upon cross-priming, few CD8 + T cells are activated, and the response remains abortive in the absence of Th, whereas during antiviral immune responses, a sufficient number of CD8 + T cells becomes triggered to render the response Th independent. In this model, VSV may represent an intermediate case. It induces only weak upregulation of costimulatory molecules in vivo. Correspondingly, CTL responses partly depend on the presence of Th. Interestingly, the requirements for activation of DCs were very stringent. Peptide A4Y, which is a relatively weak agonist that nevertheless stimulates efficient proliferation of specific T cells in vitro, almost completely failed to trigger the activation program in DCs in vivo. Thus, only T cells interacting with strong agonists are able to stimulate maturation of DCs, offering an explanation for why many model antigens that are often weak agonists compared with viral peptides require Th for induction of CTL responses. This study demonstrates that CD8 + T cells can mediate activation of DCs in complete absence of viral infection. However, it should be noted that a cross-talk between the innate and specific immune systems during viral infection may also facilitate the induction of Th-independent CTL responses. Thus, although CD8 + T cells can solicit their own help by themselves inducing maturation of DCs, other factors are likely to contribute to the efficiency of antiviral CTL responses ( 11 ). Viral infections are thought to be an important cause for the induction or exacerbation of autoimmune diseases ( 43 ). It has been argued that cross-reactive T cells are responsible for disease, and it has been shown that viral peptides can stimulate autoreactive T cell clones ( 44 , 45 ). Moreover, there is good evidence that Herpes Simplex virus causes autoimmune stromal keratitis by activating self-specific, cross-reactive T cells ( 46 ). However, cross-reactive T cells are not always the critical factor for disease. In the case of Coxsackie virus–induced diabetes, it has been shown that the disease is mediated by T cells recognizing self-antigens that do not cross-react with viral proteins. These self-antigens were apparently released from lysed virus-infected cells and subsequently activated the self-specific T cells ( 47 ). However, presence of these antigens alone may not be sufficient for the induction of self-specific T cells, because low amounts of most self-antigens also reach lymphoid organs in the absence of infection due to physiological cell turnover ( 48 ). Thus, the presentation of self-antigens in lymphoid organs per se does not seem to be responsible for the induction of autoimmunity, likely because immature DCs in lymphoid organs that process those antigens most efficiently are inefficient at stimulating naive T cells. However, the nonspecific maturation of DCs and the concomitant upregulation of costimulatory molecules triggered by the antiviral immune response may be responsible for the activation of self-specific T cells that usually ignore their antigens in vivo. The generalized activation of DCs upon viral infection may therefore not only boost virus-specific T cell responses and render them independent of Th but may also shift the balance from ignorance to autoimmunity. | Study | biomedical | en | 0.999997 |
10377184 | 40 cat-allergic asthmatic patients were recruited from the Allergy Clinic, Royal Brompton Hospital, London and by advertisement. The study was approved by the Royal Brompton Hospital Ethics Committee, and informed consent was obtained from all patients. Subjects were nonsmokers and had no other significant illness. Inhaled corticosteroids were discontinued at least 2 d before assessment, and oral corticosteroids were discontinued 3 mo before assessment. Patients were specifically instructed not to take antihistamines, corticosteroids, or any other medication for the duration of the study. Volunteers had a history of wheezing on exposure to cats and demonstrated airway hyperresponsiveness, with a provocative concentration (PC) 20 of <4 mg/ml methacholine, and >20% reversibility with inhaled β 2 agonists. Cat allergy was confirmed by a positive radio allergo sorbent test (>0.35 IU/ml; Pharmacia ) and skin prick test to whole cat dander (ALK) and a late phase skin reaction of >3 cm diameter at 6 h, after the intradermal testing of 30 biological units of whole cat dander (containing 4.5 ng Fel d 1) in 0.03 ml. The vehicle for whole cat dander extract contained 0.3 mg/ml human serum albumin and 4 mg/ml phenol (ALK diluent; ALK). On the first visit (control day), subjects were skin tested with intradermal cat dander. Peripheral blood (150 ml) was taken for HLA typing, histamine release assays, and proliferation assays. Local reactions were observed after 5, 15, and 30 min and hourly for 6 h thereafter. At these times, FEV 1 and peak expiratory flow rate were also recorded. On the second visit, patients received 40 μg intradermally of the three peptides comprising FC1P, contained in 25 μl, into each forearm. Local reactions were observed at the same time points as the control day. A positive reaction was defined as a fall in forced volume in 1 s (FEV 1 ) of 20% or more. The time interval between the two study days ranged from 4 to 56 d (average, 17 d). Six of the nine subjects who developed LARs after peptide injections had a further visit in which 0.03 ml of diluent (vehicle) was injected intradermally. Three subjects, having developed LAR after FC1P administration, were recalled for a repeat administration of the peptides 2–6 wk after the initial challenge. An additional three subjects received a repeat administration of FC1P a minimum of 12 mo after the first injection. FEV 1 data was summarized over time for each subject for the control and peptide days. Areas under each curve were calculated using the trapezoidal rule to generate area under curve (AUC). Differences in the AUC between the control day and the peptide day were analyzed by paired t test. The three FC1Ps for injection were synthesized by F-moc chemistry and supplied as acetate salts. They were purified to >95% by HPLC, subjected to sequence and mass analysis, and subjected to independent analysis for sterility and the absence of endotoxin. Peptides were reconstituted in ALK diluent at 1.6 mg/ml. Peptides for in vitro analysis were synthesized using F-moc chemistry at the Advanced Biotechnology Centre, Imperial College School of Medicine, Charing Cross Campus. Peptide sequences used in this study were: FC1P1, LFLTGTPDEYVEQVAQY; FC1P2, EQVAQYKALPVVLENA; and FC1P3, KALPVVLENARILKNCV. Cat allergen extract was a gift of ALK Abello, Copenhagen, Denmark. In 11 subjects, PBMCs isolated by density centrifugation from 50 ml of blood were washed three times with Ca 2+ - and Mg 2+ -free HBSS. 100-μl aliquots of cell suspension at 5 × 10 6 cells/ml were combined with an equal volume of peptide (0.02–100 μg/ml) or cat dander (0.02–200 μg/ml). Samples were incubated at 37°C for 60 min, followed by centrifugation. Supernatants were transferred to glass microfiber–coated microtiter plate wells (REFLAB), together with 50 μl of a histamine standard solution (50 ng/ml). Plates were incubated at 37°C for 60 min, washed twice (45 s) in distilled water, and air dried before measurement of histamine by spectrofluorimetry. Percentage of histamine release for each control and test substance was calculated from the mean histamine release (ng/ml) values using the equation: % release = [(sample − spontaneous)/(total − spontaneous)] × 100. Murine and human fibroblast cell lines transfected with HLA-DR molecules were obtained as gifts from a number of sources: DRB1*0101, 0401, 0403, 0404, and 0406 from Prof. R.I. Lechler and Dr. G. Lombardi (Imperial College School of Medicine [ICSM], Hammersmith Campus, London), DRB1*0402 and 0405 from Prof. J.R. Lamb (University of Edinburgh, UK) and Dr. A. Verhoef (ICSM at National Heart and Lung Institute [NHLI], London), and DRB1*1301, 1302, 1303, 1304, and 1305 from Drs. J.R. Richert and C. Katovich Hurley (Georgetown University Medical Center, Washington, DC). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 μg/ml G418 (all from GIBCO BRL ). PBMCs were isolated by density gradient centrifugation from all subjects at the initial screening visit. PBMCs were either frozen for subsequent use as APCs or cultured for 10 d in the presence of cat allergen extract (100 μg/ml; ALK) with addition of purified human IL-2 (Lymphocult LF; Biotest) at a final concentration of 10% on day 6 of culture. Viable cells were harvested and restimulated weekly with cat allergen extract and IL-2, with a further addition of IL-2 on day 3 of the restimulation cycle. Before assay, cells were rested overnight in culture medium in the absence of exogenous antigen or IL-2. PBMCs were separated from 100 ml whole blood in 25 patients by density centrifugation. PBMCs were washed and resuspended in RPMI supplemented with 5% vol/vol AB-serum ( Sigma Chemical Co. ), 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and cultured at 10 5 cells/well in 96-well plates. 16 replicate wells for each of three concentrations were established for each peptide. Purified protein derivative (PPD; 10 μg/ml) was used as positive control. Cells were cultured for 6 d before addition of 1 μCi (37 kBq) tritiated methyl-thymidine per well for 8 h. Cells were harvested, and thymidine incorporation was determined by liquid scintillation spectroscopy. Individual data points for 16 peptide culture wells were compared with those containing medium alone. Statistical evaluation was performed using Mann-Whitney analysis. Results are expressed as delta counts per minute (observed cpm − medium-alone cpm). Transfected fibroblast lines were cultured overnight in the presence of the relevant peptide at a concentration of 100 μg/ml before harvesting and incubation with mitomycin C (50 μg/ml) for 1 h. Cells were washed extensively before seeding in 96-well, flat-bottomed plates at 3 × 10 4 cells/well. Allergen-specific T lymphocytes were added (10 4 cells/well) and cultured for 48 h. Culture supernatants were harvested for cytokine analysis before pulsing wells with 37 kBq/well of tritiated methyl-thymidine and cultured for a further 8 h. Proliferation as correlated with thymidine incorporation was measured by scintillation spectroscopy (TopCount; Canberra Packard). IL-5 was measured in the laboratory of Dr. D. Huston (Baylor Medical Center, Houston, TX) according to a previously described technique ( 11 ). The sensitivity of the assay was 3 pg/ml. In contrast to whole cat extract (ALK cat), pooled FC1Ps did not release histamine from basophil-enriched mononuclear cells in vitro. Cells from 11 subjects (5 FC1P reactors and 6 nonreactors) were incubated with FC1Ps at five increasing concentrations up to 100 μg/ml (∼5 × 10 −5 M of each of the pooled peptides). Whole cat dander extract served as a positive control, releasing histamine in all subjects assayed . No immediate skin or asthmatic reactions were observed in vivo, further demonstrating that FC1P did not bind IgE. The ability of FC1Ps to stimulate T cell responses was assessed, initially by primary in vitro proliferation assays. All individuals assayed responded to one or more of the three peptides, although none of the peptides induced proliferation in all individuals, suggesting MHC-restricted recognition. Each of the peptides was recognized by PBMCs from both the group developing LARs ( n = 7) and the nonresponder group ( n = 19). As shown in Fig. 2 , each peptide was able to induce proliferative responses, in a dose-dependent fashion, in PBMCs from representative (26/40) cat-asthmatic subjects from whom cells were available. Proliferative responses from individuals who developed LARs were generally of greater magnitude than those that did not; however, this difference was not statistically significant. All subjects responded to the positive control recall antigen, PPD. Inhaled whole allergen induces both an immediate early asthmatic reaction and an LAR. To determine whether T cell activation could trigger an isolated LAR, sensitized volunteers were challenged with peptides derived from Fel d 1 chain 1. In 9/40 subjects, 80 μg of FC1P did not produce a visible immediate or late skin reaction but did elicit a progressive decline in FEV 1 , associated, in all responders, with symptoms of chest tightness and wheezing. The remaining subjects did not develop any immediate or late bronchial or cutaneous reactions to the peptides. The reaction commenced at hours 2–3 and reached a plateau by hour 6 . The decrease in FEV 1 observed ranged from 21 to 64% of baseline. AUC analysis to compare percentage change in FEV 1 on control and FC1P days in nine patients was statistically significant, at P = 0.0004. Subjects were monitored for a minimum of 6 h and treated with β 2 agonists and inhaled corticosteroids before leaving the hospital. The mean maximum change in FEV 1 observed on the whole cat dander control day was −3.83% (range −14 to +4%). Additionally, six of the nine reactors had a further control day in which 0.03 ml of the diluent (vehicle) was injected intradermally. The mean maximum change in FEV 1 was −6.3% (range from 0 to –13%). 31 individuals showed no evidence of LAR . To determine whether the reactions were HLA restricted, subjects were typed for HLA-DRB1. HLA analysis revealed an increased frequency of HLA-DR13 in those who developed LAR (4/9 subjects), compared with only 1/31 subjects who did not react, suggesting that DR13 may restrict T cell responses to one or more of the injected peptides ( P = 0.006, Fisher's exact test). Patient characteristics and HLA-DRB1 types are shown in Table I . As peptides can be presented in vitro without intracellular processing, we determined whether Fel d 1 chain 1 peptides could be recognized in the context of MHC molecules expressed by the subjects developing asthmatic reactions. For this purpose, fibroblast cell lines transfected with relevant HLA molecules were cultured together with each peptide and T cell lines from the responding individuals. Cat dander extract–specific T cell lines were derived from a DR13 + subject before induction of peptide-induced LAR. Culture of these cells with FC1Ps and a fibroblast cell line transfected with DRA and DRB1*1301 resulted in T cell proliferation and IL-5 secretion in response to FC1P3 but not to FC1P1 or FC1P2 . Fibroblasts transfected with DRA and DRB1*0101 were able to present FC1P3 to a cat dander extract–specific T cell line derived from subject RO2 , resulting in proliferation and IL-5 production . T cells from a cat dander extract–specific cell line from subject RO7 were induced to proliferate and release IL-5 when cultured with FC1P2 and fibroblasts expressing DRB1*0405 . Fibroblast cell lines transfected with either DRB1*1301 or DRB1*1302 were assayed for their ability to present FC1P3 to allergen extract–specific T cells derived from a DRB1*1301 donor. As previously described, FC1P3 presented by DRB1*1301 induced a proliferative response in autologous T cells . In addition, fibroblasts expressing DRB1*1302 were able to activate allergen-specific T cells from a DRB1*1301 donor . In the absence of fibroblasts expressing DRB1*0408, cells expressing the closely related microvariant DRB1*0405 were assayed for their ability to present FC1P2 to cat allergen extract–specific T cells from a DRB1*0408 donor. As shown in Fig. 5 B, DRB1*0405, which differs from DRB1*0408 by only one residue in the peptide binding groove (position 57, serine→ aspartic acid), was able to present FC1P2, inducing proliferation in T cells from a DRB1*0408 donor. To study the reproducibility of peptide-induced LAR, three subjects (RO1, RO4, and RO5) received a second intradermal dose of 80 μg of FC1P between 2 and 6 wk after the first injection. A marked attenuation of the initial LAR was observed. The maximum percentage changes in FEV 1 on the first peptide versus the second peptide challenge were −28 vs. −9 (R01), −21 vs. −10 (R04), and −51 vs. −9 (R05), respectively . After an interval of at least 12 mo after the initial FC1P challenge, three subjects received a further challenge of the same dose. All experienced LARs of similar magnitude to those after the initial injection . In this study, we have demonstrated that peptides derived from a major allergen elicited an LAR in a proportion of sensitized individuals. That the peptides were most likely to initiate these reactions by direct activation of T cells, rather than mast cells or basophils, is supported by (a) the lack of IgE-mediated histamine release in vitro, (b) the absence of a wheal and flare reaction at the site of injection, and (c) the absence of an early asthmatic reaction. In addition, we have demonstrated HLA restriction of T cell responses to peptides FC1P2 and FC1P3 on the basis of fibroblast presentation assays. These observations provide direct evidence for the central role of T lymphocytes in the induction of the late asthmatic response and indicate that these reactions are MHC-restricted events. Although we had a control day for six of the nine responders that included the injection of human serum albumin, we did not specifically employ a control peptide. Some small peptides cause histamine release from mast cells, but because the action of histamine is immediate, this would not be relevant to the LAR, which commences at hour 2–3 and continues to hour 6. A peptide of “irrelevant” sequence would not be informative, as the biological actions are sequence dependent. On the other hand, as we demonstrate MHC restriction of T cell recognition, control peptide(s) would need to be immunologically inactive but capable of binding to the same range of MHC molecules as the FC1Ps and with similar affinities. There is strong circumstantial evidence to support a role for T lymphocytes in the pathogenesis of asthma ( 12 ). This includes elevated numbers of activated T cells in the blood of patients with acute severe asthma ( 8 ), as well as studies using bronchoalveolar lavage (BAL) and bronchial biopsies from mild asthmatics in which increases in CD3/CD4 + cells expressing mRNA positive for IL-4 and IL-5 cells were demonstrated ( 3 , 7 , 13 ). In a provoked model of asthma, increased numbers of CD4 + cells in BAL after allergen challenge were also observed ( 14 ). Furthermore, the allergen-induced late-phase, but not the early-phase, reaction was inhibited by prior administration of cyclosporin A, a compound with major inhibitory effects on CD4 + cells ( 15 ). More recently, a single infusion of humanized anti-CD4 mAbs into patients with chronic asthma was associated with significant improvements in lung function ( 16 ). In Brown Norway rats, Watanabe et al. showed increases in airway resistance up to 8 h after adoptive transfer of CD4 but not CD8 T cells and subsequent allergen challenge ( 9 ). Adoptive transfer experiments in mice have also demonstrated that baseline airway hyperresponsiveness was T cell dependent ( 10 ). In animal models, initial administration of peptides in sensitized mice can induce activation of primed T cells ( 17 ). However, subsequent administration leads to the induction of tolerance, not only to the peptide but to the whole allergen ( 18 ). Using cat-sensitive asthmatics, Norman et al. ( 19 ) attempted to induce the counterpart of murine experimental T cell tolerance in human atopic allergic individuals by subcutaneous injection of two peptides (termed IPC1 and IPC2) spanning a large proportion of chain 1 of Fel d 1. Although there was some subjective symptomatic improvement, many patients reported “allergic” symptoms that occurred between 10 min and 6 h after subcutaneous injections. Other groups, using the same peptides, reported similar results ( 20 , 21 ). As IPC1 and IPC2 are 27 residues long ( 22 ), it is possible that the immediate reactions were the result of IgE binding, and the late reactions were due to T cell activation. For this reason, in this study, we specifically designed peptides of relatively small size (16/17 residues) and linear configuration to enable them to be presented to T cells in the absence of antigen processing and without binding to IgE. This approach, using T cell–reactive peptide epitopes that do not cross-link IgE, provides evidence that asthma can be provoked without initial mast cell activation. The mechanism of T cell peptide–induced LPRs, however, remains to be elucidated. One explanation is that T cell activation in the airways leads to mobilization of eosinophils through the elaboration of type 2 and eosinophil active cytokines (i.e., IL-3, IL-4, IL-5, and GM-CSF). Together, these are known to promote selective eosinophil accumulation, migration, activation, and subsequent tissue injury, possibly through the generation of membrane-derived sulphidopeptide leukotrienes as well as the release of basic proteins from the eosinophil crystalloid granule ( 23 ). Alternatively, or in addition, T cell activation may be associated with the generation of histamine releasing factors, which in turn activate basophils and/or mast cells to release histamine with subsequent narrowing of already hyperresponsive airways ( 24 ). Direct triggering of bronchoconstriction by release of an unidentified T cell mediator may be another possible mechanism. Studies currently underway involving fiberoptic bronchoscopy with BAL and bronchial biopsy, performed at time intervals after peptide-induced LAR, will enable us to test these hypotheses. Our findings are thus of clinical significance, because they may help to identify the critical mediator(s) involved in T cell– as opposed to mast cell–dependent airway narrowing. This may have particular relevance to ongoing chronic asthma, which is believed to have an important T cell component ( 15 , 16 ) but where the sequence of events between T cell activation and bronchial obstruction are incompletely understood. It is intriguing that intradermal injection of peptides produced no visible local reaction in the nine subjects but caused marked changes in the airways. One explanation for a reaction at a distal site is that peptides are presented by skin Langerhans cells to circulating T lymphocytes that bear putative T cell homing receptors (analogous to the cutaneous lymphocyte-associated antigen described in patients with atopic dermatitis; reference 25 ) and that these then traffic to the airways. Alternatively, the peptide may be absorbed via the circulation or lymphatics and reach the airways, where they are taken up by bronchial APCs that in turn interact with locally sensitized T cells. Related to the mechanism of T cell peptide epitope– induced asthma is the variation in responsiveness amongst the subjects studied, with only 9/40 responding with an isolated LAR. There was no association between the ability of a particular peptide to elicit a T cell–dependent proliferative response in vitro and its capacity to evoke LAR after intradermal injection in vivo, i.e., in vitro mitogenic responses were observed in the absence of an elicited in vivo effect . These findings cannot be explained solely by the lack of the appropriate HLA haplotype in the nonresponders, as many of these also expressed DR1, DR4, or DR13 (Table I ). We believe that responsiveness or nonresponsiveness was likely to have been a reflection of the dose of peptide administered, with the threshold for developing an LAR varying amongst individuals. It is clear that T cell reactivity is likely to have been dependent on HLA molecules restricting peptide presentation and T cell activation ( 26 ) to FC1P, with subsequent induction of isolated LARs in subjects in which a threshold dose was achieved. Of interest in this regard was the apparent clustering of HLA-DR13 amongst the subjects in whom these LPRs were observed. Haplotyping of reactors indicated that 4/9 reactors (44%) possessed a DR13 allele, compared with only one 1/30 (3%) nonreactors. Using transfected fibroblast cell lines, the ability of a variety of HLA-DR molecules to present FC1Ps to allergen-specific T cell lines raised from subjects who developed LARs has been assessed. Initially, based upon the high frequency of DR13 among those who developed LARs, we examined the ability of DRB1*1301 and 1302 to present one or more of the FC1Ps to cat allergen extract– specific T cell lines. We demonstrated that FC1P3 could be presented by both DRB1*1301 and 1302. These microvariants of DR13 differ by only one residue at position 86 , and our results suggest, therefore, that this residue is not critical for the binding of this peptide. Furthermore, we have demonstrated the ability of DRB1*1302 to present FC1P3 to T cells from a DRB1*1301 donor, indicating a degree of degeneracy in T cell recognition of this peptide in the context of closely related microvariants of DR13. The ability of DRB1*1303, which differs from 1301 and 1302 more substantially, to present peptide FC1P3 is currently under investigation. Investigation of the ability of DRB1*0101 to present FC1Ps revealed promiscuous binding of FC1P3 to both DR1 and DR13 molecules. As shown in Fig. 5 a, the sequences of DRB1*0101 and DRB1*1301/1302 differ substantially within the peptide binding region of the molecules. However, a precedent for promiscuous binding of a single peptide (influenza HA 307–319) to both DR1 and DR13 alleles has been described by Hickling et al. ( 27 ). Three of the nine individuals who developed peptide- induced LAR were DR4 + , although each expressed a different microvariant of DR4 (Table I ). The ability of DR4 microvariants to present FC1Ps was investigated, and it was established that DRB1*0405 was able to present FC1P2 to cat allergen extract–specific T cells from both DRB1*0405 (autologous) and DRB1*0408 individuals. Thus, in common with FC1P3, the second FC1P appears to be capable of binding to more than one HLA molecule. The ability of this peptide to bind to other DR4 microvariants is currently under investigation. Our findings suggest that induction of the LAR results from MHC-restricted T cell activation. The limited clinical benefit that has so far been observed with peptide immunotherapy may therefore be explained by the fact that only a proportion of patients treated will have been capable of reacting to the peptides, as MHC restriction has not been accounted for ( 19 – 21 ). Furthermore, the observation that individual allergen peptides are capable of promiscuous HLA binding may hold promise for the development of MHC-restricted peptide-based therapies. All of the subjects developing asthmatic reactions expressed DR1, 4, or 13. However, a significant proportion of individuals who did not react also expressed these HLA types. The fact that these subjects did not progress to develop LARs may be due to a deficit in the subjects' T cell repertoire. Alternatively, the lowest possible dose that was found to induce an LAR was used in all individuals, and some of the volunteers who did not respond may have required a higher dose of FC1P to trigger an asthmatic reaction. It should be noted that the amount of peptide required for systemic administration was ∼10,000 times that of the intact protein. However, there is clearly a dilutional effect in a situation where an intradermal injection elicits a reaction at a distal site. The minimum dose required to produce an LAR will be calculated more accurately in studies involving direct application of peptides into the airways. It should also be appreciated that any bronchial reaction elicited by intradermal injection of whole cat extract (which can very occasionally occur but did not in this study) would be predominantly anaphylactic in type, with an IgE/ mast cell–dependent bronchospasm occurring within 30 min and usually sooner ( 28 ). Recent studies have demonstrated that an initial transient activation of T cells precedes the development of hyporesponsiveness ( 17 ). In support of this concept was the observation that in three of the patients studied, there was abrogation of the LAR on repeat administration of the same dose of peptide that previously caused a 20% or greater fall in FEV 1 . This is similar to the experience of Norman et al. ( 19 ), who also observed that there was a progressive decline in the incidence and severity of untoward symptoms after continued treatment of cat allergy with IPC1/IPC2, suggesting but not proving that some form of tolerance may have developed after repeated administration. In addition, three subjects who developed LARs in response to FC1P were readministered the same dose at an interval of greater than one year. All three developed LARs of approximately the same magnitude as those that accompanied the initial challenge . These observations suggest that the induction of hyporesponsiveness after a single dose of FC1P is transient, lasting somewhere between a few weeks and one year. Further studies are currently underway to define the precise duration of peptide-induced hyporesponsiveness in humans. This may have further clinical significance if repeated administration of MHC class II–restricted T cell peptide epitopes produce safe and effective long-term “tolerance.” In conclusion, we have demonstrated that intradermal injection of a linear peptide sequence within an allergen at a high dose can elicit a late-phase response by a presumed T cell–dependent mechanism in the absence of an IgE-mediated reaction. This appears to be the first demonstration of asthma provoked by MHC-restricted T cell activation. | Study | biomedical | en | 0.999996 |
10377185 | 4–6-wk-old female C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory and housed in the Case Western University animal facility under specific pathogen-free conditions. L. major (WHO strain WHOM/IR/−/173) were grown in M199 medium ( GIBCO BRL ) containing antibiotics, supplemental glutamine, and 30% FCS (HyClone Labs.), as previously described ( 24 ). Stationary phase promastigotes were injected into the hind feet of recipient mice at a dose of 2 × 10 6 organisms per footpad to initiate infection. The course of infection was monitored by measuring the thickness of footpad swelling weekly using a dial gauge caliper. Soluble L. major antigen was made as previously described ( 24 ). Hybridomas producing cytotoxic anti-CD4 mAb GK1.5 (rat IgG2b), anti-CD8 mAb 2.43 (rat IgG2b), neutralizing anti–I-A d /I-E d M5/114 (rat IgG2b), and neutralizing anti–IL-4 mAb 11B11 (rat IgG1) were obtained from the American Type Culture Collection. Noncytotoxic anti-CD4 mAb YTS 177.9 (rat IgG2a) was obtained from Dr. Shixin Qin (Leukosite Inc., Boston, MA). Antibodies were generated using serum-free media supplemented with 1% Nutridoma-NS ( Boehringer Mannheim ) or by induction of nude mouse ascites, and were purified using HiTrap Protein G columns ( Pharmacia ). Recombinant IL-12 was generated in dihydrofolate reductase–deficient CHO-DUXB11 cells transfected with mouse p35 and p40 IL-12 cDNA coexpressing dihydrofolate reductase (Genetics Institute), and subsequently was incubated in increasing concentrations of methotrexate to amplify transgene expression, as previously described ( 25 ). IL-12 was purified from serum-free culture supernatant by sequential Mono Q and heparin-Sepharose ( Pharmacia ) affinity chromatography, and the specific IFN-γ–inducing activity (1.1 × 10 6 U/mg) was determined in cultures of normal splenocytes, as previously described ( 26 ). As indicated in the figure legends, we used rIL-12 (sp. act. 2.1 × 10 6 IFN-γ–inducing U/mg) provided by Dr. M. Gately (Hoffmann-LaRoche, Nutley, NJ) as an alternative. Lymph node cells were washed three times in HBSS, counted, and suspended in DMEM/10% fetal bovine serum (FBS) 1 (DMEM supplemented with 100 μg/ml of penicillin and streptomycin, 2 mM glutamine, 0.1 mM nonessential amino acids, and 10% FBS, buffered at pH 7.4 with 10 mM Hepes). Cells were aliquoted into flat-bottomed 96-well culture plates at 10 6 cells per well and cultured for 48 h in DMEM/10% FBS. Stimuli included media alone or media containing soluble leishmania antigen at 10–20 μg/ml. Where indicated, 10 μg/ml of M-1 anti–IL-4 receptor mAb ( Genzyme ) was added to culture to prevent IL-4 sequestration by soluble or cell-associated IL-4 receptor ( 27 ). Anti-MHC II mAb was added at 10 μg/ml to determine the MHC II dependence of antigen-inducible cytokine responses ( 28 ). Culture supernatants were assayed for murine cytokines using double sandwich mAb ELISA techniques as previously described ( 27 ). Antibodies used for capture of IL-4, IFN-γ, and IL-12 p40 were BVD-6, R46A2, and 15.6, whereas captured cytokine was detected using biotinylated antibodies BVD-4, XMG1.2, and 17.8, respectively ( PharMingen ). Recombinant mouse IFN-γ, IL-4, and IL-12 used as standards in ELISA were purchased from PharMingen or Genzyme . Lymph node mRNA was isolated using STAT-60 (TelTest) and analyzed by oligo-dT primed reverse transcription and subsequent PCR. The techniques and primers used have been published previously ( 29 ). Approximately 0.2–0.3 gram of footpad tissue was minced in 2 ml of M199/30 FCS medium, crushed through a No. 200 stainless steel screen (Small Parts, Inc.), and disrupted using a Ten-Broeck homogenizer. 50-μl aliquots of footpad or lymph node suspension were serially diluted fivefold in promastigote growth medium and incubated in flat-bottomed 96-well plates at 26°C in humidified room air. Individual wells were examined using an inverted microscope at 200× at 2-d intervals for the presence of motile promastigotes. Data represent the geometric mean ± SEM of the last positive reciprocal dilution for each experimental group. For comparisons of ELISA data, significance was assessed using the nonparametric Mann-Whitney rank sum test. Significant differences in disease outcome were determined by contingency table analysis (Fisher exact test). Since IL-4 antagonizes the immunoregulatory effects of IL-12 ( 30 , 31 ), we tested whether neutralization of IL-4 by anti–IL-4 mAb 11B11 would restore the therapeutic benefit provided of rIL-12 in BALB/c mice infected with L . major for 2 wk, a time at which these agents are individually ineffective ( 16 , 17 , 32 ). Mice treated daily with 1 μg of intraperitoneal rIL-12 on days 7–21 only demonstrated nonsignificant decreases in footpad swelling . In contrast, lesional development was significantly delayed in mice injected intraperitoneally with 1 mg of anti– IL-4 mAb 11B11 in combination with intraperitoneal rIL-12 treatments on days 7, 14, and 21 of infection. However, all mice treated with this combination still displayed progressive footpad swelling leading to ulceration and limb necrosis. Similar results were obtained in a second experiment where 1 mg of anti–IL-4 mAb was administered intraperitoneally starting at wk 3 of infection and in combination with 10 d of intralesional rIL-12 injection . As previously reported ( 17 ), treatment at wk 3 with anti–IL-4 mAb alone only delayed disease progression without affecting final outcomes (data not shown). Similarly, delayed treatment of established leishmaniasis with anti–IL-2 mAb, which is normally curative when started early in infection ( 33 ), also failed to restore protective immunity when combined with rIL-12 (data not shown). These studies indicated that the neutralization of Th2-promoting cytokines alone was insufficient to restore the therapeutic effects of rIL-12 in L . major –susceptible mice with advanced disease. Because established BALB/c Th2-biased T cell responses proved highly resistant to cytokine-induced phenotypic reversal, we next tested whether transient CD4 + T cell depletion might remove pathogenic immune responses, while also providing a newly emergent population of T cells susceptible to cytokine-directed differentiation towards the curative Th1 phenotype. Treatment of BALB/c mice with the cytolytic antibody GK1.5 given in two doses of 0.5 mg each on days 21 and 22 of L . major infection resulted in CD4 + lymphopenia lasting ∼2–3 wk (Table I ). Although this dose of antibody is curative when given during wk 1 of infection, we confirmed that delayed therapy with CD4 cytolytic antibody GK1.5 alone had no effect on outcome . Continued progression of disease was associated with the re-emergence of lymph node CD4 + T cells and IL-4 production in response to L . major antigen (data not shown). This suggested that the intrinsic bias of BALB/c mice towards Th2 development in this disease was not disrupted by transient CD4 + T cell depletion alone. Therefore, we examined the course of cutaneous leishmaniasis in 3 wk–infected BALB/c mice treated with GK1.5 in combination with anti–IL-4 11B11 mAb and intralesional rIL-12 administered during the 2-wk period leading up to CD4 + T cell recovery. In control BALB/c mice treated with nonspecific rat IgG, the cutaneous lesions progressed rapidly, as indicated by increasing footpad thickness and the development of ulceration and necrosis . In contrast, mice treated with combined GK1.5, rIL-12, and anti–IL-4 mAb were able to limit and then reverse footpad swelling, with all five mice showing complete resolution of disease . By wk 14 of infection, the hindlimb swelling of mice receiving GK1.5-based immunotherapy had diminished to an average thickness compatible with that of age-matched normal feet. To determine if successful immunotherapy provided long-lasting immunity against reinfection with L . major , these cured mice and a group of normal BALB/c mice were infected with 2 × 10 6 L . major promastigotes in each hind foot. The immunotherapy-cured mice rapidly contained reinfection by 4 wk, as indicated by the transience of footpad swelling, whereas control BALB/c mice developed progressive footpad swelling characteristic of nonhealing leishmaniasis . The protective effects of GK1.5-based immunotherapy were subsequently reproduced in four additional studies where advanced cutaneous lesions were durably cured in 20 out of 25 BALB/c mice (80%). None of the 27 control BALB/c mice recovered from infection (difference significant at P < 0.01, Fisher exact test). Decreased footpad swelling was associated with reduced parasite load within the cutaneous lesions, with 42-fold decreases apparent at 3 wk after treatment (10 9.59 ± 0.41 per gram of control tissue compared with 10 7.96 ± 0.24 for treated mice) and a further 10,000-fold reduction evident at 7 wk after therapy (10 3.1 ± 0.40 per gram). The draining lymph node cells from five cured and reinfected mice were harvested, and their antigen-specific cytokine responses were compared with infected, untreated BALB/c mice (Table II ). Although the amounts of IFN-γ stimulated by antigen were not significantly different between the two groups, immunotherapy-cured mice produced concentrations of IL-4 that were ≥20-fold reduced relative to control BALB/c mice (P < 0.05). In a second experiment in which two out of five treated mice failed to heal, therapeutic success and failure correlated with the absence and presence of IL-4 production, respectively (Table II ). There was evidence that the restored CD4 + T cell populations contributed to IFN-γ production, as synthesis of this cytokine in treated and control mice was 46 and 60% MHC II dependent, respectively. IL-4 production was reduced to undetectable levels in the presence of anti–MHC II mAb. These findings indicated that the immunotherapy-induced cure gave rise to antigen-specific CD4 + T cell populations producing IFN-γ without IL-4, consistent with protective immune deviation towards a unipolar Th1 phenotype. Although we hypothesized that the protective effects of GK1.5-based immunotherapy were dependent on the removal of IL-4–producing cells present in the CD4 + T cell population, noncytolytic effects of anti-CD4 antibody on the function of MHC II–restricted T cells may have affected the outcome separately. For instance, in vivo and in vitro Th2 development requires that CD4 bind to MHC II during antigen presentation, suggesting that neutralization of CD4 function alone might effectively substitute for cellular depletion in the immunotherapy of established leishmaniasis ( 34 , 35 ). Another possibility requiring experimental exclusion was that the process of in vivo cellular depletion, regardless of the population targeted, was contributing directly or indirectly to cure of leishmaniasis and reversal of Th2 dominance. To test these alternative hypotheses, separate groups of BALB/c mice infected for 3 wk with L . major were treated with anti–IL-4 antibody and intralesional rIL-12 after receiving 0.5 mg of either depleting or nondepleting anti-CD4 mAb (GK1.5 and YTS177, respectively) or after being treated with depleting anti-CD8 mAb 2.43. In pilot studies, YTS177 failed to reduce peripheral blood CD4 + counts by >15%, as previously described ( 36 ). Only the immunotherapy regimen based on depleting GK1.5 antibody was capable of healing the cutaneous lesions of progressive leishmaniasis (four out of six mice) . These experiments clearly indicated that specific depletion of CD4 + T cells was required to mediate the cure by combined treatment with rIL-12 and anti–IL-4 and that depletion of an alternative T cell subset did not invoke nonspecific protective responses. We next determined how immunotherapy affected the in vitro and in vivo cytokine responses of infected mice at 7 and 21 d after treatment. The first time point corresponds to the inductive stage of immunotherapy, when CD4 + T cells were freshly depleted and the mice were receiving intralesional therapy with rIL-12. The second time point corresponds to the subsequent repopulation of the lymph node by CD4 + cells. At 1 and 3 wk after the start of immunotherapy, the popliteal lymph node cells of treated mice produced approximately three- and sevenfold less IL-4 in response to soluble leishmania antigen than did the lymph node cells of control mice . Consistent with a predominantly CD4 + T cell source for IL-4 in control mice, IL-4 levels were reduced by ∼80% in the presence of anti–MHC II antibodies capable of blocking both I-A d and I-E d (data not shown). The small amounts of IL-4 produced by CD4 + -depleted mice also remained MHC II dependent, indicating that the residual IL-4 response was derived from CD4 + T cells that had escaped depletion. The lymph node cells of treated mice produced twofold more IFN-γ than controls in the first week of therapy. Some of this response may have been mediated by ongoing rIL-12 therapy, as the majority of the IFN-γ response was spontaneous. By 3 wk after therapy, control and treated mice produced similar amounts of IFN-γ in response to antigen. Although production of IFN-γ in control lymph node cultures was reduced by 60% in the presence of anti– MHC II antibodies (difference significant at P < 0.05), IFN-γ declined by only 18% ( P > 0.10) when lymph node cells from mice were cultured under the same conditions at 1 wk after treatment (data not shown). These findings indicate that starting in the first week of immunotherapy treated mice were markedly less able to produce MHC II– dependent IL-4, but maintained antigen-specific IFN-γ synthesis by MHC II–unrestricted mechanisms before CD4 + T cell recovery. In contrast, IFN-γ responses were reduced 46.3 ± 7.5% in the presence of anti–MHC II at 7–8 wk after immunotherapy (Table II ), indicating recovery of CD4 + T cell populations responsive to leishmania antigen and biased towards the production of IFN-γ without IL-4. Compared with control mice, expression of IFN-γ mRNA increased twofold and IL-4 mRNA expression decreased approximately threefold in the lymph nodes of treated mice at 7 and 21 d after the start of immunotherapy . A separate group of 3 wk– infected BALB/c mice treated with GK1.5 anti-CD4 alone also demonstrated threefold decreases in IL-4 mRNA after 3 d (data not shown), consistent with reduced IL-4 production as a result of CD4 + depletion. Although the extent to which IL-4 mRNA was decreased in vivo was less than that measured for IL-4 protein in antigen-stimulated lymph node cultures, treated mice also expressed ninefold more inducible nitric oxide synthase (iNOS) mRNA relative to control nodes. Because iNOS production is stimulated by IFN-γ, reciprocally inhibited by IL-4, and necessary for curing of infection, increased iNOS mRNA expression in treated mice confirms both the overall Th1 predominance of the in vivo cytokine response and its significance with regards to the development of leishmanicidal activity. The induction and expansion of protective Th1 responses in leishmaniasis is dependent on IL-12 synthesis by accessory cells and functional IL-12 receptor expression by responder T cells. In this regard, the lymph nodes of treated mice expressed threefold more IL-12 p40 mRNA than did those of control mice at 7 and 21 d after immunotherapy. This correlated with increased spontaneous secretion of IL-12 p40 protein in lymph node cultures at 21 d after immunotherapy (0.37 ± 0.11 ng/ml for treated compared with 0.19 ± 0.01 ng/ml for control mice; P < 0.05). Finally, IL-12 receptor β2 subunit mRNA was increased threefold in the lymph node tissue of treated compared with control mice. The IL-12Rβ2 subunit is required for transduction of IL-12–dependent signals in CD4 + T cells and its rapid downregulation in BALB/c mice under neutral conditions is thought to be essential to the preferential development of Th2 cells when mice of this strain are infected with L . major ( 18 , 37 ). Therefore, these in vivo– based findings confirm that the shift towards Th1-polarized cytokine responses after GK1.5 based immunotherapy is accompanied by enhanced IL-12 production and IL-12 responsiveness necessary for the induction and maintenance of protective immunity. Although these studies were initially designed to reconstitute the therapeutic effect of rIL-12 in the setting of progressive leishmaniasis, we also addressed the possibility that CD4 + depletion followed by neutralization of IL-4 alone was sufficient to achieve a cure. Groups of BALB/c mice infected for 3 wk with L . major were treated with GK1.5 and 11B11 with or without a 10-d course of intralesional injection with rIL-12 . Surprisingly, mice treated with GK1.5 and anti–IL-4 mAb healed at the same rate regardless of the presence or absence of intralesional rIL-12 in the immunotherapeutic protocol. The only notable difference between the two groups was the transient suppression of footpad swelling during the 2-wk period in which mice were actively treated with rIL-12. Furthermore, rIL-12 could not substitute for anti–IL-4 in the restoration of curative immunity in GK1.5-treated mice. In a separate experiment (data not shown), the combination of GK1.5 and rIL-12 cured only one out of six mice previously infected for 3 wk, whereas a combination of GK1.5, anti–IL-4 11B11 mAb, and rIL-12 cured four out of six mice. These data indicated that suppression of IL-4 bioactivity alone was sufficient to permit the establishment of unipolar Th1 cytokine responses after GK1.5 treatment of BALB/c mice with advanced infection with L . major . In these studies, we demonstrate that transient depletion of Th2-biased CD4 + T cell responses in 3-wk L . major - infected BALB/c mice restores the therapeutic Th1-differentiating effects of anti–IL-4 mAb, with or without the addition of rIL-12. The mechanisms mediating protection included a requirement for specific depletion of CD4 + T cells, as merely blocking CD4 function with nondepleting anti-CD4 antibody did not change the course of disease. Similarly, selective depletion of CD8 cells also failed to restore the cure in response to cytokine therapy and it is unlikely that the process of cellular depletion itself nonspecifically contributed to parasite clearance. Therefore, GK1.5-based immunotherapy appears to mediate protection by durably ablating Th2 CD4 + T cells responsible for the progression of leishmaniasis, while permitting the eventual recovery of curative, MHC II-dependent IFN-γ responses—effectively “resetting” the CD4 + T cell immune phenotype at the populational level. The protective effects of GK1.5-based immunotherapy were reproduced in five groups of mice and resulted in the cure of advanced cutaneous lesions in 20 out of 25 animals (80%). Once cured, these cutaneous lesions did not recur during a 14-wk period of observation and mice that had recovered after immunotherapy were fully resistant to reinfection with a large inoculum of L . major . These findings confirm the stable reversal of antigen-specific Th2 responses by a combination of CD4 + elimination and therapy-directed Th1 expansion. Surprisingly, BALB/c mice with advanced infection and genetic predispositions towards Th2 development were fully capable of supporting unipolar Th1 responses provided only that IL-4 activity was neutralized at the time of CD4 + T cell recovery. Recovery of leishmanial skin lesions was notably delayed and coincided with the onset of lymphoid tissue repopulation by CD4 + T cells expressing a Th1 cytokine phenotype. Footpad thickening initially continued for up to 4 wk after treatment with GK1.5, anti–IL-4 mAb, and rIL-12, with ∼25% of the cutaneous lesions progressing to shallow ulceration before healing began. The parasite load within the cutaneous lesions also declined after a significant delay, with relatively minor 42-fold decreases apparent at 3 wk after treatment and a more substantial 10,000-fold reduction apparent at 7 wk after therapy. Although T cell populations of untreated animals produced both IL-4 and IFN-γ in response to L . major antigen, lymph node cells from BALB/c mice treated 3 wk previously with GK1.5, 11B11, and rIL-12 produced IFN-γ in similar quantities, but with sevenfold reduced synthesis of IL-4. IL-4 production was undetectable in BALB/c mice that had been cured for 19 wk and that demonstrated resistance to infection. This was observed not only in comparison to control BALB/c mice infected with L . major , but also in comparison to the few mice that failed to heal after immunotherapy and that produced amounts of IL-4 no different than untreated controls. IL-4 productive capacity was similarly unchanged after ineffective treatment with anti–IL-4 and rIL-12 alone or with GK1.5 alone (data not shown). Treatment also increased expression of IL-12 p40, IL-12 receptor, and iNOS mRNA in the draining lymph nodes, indicating the biologically significant restoration of IL-12–dependent cytokine cascades and Th1-dependent leishmanicidal defenses. Because IFN-γ synthesis was greater than 50% MHC II-dependent in healing mice, these findings are most consistent with the delayed recovery of advanced lesions in response to repletion of a CD4 + T cell population expressing a unipolar Th1 phenotype capable of activating nitric oxide– dependent parasite killing. We first confirmed that monotherapy with rIL-12, anti– IL-4 mAb, or GK1.5 at wk 2–3 of infection had no lasting effect on the progression of disease, although they are curative when given in wk 1 of infection. Because rIL-12, GK1.5, and anti–IL-4 have been reported to alter the course of leishmaniasis when given as late as 14 d after infection in other studies ( 16 , 17 , 38 ), we studied the effects of immunotherapy at wk 3 to further ensure that monotherapy would be ineffective. Under these stringent conditions, none out of 18 mice treated with rIL-12, anti–IL-4 mAb, or GK1.5 alone were able to cure disease. Combinations of anti–IL-4 and rIL-12 therapies were also ineffective when started as early as day 7 of infection. Although IL-4 is necessary for Th2 development in L . major– infected BALB/c mice, the well-characterized inhibitory effects of this cytokine on IL-12 receptor expression and rIL-12–directed Th1 cell differentiation ( 18 , 30 , 31 ) do not appear to be necessary for maintaining the in vivo resistance of established Th2 CD4 + T cells to rIL-12 therapy. Once developed, the Th2 CD4 + T cells of BALB/c mice either do not require IL-4 to maintain numbers and function, or else impractically large doses of anti–IL-4 antibody are necessary to adequately neutralize IL-4 bioactivity in vivo. However, relatively low doses of anti–IL-4 mAb were both sufficient and essential for establishing curative immunity after GK1.5 treatment of L . major –infected BALB/c mice. In this regard, the recovering CD4 + T cells of GK1.5-treated mice appear to be functionally similar to those present in wk 1 of infection, when anti–IL-4 mAb prevents the expansion of Th2 cells by preserving IL-12 responsiveness ( 30 ). We observed residual IL-4 in GK1.5-treated animals. This probably derived from cytolysis-resistant CD4 + T cells, as antigen-induced IL-4 production remained MHC II dependent in these mice. The failure to eradicate >85% of CD4 + T cells in infected mice, compared with >95% in uninfected mice (data not shown), is compatible with previous demonstrations of the relative resistance to GK1.5-mediated depletion of T cells with a memory/effector phenotype ( 39 ). The residual IL-4 production by CD4 + T cell–depleted mice was not immediately affected by additional treatments with rIL-12 and anti–IL-4 mAb (data not shown), but declined gradually over a 3-wk period. This suggests that anti–IL-4 mAb limited re-expansion of Th2-committed T cells in infected mice without directly inhibiting residual IL-4 synthesis, consistent with recent demonstrations of cytokine-autonomous production of IL-4 in differentiated Th2 cells ( 40 , 41 ). These data otherwise do not determine whether the GK1.5-resistant cellular sources of IL-4 eventually disappear due to attrition, suppression, or phenotypic reversal at the cellular level. Instead, the recovering CD4 + T cell population was biased towards production of IFN-γ. Although Th1-biasing cytokine therapy may have preferentially affected undifferentiated precursor CD4 + T cells that were recent thymic emigrants ( 42 ), a subset of peripheral memory/effector cells susceptible to differentiating cytokines may have provided an alternative source for the reconstituted Th1 response ( 43 ). Further investigations are needed to distinguish between these mechanisms. Other mechanisms may have contributed to the GK1.5-induced cure of BALB/c leishmaniasis. Our studies suggest that CD8 + T cells and/or NK cells were interim sources of IFN-γ in the first week after CD4 + depletion and cytokine therapy, when IFN-γ synthesis was maintained and was transiently MHC II independent. Although resistant strains of mice deficient in either β2-microglobulin or CD8 + T cells remain capable of curing primary infection with L . major ( 44 , 45 ), MHC I–restricted responses have been implicated in the late recovery and subsequent resistance to reinfection ( 38 ). Detailed studies beyond the scope of the current report will be required to determine if the CD8 + T cells of disease-susceptible BALB/c mice mediate beneficial functions that are otherwise silenced in the face of functionally dominant Th2-type CD4 + T cell responses. These studies also do not exclude selective tolerization of parasite-specific Th2 cells as an alternative curative mechanism for GK1.5-based immunotherapy. Treatment with cytolytic anti-CD4 mAb establishes antigen-specific immunologic tolerance and protects against Th1-mediated autoimmune pathologies in mice ( 46 ). The preservation of MHC II–dependent, L . major– specific IFN-γ production in GK1.5/11B11/rIL-12–treated BALB/c mice indicates that any tolerizing effects were limited to Th2-inducing antigens, which are known to protect against disease when induced by other methods ( 47 ). However, noncytolytic anti-CD4 mAb YTS177 that tolerizes developing immune responses in other disease models ( 36 ) failed to cure leishmaniasis in combination with anti–IL-4 mAb and rIL-12. These findings therefore favor depletion as the relevant protective mechanism. The cure of established murine leishmaniasis by depletion-induced phenotypic “resetting” of the CD4 + T cell population is not the first form of successful immunotherapy in this model of T cell–dependent pathology, but the mechanisms involved appear to be distinct. For instance, cure by combined treatment with rIL-12 and sodium stibogluconate was associated with a Th2 to Th1 shift only after parasite burden was reduced ( 21 ). Although the central role of Th2 cytokines in the initiation of progressive leishmaniasis is well characterized, increased parasite burdens may subsequently contribute to sustaining the susceptible state by independently biasing towards Th2 differentiation and function. For instance, leishmania-infected accessory cells are less able to produce IL-12 and to respond to IFN-γ ( 19 , 48 ). Increased antigenic stimulation of BALB/c T cell receptors also independently favors the differentiation of T cells with Th2 phenotypes ( 20 ). Finally, CD28-directed costimulatory signals are necessary for Th2, but not Th1 immune responses in murine leishmaniasis and further implicate accessory cell function in regulating CD4 + cytokine polarity ( 49 , 50 ). However, mice cured by “CD4 reset” showed their greatest declines in parasite burden after the recovery of Th1 CD4 + T cell responses at wk 3 of therapy. This is most consistent with parasite killing as a result of Th1 immune deviation within the CD4 + T cell population, rather than immune deviation in response to reduced infectious burden. Therefore, these findings suggest that increased parasite load, increased antigen presentation, or altered accessory cell costimulatory function are not sufficient by themselves to reconstitute a Th2-biased response after CD4 + T cell depletion. We conclude that the perpetuation of cytokine-resistant Th2 T cell responses in progressive leishmaniasis is dependent on properties of the CD4 + T cell and is not independently determined by infection-induced changes in the non-CD4 + T cell immune environment. These studies are significant more generally in that they demonstrate the potential to reverse established, pathologic CD4 + T cell responses by combined antibody-directed cytolysis and subsequent treatment with differentiating cytokines or anticytokine antibodies. Our ability to modulate Th2 responses is especially remarkable because of the well-recognized stability of this phenotype to immunotherapeutic reversal ( 16 , 32 , 51 , 52 ) and indicates the potential for use as immune-deviating therapy in other Th2-dependent experimental and clinical diseases. A similar approach to the reversal of severe allergic disorders is technically feasible, as cytolytic anti–human CD4 antibodies are already in experimental use for autoimmunity, and Th1-deviating agents, such as soluble IL-4 receptor and rIL-12, are in various stages of clinical investigation. By analogy, Th1-dependent autoimmune diseases might also be reversible if stable deviation towards a Th2 phenotype results in disease suppression ( 53 , 54 ). The pathogenic Th1 responses of murine autoimmune encephalomyelitis, lupus nephritis, and collagen-induced arthritis can be prevented by pretreatment with anti-CD4 antibodies that tolerize against the inducing antigen. However, as in leishmaniasis, established and progressive forms of disease only briefly remit after short-term treatment with anti-CD4 mAb ( 10 , 36 ). We speculate that GK1.5 treatment of advanced autoimmunity might restore CD4 + T cell populations with naive-like susceptibility to the Th2-deviating effects of anti–IL-12 antibodies or rIL-4 that otherwise only prevent disease expression when given at the time of sensitization to self-antigen ( 55 , 56 ). In conclusion, these studies show that curative and unipolar Th1 cellular responses are recovered in heavily parasitized, disease-susceptible mice merely by transient depletion of CD4 + T cells and neutralization of residual IL-4 bioactivity. Because CD4 + T cell depletion provides a unique window of opportunity to durably reprogram the cytokine polarity of an established and pathogenic T cell response, these findings suggest a novel immunotherapeutic strategy potentially applicable to the clinical amelioration of progressive allergic and autoimmune disorders. | Other | biomedical | en | 0.999997 |
10377186 | In humans, the following antibodies, anti-CD28– PE, anti-CD45RA–PE, anti-CD45RO–PE, anti-CD57–FITC, anti-CD56–PE, anti-α/β TCR–allophycocyanin (APC), anti-α/β TCR–PE, anti-CD8β–PE, biotinylated anti-CD4, and anti-CD8α, were obtained from Immunotech; anti-CD27–FITC and anti-α/β TCR–FITC were purchased from PharMingen . Anti-CD8α–FITC and –Tricolor (TC), anti-CD4–FITC and –PE, and anti-CD3–TC were obtained from Caltag Laboratories. An anti-CD4–FITC (Diaclone) recognizing an epitope distinct from the anti-CD4 magnetic beads was also used. Streptavidin–Red 613 was purchased from GIBCO BRL . Streptavidin-PE and -TC were obtained from Caltag Laboratories. In mice, anti-CD4 (RM4-5 or RM4-4)–PE, anti-CD8α– or anti-CD8β–FITC, biotinylated or Cychrome-conjugated anti-α/β TCR and streptavidin-APC were obtained from PharMingen . Anti-α/β TCR–TC was obtained from Caltag Laboratories. Bovine anti-CD4 and anti-CD8α antibodies were provided by J. Naessens, International Laboratory for Research on Animal Diseases (ILRAD, Nairobi, Kenya). Heparinized blood was obtained from healthy donors or from MHC-deficient patients, and PBMCs were purified by Ficoll-Hypaque ( Amersham Pharmacia Biotech ) gradient centrifugation. Cells were frozen in liquid nitrogen until use. Bovine blood was provided by Mr. Noé from the GENA Laboratory, Institut National de la Recherche Agronomique (INRA, Jouy en Josas, France). After Ficoll-Hypaque separation, PBLs were labeled with anti-CD4 and/or anti-CD8 (both Ig2a), which were revealed with an FITC-conjugated goat anti-Ig2a serum. Cells were FACS ® sorted into CD4 or CD4/CD8 positive or negative fractions as indicated, and the relative amounts of boCα- or boAV19-boAJ33 transcripts were estimated by kinetic PCR (see below). C57BL/6 (H-2 b ) (B6), BALB/c (H-2 d ), 129 (H-2 b ), DBA/2 (H-2 d ), and CBA (H-2 k ) mice were bred in our own specific pathogen–free animal facility. β2m −/− and I-A b−/− mice, backcrossed nine times to B6, were obtained from the Centre National de la Recherche Scientifique central animal facility (CNRS, Orleans, France). TAP −/− B6/129 and CD8 −/− deficient mice were obtained from The Jackson Laboratory . K b−/− D b−/− mice have been described ( 33 ). CD1 −/− mice were generated by S.-H. Park and A. Bendelac (unpublished results). Surface phenotyping was carried out on a FACSCalibur™ cytometer ( Becton Dickinson ). Three-color sorting (CD4, CD8α, and α/β TCR staining) was carried out on a FACS Vantage™ ( Becton Dickinson ) to obtain α/β + DN, -CD8α + , or -CD4 + cells. Reanalyzed fractions were >99% pure. In some experiments, DN cells were seeded into 96-well PCR microplates at the indicated cell concentrations using a single cell deposition unit ( Becton Dickinson ). DN/CD8 + fractions, enriched or depleted for a given marker, were obtained by FACS ® sorting negative and positive fractions for this marker after quadruple staining with anti-CD4, anti-CD8α, anti-α/β TCR, and the relevant marker (either CD8β, CD45RA, CD45R0, CD56, CD57, CD28, or CD27). In some experiments, DN and CD8α + enriched fractions were obtained using paramagnetic beads and the VarioMacs system (Miltenyi Biotec). In brief, cells at 10 7 /100 μl were resuspended in PBS, 0.5% BSA, 5 mM EDTA and incubated with anti-CD4 paramagnetic microbeads, washed once, resuspended in 500 μl of the same buffer, and passed through an RS column. Effluent was collected as the DN + CD8 + fraction. After thorough washes of the column, the eluate was collected as CD4 + fraction. In these conditions, the CD4 + fraction was >95% pure. All primers and probes were obtained from Genosys Biotechnologies and used without further purification. Probes were FITC conjugated. The following primers have been described previously ( 14 ): mAV14, mAJ18, mAC-5′, mACV, mAC-3′, hACV, hAC-5′, and hAC-3′. The following primers were used (m, bo, and h stand for murine, bovine, and human; p is for probe; in and out are for the inner and outer primer, respectively, in the case of nested PCR): mAV19, CACTTTCCTGAGCCGCTCGAA; mAJ33, biotin-TTAGCTTGGTCCCAGAGCCCC; pmAV19, GCTTCTGACAGAGCTCCAG-FITC. The mouse-specific Vβ and Vα primers were slightly modified from Casanova et al. ( 34 ). The human Vβ primers were derived from Puisieux et al. ( 35 ) as modified by Martinon et al. ( 36 ). The other human primers were as follows: hAV7S2, biotin-CCTTAGTCGGTCTAAAGGGTACAG; hAJ33, CCCAGCGCCCCAGATTAA; hBJ1S2, GTCTCCCTCAGTCTGGCTCCG; hBJ1S3in, GGTGGTAAGGTCAGAGGCTCTTTTATC; hBJ1S3- out, TCTGTGTGAGGGAGAGAAACG; hBJ1S5, CAAGACACACCAAAGGGAACGC; hBJ2S2, CTCTCCCAGCACCCAGAACCA; hBJ2S4, CGCACAAAAACCCGAGCGCAG; hBJ1S6in, CCAGGCACCCCCGAGTCAAGA; hBJ1S6out, GGCAACACAGCAGAGCAACAA; hBJ2S7out, TCGTCCTCTCCAGTCTCGCCT; hBJ2S7in, CGCCCTCTGCTCAGCTTTCCG; and hAJ33in, CAAAAGCTGACTTGCAGGCATCG. In cattle, the following primers were used: boAV19, biotin-CATTCCTTAGACGCTCTGATGCACA; boAJ33, GCCCCAGATCCACTGATAGTTGC; pboAV19, FITC-GTGGAGTTCCTTCAGAAGGAG; bo5′CA, biotin-AGGACCCCAACCCCACTGTGT; bo3′CA, CTTCAGGAGGAGGATGCGGAAC; and pboCA, FITC-CACTGGATTGGGGGCTTC. Genomic DNA was obtained as crude cell lysate by adding to the cell pellets (1–10 6 cells) a solution containing 200 μg/ml proteinase K ( Promega Corp. ), 0.5% Tween 20 ( Sigma -Aldrich), 10 mM Tris-HCl, pH 9, 50 mM KCl, 2.5 mM MgCl 2 . After resuspension, this solution was incubated for 2 h at 56°C, and the proteinase K was denatured by incubation at 95°C for 20 min. Total RNA was extracted from 1–5 × 10 5 cells with the RNAble solution (Eurobio), ethanol precipitated with addition of 2 μl Pellet Paint coprecipitant (Novagen), and resuspended in 20 μl sterile water. Reverse transcription was carried out as described with random hexamers and oligo-dT priming ( 37 ). Quantitative kinetic ELISA PCR was carried out as described ( 37 ). Polyclonal samples were divided into two aliquots before RNA extraction except in a few instances in which the amount of sorted cells was too low (<2 × 10 4 ). Average values are shown. In the most recent experiments, an ABI prism 7700 ( Perkin-Elmer ) apparatus was used instead of the ELISA assay to monitor the amount of amplicon during the PCRs. This apparatus is based on the 5′ to 3′ nuclease activity of Taq polymerase ( 38 ), which allows the release of a fluorescent reporter during the PCR. A probe labeled with both a reporter and a quencher dye is spiked into the PCR mix at the beginning of the reaction. The sequences of the Taqman probes used in this study are the following: hCA, mGCATGTGCAAACGCCTTCAACAACAqp; hAV7.2, mTGAAAGACTCTGCCTCTTACCTCTGTGCqp; mAC, mCTCCCAAATCAATGTGCCGAAAACCAqp; mAV19, mTCCAGATCAAAGACTCTGCCTCATACCTCTGqp; and mAV14, mCACCCTGCTGGATGACACTGCCACqp; where m preceding each sequence stands for FAM and qp following for TAMRA blocked with a phosphate. When using the ABI prism 7700 apparatus, the following primers were also used: hAV7S2, TCCTTAGTCGGTCTAAAGGGTACAG; hAJ33, CCAGCGCCCCAGATTAA; hAC-5′, ACCCTGACCCTGCCGTGT; hAC-3′, GGCTGGGGAAGAAGGTGTCTT; mAV14, TGGGAGATACTCAGCAACTCTGG; mAJ18, CCAGCTCCAAAATGCAGCC; mAV19, CTTTCCTGAGCCGCTCGAA; mAJ33, CTTGGTCCCAGAGCCCC; mAC-5′, CCTCTGCCTGTTCACCGACTT; and mAC-3′, CGGTCAACGTGGCATCACA. In these quantitative kinetic PCR methods, for two samples containing the same amount of Cα, a shift of n cycles along the x-axis of the two amplification curves represents an ∼1.8 n -fold difference in the two samples studied. Single human DN α/β T cells were sorted into 84 wells of PCR plate. 11 of the remaining wells were used as negative controls and 1 as positive. After cell lysis, PCR amplification was carried out for 25 cycles using the following primers: 29 Vβ, 7 Jβ, AV7S2out, AJ33out, and as positive control AV24 and AJ18out. The resulting reaction mix was diluted fivefold, and aliquots were amplified in a second PCR reaction with an AV7S2/AJ33 primer pair for 40 cycles. The specific AV7S2-AJ33 amplicons were detected by ELISA or using the Taqman assay. Aliquots of the AV7S2-AJ33–positive wells from the first PCR reaction were then PCR amplified in 24 individual PCR reactions with 1 (or 2) specific Vβ primer(s) and a mixture of 7 Jβ primers for 45 cycles. The positive reactions were detected by agarose gel electrophoresis, and the amplicons were sequenced as described below. Polyclonal sequencing was performed after amplification for 42 cycles with hAV7S2, boAV19, or mAV19 and either AJ33 or Cα primer pairs. The amplicons were purified using the Wizard PCR clean-up system ( Promega Corp. ) and sequenced using either a nested primer in the Cα constant region (in mouse) or the same AV19 or AV7S2 primer as used for amplification, as appropriate. Sequences were performed with the Thermosequenase radiolabeled terminator cycle sequencing kit ( Amersham Pharmacia Biotech ) with 33 P-ddNTP. DN and CD8 + T cells were obtained from TAP −/− mice after depleting CD4 + T cells with anti-CD4 antibody (GK1.5 and 172.4) plus rabbit complement (Behring AG) followed by centrifugation over a density gradient (Lympholyte M; Cedarlane Laboratories). 2 × 10 6 cells were stimulated, in the presence of 4 × 10 6 γ-irradiated (2,500 cGy) splenic cells, for 2 d with soluble anti-α/β TCR (H57) at 7.5 μg/ml which had been precoated for 1 h at 37°C in flat-bottomed 96-well plates. Cells were cultured for an additional 2 d with IL-2 (100 U/ml) in complete culture medium: RPMI 1640, 15% FCS, 2 mM l -glutamine ( GIBCO BRL ), penicillin 100 U/ml, streptomycin 100 μg/ml, 50 μM β-ME. At day 4, 8 × 10 6 cells were fused with 2.5 × 10 7 TCR α/β − BW5147 thymoma cells using standard procedures, plated at 4 × 10 4 cells per well in 96-well microplates, and selected in HAT medium. Resulting clones were screened for expression of AV19-AJ33 rearrangement on genomic DNA followed by sequencing. Vβ were characterized by 17 Vβ-specific PCR reactions, and the resulting amplicons were then sequenced. Results were verified by immunofluorescence with specific anti-Vβ antibodies. A previous analysis of TCR α chain repertoire of human DN T cells indicated that besides the invariant hAV24AJ18 TCR α chain, another TCR α chain was frequently expressed that also had a restricted AVAJ usage and recurrent junctional features ( 39 ). This α chain, which was encoded by rearranged hAV7S2AJ33 elements, had a CDR3 of constant length but some variability in the two junctional codons. A GenBank search showed that among the 20 TCR α chains sequenced in cattle, one chain (BOTCRA14) used the homologous Vα and Jα segments with a CDR3 of the same length ( 40 ). In mice, no homologous invariant α chain had been described, but the corresponding Vα (mAV19) and Jα (mAJ33) segments display high homology with their human counterparts, as there is only one amino acid difference between the murine AJ33 and its human counterpart ( 41 , 42 ). Based on these observations, which suggested the existence of another conserved T cell population in mammals, we undertook an extensive analysis of the frequency and repertoire of the T cells bearing this particular combination of AVAJ elements in these three species. To determine the frequency and coreceptor phenotype of T cells bearing hAV7S2AJ33 TCR α chains, PBLs were separated into highly purified α/β DN, CD4 + , or CD8α + (i.e., CD8αα + and CD8αβ + ) subsets, and the amount of hAV7S2AJ33-encoded transcripts in duplicate fractions was quantified by kinetic PCR. Although the amounts of Cα chain were similar in all samples , there was a large shift to the left of the hAV7S2AJ33 amplification curves for the DN (8.5 cycles) and the CD8α + fractions (5 cycles) compared with the CD4 + fractions, indicating that DN and CD8α + cells contained ∼150–360- and 19–32-fold more hAV7S2AJ33 transcripts, respectively, than CD4 + lymphocytes. hAV7S2AJ33 transcripts were enriched in DN and CD8α + T cell preparations from all individuals tested, and on average the enrichment levels of hAV7S2AJ33 transcripts in DN T cells were much higher than those observed for the NK1 T cell–specific invariant α chain hAV24AJ18 (data not shown). Because a similar invariant α chain had been sequenced once in cattle ( 40 ), we examined its distribution within CD4 + , CD8 + , DN, or (CD4 + plus CD8α + ) sorted cells obtained from bovine blood. As shown in Fig. 1 , C and D, the levels of boAV19AJ33 transcripts in the DN fractions were as high as those observed in humans. No boAV19AJ33 amplification could be obtained with the CD8α + fraction, and the comparison of CD4 + and CD4 + plus CD8α + amplification curves suggested that bovine CD8α + cells did not express high amounts of boAV19AJ33 transcripts. In mice, no canonical TCR α chain rearrangement involving mAV19 and mAJ33, the murine elements that are homologous to hAV7S2 and hAJ33, respectively, had been described. However, quantitative PCR analysis of mAV19AJ33 transcripts within highly purified murine α/β DN, CD4 + , and CD8α + lymphocytes indicated that, as in human and bovine blood, increased mAV19AJ33 expression was observed in the DN fraction from lymph nodes . The shift of the amplification curves between DN and CD4 + cell preparations was lower than in humans and cattle, suggesting that mAV19AJ33-bearing cells were less abundant in mice. To examine the length of the CDR3 regions of the hAV7S2AJ33 transcripts and their homologues in mice and cattle, we carried out polyclonal sequencing of Vα-Cα or Vα-Jα amplicons obtained from the various lymphocyte subpopulations in these species. As shown in Fig. 2 A, a readable hAV7S2AJ33 sequence was obtained from hAV7S2-Cα amplicons derived from DN and CD8α + samples but not from CD4 + samples. Therefore, this indicated that in the former but not the latter cells, the hAV7S2 segment was predominantly rearranged to the hAJ33 segment and comprised a CDR3 of constant length. The polyclonal hAV7S2AJ33 sequence displayed some heterogeneity at the VJ junction, in agreement with previous results from Porcelli et al. showing some variability of the two N-encoded amino acids ( 39 ). In cattle, the picture was slightly different, as polyclonal sequencing of boAV19-Cα amplicons demonstrated the predominant presence of the invariant boAV19AJ33 chain in DN cells, but not in CD4 + or CD8 + cells (data not shown). At the VJ junction, the polyclonal sequence derived from DN cells displayed some junctional heterogeneity, indicating also some variability of the two amino acids encoded by N additions. In mice, polyclonal sequencing of the mAV19-Cα amplicons showed a fully readable sequence for the DN but not for the CD8α + fraction (data not shown). Moreover, the VJ sequence displayed no heterogeneity and corresponded to a “germline” junction without nucleotide trimming or N addition (see also Table II below). Altogether, these results demonstrated that in the three species, DN α/β + T cells were enriched for cells bearing TCR α chains with highly homologous AVAJ elements (hAV7S2AJ33, mAV19AJ33, and boAV19AJ33) and constant CDR3 length as shown in the multiple alignment displayed in Fig. 3 . This novel canonical TCR α chain will be referred to hereafter as the “invariant” Vα7/19-Jα33 TCR α chain. A large amount of invariant α chain transcripts does not formally demonstrate the existence of a large number of invariant TCR α chain–bearing cells, as this transcript could have been expressed at very high levels in a few cells. To more directly estimate the number of cells using the invariant Vα7-Jα33 chain, single α/β + DN cells from three different subjects were sorted into PCR plates using a single cell deposition unit. FACS ® -sorted α/β + TCR CD8α + or CD4 + cell suspensions were also serially diluted into PCR plates to get 12 replicates of the indicated cell concentrations. After cell lysis, hAV7S2AJ33 PCR amplification was carried out on genomic DNA with luminometry detection of the amplicons. As shown in Fig. 4 A, the assay was sensitive enough to detect a single cell harboring the relevant rearrangement and yielded a frequency of Vα7-Jα33 + DN cells of ∼1/7.4. Using limiting dilution analysis (LDA), the frequency observed for CD8α + cells was 1/36 . Sequence analysis of amplicons from single cell preparations obtained with DN and CD8α + cells demonstrated the presence of invariant Vα7-Jα33 transcripts in all cases. A similar analysis on CD4 + cells yielded a frequency of Vα7-Jα33 + cells of 1/1,093 . Importantly, sequencing of the positive wells at concentrations where the Vα7-Jα33 amplicons were derived from one cell showed that out of six positive wells, two corresponded to a rearrangement of the hAV7S2 to hAJ34 (which were amplified because of the short genomic distance [∼700 bp] separating hAJ34 from hAJ33), three corresponded to a rearranged Vα7-Jα33 chain with a different CDR3 length, and only one corresponded to the invariant Vα7-Jα33 chain. Thus, the actual frequency of CD4 + cells harboring the invariant Vα7-Jα33 chain should be ∼1/6,000. In mice, Vα19-Jα33 + cells were less numerous than in humans. Their frequency within α/β DN lymph node cells was 1/56 , compared with ∼1/7.5 in humans . In the absence of anti-hAV7S2 or anti-mAV19 antibody, we used the following strategy to characterize the surface phenotype of the population expressing the invariant Vα7-Jα33 chain. α/β DN and CD8α + cells were depleted or enriched for a given marker by FACS ® sorting, and the amounts of Vα7-Jα33 transcripts in the positive and negative fractions were quantified and compared with the amounts of amplified Cα transcripts, in order to take into account variations in cell number. Fig. 6 displays an example of such an experiment, where we examined whether the CD8 molecules expressed by Vα7-Jα33 + cells were either heterodimeric (αβ) or homodimeric (αα), since CD8αα + cells represent between 5 and 25% of CD8α + TCR α/β + lymphocytes in human PBLs (data not shown). There were ∼45-fold more Vα7-Jα33 transcripts in the CD8αα + fraction than in the CD8αβ + fraction. This result was confirmed by polyclonal sequencing of the amplicons obtained after amplification with hAV7S2/Cα (V-C) or hAV7S2/hAJ33 (V-J) primers: no readable sequence was obtained from the CD8αβ + fraction, whereas the invariant α chain was present in the CD8αα + fraction . The absence of a readable sequence in the CD8αβ + fraction using V-J amplification indicates the near absence of the invariant α chain in this fraction. Using this strategy, it could be established that Vα7-Jα33 + cells had mainly a memory phenotype (i.e., CD45R0 + CD45RA − ) and were CD56 − CD57 − CD28 + CD27 + (data not shown). Despite many attempts using different lymphokine mixtures, we were unable to obtain a large enough number of T cell clones expressing the invariant Vα7-Jα33 chain. Therefore, the TCR β chain repertoire of DN cells bearing invariant Vα7-Jα33 chain was directly estimated by single cell PCR analysis. After single cell deposition of DN α/β cells into PCR plates, cells were lysed and a first PCR reaction was carried out using hAV7S2, hAJ33, and a mixture of 29 Vβ and 7 Jβ. This first reaction was then amplified in a second PCR with nested AV7S2/AJ33 primers allowing us to find the wells containing Vα7-Jα33 + cells. Using the reaction mixture of the first PCR corresponding to the positive wells, 24 individual PCR reactions were set up using 1 (or 2) Vβ and a mixture of 7 Jβ primers. This allowed us to get a specific band for 0–2 Vβ which was then sequenced using the relevant Vβ primer. In 9 independent experiments carried out on DN cells obtained from 3 different subjects, the frequency of Vα7-Jα33 + wells ranged between 6 and 16/84. Among the 92 Vα7-Jα33 + wells studied, a β chain could be assigned and sequenced in 37 cases (40%), and we found that there was a heavy bias toward Vβ13 and Vβ2. Out of 13 Vβ sequenced in donor A, 7 were BV13 + and 3 were BV2 + . In donor B, 8/12 were BV13 + and 4/12 were BV2 + . In donor C, 7/12 were BV13 + and 2/12 were BV2 + . However, despite this biased usage of BV13 or BV2 regions, the TCR β chains derived from Vα7-Jα33 + cells did not show obvious restrictions in Jβ usage or in CDR3 length (Table I ). Sequence analysis of VαJα junctions confirmed the presence of Vα7-Jα33 chains with the canonical CDR3 length and revealed significant variations in amino acid composition at the two N-encoded positions (Table I ). Moreover, the presence of repeated TCR α and β chain junctional sequences in different cells from the same subject cloned in different PCR plates and the fact that these TCR chains were not found in other subjects indicated that this subset had undergone clonal expansion in vivo, an interpretation that is consistent with its memory phenotype (see above). Overall, Vα7-Jα33 + cells appear to use a semirestricted repertoire that is expanded as needed. Because the frequency of Vα19-Jα33 + cells is not as high in mice as in humans, the strategy followed to characterize the surface phenotype of human Vα7-Jα33 + cells was not successful in mice. Therefore, to characterize the TCR β chain repertoire of this subset, we took advantage of the fact that Vα19-Jα33 + cells are enriched in TAP −/− mice (see below). We generated T–T hybridomas from DN enriched lymph node cells from TAP −/− mice, and screened for expression of Vα19-Jα33 transcripts. In agreement with the single cell analysis , 16/168 hybridomas studied were Vα19-Jα33 + . As shown in Table II , in all cases, these hybridomas carried Vα19-Jα33 chains with the canonical CDR3 length and in all but two cases, the Vα19-Jα33 junction was germline encoded. The Vβ segments used were predominantly BV8 + (7/16) and BV6 + (4/16). No restriction in the Jβ repertoire was apparent. NK1 T cells are not found in neonates and accumulate after birth ( 5 ). Therefore, we examined cord blood lymphocytes for Vα7/ 19-Jα33 sequences by quantitative PCR and polyclonal sequencing of Vα7-Cα or Vα7-Jα33 amplicons on cDNA obtained from enriched DN/CD8 + or CD4 + fractions. We were unable to find significant amounts of invariant Vα7-Jα33 transcripts in cord blood in the six samples studied. However, significant amounts of this chain could be obtained in young children, albeit in lower amounts than in adults (data not shown). Similarly, Vα19-Jα33–bearing cells were not found in spleen or thymus of mouse neonates (data not shown). They were present in lymph nodes and blood in large amounts and in smaller amounts in the spleen in the five mouse strains tested, as shown in Table III (top), which displays a summary of the studies examining the tissue distribution of Vα19-Jα33–bearing cells. Importantly, although more than half of the human invariant Vα7-Jα33 + cells were CD8αα + cells, which might suggest an extrathymic development pathway ( 43 ), invariant Vα19-Jα33 rearrangements were not detected in nude mice either by quantitative PCR (Table III , top) or polyclonal sequencing (data not shown), indicating that the thymus is required for their development. The restriction element of Vα7-Jα33 + cells could be either a class I or class II MHC molecule, as they do not express CD4 or CD8αβ accessory molecules. To address this issue, we quantified the expression of the Vα7-Jα33 invariant chain in PBLs obtained from MHC-deficient patients. MHC class II– deficient patients harbor low but significant numbers of CD4 + α/β lymphocytes whose Vβ repertoire is grossly normal ( 44 ). CD4 + , CD8α + , and DN enriched fractions were prepared and analyzed by quantitative PCR. Although the amount of Cα was much lower in the DN than in the CD4 + samples , the amounts of Vα7-Jα33 transcripts were comparable in both samples , therefore indicating an enrichment of Vα7-Jα33 transcripts within DN cells and, presumably, selection of invariant Vα7-Jα33–bearing cells in this patient. Accordingly, the predominance of the invariant Vα7-Jα33 sequence within DN cells was confirmed by polyclonal sequencing (data not shown). Similar results were obtained in the two other patients studied. Taken together, these results strongly suggest that the restriction element of the invariant Vα7-Jα33 + cells is not an MHC class II molecule. There is no MHC class I–deficient patient described to date, but we could obtain PBLs from two TAP-deficient siblings ( 45 ). In these patients, α/β + TCR CD8α + cells are reduced in numbers at birth but may expand with age. α/β + TCR CD4 + and CD8α + cells were separated in one of these patients, and the amount of Cα and Vα7-Jα33 transcripts was quantified in the two fractions. Despite lower amounts of Cα in the CD8α + fraction, the amount of Vα7-Jα33 transcripts was much higher in the CD8α + fraction . Enrichment for Vα7-Jα33 transcripts was also demonstrated in a PHA-stimulated CD8α + line derived from the other TAP-deficient patient, when compared with either unsorted PBL-derived cell line from the same patient or to CD8α + cells from healthy donors (data not shown). Actually, the larger amounts of Vα7-Jα33 transcripts in the CD8α + α/β T cells of TAP-deficient patients is in agreement with the higher proportion of CD8αα + cells in these patients (data not shown). This indicates that T cells bearing this invariant α chain are probably not selected by a TAP-dependent MHC class I molecule. Thus, the selecting molecule for the invariant Vα7-Jα33 + cells appeared to be neither an MHC class II nor a classical MHC class I molecule. However, because the defect in MHC class II expression may not be complete and the level of MHC class I expression in TAP-deficient patients is still 1% of normal, no definitive conclusion could be drawn from these studies. Therefore, we turned to the mouse, where carefully controlled studies using well-characterized MHC-deficient mice can be performed. The amount of Vα19-Jα33 transcripts was examined in α/β + DN lymph node cells from several MHC-deficient strains. Because the frequency of Vα19-Jα33 + cells and the percentage of DN cells are lower in mice, the differences between positive and negative samples are smaller in mice than in humans. However, despite higher levels of Cα in the β2m −/− samples, there were fewer Vα19-Jα33 transcripts than in control or I-A b−/− mice . Polyclonal sequencing of Vα19-Jα33 amplicons from β2m −/− samples showed the complete absence of invariant Vα19-Jα33 transcripts. This was consistent with an MHC class I or β2m-dependent class I–like selection of the invariant Vα19-Jα33 + cells. However, the relevant molecule is not a classical MHC class I molecule because the amount of invariant Vα19-Jα33 transcripts was higher in the DN cells from K b−/− D b−/− mice than in controls, though the Cα levels were lower. These results suggest that the MHC class I selecting molecule is not a classical one and that, in the absence of classical class I molecules, the invariant chain–bearing DN cells are less diluted by mainstream cells. In Fig. 8 , C and D, Vα19-Jα33 transcripts were quantified in CD8-, CD1-, and TAP-deficient mice, B6 and β2m −/− mice being used as positive and negative controls, respectively. There was no decrease in the amounts of Vα19-Jα33 transcripts in CD1-deficient mice, indicating that Vα19-Jα33 + cells were not selected by CD1 or a CD1-bound ligand. The curves for TAP −/− samples were actually shifted to the left, indicating an increased frequency of Vα19-Jα33 + cells in TAP −/− mice, in agreement with the result shown in Fig. 5 B where the frequency of Vα19-Jα33 + cells in DN TAP −/− mice was 1/8.5 . The Vα19-Jα33 curve corresponding to the CD8-deficient mice is similar to that of B6, indicating the presence of Vα19-Jα33 invariant chains in CD8 −/− mice. This was confirmed by polyclonal sequencing (data not shown). Altogether, these results, summarized in Table III (bottom), indicate that Vα19-Jα33 + cells do not require CD8 for their selection and that the selecting molecule is a β2m-dependent, TAP-independent molecule distinct from CD1. Invariant Vα7.2-Jα33 TCR α chain and its murine and bovine homologues were found in three different mammalian species and, thus, define a new phylogenetically conserved T cell population using a canonical TCR α repertoire. In humans, DN and CD8αα + cells bearing invariant Vα7-Jα33 chain accumulate after birth and become quite abundant, as they represent ∼0.1–0.2% of all human PBLs. Therefore, it is legitimate to wonder why such cells have not been previously reported. This may be due to the lack of a stringent Vβ repertoire restriction, or to the difficulties in growing clones expressing this invariant chain (our unpublished observations), together with methodological limitations linked to TCR α chain repertoire analysis (e.g., lack of allelic exclusion ). However, this invariant α chain had already been described in DN cells by Porcelli et al. ( 39 ) and, among the 317 random CDR3 sequences reported by Moss and Bell ( 47 ), 4 (1.2%) had a sequence corresponding to the Vα7-Jα33 invariant α chain. In the same study, this canonical sequence was not found in cord blood samples or in CD4 + cells, but represented 3/40 (7.5%) CDR3 sequences derived from CD8α + cells. This age and cell distribution is in agreement with our own measurements (data not shown). In cattle, this CDR3 was found in 1/20 TCR α chains randomly sequenced ( 40 ). In mice, random cloning of TCR α chains revealed one example of this invariant Vα19-Jα33 α chain ( 48 ). It should be stressed that as shown in Fig. 3 , the AJ33 segments are quasi-identical in the three species. The mouse and human Jα loci are entirely homologous, with the same genomic organization and an average similarity of 71% including the Jα coding sequences ( 41 , 42 , 49 ). The AJ33 is the most similar Jα segment between mouse and human sequences with only one amino acid difference; the two other most similar human/ mouse pairs (AJ23 and AJ24) have four and three amino acid differences, respectively ( 41 ). This quasi identity between mouse and human AJ33 segments suggests a strong selective pressure for these segments. In humans and cattle, the TCR α chain displayed some junctional diversity, whereas in mice a genomic sequence without trimming or N additions was found in most cases (Table II ). However, a murine sequence made by trimming and reconstitution of the canonical sequence through N additions was found in two hybridomas (11F1 and 4H1 in Table II ), indicating that the TCR in mice is selected at the protein level. In humans, the invariant TCR α chain was associated with a limited number of Vβ segments (mostly hBV2 and hBV13), and the same TCR with the same nucleotide sequence was present in different cells from two subjects, suggesting oligoclonal expansions. Despite this combinatorial restriction, TCR β chain diversity of invariant Vα7-Jα33 + cells remained extensive, since most Jβ segments and seven Vβs were found associated with CDR3s of variable length. In mice, five different Vβs were observed with a high proportion of mBV6 and mBV8. Importantly, the murine orthologous segments of hBV13 are mBV8 and mBV6 ( 50 – 52 ). This semi-invariant repertoire selected at the protein level is reminiscent of the repertoire observed in murine epithelial DECs and in murine and human NK1 T cells. In DECs, an invariant repertoire generated from genomic sequences is selected at the protein level, as the same epitope recognizable by an mAb can be reconstituted by different Vγ segments when the original GV1 and GV2 segments are inactivated by homologous recombination ( 13 ). For NK1 T cells, selection at the protein level was also apparent ( 14 ), and the transgenic overexpression of the invariant mAV14AJ18 was sufficient to induce a large increase in the number of NK1 T cells ( 53 ). The restriction element of Vα7/19-Jα33 + cells is probably an MHC class Ib molecule distinct from CD1d, though a β2m-derived peptide presented by a “nonclassical” class II molecule cannot be formally excluded. The selecting molecule should be present in both mice and humans. Murine Qa-1 and human HLA-E might be good candidates, given their structural homology and their ability to bind to homologous CD94/NKG2 receptors in both species ( 54 – 56 ). However, when using HLA-E tetramers complexed with HLA-G leader sequence–derived peptide ( 56 ), we were unable to find any difference in expression levels of the invariant Vα7-Jα33 α chain between FACS ® -sorted HLA-E tetramer–positive and –negative (DN plus CD8α + ) α/β + TCR PBL fractions (data not shown). Moreover, the invariant Vα19-Jα33 chain was not expressed by three TAP-independent anti–Qa-1 clones provided by J. Forman (University of Texas, Dallas, TX ; data not shown). Nonetheless, no definitive conclusions as to the HLA-E/ Qa-1 specificity of the invariant TCR can be drawn from these negative results because the invariant TCR may recognize HLA-E complexed with another peptide. Another candidate class I molecule that is widely expressed in both mice and humans is the recently described MR1 molecule ( 58 , 59 ). Identification of the selecting element is underway by testing reactivity of mouse hybridomas against different cell types and MHC class I/Ib transfectants. The expression of CD8αα on Vα7-Jα33 + cells could argue for an extrathymic development pathway ( 43 ). However, these cells were not found in nude mice (Table III , top), in human intraepithelial lymphocytes (IELs) (Cerf-Bensussan, N., E. Treiner, and O. Lantz, unpublished results), or in murine IELs or lamina propria lymphocytes (Guy-Grand, D., F. Tilloy, and O. Lantz, unpublished results), and their frequency was not particularly high in mouse bone marrow (Table III , top). Thus, Vα7/19-Jα33 + cells seem to be mainly thymus dependent. In this respect, the fact that we have been unable to find large numbers of these cells in human or mouse thymus (data not shown) does not preclude an intrathymic development. Indeed, if the antigen is not present in sufficient amounts in the thymus, these cells may not accumulate locally and may be diluted out by mainstream α/β T cells in the periphery. The absence of significant numbers of Vα19-J33 + cells in mouse CD8α + cells is in agreement with the extremely low numbers (<0.4%) of CD8αα + cells in murine blood or lymph nodes (data not shown). The CD8αα expression on some invariant Vα7-Jα33 + cells and its complete absence on Vα19-Jα33 + counterparts is reminiscent of the phenotype of NK1 T cells, which may express CD8αα + in humans ( 28 ) but never in mice. The reasons for such phenotypic differences between mice/cattle and humans as well as the role of CD8αα expression in human PBLs are not clear. There have been contradictory reports concerning the role of homo- versus heterodimeric CD8 ( 60 – 63 ). In addition, CD8αα expression in human PBLs might reflect an activated status rather than a particular differentiation pathway ( 64 ). Concerning the development pathway of the Vα7/19-Jα33 cells, in the three species there seems to be no expression of the invariant α chain in the CD8αβ + T cells, strong enrichment for this sequence within DN cells (as well as within human CD8αα + cells), and persistence of the invariant sequence in the CD4 + subset. Indeed, in humans, the frequency of invariant α chain–bearing cells was ∼1/6,000 in the CD4 + fraction. Moreover, polyclonal sequencing of Vα7-Jα33 amplicons from CD4 + cells revealed the presence of canonical CDR3 at a low, though readable, frequency, whereas the canonical CDR3 was undetectable in CD8αβ + samples . In mice and cattle, there was also a small but reproducible shift of the CD4 + amplification curves to the left compared with the CD8α + curves, suggesting some expression of the invariant α chain in murine or bovine CD4 + cells. Accordingly, a readable canonical sequence in Vα19-Jα33 amplicons was detected in the CD4 + but not in the CD8α + fraction in both species (data not shown). The absence of any readable sequence after V-J amplification of CD8α/β + cells suggests that the CD8αβ + Vα7/19-Jα33– bearing cells are deleted by negative selection, as are NK1 T cells in CD8 transgenic mice ( 14 ). These results are compatible with the hypothesis that Vα7/19-Jα33 cells follow a normal T cell development pathway but that, in contrast with NK1 T cells where the numbers of CD4 and DN NK1 T cells are similar, CD4 + T cells bearing the Vα7/19-Jα33 α chain are diluted out by mainstream lymphocytes. A memory phenotype associated with an oligoclonal repertoire may be related to recognition of either an endogenous ligand or a ubiquitous pathogen. In favor of an endogenous ligand is the finding that invariant Vα7-Jα33 + cells existed in an 18-mo-old child who had not been vaccinated with Calmette-Guérin bacillus (BCG) and in several subjects who had negative EBV and CMV serologies (data not shown). On the other hand, the abundance of Vα7/19-Jα33–bearing cells in humans and cattle contrasting with a lower number of these cells in mice could be related to the clean environment in which laboratory mice are housed. The two hypotheses are not mutually exclusive, as cells selected by an endogenous ligand could be ready to react against a highly prevalent pathogen. Both NK1 T cells and Vα7/19-Jα33–bearing cells display semi-invariant repertoires (i.e., one monomorphic α chain and a biased Vβ chain repertoire), suggesting that their TCRs may have similar characteristics, probably a high affinity for a selecting ligand by the invariant α chains, as the transgenic overexpression of AV14AJ18 is sufficient to greatly increase the number of NK1 T cells ( 53 ). Together with the similarities in their restricting elements (a TAP-independent, β2m-dependent nonclassical MHC class I–like molecule), the absence of accessory molecule (CD4 or CD8) involvement suggests that they may recognize a high-density ligand that might therefore be a saccharide or a glycolipid. Are there other invariant TCR α chains defining other T cell subpopulations? We did not find any after measuring the amounts of several other α chains which had been found either in DN T cells ( 39 ) or in a “regulatory” subpopulation (65–67; Tilloy, F., and O. Lantz, unpublished observations). However, the definite resolution of this issue awaits a systematic study of the expression of all AV-AJ combinations, and other populations may exist in other organs in the same way that γ/δ subpopulations are distributed. Concerning the functions of Vα7/19-Jα33 + lymphocytes, future analysis of the mAV19AJ33 transgenic mice we have made will certainly help address this point. The high frequency of Vα7/19-Jα33 + T cells and their extensive phylogenetic conservation in mammals both argue for an important physiological function. Because Vα7/19-Jα33 + and NK1 T cells are selected by distinct ligands, they may complement each other and act synergistically either in defense mechanisms against broadly distributed pathogens or in immune/nonimmune homeostatic processes. | Other | biomedical | en | 0.999998 |
10377187 | Human and mouse rTNF-α and human rGM-CSF were obtained from Boehringer Mannheim . Dulbecco's PBS (Mg 2+ - and Ca 2+ -free), RPMI 1640, and FCS were purchased from BioWhittaker, Inc. Ficoll-Hypaque was from Organon Teknika Corp., and HBSS was purchased from GIBCO BRL . BSA, dextran, antibiotics, l -glutamine, cytochrome C, superoxide dismutase, and zymosan were obtained from Sigma Chemical Co. The assessment of human IL-1β in supernatants was performed by using an immunometric assay with acetylcholine esterase (Cayman Chemical). Murine IL-1β was assessed using an ELISA from Endogen . ELISAs for IL-4 and IL-10 were from Amersham Corp. ; MIP-2 and IL-13 ELISAs were from R & D Systems, Inc. LXA 4 and ATL metabolically stable analogues were prepared and characterized, including nuclear magnetic resonance spectroscopy, as in reference 14 . Concentrations of each LX analogue were determined using an extinction coefficient of 50,000/M/cm just before each experiment and used as methyl esters. Where indicated, statistical analyses were performed using nonpaired t test (two-tailed), and significance was considered to be attained when P < 0.05. Venous blood from healthy donors was collected under sterile conditions using acid citrate dextrose as an anticoagulant, and PMN were isolated as in reference 15 . PMN were suspended in cold (4°C) Hank's medium (supplemented with 1.6 mM Ca 2+ , 0.1% FCS, 2 mM l -glutamine, 1% penicillin, and 2% streptomycin, pH 7.4). Cell preparations were >98% PMN, as determined by Giemsa-Wright staining. Cell viability was >98% for freshly isolated PMN and ≥92% for PMN incubated for 20 h, as determined by trypan blue exclusion using light microscopy. To examine superoxide production, PMN (10 6 /ml) were placed at 37°C (3 min) and then exposed to vehicle (0.1% ethanol) or synthetic LXA 4 , 15 R/S -methyl LXA 4 , or 16-phenoxy-LXA 4 for 5 min at 37°C. Before adding TNF-α (50 ng/ml), PMN were incubated with cytochrome C (0.7 mg/ml) for 10 min at 37°C. Superoxide dismutase–dependent reduction of cytochrome C was terminated by rapidly placing tubes in an ice water bath. The extent of cytochrome C reduction in each supernatant was determined at 550 nm in reference and compared with control values obtained when superoxide dismutase was added before a stimulus or vehicle control. Cytochrome C reduction was quantitated using the extinction coefficient of 21.1/mmol/liter. Total RNA extraction and Northern blot analyses were performed as in reference 7 . pSM320 vector containing cDNA for IL-1β was purchased from American Type Culture Collection. 6–8-wk-old male BALB/c mice were obtained from Taconic Farms, Inc. Air pouches were raised on the dorsum by subcutaneous injection of 3 ml of sterile air on days 0 and 3. All experiments were conducted on day 6 ( 16 ). Individual air pouches (one per mouse) were injected with vehicle alone (0.1% ethanol), TNF-α, 15 R/S -methyl-LXA 4 , or TNF-α plus 15 R/S -methyl-LXA 4 , and each was suspended in 1 ml endotoxin-free PBS immediately before injection into pouch cavities. At given intervals, the mice were killed, and individual air pouches were lavaged three times with sterile PBS (1 ml). The exudates were centrifuged at 2,000 rpm (5 min), and the supernatants were removed. Cell pellets were suspended in PBS (200 μl) for enumeration and assessed for viability. 50 μl of each cell suspension was mixed with 150 μl 30% BSA and then centrifuged onto microscope slides at 500 rpm for 5 min using a cytospin centrifuge, air dried, and stained with Giemsa-Wright. TNF-α, although a modest agonist of O 2 − generation by human PMN, is a physiologically relevant stimulus for the generation of ROS by nonadherent human PMN ( 17 ) that can play critical roles in local tissue injury during both inflammation and reperfusion ( 17 – 19 ). In Fig. 1 , we evaluated the impact of LXA 4 - and ATL-related bioactive stable analogues on TNF-α–stimulated superoxide anion production. TNF-α gave a concentration-dependent increase in superoxide anion dependence with nonadherent PMN; therefore, TNF-α (50 ng/ml) was used to examine the analogues. Native LXA 4 and the analogues (15 R/S -methyl-LXA 4 and 16 phenoxy-LXA 4 ) inhibited TNF-α–stimulated superoxide anion generation in a concentration-dependent fashion. Their rank order of potency at 10 nM was 15 R/S -methyl-LXA 4 (81.3 ± 14.1% inhibition) ≈ 16-phenoxy-LXA 4 (93.7 ± 3.2%) > LXA 4 (34.3 ± 2.3%). 15 R/S -methyl-LXA 4 covers both LXA 4 and ATL in structure, and 16-phenoxy-LXA 4 is an LXA 4 analogue . Each analogue competes at the LXA 4 R ( 7 ). LXA 4 , 15 R/S -methyl-LXA 4 , and 16 phenoxy-LXA 4 , at concentrations up to 1 μM added to cells alone, did not stimulate generation of ROS (data not shown). 15 R/S -methyl-LXA 4 and 16-phenoxy-LXA 4 were approximately three times more potent than native LXA 4 and proved to be powerful inhibitors of TNF-α–stimulated superoxide generation by PMN. However, neither LXA 4 nor its analogues inhibit PMA (100 nM)- or fMLP (100 nM)-stimulated O 2 − production ( n = 3; data not shown). Inhibition of ROS by LXA 4 and its analogues is of interest in a context of ischemia/reperfusion, where ROS are held to be primary mediators of tissue injury ( 15 ). PMN express and release interleukin-1β, which is a potent pro-inflammatory cytokine ( 20 ). Therefore, we next investigated the actions of native LXA 4 and its analogues on TNF-α–induced IL-1β release. Incubation of PMN with physiologically relevant concentrations of TNF-α, GM-CSF, or phagocytic particles (zymosan) resulted in a concentration-dependent increase in the levels of IL-1β present in supernatants. Approximate EC 50 for each agonist were: TNF-α, 10 ng/ml; GM-CSF, 10 U/ml; and zymosan, 100 μg/ml. Native LXA 4 specifically inhibited TNF-α–induced IL-1β release , whereas similar amounts of IL-1β were released in the presence or absence of LXA 4 when PMN were exposed to either GM-CSF or zymosan. The viability of PMN exposed to ATL or TNF-α was examined using trypan blue exclusion. PMN exposed to these agents did not dramatically increase their staining , suggesting that the ATL did not reduce PMN viability during the time courses of these experiments. PMN were exposed to increasing concentrations of 15 R/S -methyl-LXA 4 , 16-phenoxy-LXA 4 , or native LXA 4 in the presence of TNF-α (10 ng/ml) or vehicle alone. At a concentration of 100 nM, 15 R/S -methyl-LXA 4 inhibited ∼60% of IL-1β release, and 16-phenoxy-LXA 4 at equimolar levels gave ∼40% inhibition (values comparable to those obtained with native LXA 4 ; data not shown). Time course and concentration dependence were carried out with 15 R/S -methyl LXA 4 . At 10 nM, 15 R/S -methyl-LXA 4 gave clear, statistically significant inhibition, which was evident within 6 h and more prominent after 24 h . Inhibition of IL-1β by these LX analogues was, at least in part, the result of a downregulation in gene expression, because the IL-1β messenger RNA levels in cells treated with TNF-α (10 ng/ml) plus 15 R/S -methyl-LXA 4 (100 nM) were decreased by ∼60% when compared with cells treated with TNF-α alone . Therefore, as IL-1β and TNF-α are two cytokines that are considered important in inflammation, the inhibition of IL-1β observed suggested that 15 R/S -methyl-LXA 4 might exert a potent in vivo anticytokine action (vide infra). To investigate whether LXA 4 R was involved in the regulation of TNF-α–stimulated IL-1β release, the rabbit polyclonal antibodies against a portion of the third extracellular domain (ASWGGTPEERLK) of LXA 4 R prepared earlier ( 21 ) were used. PMN were incubated with ∼50 μg/ml of either preimmune protein A–purified IgG or IgG directed against LXA 4 R for 1 h at 4°C before exposure to TNF-α (10 ng/ml) and 15 R/S -methyl-LXA 4 (100 nM). Anti-LXA 4 R antibodies prevented IL-1β release by TNF-α, suggesting that the third extracellular loop plays a crucial role in LXA 4 R activation . 15 R/S -methyl-LXA 4 inhibited ∼50% of IL-1β release. When added together, anti-LXA 4 R antibodies and 15 R/S -methyl-LXA 4 in the presence of TNF-α did not further inhibit IL-1β appearance, and neither anti-LXA 4 R antibodies nor 15 R/S -LXA 4 alone stimulated significant amounts of IL-1β to appear in supernatants. The results of these experiments are twofold: first, they indicated that the inhibitory action of 15 R/S -methyl-LXA 4 is transduced via LXA 4 R and second, that the anti-LXA 4 R antibodies alone activate LXA 4 R and lead to inhibition of IL-1β release. As TNF-α evokes leukocyte infiltration in a chemokine-dependent fashion in the murine six-day air pouch ( 16 , 22 ), we evaluated the impact of 15 R/S -methyl-LXA 4 in this model to determine whether LXA 4 or ATL also intersects the cytokine–chemokine axis in vivo. 15 R/S -methyl-LXA 4 is the most subtle modification to native LXA 4 and ATL structure, with addition of a methyl at carbon 15. Murine TNF-α (10 ng/ml) caused a transient infiltration of leukocytes to the air pouch in a time-dependent fashion, with maximal accumulation at 4 h. 15 R/S -methyl-LXA 4 at 25 nmol inhibited the TNF-α–stimulated recruitment of leukocytes to the air pouch by 62% . Inhibition was evident at 1 h and maximal between 2 and 4 h. At these intervals, a >60% reduction in leukocyte infiltration was noted that remained significantly reduced at 8 h . Injection of pouches either with vehicle or the analogue alone did not cause a significant leukocyte infiltration. Also, inflammatory exudates were collected 4 h after injection with vehicle alone, TNF-α, 15 R/S -methyl-LXA 4 alone, or TNF-α plus 15 R/S -methyl-LXA 4 , and cell types were enumerated. In the six-day pouches given TNF-α, PMN constituted the major cell type present within the exudates at 4 h and ranged from 80 to 85% of total cell number. Administration of both 15 R/S -methyl-LXA 4 and TNF-α into the six-day air pouch cavity inhibited migration of PMN and eosinophils/basophils as well as mononuclear cells (Table I ). Of interest is the finding that administration of 15 R/S - methyl-LXA 4 alone evoked a small but statistically significant increase in mononuclear cell influx (Table I ), a result that is consistent with earlier in vitro observations ( 23 ) in which specific stimulation of monocyte and inhibition of PMN chemotaxis have been observed. Because MIP-2 is the major chemokine involved in recruiting PMN to the air pouch after injection of TNF-α ( 16 ), we determined the action of 15 R/S -methyl-LXA 4 in this TNF-α–induced chemokine– cytokine axis. MIP-2 and IL-1β are important proinflammatory cytokines, and IL-4, IL-10, and IL-13 possess immunomodulatory properties ( 24 , 25 ). Exudates from selected time intervals were collected, and cell-free supernatants were assessed for the presence of these murine cytokines. TNF-α induced maximal detectable amounts of MIP-2 and IL-1β within 90 min (data not shown). 15 R/S -methyl-LXA 4 (25 nmol) inhibited TNF-α–stimulated MIP-2 and IL-1β release by 48 and 30%, respectively . 15 R/S -methyl-LXA 4 alone in the air pouch did not stimulate MIP-2 or IL-1β release. In sharp contrast, 15 R/S -methyl-LXA 4 stimulated the appearance of IL-4 within the exudates. This stimulation of IL-4 was observed both in the absence as well as the presence of TNF-α. Neither IL-10 nor IL-13 was detected within the pouch exudates. These results demonstrate that administration of 15 R/S -methyl-LXA 4 modified the cytokine–chemokine axis in TNF-α–initiated acute inflammation, and, interestingly, this reorientation of the cytokine–chemokine axis paralleled the reduction in leukocyte infiltration. Several different strategies have been explored in an attempt to attenuate nondesirable action of TNF-α in inflammatory diseases and ischemia/reperfusion injury, including treatment of patients suffering from RA with rTNF-αR linked to human Ig as a fusion protein ( 26 ). Different steroidal and nonsteroidal drugs ( 27 ) to alleviate the pain and the severity of inflammatory responses are extensively used. However, certain clinical settings, such as reperfusion injury, are still not well controlled, and new therapeutic agents are needed. Our results indicate that LXA 4 and ATL, as evidenced by the actions of their metabolically stable analogues (16-phenoxy-LXA 4 and 15 R/S -methyl-LXA 4 ), are potent cytokine-regulating lipid mediators that can also impact the course of inflammation initiated by TNF-α and IL-1β. These two cytokines are considered to be key components in orchestrating the rapid inflammatory-like events in ischemia/reperfusion (within minutes to hours) and are major cytokines in RA and many other chronic diseases. Interestingly, in an exudate and skin wound model, 15 R/S -methyl-LXA 4 not only inhibited the TNF-α–elicited appearance of IL-1β and MIP-2 but also concomitantly stimulated IL-4 . This represents the first observation that lipoxins induce upregulation of a potential “antiinflammatory” cytokine such as IL-4. Hence, it is of particular interest that IL-4 inhibits PMN influx in acute antibody-mediated inflammation ( 28 ) and inhibits H 2 O 2 production by IFN-γ–treated human monocytes ( 29 ). IL-4 is also an active antitumor agent and, most recently, was shown to be a potent inhibitor of angiogenesis ( 25 ). It is thus likely that the increase in IL-4 levels stimulated by metabolically stable LX analogues may in part mediate some of the in vivo impact of LXA 4 and aspirin-triggered 15-epi-LXA 4 , a finding that provides a new understanding of the relationship between antiinflammatory cytokines and lipid mediators. In conclusion, LXA 4 and ATL appear to be involved in controlling both acute as well as chronic inflammatory responses. The results presented here support the notion that aspirin may exert its beneficial action in part via the biosynthesis of endogenous ATL that can in turn act directly on PMN and/or the appearance of IL-4. Thus, LX-ATL can protect host tissues via multilevel regulation of proinflammatory signals. | Study | biomedical | en | 0.999998 |
10377188 | The following human cell lines were used: MCF-7 (a mammary epithelium line, provided by Dr. Arnold Greenberg of the Manitoba Cancer Foundation); MRC-5 (fibroblast; American Type Culture Collection [ATCC]); 2C4 (fibroblast; provided by Dr. George Stark of the Cleveland Clinic Foundation); and HeLa (cervical epithelium; ATCC). Cells were infected with Chlamydia trachomatis LGV2 strain at a multiplicity of infection (MOI) 1 of 5 or as indicated and for 24 h or as indicated in individual experiments ( 24 ). Cells with or without infection were stimulated with human IFN-γ ( PharMingen ) at 200 U/ml or as indicated for another 10 h (for reverse transcriptase [RT]-PCR analysis) or 20–24 h (for flow cytometry and Western blot analysis). Cell samples were stained with mouse anti– HLA-DRα (L243; ATCC), mouse anti–human intercellular adhesion molecule (ICAM)-1 (HA58; PharMingen ), or normal mouse IgG ( Zymed Labs., Inc. ). Primary antibody binding was detected using goat anti–mouse IgG conjugated with FITC (Caltag Labs.) and analyzed with a FACSCalibur™ equipped with CellQuest software ( Becton Dickinson ). Dead cells were excluded by propidium iodine staining. Western blot assay was carried out as we previously described ( 24 ). Rabbit antibodies were used to detect IFN-γR (SC-700; Santa Cruz Biotechnology ), tyrosine-phosphorylated signal transducers and activators of transcription (STAT)1α , IFN regulatory factor (IRF)-1 (SC-497), upstream stimulatory factor (USF)-1 (SC-229) and USF-2 (SC-862; all from Santa Cruz Biotechnology ). Mouse antibodies were used to detect Janus tyrosine kinase (JAK)-1 and STAT1α (SC-464; Santa Cruz Biotechnology ), HLA-DRα (DA6.147; provided by Dr. Peter Cresswell, Yale University; reference 27 ), and a chlamydial major outer membrane protein (MOMP; clone MC22, our unpublished data). Primary antibody binding was detected with horseradish peroxidase–conjugated goat anti–mouse IgG or –rabbit IgG, depending on the source of the primary antibodies, and visualized using an ECL kit ( Amersham Corp. ). Cell samples were collected for RNA extraction using the Rneasy Mini Kit from QIAGEN, Inc. 2 μg of total RNA was used for each cDNA synthesis with random primers and the 1 st Strand cDNA synthesis kit from Boehringer Mannheim . Aliquots of the cDNA samples were used as a template for amplifying specific gene fragments by PCR reactions ( 28 , 29 ). The primers used for amplification of DRα ( 18 ), DMα ( 29 ), invariant chain p41 (IP41) ( 29 ), and IRF-1 ( 18 ) were previously described. The other primers used in this study were: for class II transactivator (CIITA) amplification, 5′-GACACGGTGGCGCTGTGGGAGTC-3′ (forward) and 5′-GGCAGCCGTGAACTTGTTGTACTGG-3′ (reverse); for USF-1 amplification, 5′-TGGCACTGGTCAATTCTTTGTG (forward) and 5′-GTTGCTGTCATTCTTGATGAC (reverse); for STAT1 amplification, 5′-TAGAGTTGCTGAATGTCACTG-3′ (forward) and 5′-GGAGTGAAGCTCTTCAGTAAC-3′ (reverse); for indoleamine 2,3-dioxygenase (IDO) gene amplification, 5′-ATGCATCACCATGGCATA-3′ (forward) and 5′-GCTTCCCGCAGGCCAGCATCA-3′ (reverse); and for β-actin amplification, 5′-GTGGGGCGCCCCAGGCACCA-3′ (forward) and 5′-CTCCTTAATGTCACGCACGATTTC-3′ (reverse). β-actin mRNA detection was used as an internal control for the amount of cDNA synthesized. To ensure the specificity of the mRNA detection, all primers were designed to cover at least two exons, and parallel samples without RT were run as negative controls. The amplified DNA products were run on an agarose gel and visualized with ethidium bromide staining. To investigate whether chlamydia possesses the ability to evade the IFN-γ–induced immune recognition mechanism, we evaluated IFN-γ–inducible MHC class II antigen expression in cells with or without chlamydial infection. IFN-γ significantly upregulated HLA-DR surface expression on uninfected cells, whereas the chlamydia- infected cells displayed a minimal level of DR, regardless of IFN-γ exposure . However, chlamydial infection did not affect the IFN-γ–induced ICAM-1 surface expression . These observations suggest that chlamydia selectively inhibits IFN-γ–inducible DR expression rather than preventing all IFN-γ dependent signaling or generally suppressing surface protein expression. Furthermore, the total cellular protein level of IFN-γ–induced HLA-DRα was also significantly diminished in chlamydia-infected cells as compared with uninfected cells , suggesting that the suppression of surface expression of HLA-DR was not due to an alteration in intracellular trafficking. The chlamydial inhibition of IFN-γ–inducible HLA-DRα was reproduced in many other human cell lines, including HeLa, MRC-5, and 2C4 , demonstrating that the inhibitory effect is not a cell line–specific phenomenon. To determine whether the chlamydial inhibition of HLA-DR expression occurs at the transcription or translation level, MHC class II mRNA levels were evaluated by semiquantitative RT-PCR. IFN-γ dramatically induced the expression of DRα, DMα, and Ip41 mRNA in the uninfected but not the infected cells , suggesting that chlamydial inhibition of MHC class II occurred at the transcription level. Because the genes encoding the MHC class II presentation–related molecules DRα, DMα, and Ip41 share similar promoter structures and CIITA is a master regulator for the expression of these genes ( 30 ), we hypothesize that chlamydia may inhibit CIITA function or CIITA gene expression. Although CIITA is constitutively expressed in professional APCs, such as dendritic cells and B cells, IFN-γ stimulation is often required for the expression of CIITA in nonprofessional APCs, such as epithelial cells ( 31 ). CIITA mRNA expression was induced by IFN-γ in uninfected MCF-7 cells . However, CIITA mRNA expression was significantly lower in chlamydia- infected and IFN-γ–treated cells , in accord with chlamydial inhibition of MHC class II gene expression. To investigate how CIITA gene expression was inhibited, we measured mRNA levels for three transcription factors, USF-1, STAT1, and IRF-1, all of which are required for IFN-γ–inducible transcription of the CIITA gene ( 32 ). Both USF-1 and STAT1 mRNAs were constitutively expressed, and IRF-1 mRNA was induced by IFN-γ in MCF-7 cells regardless of chlamydial infection , suggesting that chlamydial infection did not affect transcription of the genes for these nuclear factors. Because the three transcription factors are considered to be sufficient and necessary for the IFN-γ induction of CIITA ( 32 ), we evaluated the protein levels of these transcription factors as well as upstream molecules in the IFN-γ signaling pathway. We found that IFN-γR, JAK-1, and STAT1 protein levels were not altered by chlamydial infection . Chlamydia did not affect IFN-γ–induced STAT1 tyrosine phosphorylation . Furthermore, IFN-γ induced both IRF-1 and ICAM-1 expression in chlamydia-infected cells . As STAT1 is required for the expression of both IRF-1 and ICAM-1 genes ( 32 , 33 ), we conclude that STAT1 is transcriptionally functional in chlamydia-infected cells. The IFN-γ–induced IRF-1 in chlamydia-infected cells may also be transcriptionally functional, as we found that IFN-γ induced expression of IDO gene in chlamydia-infected cells (data not shown), and it is known that IRF-1 is required for IFN-γ induction of IDO ( 34 ). Therefore, the failure of the IFN-γ–inducible CIITA expression in chlamydia-infected cells is likely due to the deficiency in USF-1. We found that the USF-1 protein was not detectable in chlamydia-infected cells , despite normal USF-1 mRNA expression . We next determined the cause of the USF-1 protein loss. Because USF-1 mRNA is expressed in chlamydia-infected cells with or without IFN-γ stimulation, the lack of USF-1 protein may be due to either the inhibition of translation of USF-1 mRNA or the accelerated degradation of USF-1 protein. We tested the protein degradation hypothesis by using protease inhibitors. We found that the proteasome inhibitor lactacystin prevented USF-1 protein degradation in chlamydia-infected cells . Furthermore, the lactacystin treatment also preserved the IFN-γ–inducible HLA-DR expression in chlamydia-infected cells . These observations not only demonstrate that a proteasome-like activity is responsible for the loss of USF-1 protein in chlamydia-infected cells but also suggest that USF-1 degradation may be responsible for the chlamydial suppression of MHC class II expression. To examine whether the USF-1 protein degradation in chlamydia-infected cells is dependent on chlamydial growth and protein synthesis, we first evaluated the relationship between the chlamydial infection dose and USF-1 degradation. As MOI (ratio of number of organisms versus number of host cells) increased, more chlamydial protein was produced and less USF-1 protein was detected . This effect was selective, as USF-2 was not degraded, regardless of the infection dose . The time course relationship between chlamydial growth and USF-1 degradation was also analyzed . Although the STAT1 protein level was not affected by chlamydial infection at any time points examined, significant USF-1 degradation was detected 17 h after chlamydial infection, when chlamydial protein synthesis approached its maximum . The role of chlamydial and host protein synthesis in USF-1 degradation was examined using antibiotics specifically inhibiting either prokaryotic or eukaryotic protein synthesis. We found that both rifampin (inhibiting prokaryotic transcription) and chloramphenicol (inhibiting prokaryotic translation) blocked chlamydial protein synthesis . More importantly, these antibiotics also prevented USF-1 degradation and preserved HLA-DRα expression in chlamydia-infected cells . However, treatment with penicillin failed to prevent USF-1 degradation and preserve HLA-DRα expression . Penicillin only blocks chlamydial particle assembly without inhibiting chlamydial protein synthesis ( 35 ). Penicillin did not alter the constitutively expressed USF-1 protein level and IFN-γ–inducible HLA-DRα expression in uninfected cells . Together, these observations demonstrate that chlamydial protein synthesis, but not particle assembly, is necessary for chlamydia-induced degradation of USF-1 protein and suppression of HLA-DRα expression. Finally, cycloheximide treatment did not affect the chlamydia- induced degradation of USF-1 . Because cycloheximide did not affect chlamydial protein synthesis but completely inhibited new protein synthesis by the host cell, for example, production of IFN-γ–induced HLA-DRα , we conclude that newly synthesized host proteins are not required for chlamydia-induced degradation of USF-1. We have demonstrated that the obligate intracellular bacterial pathogen chlamydia can inhibit IFN-γ–inducible MHC class II expression. This inhibitory effect has also been found with other intracellular pathogens, including leishmania ( 13 ), listeria ( 14 ), cowdria ( 15 ), and cytomegalovirus ( 16 – 18 ). CD4 + T cell–mediated immunity plays an important role in host defense against various intracellular infections ( 36 – 38 ). Recognition of the infected cells by CD4 + T cells often requires IFN-γ induction of MHC class II expression, because many pathogen-targeted cells, such as epithelial cells, are generally MHC class II–negative. Suppression of IFN-γ–inducible MHC class II expression may represent an efficient immune evasion strategy used by intracellular pathogens to escape host defenses. Thus, chlamydial inhibition of IFN-γ–inducible MHC class II may contribute to the persistent infection caused by chlamydia in humans ( 22 ). It has been demonstrated that cytomegalovirus can prevent IFN-γ–inducible class II expression in infected cells by both IFN-β–mediated inhibition ( 17 , 39 ) and disruption of IFN-γ intracellular signaling pathways ( 16 , 18 ). However, mechanisms of IFN-γ–inducible MHC class II inhibition by many other intracellular pathogens are still not clear ( 13 – 15 ). It was recently proposed that chlamydia may suppress IFN-γ–inducible MHC class II expression by stimulating host cells to release IFN-β ( 40 ). Here we show that the intracellular bacterial pathogen chlamydia has evolved a more specific mechanism for disrupting IFN-γ signaling pathways and inhibiting MHC class II expression. Chlamydia degrades USF-1, a downstream transcription factor required for IFN-γ–inducible MHC class II but not IRF-1 and ICAM-1 expression . USF-1 degradation may represent an efficient means of interrupting IFN-γ– inducible MHC class II expression by chlamydia. Although the constitutively and ubiquitously expressed USF-1 is a member of the basic helix-loop-helix family consisting of multiple transcription factors, including Myc and USF-2, only USF-1 is both necessary and sufficient for binding to the E box within the CIITA promoter IV and cooperating with STAT1 and IRF-1 for promoting transcription of CIITA ( 32 ). Therefore, the constitutively and ubiquitously expressed USF-1 may serve as a convenient and efficient target for chlamydia-induced degradation. The correlation between the degradation of USF-1 and the suppression of IFN-γ–inducible MHC class II further confirms that USF-1 plays a critical role in IFN-γ induction of MHC class II ( 32 ). Besides its involvement in MHC class II expression, USF-1 also participates in many other cellular activities, including promoting the transcription of fatty acid synthase in response to insulin regulation ( 41 ), interfering with Ras transformation ( 42 ), and transactivating the promoter of the p53 tumor suppressor gene ( 43 ). Depletion of USF-1 may cause inhibition of host cell lipid biosynthesis and promotion of host cell survival, both of which are likely beneficial to the intracellular chlamydia organisms. Proteolysis is an important aspect of normal cellular physiology ( 44 – 46 ). Many viruses can take advantage of host proteolysis for the purposes of evading host defenses ( 2 , 47 ). For example, human cytomegalovirus infection can induce degradation of JAK-1, a critical upstream kinase required for IFN-γ JAK/STAT signaling pathways, to suppress IFN-γ–inducible MHC class II on the infected cells ( 18 ). Furthermore, the cytomegalovirus-induced degradation can be inhibited by the proteasome inhibitor Z-L 3 VS ( 18 , 48 ), suggesting that cytomegalovirus may be able to manipulate host proteasome activity. Because USF-1 degradation by chlamydia is inhibitable by lactacystin and lactacystin is a potent proteasome inhibitor ( 48 , 49 ), we propose that chlamydia may also produce a factor(s) for manipulating host proteasomes. Efforts to identify the chlamydial factor(s) are underway. | Study | biomedical | en | 0.999995 |
10377189 | The cell lines were grown in IMDM medium supplemented with penicillin, streptomycin, and 10% FCS. The cell lines HDLM2 and LCL-HO were purchased from the German Collection of Microorganisms and Cell Cultures. The cell line LCL-GK was a gift from Dr. M. Dosch (Hospital for Sick Children, Toronto, Ontario, Canada), and the EBV-negative non-Hodgkin lymphoma (NHL)-derived cell lines Ly4, Ly7 and Ly13.2 were provided by Dr. H. Messner (Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario, Canada). Polyadenylated [poly(A) + ] mRNA was prepared from total RNA on Oligotex-dT resin (Qiagen). Each mRNA sample was converted into fluorescent-labeled cDNA probes using the microarray (GEM) probe labeling kit (Synteni). The microarray used in these experiments contained 950 human genes involved in inflammation and neoplasia (Synteni). Two microarrays were independently probed with 200 ng of a 1:1 mixture of fluorescein-labeled cDNA from HD-derived L428 cells and control LCL-GK cells on Microarray 1 (M1) or from HD-derived KMH2 cells and LCL-GK cells on microarray 2 (M2), respectively. Known concentrations of mRNA synthesized from inter–open reading frame regions from Saccharomyces cerevisiae were used as quantitation standards. The hybridization was performed as previously described ( 7 ). Total RNA was isolated using Trizol reagent ( GIBCO BRL ). 15 μg total RNA was separated on a 1% formaldehyde agarose gel. Samples were run in 0.02 M MOPS (3-[N-morpholino]propanesulfonic acid), pH 7.0, 8 mM sodium acetate, 1 mM EDTA. The gel was blotted overnight in 20× SSC on a Hybond N+ nylon membrane ( Amersham Pharmacia Biotech ) and cross-linked using UV irradiation. Membranes were hybridized at 65°C to [α- 32 P]dATP-labeled (Mutiprime DNA Labeling System, Amersham Pharmacia Biotech ) cDNA fragments specific for the human Notch2, NF-IL3, urokinase, IL-13, and human β-actin genes. Membranes were washed first in 0.1% SDS/2× SSC (30 min, 65°C) and then in 0.1% SDS/0.2× SSC (30 min, 65°C). Supernatants of cell cultures (10 5 cells/ml) were recovered 48 h after medium exchange and assayed for IL-5, IL-13, and GM-CSF production by specific ELISA using Quantikine-kits (R&D Systems). Freshly biopsied lymph nodes were fixed in 10% formalin for 24 h before overnight processing and paraffin embedding. For in situ hybridization, paraffin sections were mounted and fixed according to standard protocols. The IL-13 probe used was a 388-bp human cDNA probe synthesized using reverse transcriptase PCR and IL-13–specific primers as follows: 5′ GTTGACCACGGTCATTGCTCTCACT and 3′ TTCAGTTGAACCGTCCCTCGCGAA. The cDNA was cloned into the pCRII vector (TA Cloning Kit; Invitrogen). Sense and antisense probes were synthesized from the linearized vector with SP6 or T7 polymerase, labeled with [ 33 P]UTP, and processed as previously described ( 10 ). The sections were counterstained with toluidine blue using a standard protocol. Immunohistochemistry was performed using mAbs against CD30 (Dako) and CD15 ( Becton Dickinson ). Formalin-fixed paraffin sections were digested with pepsin and incubated (1 h) with the primary antibody. The slides were washed in PBS and incubated with biotinylated rabbit anti– mouse IgG (20 min) and with streptavidin–biotin complex labeled with horseradish peroxidase (20 min) after a second washing step (Ultra Streptavidin Detection System; Signet). The enzyme reaction was developed with AEC (3-amino-9-ethyl carbazole) and the slides were counterstained with hematoxylin. HD-derived L428, KMH2, and HDLM2 cells and LCL control cells (2 × 10 4 /well) were cultured in 96-well flat-bottomed plates for 24, 48, or 72 h in the presence of anti–IL-13, anti–IL-5, or isotype control antibodies ( PharMingen ) at 5, 10, 20, 30, 50, 100, or 150 μg/ml. Cells were treated either with 0.5, 1, 5, 10, 50, 100, and 200 ng/ml IL-13 (R&D Systems) or with these doses of IL-13 combined with 20 μg/ml anti–IL-13. [ 3 H]thymidine (1 μCi/ well) was added to each well for an 8-h incubation. The cells were harvested on filters and the incorporation of [ 3 H]thymidine into cellular DNA was measured as previously described ( 11 ). Of 950 genes displayed on the microchip, the genes showing a greater than threefold difference in expression in both HD-derived cell lines were IL-13; IL-5; ornithine decarboxylase (ODC); ICAM-3; urokinase plasminogen activator (UPA); IgE Fc receptor II; insulin-like growth factor (IGF) II; NF-IL3/E4BP4; Notch2; GM-CSF; interferon regulatory factor (IRF)-1, IRF-5, and IRF-6; nitric oxide synthase (NOS) 3; and the TCR δ chain . IL-13 expression in HD-derived L428 and KMH2 cells was increased compared with that in control LCL-GK cells by 26.7- and 17.1-fold, respectively, and IL-5 expression was increased by 14.2- and 18.5-fold . To evaluate the significance of these results, we used Northern blots and ELISA to compare the expression of IL-13, IL-5, GM-CSF, NF-IL3, Notch2, and urokinase in the HD-derived EBV-negative cell lines L428, KMH2, and HDLM2 ( 2 ) with that in the lymphoblastoid EBV- infected B cell lines LCL-GK and LCL-HO, and in three EBV-negative NHL-derived cell lines with either B cell (Ly4, Ly7) or T cell (Ly13.2) phenotype. Notch2, urokinase, and NF-IL3 were upregulated in LCL or NHL cell lines as well as in the HD-derived cell lines, but the overexpression of IL-13, IL-5, and GM-CSF was restricted to the HD-derived cell lines . Although IL-5 and GM-CSF expression and secretion could be demonstrated only in L428 and KMH2 cells, mRNA expression and secretion of IL-13 could be detected in all three HD-derived cell lines. HDLM2 cells secreted a moderate amount of IL-13 (27 pg/ml), whereas L428 and KMH2 cells demonstrated intense IL-13 (4,800 and 6,100 pg/ml, respectively) secretion . To confirm that IL-13 was expressed by the H/RS tumor cells, in situ hybridization using sense and antisense RNA probes for IL-13 was performed on lymph node biopsies from four untreated patients newly diagnosed with classical nodular sclerosis HD. The diagnosis was based on the nodular sclerosis architecture of the lymph nodes, the presence of H/RS cells in the appropriate context, and the immunohistological detection of CD30 and CD15 expression by cells with H/RS morphology. In all four patients, significant numbers of cells expressing IL-13 were detected . Virtually all IL-13–expressing cells displayed morphological features of H/RS cells such as large size, prominent nucleoli, and multinuclear and/or lacunar cell morphology. Also, some IL-13–expressing cells were mitotic . To test whether cells with a positive signal for expression of IL-13 express the HD markers CD30 and CD15, serial sections from two patients were cut and stained by immunohistochemistry for CD30 or CD15, or used for in situ hybridization with an IL-13 antisense probe. On these serial sections, it was possible to correlate a high proportion of IL-13 mRNA–containing cells with the presence of CD30 and CD15 expression by the same tumor cell . A benign reactive lymph node hybridized in parallel with the IL-13 probe as well as a lymph node from a HD patient hybridized with an IL-13 sense probe did not yield any signal (data not shown), illustrating the specificity of IL-13 expression by H/RS cells in lymph nodes from HD patients. To investigate the effects of IL-13 and IL-5 on the proliferation of H/RS cells, L428, KMH2, and HDLM2 cells were incubated with medium alone, medium containing neutralizing antibodies to IL-13 or IL-5, or isotype controls. Proliferation was measured by determining [ 3 H]thymidine incorporation at 24, 48, or 72 h after treatment. The viability of cultured cell lines after treatment with anti–IL-13 neutralizing antibodies was examined by 7-aminoactinomycin D (7-AAD) staining; no significant differences between HD-derived cells and controls were detected (data not shown). Neutralizing antibodies against IL-13 and IL-5 had no effect on the proliferation of control LCL-HO cells . However, after 72 h of treatment with 20 μg/ml anti–IL-13 neutralizing antibody, the proliferation of HDLM2 cells (which secrete moderate levels of IL-13) was suppressed to 27% of that of untreated control cells . Treatment of L428 and KMH2 cells with ≤150 μg/ml anti–IL-13 antibody had no effect on the proliferation of the cell lines (data not shown), perhaps because of their vigorous secretion of IL-13 . No significant changes in proliferation were observed in control groups of HDLM2 cells treated with an anti–IL-5 neutralizing antibody or isotype control antibodies . A combination of anti–IL-13 and anti–IL-5 antibodies did not inhibit proliferation to any greater extent than anti–IL-13 alone (data not shown). Treatment with increasing concentrations of anti–IL-13 antibody demonstrated that the effect on the proliferation of HD-derived cells was dose dependent . Furthermore, the antiproliferative effect of anti–IL-13 on HDLM2 cells could be overcome by the addition of exogenous IL-13 . Treatment of HDLM2 cells with exogenous IL-13 alone did not result in an increase in proliferation over that of untreated cells , suggesting that the cells produce saturating levels of IL-13 sufficient to support maximal proliferation. The systemic symptoms of HD and the unique reactive cellular background exhibited in HD biopsies suggest that cytokines are involved in HD pathogenesis. The elevated expression of various cytokines has been reported in HD biopsies and several HD-derived cell lines ( 1 , 2 ). Overexpression of IL-5 in H/RS cells has been demonstrated previously by in situ hybridization, but only in HD patients exhibiting eosinophilia ( 12 , 13 ). To date, no cytokine has been consistently reported as being overexpressed in HD-derived cell lines or in primary H/RS cells. Of 950 genes tested, we found that only IL-13 was consistently and specifically overexpressed in H/RS tumor cells. IL-13 is a T cell–derived cytokine with immunomodulatory and antiinflammatory properties ( 14 ). The biological effects of IL-13 on B cells, macrophages, and monocytes are very similar to those of IL-4, probably because the IL-4 and IL-13 receptors share a common α chain. In B cells, IL-13 promotes proliferation, differentiation, and Ig heavy chain class switching to IgE and IgG4 ( 15 ). Proliferation results from a signaling pathway in which the engagement of the IL-13 receptor activates JAK1, which in turn activates STAT6 ( 16 ). Our results suggest that a similar mechanism may operate in the B cell–like H/RS cells, since neutralization of IL-13 dramatically inhibited the proliferation of HD-derived HDLM2 cells . Indeed, in the microarray experiments, mRNA for the IL-13 receptor was found to be expressed in both of the HD-derived cell lines tested (data not shown). The secretion of IL-13 by H/RS cells, the expression of mRNA for the IL-13 receptor, and the specific effect of the neutralizing anti–IL-13 antibody suggest that an autocrine mechanism may control the growth of H/RS tumor cells. Evidence for a role for IL-13 in the etiology of HD is indirect but consistent. IgE is elevated in HD tissues and serum samples from HD patients ( 13 , 17 ), and IL-13 is known to promote Ig class switching to IgE. IL-13–deficient mice exhibit lower basal levels of serum IgE ( 18 ). Furthermore, studies of IL-4–deficient, IL-13–transgenic mice have demonstrated that IL-13 can promote class switching to IgE independently of IL-4 ( 19 ), emphasizing that IL-4 and IL-13 have distinct roles in regulating B cell functions. Our results have clearly demonstrated that HD-derived cell lines and H/RS tumor cells express elevated levels of IL-13. The elevation of IgE in H/RS cells and in the serum of HD patients therefore could be explained if IL-13 secreted by H/RS cells affects both the H/RS cells themselves and the bystander B cells. Other aspects of the HD phenotype may also be attributable to the effects of IL-13. A recent study of IL-13–deficient mice has shown that cultures of Th2 cells from these animals produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with the results from the wild-type animals, suggesting an important role for IL-13 as a regulator of Th2 cell commitment ( 18 ). If IL-13 is also important for promoting the differentiation of Th2 cells in humans, it could explain why H/RS cells (which secrete IL-13) are surrounded by Th2 cells in HD biopsies. In addition, because fibroblasts express the IL-13 receptor and can be activated by IL-13 ( 20 ), the secretion of IL-13 by H/RS cells may underlie the pathogenesis of the fibrosis observed in nodular sclerosis HD. Our microarray hybridization also showed that the expression of NF-IL3 and Notch2 was upregulated by more than threefold in HD-derived cell lines . Northern blot analysis revealed NF-IL3 and Notch2 expression also in NHL-derived cell lines . The basic leucine zipper transcription factor NF-IL3 acts downstream of IL-3 and has been shown to prevent apoptosis after forced expression in an IL-3–dependent pro-B cell line ( 21 ). The relevance of NF-IL3 in HD is unknown. Notch2 is a transmembrane receptor that has been shown to be involved in cell fate decisions ( 22 ). The human Notch2 gene has been mapped to chromosome 1 at position 1p13-p11, which is a region of translocations associated with neoplasia ( 23 ). Chromosome 1 has also been linked to the HD phenotype, since structural rearrangements of chromosome 1 are frequently observed in HD ( 24 ). Interestingly, the gene for CD30, which was first identified in the HD-derived cell line L428 ( 25 ) and is considered a marker for the disease, is also located on chromosome 1 at 1p36 ( 26 ). CD30 is highly expressed in lymphoblastoid cells and H/RS cells and has been shown to promote a Th2 phenotype ( 27 ). The possible roles of NF-IL3 and Notch2 and their relationship to IL-13 and the CD30 antigen in the pathogenesis of HD lymphoma require further investigation. In conclusion, our data combined with recent reports on the function of IL-13 indicate that IL-13 may be a critical cytokine in the etiology of HD. We have shown both that IL-13 is secreted by HD tumor cells and that IL-13 specifically promotes the proliferation of these cells. Our results are consistent with a role for IL-13 in the pathogenesis of the fibrosis, increased IgE production, and bias towards Th2 cells, all of which are typical of classical HD. | Study | biomedical | en | 0.999997 |
10377190 | Balb/c mice infected with P . berghei ANKA strain were prepared by a peritoneal injection of the infected blood samples maintained at −70°C. Infected mice were used within one blood passage for ookinete culture. The culture conditions were as described ( 9 ). Parasites were collected from the culture at different time intervals, purified by erythrocyte lysis in 0.83% NH 4 Cl, and used for further analysis. Infected blood was collected from anesthetized rats by a cardiac puncture and applied to a CF 11 column (Whatman Biosystems) to remove white blood cells. This blood was cultured by a candle-jar method for 24 h, and erythrocytes containing merozoites were purified by density gradient ( 10 ). Parasites were purified by erythrocyte lysis, and genomic DNA was extracted. The genomic DNA was partially digested by restriction enzyme Sau3AI, ligated to BamHI-digested arms of lambda phage vector, λFix II (Stratagene, Inc.), by a partial fill-in method, and packed in vitro. Degenerated primers were designed based on amino acid sequences of CTRP (GDDCFC and APKVTILF), and related sequences were amplified from the genomic DNA of P . berghei by PCR under low-stringency conditions (annealing conditions, 47°C for 1 min). The amplified fragment (380 bp) was subcloned to a plasmid vector, pBluescript II (Stratagene, Inc.), and the DNA sequence was determined. The deduced amino acid sequence of this fragment has 68% identity with the second integrin-like domain of CTRP and ∼30% identity with plasmodial TRAPs ( 5 , 8 ). It was labeled with [ 32 P] dCTP and used for screening of a genomic library of P . berghei . Screening of the library yielded four positive phage clones. The insert DNA of a positive phage was digested into two fragments with restriction enzymes, SalI and EcoRI, separately subcloned into the plasmid vector, pBluescript II, and sequenced. Genomic DNA of P . berghei was digested with restriction enzymes, separated on an agarose gel, fragmented in 0.25 M HCl, and transferred to nylon membrane. The blot was hybridized with a [ 32 P]dCTP-labeled HindIII/MunI- digested DNA fragment (0.8 kb) of PbCTRP, and visualized by the BAS 2000 system (Fuji Film and Photo, Inc.). Poly A(+) RNA was extracted using a microprep mRNA purification kit ( Amersham Pharmacia Biotech ) from blood-stage parasites and ookinetes cultured for 16 h after fertilization from the same infected blood. Poly A(+) RNA (0.1 μg per lane) was separated on an agarose gel and transferred to nylon membrane. The blot was hybridized with a [ 32 P]dCTP-labeled HindIII/EcoRI-digested DNA fragment of PbCTRP (3.2 kb) and rehybridized with a control probe for Pbs 21, the transcript of which is known to be present in both gametocytes and ookinetes ( 11 ). The DNA fragment encoding the NH 2 -terminal region of PbCTRP (amino acids 1–270) was subcloned into the baculovirus transfer vector, pAcYM1. The construction of recombinant virus and production of recombinant protein were performed as described ( 12 ). Recombinant protein was purified from the culture medium by gel-filtration HPLC and anion exchange HPLC as described ( 12 ). The DNA fragment encoding the putative cytoplasmic domain was subcloned into Escherichia coli expression vector, pGEX 2T ( Amersham Pharmacia Biotech ), and produced as a glutathione S -transferase (GST) fusion protein. This recombinant protein was affinity purified on glutathione-Sepharose ( Amersham Pharmacia Biotech ), emulsified in TiterMax Gold ( CytRx Corp. ), and used for immunizing rabbits. Antibodies against GST were removed from the rabbit antisera by incubation with GST-conjugated Sepharose. Specific antibodies were affinity purified on GST fusion protein–conjugated Sepharose. The specificity of purified antibodies was checked by Western blot analysis. Parasites were cultured in ookinete culture medium without methionine and cysteine. Pro-mix [ 35 S] ( Amersham Pharmacia Biotech ) was added to medium (50 μCi/ml in the final concentration) 5 h after exflagellation and cultured for an additional 17 h. Parasites were collected by centrifugation (400 g , 6 min), washed in PBS, and homogenized in extraction buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin). After centrifugation (15,000 g , 30 min), supernatant was recovered. Labeled PbCTRP was bound to antibody-conjugated protein G–Sepharose ( Amersham Pharmacia Biotech ) and eluted by sample buffer for SDS-PAGE. Samples were separated by SDS-PAGE in a linear gradient (5–20%) and visualized by the BAS 2000 system. Parasites were purified by erythrocyte lysis and homogenized in sample buffer. Parasite proteins were separated by SDS-PAGE in a linear gradient (5–20%) and transferred to nitrocellulose membrane. Blotted membrane was blocked in PBS containing 5% skim milk, and incubated with primary antibody against PbCTRP (10 μg/ml in PBS containing 0.05% Tween 20) and then with alkaline phosphatase–conjugated anti– rabbit IgG ( GIBCO BRL ). After culture for ookinete development, parasites were purified by erythrocyte lysis and fixed in acetone for 2 min on a glass slide. The samples were washed three times in PBS, preincubated in PBS containing 1% BSA for 10 min, and incubated with primary antibody against PbCTRP (6 μg/ml in PBS containing 1% BSA) for 1 h at room temperature. After washing three times in PBS, samples were incubated with FITC-conjugated goat anti–rabbit IgG ( GIBCO BRL ) for 1 h at room temperature, washed three times in PBS again, and examined under a fluorescence microscope. For control, nonimmune rabbit serum (diluted 1:100) was used as the primary antibody instead of anti-PbCTRP antibody. Cloned genomic DNA contains a single open reading frame of 5715 bp, which encodes a 1905–amino acid protein. Southern blot analysis showed it was a single-copy gene . The hydropathy plot and the TMpred prediction ( 13 , 14 ) showed that this protein has a transmembrane-like structure composed of a large putative extracellular region, a transmembrane-like domain, and a small putative cytoplasmic domain . The putative extracellular region of PbCTRP is composed of six integrin I region–like domains and seven TSP-like domains. The NH 2 -terminal 21 amino acid residues of this protein probably encode a signal peptide. This is supported by the observation that the recombinant protein (amino acids 1–270) produced using the baculovirus–insect cell system is cleaved at this site (data not shown). The molecular mass of the mature protein is estimated to be 212 kD. We named this protein PbCTRP, because, in the overall structure and amino acid sequence, it showed striking similarity to CTRP of P. falciparum (PfCTRP). For example, amino acid sequence identities between PbCTRP and PfCTRP in the first integrin I region–like domain, the first TSP-like domain, and the putative cytoplasmic domain are 62.7, 64.9, and 80.5%, respectively. Conservation of amino acid sequences was also found among the putative cytoplasmic and transmembrane domains of PbCTRP, TRAPs, and PfCTRP . To investigate expression of PbCTRP, we performed Northern blot analysis of RNA preparations obtained from intraerythrocytic-stage parasites and ookinetes. Transcripts of PbCTRP were absent from blood-stage parasites but were detected in ookinetes . The size of the transcript was 7.5 kb. We prepared polyclonal antibodies against recombinant protein . Western blot analysis using this antibody showed that PbCTRP was abundantly present 10 h after exflagellation when zygotes had just begun transformation to ookinetes . After this, PbCTRP gradually increased during ookinete maturation. The apparent molecular mass of a detected protein was 210 kD, which is in good agreement with that estimated from the amino acid sequence. In addition, we metabolically labeled ookinete proteins by adding [ 35 S]methionine/cysteine to the culture medium, immunoprecipitated labeled PbCTRP, and analyzed it on SDS-PAGE . PbCTRP was identified as a distinct band of 210 kD among labeled proteins, which showed that it was actively produced during ookinete development. By SDS-PAGE analysis under reducing and nonreducing conditions, no apparent mobility change of the purified protein was observed (data not shown). When cultured for 10 h after exflagellation, most zygotes showed an oval or budding shape, indicating they had just started transformation into the invasive form. In this stage, a strong signal was already detected over the whole zygote . When cultured for an additional 12 h, most parasites had transformed to ookinetes and a strong signal was detected in the cytoplasm mainly anterior to the nucleus . This staining pattern was similar to that reported for TRAP in the sporozoite ( 5 , 15 ). In the retort form, the incompletely mature ookinete with a remnant of the spherical zygote, the signal was observed only in the posterior (zygote) side, but not in the anterior (ookinete) side (data not shown). In this study, we report the structure and expression profile of PbCTRP. PbCTRP is structurally homologous to CTRP of the human malarial parasite, P. falciparum , and is expressed in the mosquito invasive-stage parasite, the ookinete. The expression profile of PfCTRP has not yet been clearly established ( 5 ). However, striking amino acid identities between PbCTRP and PfCTRP indicate they may play identical roles in the ookinete stage. The putative extracellular region of PbCTRP is composed of adhesive protein–related domains, that is, six integrin I region–like domains and seven TSP-like domains. The first four integrin I region–like domains contain a metal ion–dependent adhesion site (MIDAS) motif. In integrins, this motif is known to mediate the divalent cation binding of the I domain and play a role in ligand binding of I domain–containing integrins ( 16 ). It is also found in a malaria sporozoite protein, TRAP ( 5 , 8 ). TSP-like domains contain a similar amino acid sequence to the TSP 1 motif in different identities, especially around the sequence, WSXWXXCSXTCGXXXRXR. TSP-like domains are also found in mammalian adhesive proteins like properdin ( 17 ) and F-spondin ( 18 ) and malarial proteins, CS proteins, and TRAPs ( 5 , 8 , 19 ). These structural relationships to adhesive proteins indicate its possible role in attachment to the mosquito midgut epithelium. In addition, the structural similarity of PbCTRP to other apicomplexan proteins indicates its involvement in ookinete motility and in the invasion of the mosquito midgut. As shown in Fig. 6 , PbCTRP bears a resemblance to TRAPs, the micronemal protein 2 (MIC 2) of Toxoplasma gondii , and the micronemal protein ETP 100 of Eimeria tenella ( 5 – 8 ). These proteins have single putative transmembrane structures and cytoplasmic domains. The extracellular regions are composed of tandemly aligned integrin I region– like domains and TSP-like domains. These proteins are specifically expressed in the invasive-stage parasites and are thought to play an essential role in invasive motility called gliding motility ( 1 ). It has been reported that the TRAP gene–disrupted malaria parasite lost its gliding motility and cell invasion abilities. PbCTRP is also expressed in the motile-stage parasite, the ookinete, indicating its possible role in motility through the mosquito midgut epithelium. The amino acid conservation observed in the transmembrane and cytoplasmic domains of PbCTRP and P. berghei TRAP might indicate that these domains carry important functions related to the movement mechanism common to these proteins . Despite intensive studies, the invasive mechanisms of the mosquito midgut by malarial parasites remain unknown. PbCTRP is the first identified malaria protein that may be involved in ookinete invasion into the mosquito midgut. We think PbCTRP may be a clue for elucidating the invasive mechanisms of the mosquito midgut by malarial parasites, and we here establish the molecular basis of malarial parasite–mosquito cell interactions. | Study | biomedical | en | 0.999996 |
10377191 | SCID-hu Thy/Liv mice were obtained by coimplanting small pieces of human second trimester fetal liver and thymus under the kidney capsule of male homozygous CB-17 scid/scid (SCID) mice ( 20 ). Thy/Liv implants were infected 4–7 mo after implantation, as described ( 23 ). In brief, mice were anesthetized, the left kidney was surgically exposed, and the implant was inoculated with 25– 50 μl of viral stock with a titer of 10 5 (strain GS) or 10 4 (strain PL-1) 50% tissue culture infectious doses (TCID 50 ) per 50 μl. UV-inactivated viral stock and culture medium were used for mock inoculations. The abdominal wall incision was sutured, and the skin was closed with staples. All procedures and practices were approved by the University of California, San Francisco Committee on Human Research or by the University of California, San Francisco Committee on Animal Research. HHV-6A GS , a primary subgroup A isolate, was obtained from cultures of purified human cord blood CD4 + T cells infected with cell-free viral stock at a multiplicity of infection of 0.5. At day 6 after infection, the supernatants were collected, centrifuged at 2,500 g , and stored at −80°C. HHV-6B PL-1 , a primary subgroup B isolate, was obtained from in vitro–activated peripheral blood cells of a healthy adult blood donor and propagated for a single passage in activated cord blood CD4 + T cells. No HHV-7 contamination was detected by nested PCR assay, and the stocks were also negative for Mycoplasma . Virus titration was performed by infecting triplicate cultures of phytohemagglutinin-activated human peripheral blood mononuclear cells with serial 10-fold dilutions of the viral stock. SCID-hu mice were killed by CO 2 inhalation, and implants were removed at different times after inoculation. Implants were processed for histopathology and electron microscopy as detailed below. For cytofluorimetric analysis, cell sorting, and quantitative PCR, single-cell suspensions were obtained by grinding the implants in a nylon bag. The cells were counted in a Coulter counter and divided into aliquots for the different assays. Thy/Liv implants were fixed in 10% buffered formalin, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin (H&E). For immunohistochemistry, unstained sections were pretreated with trypsin and pronase. The sections were then stained with a polyclonal antibody specific for CD3 (Dako) or an mAb specific for CD68 (Dako), using a standard peroxidase-labeled streptavidin-biotin (LSAB-peroxidase) method (LSAB2; Dako), followed by visualization with diaminobenzidine (DAB) and CuSO 4 enhancement ( 24 ). Before the second phase of staining, the samples were denatured in 2 N HCl for 10 min. The sections were then stained with 9A5D12, an mAb specific for the p41 early protein of HHV-6 ( 25 ). This antibody was revealed by a standard LSAB-alkaline phosphatase method (LSAB2; Dako) followed by visualization with Vector Red (Vector Laboratories). Negative controls included purified nonimmune mouse or rabbit serum (Dako), followed by LSAB-peroxidase staining. The sections were then denatured in HCl, and LSAB-alkaline phosphatase staining was carried out after incubation with either mouse serum (no stain) or 9A5D12 (red staining only). Small pieces of Thy/Liv implants were fixed in 2.5% glutaraldehyde, and samples were postfixed in osmium tetroxide, dehydrated in 2,2-dimethoxypropane, and embedded in Poly/Bed 812 resin (Polysciences, Inc.). Thin sections (600–900 Å) were cut with a diamond knife, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined on a JEOL 100 SX electron microscope. Single-cell suspensions were stained with an mAb cocktail containing CD4-FITC ( Becton Dickinson ), CD8-PE ( Becton Dickinson ), and CD3-tricolor (CD3-TC) (Caltag). Cells were washed, resuspended in PBS containing 1% paraformaldehyde, and analyzed on a FACScan ® ( Becton Dickinson ) for CD4, CD8, and CD3 expression after gating on a live-cell lymphoid population identified by forward- and side-scatter characteristics. For cell sorting, unfixed cells were stained as for FACS ® analysis. A FACS Vantage™ ( Becton Dickinson ) was used to purify four populations of thymocytes: CD3 + CD4 + CD8 − (SP4), CD3 + CD4 − CD8 + (SP8), CD3 + CD4 + CD8 + (DP), and CD3 − CD4 + CD8 − (ITTP). Sorted cells were frozen as dry pellets and stored at −80°C for DNA extraction. DNA was extracted from frozen pellets by proteinase K digestion and purified by phenol-chloroform extraction by standard techniques. Viral load was quantitated with the TaqMan ® fluorogenic detection system on an ABI Prism 7700 Sequence Detector ® ( Perkin-Elmer Applied Biosystems ). PCR was performed with primers 5′-CAAAGCCAAATTATCCAGAGCG-3′ and 5′-CGCTAGGTTGAGGATGATCGA-3′ and a probe located in the highly conserved open reading frame U67 of HHV-6 ( 27 ): 5′-CACCAGACGTCACACCCGAAGGAAT-3′. PCR amplification consisted of denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 58°C for 60 s (Locatelli, G., F. Santoro, A. Gobbi, F. Veglia, P. Lusso, and M. Malnati, manuscript in preparation). Nonparametric statistical analyses were performed using the Mann-Whitney U test (StatView 5.0; Abacus Concepts). 58 SCID-hu Thy/Liv mice from 6 different cohorts, each made with tissues from a single fetal donor, were inoculated with HHV-6 by direct intrathymic injection of 10 5 TCID 50 of strain GS or mock infected with UV-inactivated viral stock or with cell culture medium. Mice were killed 4, 7, 11, 14, and 27 d after inoculation, and the implants were removed. Viral replication was monitored by quantitative PCR on total thymocyte suspensions with the TaqMan ® real time detection system. HHV-6 DNA was detected as early as 4 d after inoculation in each of the three implants examined . The viral load peaked on day 14 with a mean value of ∼6,000 copies/10 3 cells. In the implants harvested 27 d after inoculation, a drastic reduction in the number of genomic copies, with a mean of ∼400 copies/ 10 3 cells, was observed . No HHV-6 DNA was detected in the mock-infected control implants. The productive nature of HHV-6A GS infection in Thy/Liv implants was demonstrated by transmission electron microscopy showing viral particles at different stages of maturation . In particular, naked viral nucleocapsids were observed in the nucleus, tegument-coated particles in the cytoplasm (not shown), and mature, enveloped virions in cytoplasmic vacuoles. This pattern is consistent with the documented ultrastructural development of HHV-6 in cells infected in vitro ( 28 ). HHV-6A GS infection of Thy/Liv implants was associated with a marked, progressive depletion of thymocytes . The number of thymocytes harvested from the HHV-6A GS –infected implants was significantly reduced at day 14 ( P < 0.01) compared with mock-infected implants and was reduced by >98% by day 27 ( P < 0.001). A representative HHV-6A GS –infected Thy/Liv implant is shown in Fig. 3 A. Compared with the mock-infected implant shown in Fig. 3 B, the HHV-6A GS –infected thymus had a marked expansion of the medullary zone, apparent destruction or compression of the cortical zone, and destruction of the corticomedullary junction . The medullary expansion appeared to be driven by endothelial proliferation or by vascular ectasia and fibrocyte proliferation. The endothelial cells lining medullary vessels appeared to be activated, in a fashion resembling vascular endothelium in the setting of inflammation. The expanded medullary zone contained atypical cells with prominent nuclear inclusions . Immunohistochemical staining with an mAb specific for the p41 early protein of HHV-6 demonstrated that most of these atypical cells were infected . Double-staining analysis showed that virtually all the infected cells were CD3 + , albeit with varying degrees of intensity . By contrast, double-label staining with CD68 did not indicate a tropism of HHV-6A GS for macrophages in this model . To evaluate the effects of viral replication on different thymocyte subpopulations, we performed quantitative flow cytometry on total thymocyte suspensions obtained from six different cohorts of SCID-hu Thy/Liv mice in three independent experiments. HHV-6A GS –infected implants were harvested 4, 7, 11, 14, and 27 d after inoculation and examined for expression of CD3, CD4, and CD8. 4 and 7 d after inoculation, the percentages of the different thymocyte subsets were unchanged compared with mock-infected implants (Table I ). The earliest detectable phenotypic change was a reduced percentage of ITTPs at day 11 ( P < 0.05), a time at which the percentages of the other thymocyte subpopulations were not yet significantly changed (Table I ). By day 14, a reduction in the percentage of live thymocytes, identified by forward- and side-scatter profiles, was observed (Table I ). At this time point, ITTPs remained significantly depleted ( P < 0.01), and there was also a reduction in the percentage of DP thymocytes ( P < 0.001) and an increase in the percentage of SP4 ( P < 0.05) and SP8 ( P < 0.01) thymocytes (Table I ). By day 27, most of the thymocytes were dead or dying, and it was no longer possible to analyze the different subpopulations (Table I ). To study the cellular tropism of HHV-6A GS in Thy/Liv implants, the distribution of viral DNA was quantitated in the different subpopulations of thymocytes as a function of time after inoculation. Total thymocytes were harvested at 4, 7, 11, and 14 d after inoculation and sorted into DP, SP4, SP8, and ITTP subpopulations. The viral load in each subpopulation was measured by quantitative PCR (Table II ). At day 4 after inoculation, viral DNA was detectable in ITTPs but was less abundant or undetectable in the other subpopulations. By day 7, HHV-6 DNA was detectable in all the thymocyte subsets, but was significantly more abundant in ITTPs ( P < 0.05) and remained so at 11 and 14 d after inoculation. No HHV-6 DNA was detected in any of the thymocyte subpopulations from Thy/Liv implants infected with UV-inactivated viral stock (data not shown). To study the effects of HHV-6 subgroup B on Thy/Liv implants, six SCID-hu Thy/Liv mice were inoculated with 10 4 TCID 50 of strain PL-1 (a primary isolate passaged in vitro only twice), or mock infected with cell culture medium. Mice were killed 10 d after inoculation, and the implants were removed. Quantitative PCR analysis revealed efficient replication of HHV-6B PL-1 (mean viral load of ∼2,200 copies/10 3 cells) similar to that found in HHV-6A GS –infected implants (Table III ). The effects of HHV-6B PL-1 replication on thymocyte subpopulations were assessed by flow cytometry. As observed for strain GS at a similar time point (day 11; Table I ), the main effect of HHV-6B PL-1 on thymocyte subpopulations was a reduction in the percentage of ITTPs (Table III ). At this relatively early time point, the percentage of live thymocytes was also decreased (Table III ). This study provides the first evidence that HHV-6 can replicate in the human thymus in vivo, leading to a progressive destruction of the organ. The severity of the lesions caused by HHV-6 in Thy/Liv implants supports the hypothesis that this virus may be immunosuppressive, as previously suggested by its tropism for CD4 + T cells and by the severe cytopathic effects it exerts on infected cells in vitro. Interestingly, the histological lesions we observed in HHV-6–infected Thy/Liv implants are similar to those recently described in the thymus of an HIV-1–negative infant with immunosuppression associated with widespread HHV-6 infection ( 5 ), suggesting that SCID-hu Thy/ Liv mice may reproduce the pathological mechanisms of HHV-6 infection in humans. However, despite the high prevalence of HHV-6 infection in the human population, only rare cases of putative HHV-6–related immunosuppression have been reported to date. It remains to be determined whether thymic infection by HHV-6 is an uncommon occurrence or, alternatively, whether its pathological consequences in vivo may be attenuated by host immune responses. The experiments reported here were focused mainly on HHV-6A GS , but limited studies with a subgroup B virus (PL-1) indicate that viruses of both subgroups can replicate in the Thy/Liv organ and induce cytopathicity. Clinical and epidemiological studies have shown an association of both subgroup A and B viruses with immunodeficiency ( 2 ), although subgroup B viruses have also been frequently isolated from the general population of nonimmunocompromised subjects. Coinfection by A and B strains was also documented in the infant with HHV-6–associated immunosuppression mentioned above ( 5 ). Further work will be required, both in humans and in SCID-hu Thy/Liv mice, to determine whether there are any intrinsic differences between the two HHV-6 subgroups in their propensity to induce immunosuppression in vivo. The immunohistochemical detection of the CD3 antigen on the majority of the infected cells suggests that mature thymocytes can be infected by HHV-6 in vivo. However, the early and persistent depletion of ITTPs indicates that HHV-6 has a particular tropism for these immature cells. Indeed, quantitative PCR on sorted thymocyte subpopulations showed a markedly higher HHV-6 DNA load in ITTPs than in the other subpopulations. ITTPs are a relatively rare but rapidly dividing subpopulation that represents an early step of thymocyte maturation and gives rise to DP thymocytes and their mature SP progeny ( 21 ). Destruction of ITTPs may result in the generalized suppression of thymopoiesis and ultimately in the depletion of the thymus. The thymus is the primary site of T lymphocyte development in utero and in early life. Although its activity diminishes progressively with age, this organ may still be functional in adults and, during conditions of severe T cell depletion (including HIV-1 infection), it may play an important role in maintaining and reconstituting the peripheral immune system ( 29 – 32 ). Accordingly, abundant thymic tissue has been detected by chest computed tomography in HIV-1–seropositive adults, suggesting that thymic function may be enhanced, by a compensatory mechanism, in some HIV-1–infected patients ( 32 ). However, persistent thymic function in an adult could be quickly abolished by thymic infection with HIV or other T cell–tropic viruses such as HHV-6. Because of its tropism for CD4 + T lymphocytes and its positive interactions in vitro with HIV-1, HHV-6 has been suggested as a possible cofactor in AIDS ( 6 ). This hypothesis is supported by clinical evidence of active and widespread HHV-6 infection in symptomatic AIDS patients ( 7 – 9 ). HHV-6 infects human thymocytes in vitro ( 3 ) and has been detected in the thymus in vivo ( 5 , 33 ). Thus, HHV-6 infection of the thymus may play a role in the immunodeficiency associated with HIV-1 infection. However, it is technically difficult to study the thymus in humans, and no reports are currently available on the presence of thymic HHV-6 infection in patients with HIV disease. The SCID-hu Thy/Liv mouse model, carrying a human graft that is morphologically and functionally equivalent to the human thymus, provides a unique system to study the pathogenesis of HHV-6 and HIV-1 infections in vivo ( 10 – 14 ). Interestingly, some chemokine receptor CXCR4-using strains of HIV-1, like NL4-3, suppress thymopoiesis in SCID-hu Thy/Liv mice by direct infection and destruction of ITTPs ( 11 ). The tropism of HHV-6 and HIV-1 for the same thymocyte subset raises the possibility of synergistic interactions in the thymic environment. Future studies of coinfection with HHV-6 and HIV-1 in SCID-hu Thy/Liv mice may provide important insights into the role of HHV-6 in HIV-1 disease. | Study | biomedical | en | 0.999998 |
10377192 | BALB/c and sv/ev129 mice were bred at the Sir William Dunn School of Pathology, and males and females were used at 8–10 wk of age. Primary Abs used in this study are described in Table I . MR polyclonal Abs raised against MR purified from the J774e cell line and mAbs F4/80, FA.11, and 3D6 were prepared in-house. CR-Fc, a recombinant protein consisting of the CR of mouse MR fused to the Fc region of human IgG1, was also prepared in our laboratory ( 30 ). The ERTR-9 mAb was a gift of Dr. C.D. Dijkstra (Free University, Amsterdam, The Netherlands). Other Abs were purchased as shown. N418 was biotinylated in-house for direct detection. The secondary Abs, biotinylated goat anti–rabbit IgG and biotinylated rabbit anti–rat IgG, were purchased from Sigma Chemical Co. Biotinylated mouse anti–human IgG (Fab′) 2 was purchased from Jackson ImmunoResearch Labs. Organs were collected and immersed in OCT compound (BDH Chemicals-Merck) and frozen in dry ice–cooled isopentane. Frozen sections were cut at 5 μm, air-dried for 1 h, and stored at −20°C. Before staining, slides were thawed at room temperature for 30 min, then hydrated in PBS for 5 min at room temperature followed by fixation for 10 min in 2% paraformaldehyde in Hepes-buffered isotonic saline at 4°C. The hydration step was found to be essential for binding of anti-MR to tissues, and avoids the requirement for protease treatment of sections described by Takahashi and co-workers ( 42 ). Fixed sections were washed in 0.1% (vol/vol) Triton X-100 in PBS, and endogenous peroxidase activity was blocked with 10 mM glucose, 1 mM NaN 3 , 0.4 U/ml glucose oxidase ( Sigma Chemical Co. ) in phosphate buffer for 15 min at 37°C. Blocking steps used an avidin-biotin kit (Vector Laboratories) and a 30-min incubation with 5% normal serum of the species in which the secondary Ab was raised. 5% serum was used as the diluent for primary and secondary Abs, with which the sections were incubated for 60 and 30 min, respectively. Sections were then incubated with avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories). Peroxidase activity was finally detected with 0.5 mg/ml 3,3′-diaminobenzidine tetrahydrochloride (Polysciences, Inc.) and 0.024% H 2 O 2 in 10 mM imidazole in PBS, pH 7.4. Sections were counterstained with 0.1% cresyl violet or methyl green. When double ICC was used to detect MR and other markers, the first staining step with the above protocol was used, with the substitution of 3-amino-9-ethylcarbazole (AEC) substrate kit for diaminobenzidine (Vector Laboratories). In the second step, avidin-biotin-alkaline phosphatase complex (ABC-AP) and Vector blue detection system (Vector Laboratories) were used. The probe templates were generated by subcloning regions of MR and Sn cDNA into pBS SK+/−, allowing sense and antisense transcription from T3 and T7 promoters. The 301-bp SSt1-BamH1 fragment of MR corresponding to 922– 1223 bp of the cDNA and the 311- and 357-bp BamH1-BamH1 fragments of Sn corresponding to 765–1076 and 1076–1433 bp of the cDNA, respectively, were chosen. 35 S-labeled RNA probes were transcribed from linearized plasmids using a Stratagene RNA transcription kit. Probe was used at 3 × 10 6 to 1 × 10 7 cpm/ml in 50% formamide, 10 mM Tris, pH 8.0, 300 mM NaCl, 10 mM dithiothreitol, 100 mg/ml dextran sulphate, 200 g/ml Ficoll, 200 μg/ml polyvinylpyrrolidone, 200 μg/ml BSA, and 500 μg/ml tRNA. Mouse tissue was prepared by perfusion-fixation. After perfusion with heparinized physiological saline, fixation was commenced with chilled 4% paraformaldehyde, 100 mM NaOH, 100 mM sodium acetate, pH 6.5 with acetic acid, and followed by chilled 4% paraformaldehyde, 100 mM NaOH, 100 mM sodium tetraborate, pH 9.5 with HCl. Dissected organs were postfixed at 4°C in this latter fix for 3.5 h, then placed in 20% sucrose in PBS for 16 h before freezing in OCT compound over dry ice in isopentane. Tissues were sectioned at 10 μm onto slides pretreated with Vectabond (Vector Laboratories). Before hybridization, sections were rinsed in 100 mM triethanolamine, pH 8.0, then treated by acetylation for 10 min with 2.5 μl/ml acetic anhydride in 100 mM triethanolamine, pH 8.0, rinsed in 2× SSC, and dehydrated through ethanol. Probe was heated at 65°C for 10 min, spun at 4,000 rpm for 10 min, then hybridized to tissue sections for 16 h at 56°C. Several posthybridization washes were performed, including a 30-min 20 μg/ml RNAse A treatment, and high stringency washes for 30 min at 60°C in 0.1× SSC, 1 mM dithiothreitol. Slides were exposed to Ilford nuclear research emulsion for 10–17 d, and signal was detected by Ilford PQ Universal paper developer diluted 1:4 with distilled water and Unifix ( Eastman Kodak Co. ). Slides were counterstained with 0.1% cresyl violet and photographed under bright field microscopy. The specific recognition of MR may be hampered by the tissue heterogeneity of MR, the potential cross-reactivity of reagents with other members of the family of proteins with which it shares homology, and the existence of other receptors that share a similar ligand recognition profile. We have used two independent methods to detect MR specifically: ISH to define mRNA and therefore sites of synthesis, and ICC to define protein expression in a wide range of organs of normal adult mice. Specificity of mRNA detection was confirmed by performing control ISH with sense strand probes. The specificity of the polyclonal anti-MR Ab was examined by Western blotting of tissue lysates. There was some tissue-specific heterogeneity with respect to apparent molecular weight in the protein detected, but in the absence of anti-MR, no signal was detected (not shown). The treatment of tissues for ICC was mild, allowing double ICC detection of MR with markers of Mφ, DCs, and endothelium to define expression more closely. Of particular interest, we used double ICC in lymphoid organs to compare the expression of MR with that of the putative CR ligand, Sn, and other ligands of CR-Fc. MR mRNA expression was seen in the medullary cords . The subcapsular sinus was clearly negative , although this site and the medulla were strongly labeled by the Sn probe . Control sections hybridized with sense probes of MR and Sn had low background . Although Sn is expressed by medullary and subcapsular sinus Mφ, only the latter bear ligands of CR-Fc ( 30 ). Together these data clearly indicate that MR and its Sn ligand are not produced concurrently in the lymph node. MR expression was observed throughout the red pulp by ISH, but appeared to be absent from the marginal zone and white pulp . The marginal metallophilic zone was readily identified by its high expression of Sn . The control sections for MR and Sn, respectively, probed with sense strand RNAs, had no significant background . Although precise anatomical localization of the sites of synthesis was not possible by this method, these data are highly suggestive of MR and Sn synthesis occurring at distinct sites. Discrete cells were labeled with MR probe within the thymus , with very little background in the control . Comparison with expression of Sn is not informative, since thymic Sn is not a ligand of CR-Fc. MR antigen was found on medullary Mφ (m), sinus lining Mφ, and endothelium of the marginal sinus (ms), but not in T cell areas (t) . No detectable background staining was found in these areas in the absence of MR Ab . In contrast, DEC-205 was detected on interdigitating cells throughout the T cell areas of an adjacent section . By double ICC, Sn and MR were shown to colocalize in medullary Mφ (m) , but only Sn could be detected on the subcapsular sinus Mφ . By contrast, no colocalization of CR-Fc and MR could be detected, CR-Fc being confined to subcapsular sinus Mφ and some germinal center cells . Scattered cells expressing both MHCII and MR were detected in lymph nodes in the paracortex bordering the B cell follicle defined by reactivity to anti-IgM . These cells did not express DC markers DEC-205 or CD11c, nor did they express the Mφ marker F4/80 (not shown). The elongated morphology of MR staining cells and location in the marginal sinus are characteristic of lymphatic endothelial cells, but expression of MR was restricted to a CD31 − population, indicating that high endothelial venules do not express MR . MR + endothelial cells (arrow) did not coexpress the Mφ marker macrosialin, detected with mAb FA.11, although the intimately associated sinus lining Mφ (arrowhead) expressed both of these markers . Single immunostaining of MR in spleen revealed expression in red pulp (rp) Mφ but not in the white pulp (wp) or marginal zone (mz) , while background staining in the absence of primary Ab was not detected . This is in contrast to the expression of CD11c by DCs at the border of the white and red pulp and other DC subsets of the white pulp that are detectable with Abs to MHCII and DEC-205 (not shown). Expression of Sn, a CR-Fc ligand in spleen, was compared with that of MR by double ICC . Sn alone was detected in marginal metallophilic Mφ, and there was additional low level expression in red pulp Mφ along with strong expression of MR. In contrast, CR-Fc bound to splenic marginal metallophilic Mφ, but not red pulp Mφ . Therefore, MR and its putative ligand are expressed at nonoverlapping sites, separated by a clear region in the outer marginal zone. The absence of MR expression in this compartment was confirmed by double staining with ERTR-9, an mAb that specifically recognizes Mφ of the outer marginal zone . Double ICC for MR and macrosialin with FA.11 defined two subsets of MR + cells, double-positive Mφ , and elongated venous sinus endothelial cells which did not express macrosialin . ICC revealed two distinct populations of cells that express MR. These were highly stained flattened Mφ lying beneath the capsule and along the connective tissue septa that penetrate the cortex (not shown), and less intensely stained Mφ with fine processes that were found throughout the cortex (c) and the corticomedullary junction (cmj) . Staining of Mφ in the medulla (m) was very weak or negative . A control section did not reveal any detectable background staining . Cells expressing MR were quite distinct from the DEC-205–expressing cortical epithelial cells which have extensive dendrites, and the few rounded interdigitating cells of the medulla . It seems likely that all MR + cells in the thymus are Mφ. By double ICC it is apparent that most of them coexpress the Mφ marker, F4/80 . As in spleen and lymph node, double staining with CR-Fc in thymus revealed that MR and CR-Fc ligand(s) are expressed by two distinct populations of cells . CR-Fc bound to large undefined cells of the medulla which may be part of the thymic epithelium. MR antigen appeared to be confined to the lymphatic endothelium of interfollicular areas (i) and was notably absent in follicles (f) . Mφ and DCs in the interfollicular areas and follicles of an adjacent section that were identified with FA.11 did not express MR . Control sections of Peyer's patch gave no background signal (not shown). MR was detected in dermal Mφ, but not in epidermal Langerhans cells . In contrast, F4/80 stained both Mφ and Langerhans cells . Again, no nonspecific staining was seen in control sections (not shown). We could not detect MR in isolated epidermal sheets of normal mice, using either the ICC method presented here, or the method of Takahashi and co-workers (42; data not shown). We confirmed by ISH and ICC previous studies demonstrating expression of MR in hepatic endothelium and Mφ of liver (Kupffer cells), gut, lung, and resident tissue Mφ of other organs (not shown). We describe here the novel finding of MR in perivascular microglia of brain and glomerular mesangial cells of kidney. Mφ and related cells of the brain perform specialized functions in tissue homeostasis, inflammation, and maintenance of the blood–brain barrier. They are phenotypically, functionally, and morphologically distinct, and thus deserve special attention in their expression of MR. In addition, previous studies have suggested a role for an MR on vascular endothelium in regulating blood–brain barrier function. We observed that meningeal Mφ express MR by ISH and ICC (not shown). Perivascular microglia also express MR, but adjacent vessel endothelium does not, as shown by ISH and ICC . Confirmation that these cells are perivascular microglia was deduced from their expression of F4/80 . No signal was detected in the meninges or brain parenchyma in control sections examined by ISH or ICC (not shown). Like perivascular microglia, astrocytes are also associated with vessels, whereas more differentiated microglia are deeper in the parenchyma. Neither of these cell types appeared to express MR in normal brain . MR was observed in kidney glomeruli, both by ISH and ICC in repeated experiments. Control sections for ISH and ICC have low background, verifying the authenticity of these observations. An example of a glomerulus stained for MR and observed at high magnification indicated that expression is present on the mesangial cells . No expression of Mφ markers F4/80, FA.11, or Sn was observed on glomeruli, nor did they bind CR-Fc (not shown). We have used two independent methods to examine the expression of MR mRNA and protein in normal adult mouse. In all tissues studied, sites of synthesis of mRNA, examined by ISH, and expression of antigen, detected by ICC, were identical, suggesting that protein transfer between cells did not contribute to the staining observed. Both methods demonstrated MR expression by subsets of Mφ and endothelial cells. We confirmed previous studies of mature Mφ labeling in mice ( 42 ) and humans ( 40 , 41 ), although a closer analysis of Mφ in spleen and lymph node revealed an unexpected anomaly. An MR-like binding activity has been described on marginal zone Mφ in mouse ( 16 ) and rat ( 15 ) spleen. Similarly, the subcapsular sinus Mφ of the lymph node have also been documented as having an MR-like binding activity in mice ( 14 ) and rats ( 15 ), although no specific carbohydrate receptor has been characterized structurally or antigenically in either case. Here we demonstrate clearly that MR is not responsible for these activities. Double ICC staining of MR with the marginal zone Mφ marker ERTR-9 confirmed that the cells of the marginal zone do not express MR. Similarly, we did not detect MR on subcapsular sinus Mφ by ISH or ICC. These binding activities, which are described as calcium dependent and of high affinity for ligands such as a linear β-1,2–linked tetramannose from C. albicans ( 16 ), Trypanosoma cruzi amastigotes ( 14 ), mannose/fucose/ N -acetylglucosamine-BSA ( 15 ), and mannan ( 43 ) must therefore be mediated by some additional unknown receptor(s). We found endothelium to be heterogeneous with respect to expression of MR. MR was detected on endothelial cells of spleen red pulp and liver, whereas blood vessel and high endothelial cells were negative. However, lymphatic endothelium appeared to express MR widely, consistent with a constitutive function, possibly endocytosis. Our finding contrasts with that in humans, in which lymphatic endothelium appeared negative, although coexpression of MR with endothelial markers CD31, VE-cadherin, and von Willebrand factor was observed in sinus lining cells of the spleen and lymph node ( 40 ). There may be additional phenotypic differences between human and murine endothelial cells. In contrast to humans, we noted the absence of CD31 expression by lymphatic endothelium and sinus lining cells of murine lymph node. Further studies are needed to establish the functional significance of heterogeneity in MR expression by selected vascular and lymphatic endothelium in different species. MR has been implicated in T cell immunity, after the discovery of its expression on cultured human blood monocyte–derived DCs ( 24 ) and on DCs expanded from cord blood hemopoietic progenitors ( 11 ). Isolated DCs use MR to endocytose mannosylated ligands for presentation to T cells by MHCII ( 24 ) and CD1b ( 27 ). MR-mediated antigen uptake confers a greatly enhanced efficiency of presentation to T cells, of the order of 100 ( 25 ) and 200–10,000-fold ( 26 ). MR may be a marker of immature DCs, since it is downregulated in vitro by inflammatory stimuli ( 10 ). However, we found no expression of MR on DCs in vivo in thymus, lymph node, spleen, and Peyer's patch of normal mice. In particular, the CD11c + cells of the spleen, which are thought to represent an immature population of myeloid-derived DCs, did not express MR. Likewise, we did not observe expression of MR by resting Langerhans cells of skin epidermis. This observation is consistent with the study by Reis e Sousa et al. ( 12 ), in which MR could not be detected on lysates of purified murine Langerhans cells by Western blotting, although a mannose-specific uptake by these cells was identified. Similarly, ICC studies in human tissue did not detect expression of MR in Langerhans cells ( 40 , 41 ), although freshly isolated Langerhans cells did express functional MR ( 13 ). We did detect a subpopulation of MR + cells of lymph nodes in the T cell areas bordering the B cell follicles which express MHCII, but these are unlikely to represent a known population of DCs, as they did not express DEC-205 or CD11c (not shown). Further studies are required to determine whether MR is expressed by DCs after immunization, and to characterize the mannose-specific binding activity of Langerhans cells, which may be due to a distinct receptor. The apparent lack of expression of MR on resting murine DCs in situ should be cautionary for those working on cultured DC populations. We compared expression of MR with that of the putative endogenous ligand(s) of the CR, those that bind CR-Fc. Previously we hypothesized that a soluble form of MR or MR + cells may interact with CR-Fc binding cells of spleen marginal metallophilic Mφ, lymph node subcapsular sinus Mφ, and germinal center cells ( 31 ). This would allow transfer of MR-bound carbohydrate antigen to cells strategically positioned at sites of generation of B cell responses to carbohydrate antigens. Here, we show that cells that bind CR-Fc in spleen and lymph node do not coexpress MR; indeed, the receptor and the ligand(s) are at spatially distinct sites within these organs, consistent with a transfer function via sMR. Although we did not detect sMR bound to the subcapsular sinus Mφ or marginal metallophilic Mφ, it may be present at levels below detection or may depend on immune stimulation. Intriguingly, we also observed scattered CR-Fc binding cells in the thymic medulla, where a role in capture of antigen-laden sMR would be unexpected. Thymic epithelial cells synthesize a variety of glycoprotein hormones ( 44 ), and our recombinant protein may recognize one of these in the thymus. Our CR-Fc, like the CR-Fc prepared by Fiete and co-workers ( 34 ), binds to bovine lutropin hormone, a glycoprotein bearing terminal galNAc-4-SO 4 (Linehan, S.A., and L. Martínez-Pomares, unpublished data). We have made a wider survey of MR expression than had previously been undertaken, including brain and kidney. We identified MR expression in perivascular microglia of murine brain by ISH and ICC. Perivascular microglia lie on the parenchymal side of arterioles, and MR at this location may be appropriately placed to endocytose glycoproteins that have traversed the blood–brain barrier. These specialized Mφ also express class A scavenger receptors and take up modified low density lipoprotein injected into the blood or cerebral ventricles ( 45 ). Those authors also showed that horseradish peroxidase, a known ligand of MR, can be endocytosed by perivascular microglia ( 45 ). In another study, liposomes labeled with mannose passed through the murine blood–brain barrier more efficiently than those labeled with fucose or galactose ( 46 ). Similarly, the ependymal cell layer lining the cerebral ventricles regulates solute transport between the cerebrospinal fluid and brain tissue, and in rat this can be dissociated by mannose- but not glucose- or galactose-BSA ( 47 ). However, we found that neither the ependymal cells nor the endothelial cells of the blood–brain barrier expressed MR. Astrocytes and more differentiated microglia of the parenchyma do not express MR in normal brain. Both of these cell types have a tendency to upregulate various Mφ markers when cultured in vitro or stimulated in vivo, so a definitive study of their phenotype requires further in situ analysis. We also demonstrated expression of both MR mRNA and protein in glomerular mesangial cells of the kidney in situ. The glomerulus is the site at which blood is first filtered in the kidney. MR mRNA and protein have been observed on in vitro–cultured mouse mesangial cells stimulated with the inflammatory cytokines TNF-α and IL-1α, but were absent from unstimulated cells ( 48 ). An endocytic role for MR on mesangial cells is consistent with clearance of the MR ligand COOH-terminal propeptide of type 1 procollagen labeled with nondegradable 125 I-tyramine-cellobiose, in which 20% of the label was found in the kidneys while 70% was recovered from liver ( 49 ). Glomerular mesangial cells share some features of the reticulo-endothelial system, including the ability to phagocytose apoptotic cells ( 50 , 51 ). Cultured human mesangial cells also express components of NADPH oxidase ( 52 ) and FcγRIII and FcεRIγ chain ( 53 ). However, murine mesangial cells lacked all of the Mφ markers used in this study apart from MR (not shown), and are not believed to share a common lineage with hemopoietic and endothelial cells, which can both be generated from embryonic mesodermal cells ( 54 , 55 ). Another cell type that is not hemopoietic or endothelial, but has been reported to express MR, is retinal pigment epithelium ( 7 ). Although the expression of MR in myeloid cells appears to be regulated by the transcription factors PU.1 and Sp1 ( 56 ), the detection of MR in mesangial cells and retinal pigment epithelium suggests that other transcription factors must be involved in these distantly related cell types. In conclusion, we have characterized murine MR expression in situ in subsets of Mφ and endothelial cells, but not DCs, describing novel expression in perivascular microglia and renal mesangial cells. We demonstrate that MR-like binding activities of spleen marginal zone Mφ and lymph node subcapsular sinus Mφ, and possibly Langerhans cells, in situ are not due to MR. The expression pattern of MR in lymphoid organs is consistent with a model of antigen capture by MR and transfer to sites of anticarbohydrate immunity by a soluble form of MR that may recognize cells at these sites by their expression of ligands of the cysteine-rich domain of MR. Overall, the MR is widely expressed by distinct cell types involved in potential clearance functions. Further studies are needed to investigate the regulation of MR expression by these cells and the posttranslational modification of MR protein in different tissue microenvironments, as well as to characterize other MR-like activities. | Study | biomedical | en | 0.999997 |
10377193 | 6–8-wk-old male (129/Sv,C57BL/6) TAP1 −/− ( 26 ) and control (C57BL/6 × 129/Sv) F1 or F2 mice were obtained from The Jackson Laboratory . C57BL/6 β2m-deficient mice (β2m −/− ) and control C57BL/6 mice were also obtained from The Jackson Laboratory . CD1D −/− mice and their littermate controls were used at the F2 and F6 backcross to C57BL/6 ( 27 ) or the F8 backcross to BALB/c mice ( 28 ). CD1D −/− TAP1 −/− mice were generated by backcrossing the CD1D −/− onto the TAP1 −/− background. All mice were housed in a biosafety level 3 facility under specific pathogen–free conditions at the Animal Biohazard Containment Suite (Dana-Farber Cancer Institute, Boston, MA) and used in a protocol approved by the institution. Virulent M. tuberculosis (Erdman strain; originally obtained from Barry Bloom, Albert Einstein College of Medicine, Bronx, NY) was passaged through mice and then grown in Middlebrook 7H9 supplemented with oleic acid- albumin-dextrose complex (OADC; Difco), before freezing aliquots at −80°C. Before inoculation of mice, an aliquot was thawed, diluted in normal saline (0.9% NaCl) containing 0.02% Tween 80, and sonicated twice for 10 s using a cup horn sonicator (Branson Ultrasonics Corp.). Mice were infected intravenously via the lateral tail vein with 10 6 live bacilli. The inoculum dose was confirmed by plating an aliquot onto 7H10 agar plates (Hardy or Remel). Venous blood from mice infected with M . tuberculosis was obtained by retro-orbital puncture. 50 μl of blood anti-coagulated with heparin was stained with PE-conjugated anti-CD8 antibody (clone 53-6.72) ( PharMingen ) or a control antibody. The RBCs were lysed with NH 4 Cl and after extensive washing with buffer the samples were resuspended in 1% paraformaldehyde-PBS and analyzed after 24 h using a FACSort™ ( Becton Dickinson ). The percentage of CD8 + T cells within the lymphoid gate was determined. To quantify viable mycobacteria in the infected mouse organs, the lungs, liver, and spleen were aseptically removed from each killed animal. The left lung, left lobe of the liver, and half of the spleen were homogenized in 0.02% Tween 80 in normal saline using Teflon homogenizers (Fischer). 10-fold serial dilutions were plated onto 7H10 agar plates and colonies were counted after incubation for 3 wk at 37°C. Tissues for histological studies were fixed in 10% buffered formalin and then embedded in paraffin blocks. 5-μm sections were stained with hematoxylin and eosin or by the Fite-Faraco method for acid-fast bacilli (AFB) ( 29 ). To test the hypothesis that the increased susceptibility of β2m −/− mice to M . tuberculosis was due to the absence of CD1d-restricted T cells, mice that had both the CD1D1 and CD1D2 genes disrupted by homologous recombination (CD1D −/− mice) were infected with virulent M . tuberculosis ( 27 ). No significant difference between the mortality of CD1D −/− mice and that of their heterozygous littermate controls was observed after intravenous infection with 10 6 CFU . The median survival time (MST) was 169 d for the CD1D −/− mice and 136 d for the CD1D +/− mice. These CD1D −/− mice were used after the second backcross to C57BL/6 mice, and further studies using CD1D −/− mice after the sixth backcross gave similar results (data not shown). We also considered whether deletion of CD1D increased the resistance of mice to infection with M . tuberculosis . Since both C57BL/6 and 129/Sv mice are relatively resistant to tuberculosis, an increase in resistance of CD1D −/− mice on these genetic backgrounds would be difficult to detect. Therefore, CD1D −/− mice on the susceptible BALB/c genetic background were infected. CD1D −/− mice backcrossed eight generations to BALB/c mice showed no significant differences in survival compared with CD1D +/+ BALB/c mice after infection with M . tuberculosis . Additional experiments using a higher (3 × 10 6 ) or lower (2 × 10 5 ) inoculum did not reveal any differences in survival (data not shown). Experiments done in parallel demonstrated a significant reduction in survival for β2m −/− mice compared with β2m +/+ mice (data not shown), as had been reported previously ( 12 ). These results indicate that the increased mortality of β2m −/− mice was not due to an absence of CD1d-restricted T cells. As the absence of the CD1D1 and CD1D2 genes did not significantly alter the survival of mice, TAP1 −/− mice were infected with M . tuberculosis to independently verify that the susceptibility of β2m −/− mice to tuberculosis was secondary to the absence of T cells restricted to MHC molecules loaded in the ER in a transporter-dependent manner. The vast majority of such T cells are class I MHC– restricted CD8 + T cells, and mice with disruption of the TAP1 gene are known to have a profound deficiency in CD8 + T cells ( 26 ). We confirmed the loss of CD8 + T cells from peripheral blood after intravenous infection with M . tuberculosis . We found that in infected TAP1 +/+ mice, 9.0 ± 0.7% (mean ± SEM) of PBLs were CD8 + , whereas only 1.2 ± 0.1% of PBLs were CD8 + in TAP1 −/− mice . These results were similar in uninfected TAP1 −/− and control mice ( 30 ). In three separate experiments, a total of 42 TAP1 −/− mice and 42 control mice were infected intravenously with 10 6 CFU of M . tuberculosis . It is striking that the TAP1 −/− mice were more vulnerable to death from infection than were control mice . The TAP1 −/− mice had a MST of 63 d, and with the exception of one mouse all were dead by day 91 . In contrast, the MST for the control mice was >150 d . The difference between the survival of the TAP1 −/− and the TAP1 +/+ mice was highly statistically significant, and the P values for the individual experiments were P < 0.0001, P = 0.0047, and P < 0.0001. These results demonstrate the importance of an intact TAP-dependent peptide loading antigen presentation pathway for immunity to tuberculosis and strongly supports a critical role for class I MHC–restricted CD8 + T cells in the immune response to M . tuberculosis . To exclude the possibility of a subtle CD1d-dependent effect that was obscured in the presence of CD8 + T cells, the survival of TAP1 −/− CD1D −/− mice was compared with TAP1 −/− CD1D +/+ mice, on a mixed 129/Sv and C57BL/6 genetic background. No significant difference was observed in the survival of these strains of mice . The MST was 79 d for the TAP1 −/− CD1D −/− mice and 65 d for the TAP1 −/− CD1D +/+ mice, which was similar to other experiments in which TAP1 −/− CD1D +/+ mice were infected . This experiment is consistent with the conclusion that CD1d does not contribute to a protective immune response after intravenous inoculation with M . tuberculosis . The TAP1 −/− mice were unable to control the progression of the infection. The number of bacteria deposited in the spleen, liver, and lungs was determined 1 d after infection and was comparable to the numbers reported by other investigators . The ability of the mice to limit the mycobacterial growth was studied at several time points after infection. In all three organs examined, the TAP1 −/− mice were not able to control the infection as efficiently as the control mice . For example, the TAP1 −/− mice had a 10–100-fold increase in the number of mycobacteria isolated from the lung 10 wk after infection. Similar differences were seen in the spleen and liver. In contrast, the early phase of the infection (days 1–21) was similar in TAP1 −/− and TAP1 +/+ mice . This result is consistent with the finding that protective CD4 + T cells are present by day 10 after infection, whereas protective CD8 + T cells do not become apparent until 3–4 wk after infection ( 4 ). These data indicate that the absence of TAP1 affected the adaptive immune response. We observed some variability between experiments, particularly in the colony count data. We believe that this variability arose from the use of two different batches of M. tuberculosis . One of the batches was more virulent than the other. Formalin fixed sections of lung, spleen, and liver, were stained for mycobacteria (AFB) to confirm the increased bacterial burden in the TAP1 −/− mice. In all tissues, but most dramatically in the lung, AFB were more abundant in tissue obtained from TAP1 −/− mice compared with TAP1 +/+ mice . Although enumeration of AFB was not done, the tissues from the TAP1 −/− mice had more numerous foci containing AFB, and those foci contained greater numbers of bacilli compared with tissue taken from TAP1 +/+ mice. This is consistent with the colony count data, and suggests that TAP1 −/− mice are defective in their capacity to control infectious foci. The TAP1 −/− mice had a much greater degree of hepatosplenomegaly and enlargement of the lungs compared with the control mice (data not shown). Lung tissue examined 1 d after infection appears normal by light microscopy and similar in both the TAP1 +/+ and TAP1 −/− mice . 1 mo after infection, the lungs of the TAP1 +/+ mice contain abundant foci of infection containing mixed inflammatory cells and most major blood vessels were surrounded by inflammatory cells; however, the airspaces of the lungs were well preserved . In contrast, the TAP1 −/− mice had severe pneumonia characterized by massive inflammatory cell infiltrates, and severe reductions in airspace . The inflammatory cells were chiefly mononuclear cells, with some areas of granulomatous inflammation where the predominant cell types were epithelioid cells and foamy macrophages. By wk 7, well defined granulomas were observed grossly. Microscopically, the lungs of the TAP1 +/+ mice also had severe granulomatous pneumonia with reduction of lung aeration . In the TAP1 −/− lungs, there was nearly complete obliteration of the airspace by pneumonia with spread via the large airways; neutrophilic infiltrates and early signs of tissue necrosis were apparent . Although there were similar qualitative changes in the nature of the inflammatory infiltrate in the TAP1 −/− mice compared with the control mice, the amount of cellular infiltrate was greater in the TAP1 −/− mice at every time point. In contrast, the infiltrate observed in the TAP1 +/+ mice was more focal and was distributed primarily in a perivascular location with better preservation of the alveolar air space . A similar pattern was seen with the spleens and livers. The spleens of the TAP1 −/− animals were more disrupted, and the spleens of both the TAP1 −/− and control mice had numerous giant cells. The livers of both types of mice had well-defined granulomata, with a tendency for the granulomas in the TAP1 −/− mice to be slightly more cellular. Although it is clear that CD8 + T cells play a critical role in host defense against viral infections and some intracellular infections such as Toxoplasma gondii and Listeria monocytogenes , the role of CD8 + T cells in immunity to tuberculosis remains controversial despite numerous studies that have examined this question. The 1992 study by Flynn et al. redressed the role of CD8 + T cells in M . tuberculosis infection using mice deficient in β2m ( 12 ). Since β2m forms a heterodimer with the class I MHC heavy chain, mice deficient in β2m lack surface expression of class I MHC molecules, and therefore are unable to positively select CD8 + T cells during thymic T cell development. As a result such mice are largely deficient in CD8 + T cells. When β2m −/− mice were infected with the Erdman strain of M . tuberculosis , they quickly succumbed to infection. Associated with the decreased survival time was an increased mycobacterial burden in the lungs and more severe tissue pathology compared with infected β2m +/+ mice ( 12 ). One interpretation of the increased vulnerability of the β2m −/− mice to M . tuberculosis is that class I MHC–restricted CD8 + T cells are critical in immunity to tuberculosis. However, alternative explanations exist. The β2m molecule forms heterodimers with molecules other than the class I MHC heavy chain, such as class Ib MHC heavy chains (i.e., H2-M3) and the non-MHC–encoded CD1 heavy chain, both of which are antigen-presenting molecules that present unique bacterial antigens to T cells. H2-M3 is known to specifically present N-formylated peptides derived from bacterial proteins to murine CD8 + T cells ( 31 ). CD1 is known to present antigens from M . tuberculosis and Hemophilus influenzae to human CD8 + and CD4 − 8 − T cells. For example, human CD1b and CD1c present lipid and glycolipid antigens that are unique to mycobacterial species, including mycolic acid and lipoarabinomannan to human T cells, and antigen-specific CD1-restricted human T cells are able to lyse infected cells and kill the intracellular mycobacteria ( 13 , 15 , 32 , 33 ). Thus, although the β2m −/− experiments strongly implicated a crucial role for class I MHC–restricted CD8 + T cells in immunity to M . tuberculosis , it is now clear that the effect of deleting β2m also might be mediated by T cell subsets other than class I MHC–restricted CD8 + T cells. One strategy to clarify the role of β2m-dependent T cells in immunity to tuberculosis was to examine the susceptibility of TAP1 −/− or CD1D −/− mice to infection with M . tuberculosis . TAP1 −/− mice are largely deficient in class I MHC–restricted T cells; however, because the recognition of CD1-restricted antigens is TAP independent, the CD1-restricted T cell populations should be largely unaffected ( 34 , 35 ). Conversely, CD1D −/− mice have intact CD8 + T cell populations. The finding that the absence of CD1d did not affect the outcome of M . tuberculosis infection in mice does not exclude the possibility that the human CD1 proteins play an important role in immunity to tuberculosis. Presentation of microbial antigens to T cells has been elucidated for the human group I CD1 proteins (i.e., CD1a, CD1b, and CD1c), but not for CD1d, which may have a greater role in immunoregulation ( 36 ). Furthermore, although humans are inherently more susceptible to tuberculosis than are mice, 95% of infected individuals develop long-lived immunity. If the group I CD1 proteins were to participate in the human immune response to M . tuberculosis , evolutionary selection may explain why group I CD1 genes are preserved in the human but not the murine genome. In this regard, it is of great interest that guinea pigs, another species that is highly susceptible to tuberculosis, has also retained the group I CD1 genes (Dascher, C.C., manuscript in preparation). The guinea pig may be a more suitable experimental animal for investigating the role of the group I CD1 proteins in the immune response to M . tuberculosis . We found that TAP1 −/− mice had an increased susceptibility to tuberculosis that was manifested by a decreased survival after intravenous infection, increased mycobacterial burden in the lungs, liver, and spleen, and overall more severe pathological changes in the target organs. These data establish that antigen-processing pathways that require TAP-dependent peptide loading are critical in the development and maintenance of protective immunity to virulent M . tuberculosis . In considering the β2m-associated antigen-presenting molecules, CD1 and TL are TAP independent ( 24 , 37 , 38 ). Presentation of antigens by H2-M3 and Qa-1 can be either TAP dependent or independent ( 35 , 35 , 37 , 39 ). There exist examples of antigen presentation during intracellular infection with Listeria monocytogenes that are TAP independent for H2-M3 ( 39 ) and TAP dependent for Qa-1 ( 40 ). Qa-2 is TAP dependent ( 41 ), and although Qa-2 can bind peptides, T cell recognition of Qa-2 has not been demonstrated. Therefore, the TAP-dependent antigen-processing pathway primarily activates class I MHC–restricted CD8 + T cells. CD8 + class I MHC–restricted T cells are not the only cellular subset that is abnormal in the TAP1 −/− mice. Although their numbers and capacity to kill the YAC-1 cell line are normal, the repertoire of NK cells may be altered secondary to a change in the peptides bound by the class I MHC molecules and the overall decreased surface expression of class I MHC ( 30 ). Likewise, there is a relative expansion of NK1 + T cells in TAP1 −/− mice ( 42 ), although these cells, which require CD1d1 for their positive selection, do not appear to be critical for the long-term survival of mice infected under the conditions used in this study. Further work will be needed to clarify the role of CD8 + T cells during M . tuberculosis infection. It appears that progression of M . tuberculosis infection in both perforin- and fas-deficient mice is unaltered, and suggests that the cytolytic function of CD8 + T cells is not critical in immunity to tuberculosis ( 43 , 44 ). Other work suggests that the crucial function of CD8 + T cells is mediated by IFN-γ ( 45 ). We have found that 40–60% of the CD4 + T cells in the lungs of infected mice are primed to produce IFN-γ (Chackerian, A., and S.M. Behar, manuscript in preparation), and it remains to be determined whether CD8 + T cells serve a role other than the production of IFN-γ. For example, CD8 + CTLs and NK cells produce granulysin, a protein found in cytotoxic granules that has direct microbicidal action against a variety of microorganisms ( 33 ). Granulysin does not have activity against intracellular bacteria unless it can gain access via a pore-forming molecule such as perforin ( 33 ). Although perforin-deficient mice are initially able to control mycobacterial infections ( 43 ), perforin and granulysin may have a role late in infection, for example in preventing recrudescence of disease. Immunity to intracellular bacterial infections has been shown to be a cooperative effort between the innate and adaptive immune responses. Optimum protection against M . tuberculosis , L . monocytogenes , and Listeria major requires synergism between CD4 + and CD8 + T cells. Although the unique roles of each T cell subset during the course of infection remain to be elucidated, an understanding of these roles is critical to the rational development of vaccines and immunotherapeutic strategies. | Study | biomedical | en | 0.999996 |
10377194 | B10.D2 and C57BL/6 mice were purchased from Sankyo Labo Service Co. Inc. (Japan). B10.D2–Rag-2 −/− mice, generated by backcross of Rag-2 −/− mice to B10.D2/nSnJ for 10 generations (reference 14 and unpublished data) were obtained from Taconic Farms. C57BL/6-Rag-2 −/− mice which also lack the cytokine receptor common γ chain [hereafter C57BL/6-γ c −/−(Y) Rag-2 −/− mice], were made as follows. Rag-2 −/− ( 14 ) and γ c −/−(Y) mice ( 15 ) were backcrossed to C57BL/6 for eight generations and intercrossed to generate C57BL/6-Rag-2 −/− and C57BL/6-γ c −/−(Y) mice. These mice were further bred to generate C57BL/6-γ c −/−(Y) Rag-2 −/− mice. NOD/LtSz-scid/scid mice ( 16 ) were provided by N. Hozumi of Science University of Tokyo (Tokyo, Japan) and J. Hata of Keio University School of Medicine (Tokyo, Japan). All mice were maintained in our specific pathogen-free animal facility, and experiments were performed on mice between 6 and 12 wk of age, in accordance with our Institutional Guidelines. Listeria monocytogenes EGD strain (LM) was provided by M. Mitsuyama of Kyoto University (Kyoto, Japan). The bacteria had been passed through C57BL/6 mice and colonies were obtained from the spleens of infected mice on agar plates with trypto-soy broth (Eiken Chemical Co., Japan). Bacteria were then grown in trypto-soy broth overnight at 37°C. Aliquots of bacteria suspension were stored at −80°C until use. After thawing, 2 × 10 6 LM were injected into mice intraperitoneally. In some in vitro experiments, 10 6 collagenase-treated splenocytes were cultured with 4 × 10 5 LM in a 96-well flat-bottomed plate. After 45 min, penicillin and streptomycin were added to the culture media at final concentrations of 100 U/ml and 100 μg/ml, respectively, to limit the growth of LM, and culture supernatants were collected after 72 h and subjected to ELISA. Splenocytes were prepared by homogenizing collagenase-treated spleens in all experiments. DCs were prepared from spleens as previously described ( 17 ). In brief, collagenase-treated spleens (Collagenase D; Boehringer Mannheim ) were homogenized and suspended in a dense BSA solution ( P = 1.080), overlaid with 1 ml of RPMI medium, and centrifuged in a swing bucket rotor at 9,500 g for 10 min at 4°C. DCs and monocytes at the interface were collected, washed, and allowed to adhere to plastic dishes for 2 h. Cells were incubated for an additional 18 h to allow DCs to detach from the plastic dishes. Nonadherent cells containing DCs were then collected and contaminated B cells were further excluded by anti–mouse Ig(H+L)-beads (Perseptive Biosystems) using a MACS magnet (Miltenyi Biotech). After removing DCs, adherent macrophages were detached from the plastic dishes by a cell scraper (Sumitomo Bakelite Co. Ltd., Japan). NK cells were enriched by a combination of PK136-biotin (anti-NK1.1) and Streptavidin-MicroBeads (Miltenyi Biotech). These fractions were stained with appropriate mAbs and were further purified by cell sorting on a FACS Vantage™ ( Becton Dickinson ). The following mAbs were purchased from PharMingen : 145-2C11–FITC and 145-2C11–PE (anti-CD3ε); DX5-FITC (anti-Pan NK); PK136-PE and PK136-biotin (anti-NK1.1); HL3-FITC and HL3-PE (anti-CD11c); AF6-120.1-PE (anti-I-A b ); 53-6.7-PE and 53-6.7-biotin (anti-CD8α); M1/70-biotin (anti-CD11b); and PO3-biotin (anti-CD86). F4/80-FITC (anti-pan macrophage) was purchased from Caltag Labs. An mAb, Y3P (anti-pan-I-A), was purified from hybridoma culture supernatants and conjugated with FITC. Biotinylated mAbs were detected with streptavidin–Red 670 ( GIBCO BRL ). 1–2 × 10 6 cells were stained in PBS/2% FCS, washed, and analyzed on a FACScan ® using the CELLQuest program ( Becton Dickinson ). Rabbit polyclonal anti-asialoGM1 (αASGM1) and anti–IL-12 Abs were purchased from Wako Pure Chemical Industries, Ltd. and PharMingen , respectively. To induce IFN-γ in vivo, 0.5 μg IL-12 or 2 × 10 6 LM were intraperitoneally injected into mice. In in vivo experiments, cells were cultured in the presence of 1 ng/ml IL-12 for 72 h or 4 × 10 5 LM as described above. Titers of IFN-γ in the sera and culture supernatants were determined by Quantikine M ELISA Kit (R&D Systems). Immunofluorescence staining of intracellular IFN-γ was conducted as previously described ( 18 ). Sorted DCs were grown on coverslips coated with Cell-Tak7 ( Becton Dickinson Labware ) and fixed for 15 min with 3.7% paraformaldehyde in PBS. After surface staining with FITC-conjugated mAb against CD11c, cells were permeabilized with 0.5% saponin/1% BSA in PBS for 30 min. Cells were further incubated with polyclonal rabbit anti–mouse IFN-γ Ab (Pestka Biomedical Labs.), polyclonal rabbit anti–mouse IL-12Rβ Ab ( Santa Cruz Biotechnology, Inc. ), or normal rabbit serum as a negative control. Specimens were further developed with Rhodamine-conjugated goat anti–rabbit IgG (ICN Pharmaceuticals, Inc.). Samples on coverslips were mounted onto glass slides with Mowiol ( Calbiochem Corp. ) and examined under a Fluorescence Microscope Axiovert 100 ( Carl Zeiss, Inc. ) equipped with an image analysis system (Signal Analytics Co.). Several studies have demonstrated that IFN-γ produced by NK as well as Th1 cells is a crucial cytokine for limiting and clearing infectious intracellular agents such as protozoan and bacterial pathogens ( 6 – 8 , 19 – 22 ). To examine the role of NK cells in the early production of IFN-γ, we injected polyclonal αASGM1 Abs into B10.D2 or B10.D2– Rag-2 −/− mice to deplete NK cells as previously demonstrated ( 23 ). Consistent with previous studies, NK cells were absent in the spleen of both B10.D2 and B10.D2–Rag-2 −/− mice 3 d after administration of 300 μg αASGM1 Ab . These mice were then injected with 2 × 10 6 LM or 0.5 μg IL-12, and serum IFN-γ levels were examined after 48 and 24 h, respectively. As shown in Fig. 1 B, IFN-γ was detected in the sera of NK cell–depleted B10.D2 mice at a level comparable to those in untreated B10.D2 mice upon LM infection . As both T and B cells ( 24 ) are able to produce IFN-γ, we performed the same experiment with B10.D2–Rag-2 −/− mice lacking both T and B cells. To our surprise, comparable levels of IFN-γ were induced in NK cell–depleted B10.D2–Rag-2 −/− mice and in untreated B10.D2–Rag-2 −/− mice . Because IL-12 is important in inducing IFN-γ, we also injected recombinant IL-12 into these mice and observed IFN-γ production in the sera independent of NK cell depletion . IFN-γ was not detected in the sera of mice injected with PBS or αASGM1 alone. These data suggest that the contribution of NK cells to early IFN-γ production in response to LM infection or IL-12 administration is minimal. We further examined C57BL/6-γ c −/−(Y) Rag-2 −/− mice lacking T, B, and NK cells, as well as NOD/LtSz-scid/ scid mice lacking T and B cells and having functional defects in NK cells and monocytes/macrophages ( 16 ). After 24 h of IL-12 administration, only a small amount of serum IFN-γ was detected in C57BL/6-γ c −/−(Y) Rag-2 −/− mice, whereas substantial amounts of IFN-γ were produced by NOD/LtSz-scid/scid mice. IFN-γ production in NOD/ LtSz-scid/scid mice was also unaffected by pretreatment with αASGM1 Ab . To identify the IFN-γ–producing cells in αASGM1-treated Rag-2 −/− mice, splenocytes were prepared from mice treated with αASGM1 and infected LM in vitro. As shown in Fig. 1 C, IFN-γ was produced by Rag-2 −/− splenocytes in the absence of NK cells, and the production of IFN-γ was completely blocked by the addition of anti–IL-12 Ab, indicating that IL-12 plays a critical role in IFN-γ production upon LM infection. These results further indicate the presence of IFN-γ–producing cells other than T, B, and NK cells. In contrast, amounts of IFN-γ produced by C57BL/ 6-γ c −/−(Y) Rag-2 −/− splenocytes were 1–5% of those from NK-depleted Rag-2 −/− splenocytes upon either Listeria infection or IL-12 administration, suggesting that IFN-γ production is impaired in C57BL/6-γ c −/−(Y) Rag-2 −/− mice. To further identify IFN-γ– producing cells, DCs as well as macrophages and NK cells were freshly isolated from collagenase-treated spleen cells of unprimed mice by cell sorting. Highly purified CD11c + I-A + , Mac1 + F4/80 + , and CD3 - NK1.1 + cells were used as DCs, macrophages, and NK cells, respectively . These cells were cultured for 3 d in the presence of 1 ng/ml IL-12 in vitro. As shown in Fig. 2 B, significant amounts of IFN-γ were detected in the culture supernatants of DCs and macrophages. The amounts of IFN-γ from DCs and macrophages were significantly higher than those from NK cells. DCs cultured in the absence of IL-12 produced IFN-γ to a certain level, probably due to the cross-linking of surface MHC class II molecules by the use of anti–I-A mAb for DC preparation ( 25 ). Consistent with this interpretation, DCs purified using anti-CD86 mAb ( 26 ) instead of anti–I-A mAb did not produce IFN-γ without IL-12 . There are two different types of DCs in the spleen of an adult mouse ( 27 – 30 ). They differ in surface phenotypes (CD8α − DEC-205 − CD11b + versus CD8α + DEC-205 + CD11b − ), origin (myeloid versus lymphoid), requirement of cytokines for their development (GM-CSF versus IL-3), and biological function. To this end, we examined IFN-γ production from DC subpopulations. CD8α − DCs (myeloid DCs) and CD8α + DCs (lymphoid DCs) were isolated by cell sorting and cultured with 1 ng/ml IL-12 for 3 d. As shown in Fig. 3 , CD8α + DCs were found to produce an approximately fivefold higher level of IFN-γ than do CD8α − DCs, indicating that CD8α + lymphoid DCs are the major IFN-γ producers in response to IL-12 stimulation. Immunofluorescence microscopy was conducted to directly detect the expression of IFN-γ protein in DCs. Purified CD11c + CD86 + splenic DCs were cultured in the presence of IL-12 for 3 d, fixed on coverslips, and subjected to intracellular immunofluorescence microscopic analysis. As shown in Fig. 4 , IFN-γ proteins were clearly detected in the cytoplasm of CD11c + DCs , whereas staining was undetectable with the control rabbit serum . Consistent with a previous study ( 31 ), expression of IL-12Rs was readily observed on the cell surface of DCs . IFN-γ was not detected in freshly isolated DCs but was detected in splenic macrophages upon IL-12 stimulation by immunofluorescence microscopy (data not shown). We presented here evidence that NK cells play a small role in the production of IFN-γ at early stages of LM infection or IL-12 administration, and that DCs and macrophages produce IFN-γ. Among DC subpopulations, CD8α + lymphoid DCs are major producers of IFN-γ in response to IL-12. Recent studies have also reported the ability of macrophages to produce IFN-γ ( 12 , 13 ). Amounts of IFN-γ produced by DCs and macrophages were substantially larger than the amount produced by NK cells. It has long been assumed that IL-12 is initially produced by macrophages in response to various intracellular pathogens and later by DCs ( 32 , 33 ), based on the observations that DCs produce IL-12 through ligation of CD40 on DCs by CD40L on activated T cells, or through cross-linking of MHC class II molecules by the TCR ( 25 , 34 ). However, it has been shown recently that phagocytosis of microparticle-adsorbed proteins stimulates DCs to synthesize IL-12 without interacting with T cells ( 35 ), and that DCs but not macrophages produce IL-12 in vivo in microbial infection such as Toxoplasma gondii ( 36 ). Furthermore, accumulating evidence has indicated that resting macrophages are unable to produce IL-12 in response to bacteria or microbial products such as LPS without prior activation by certain cytokines such as IFN-γ ( 4 , 37 , 38 ). In this paper we showed that DCs are able to produce IFN-γ upon IL-12 stimulation. Because DCs produce IL-12 upon phagocytosis and microbial infection, and IL-12 in turn augments the production of IL-12 itself from DCs ( 31 , 35 , 36 ), it is likely that DCs produce IL-12 and IFN-γ by an autocrine manner once they have been triggered by microbial infection. The fact that the addition of anti–IL-12 Ab completely blocked the IFN-γ production by NK cell– depleted Rag-2 −/− splenocytes upon LM infection supports this notion. In addition to IL-12, IL-18 and IL-1β are also likely to be involved in augmenting IFN-γ production from DCs in vivo, as observed in T and NK cells ( 39 , 40 ). Our results on C57BL/6-γ c −/−(Y) Rag-2 −/− mice are consistent with a recent paper by Andersson et al. that reports that γ c −/−(Y) Rag-2 −/− mice produce minimal amount of IFN-γ ( 41 ). Since these mice lack NK cells as well as T and B cells, it was concluded that NK cells are the major producers of IFN-γ. Flow cytometric analysis showed the presence of normal numbers of DCs and macrophages in the spleens of C57BL/6-γ c −/−(Y) Rag-2 −/− mice (Ohteki, T., and S. Koyasu, unpublished results). It is likely that APCs in the γ c −/−(Y) Rag-2 −/− mice have some functional rather than developmental defects that remain to be examined. It is likely that the IFN-γ derived from DCs plays a key role in priming and activating macrophages to produce IL-12 in response to intracellular pathogens. DC-derived IFN-γ, together with IL-12, may also be important in upregulation of surface molecules on DCs such as MHC class II. Once IL-12 and IFN-γ are produced by DCs, a positive feedback pathway(s) would be activated between DCs and macrophages even in the absence of NK cell–derived IFN-γ. Macrophages then secrete IFN-γ in response to IL-12 or a combination of IL-12 and IL-18 ( 12 , 13 ), which also activates macrophages in an autocrine manner to produce nitric oxide. In microbial infection such pathways would be quicker than the pathway through NK cell–derived IFN-γ, and thus important, although not sufficient, for an early stage of innate immune response. DCs are divided into at least two subpopulations by origins, surface molecules, and the requirement of cytokines for their development ( 27 – 30 ). One subpopulation is myeloid DCs without CD8α expression, and the second is lymphoid DCs expressing CD8α. It has been shown that CD8α + lymphoid DCs primarily produce IL-12 in vivo in intracellular protozoan infection ( 36 ). Given that the CD8α + DCs produce IFN-γ in response to IL-12 and predominantly localized in the T cell area of the spleen ( 30 ), lymphoid CD8α + DCs rather than myeloid CD8α − DCs are probably the most efficient initiators for innate immune response upon infection of intracellular microorganisms, as well as the directors of subsequent Th1 differentiation in vivo. | Study | biomedical | en | 0.999995 |
10377195 | Generation of CCR1 −/− and littermate control CCR1 +/+ mice has been described ( 20 ). The mice used in this study were from an F 1 or F 6 backcross of 129/Sv with C57BL/6 mice. Results with both were similar and averaged. Age- and weight-matched CCR1 −/− and CCR1 +/+ mice were used. C3H/HeJ mice were purchased from The Jackson Laboratory . Purified recombinant preparations of cytokines were used. Human (hu) and murine (mu) MIP-1α, muMIP-2, and hu preparations of monocyte chemotactic protein (MCP-1), IL-8, platelet factor 4 (PF4), growth-related oncogene (GRO)-γ, also known as MIP-2β, neutrophil-activating peptide (NAP)-2, RANTES (regulated upon activation, normal T cell expressed and secreted), MIP-1β, and muM-CSF were purchased from R&D Systems. huExodus-1 ( 21 ) was a gift from Dr. Robert Hromas (Indiana University School of Medicine). huENA-78 was a gift from Dr. M.-S. Chang (Amgen Corp., Thousand Oaks, CA). huIFN-γ inducible protein (IP)-10 was a gift from Dr. Andreas Sarris (M.D. Anderson Tumor Hospital, Houston, TX). huGRO-α, muGM-CSF, and muSLF were gifts from Immunex Corp. (Seattle, WA). huEpo was purchased from Amgen Corp. Hemin was purchased from Eastman Kodak Co. PWM mouse spleen cell–conditioned medium (PWMSCM, a source of numerous growth factors, including GM-CSF and IL-3) was prepared as described ( 22 ). Colony assays were done as described elsewhere ( 22 ). Unseparated bone marrow (5 × 10 4 cells/ml) and low-density blood cells (1–2 × 10 5 cells/ml, obtained after density cut procedure) were isolated from mice ( 22 ). To assess whether MIP-1α stimulates or enhances colony formation, marrow cells were plated in 0.3% agar (Difco) culture medium in the presence of 10% FBS (Hyclone, Inc.) with or without muGM-CSF (100 U/ml) or muM-CSF (100 U/ml) and with or without mu or huMIP-1α (100 ng/ml) ( 6 , 7 ). Marrow cells were plated in agar with or without muGM-CSF (100 U/ml) plus muSLF (50 ng/ml) and with or without chemokines (100 ng/ml each) to evaluate inhibitory effects on multi-growth factor–stimulated colony formation by CFU-GM ( 12 ). Inhibitory assays were also done on marrow cells growing in 1% methylcellulose culture medium with 30% FBS, huEpo (1 U/ml), muSLF (50 ng/ml), PWMSCM (5%), and 0.1 mM hemin for effects on colony formation by CFU-GM, BFU-E, and CFU-GEMM. Results for CFU-GM suppression were similar for assays done in agar and methylcellulose and were pooled. Absolute numbers of MPCs in the blood were calculated based on the number of viable low-density nucleated cells, and the number of colonies was scored per number of cells plated in methylcellulose culture medium with Epo, SLF, PWMSCM, and hemin at the above-noted concentrations. The concentrations of cytokines chosen were predetermined to be maximally effective. Three plates were scored per point, and colonies were scored after 7 d incubation in a humidified environment at 5% CO 2 and lowered (5%) O 2 . Mice were given either control diluent, huG-CSF, huMIP-1α, or G-CSF plus MIP-1α. Timing and dosages were based on reports by others ( 18 ) and our own preliminary studies. Mice were injected subcutaneously with either control diluent (pyrogen-free saline; used at the same volume and timing as for injections of G-CSF plus huMIP-1α), 2.5 μg G-CSF given two times per day for 2 d, or 5 μg MIP-1α administered 12 h after the last injection of either control diluent or G-CSF. Mice were bled 30 min after injection of MIP-1α (or the control diluent for MIP-1α) and then killed. Results are given as mean ± SEM, and Student's t test was used to analyze the data. P values < 0.05 designated significant differences between test points. To determine if CCR1 was a dominant receptor for MIP-1α enhancement of colony formation ( 6 , 7 ), we tested mu and hu forms of MIP-1α. As shown in Fig. 1 , mu and huMIP-1α significantly enhanced colony formation by CCR1 +/+ , but not by CCR1 −/− , marrow cells stimulated to proliferate by either GM-CSF or M-CSF. Colonies formed in the presence of GM-CSF, with or without MIP-1α, were composed mainly of granulocytes and macrophages with <20% of the colonies containing only granulocytes or macrophages. No shifts in colony types were noted in the absence or presence of MIP-1α. Colonies formed with M-CSF, with or without MIP-1α, were composed of macrophages. No colonies formed in the absence of GM-CSF or M-CSF whether or not MIP-1α was added to the plates. This suggests that CCR1 acts as a dominant receptor for the MIP-1α enhancing effects on MPCs stimulated by GM-CSF or M-CSF. MIP-1α suppresses MPCs stimulated to proliferate by combinations of growth factors ( 7 , 12 , 13 ). One report using antibodies to CCR1 suggested that the suppressing effects of MIP-1α on colony formation by BFU-E were mediated by CCR1 ( 23 ). To determine if CCR1 was a dominant receptor for MIP-1α suppression, we analyzed the effects of MIP-1α on colony formation by marrow cells from CCR1 +/+ and CCR1 −/− mice stimulated to proliferate with Epo, PWMSCM, SLF, and hemin. As shown in Fig. 2 , both mu and huMIP-1α were equally potent in suppressing colony formation by CFU-GM, BFU-E, and CFU-GEMM from CCR1 +/+ and CCR1 −/− marrow cells. As controls, other CC (MCP-1 and Exodus-1) and CXC (IL-8, muMIP-2, ENA-78, IP-10, and PF4) chemokines known to be inhibitory under these conditions ( 5 ) were tested and found to be equally suppressive on CCR1 +/+ and CCR1 −/− MPCs . Chemokines known to be nonsuppressive (5; GRO-α, GRO-γ, NAP-2, RANTES, and MIP-1β) did not inhibit colony formation of CCR1 +/+ or CCR1 −/− MPCs . These results suggest that CCR1 is not a dominant receptor for MIP-1α suppression of multi-growth factor–stimulated MPCs. G-CSF and BB10010, an MIP-1α analogue, induce mobilization of stem and MPCs to blood ( 1 , 18 ) although with different kinetics, and G-CSF and BB10010 in combination are additive or synergize in this mobilization ( 18 ). In preliminary experiments using C3H/Hej mice, we confirmed that huMIP-1α induction of mobilization of MPCs to blood was rapid (within 15 min to 1 h) and reversible. huRANTES did not have a mobilizing effect on MPCs in these same experiments. We thus assessed the in vivo MPC mobilizing effects of MIP-1α and G-CSF. As shown in Fig. 3 , MIP-1α and G-CSF each significantly mobilized MPCs to the blood of CCR1 +/+ mice, and the combination of G-CSF with MIP-1α showed greater mobilization than either cytokine alone. In contrast, MIP-1α did not significantly enhance mobilization of MPCs to the blood of CCR1 −/− mice . Moreover, MIP-1α did not act with G-CSF to enhance mobilization further in CCR1 −/− mice. However, MPCs in CCR1 −/− mice were more sensitive to the mobilizing effects of G-CSF alone than in CCR1 +/+ mice. In 3 separate experiments in which 4–5 mice per group per experiment were assessed, 1.3-, 1.7-, and 11.6-fold more CFU-GM, 1.5-, 2.3-, and 4.9-fold more BFU-E, and 2.6- and 3.4-fold more CFU-GEMM were mobilized in CCR1 −/− compared with CCR1 +/+ mice. In one experiment, we did not detect greater mobilization of CFU-GEMM in CCR1 −/− mice. These results suggest that CCR1 is a dominant receptor for the MPC mobilizing effects of MIP-1α; moreover, CCR1 appears to play a negative role in G-CSF–induced mobilization of MPCs to the blood. Since several chemokine receptors can bind more than one chemokine and some chemokines can bind more than one chemokine receptor ( 2 – 5 ), it is not always clear which chemokine receptor mediates the effects of specific chemokines. MIP-1α binds to three chemokine receptors: CCR1, CCR5, and D6. Although D6 does not elicit a Ca 2+ influx signal in response to MIP-1α or other chemokines that bind this receptor ( 19 ), it is possible that other intracellular signals are activated through D6 in response to certain chemokines, and there is the possibility that additional receptors will be identified that bind MIP-1α. The availability of CCR1 −/− mice ( 20 ) allowed us to assess if CCR1 served as a dominant receptor for three previously reported functions of MIP-1α. The results presented here clearly demonstrate that in cells without functional CCR1, MIP-1α did not enhance proliferation of CFU-GM stimulated by GM-CSF, or CFU-M stimulated by M-CSF, nor did MIP-1α induce in vivo mobilization of MPCs to the blood, implicating CCR1 as a dominant receptor for these activities. The MIP-1α– induced MPC mobilization effects complement our previous studies in which CCR1 was shown to be a dominant receptor for bacterial LPS–induced movement of MPCs between bone marrow, spleen, and blood and for MIP-1α mobilization of neutrophils to the blood ( 20 ). Our current studies with MIP-1α confirm the mobilization effects on MPCs noted by others using an MIP-1α analogue, BB10010 ( 18 ), as well as the additive/greater than additive mobilization apparent when BB10010 is given as a single injection to mice previously injected with G-CSF. G-CSF mobilizes stem and progenitor cells for autologous and allogeneic transplantation ( 1 ), and enhancement of this may be clinically important. We also found that CCR1 −/− mice were more sensitive to the MPC mobilizing effects of G-CSF than were CCR1 +/+ mice. This unexpected finding implicates CCR1 as a negative component in G-CSF– induced MPC mobilization. How CCR1 would negatively mediate such an effect is not clear. It is known through the use of G-CSF receptor (G-CSFR)–deficient mice that the G-CSFR is crucial for G-CSF–induced MPC mobilization ( 24 ). Interestingly, no increase in circulating CFU-GM was detected in G-CSFR −/− mice in the absence of added G-CSF and after administration of IL-8 ( 24 ). Thus, CXC chemokine receptors have been linked to MPC mobilization by G-CSF through a G-CSFR. Although CCR1 has been implicated in MIP-1α suppression of BFU-E proliferation in vitro ( 23 ), our studies suggest that CCR1 is not a dominant receptor for suppression of MPCs. We have previously shown no significant difference in cycling of MPCs in CCR1 −/− versus CCR1 +/+ marrow ( 20 ), consistent with CCR1 not being a dominant receptor for negative regulation of MPC proliferation. This contrasts with studies in other chemokine receptor–deficient mice. CCR2 binds several chemokines, including MCP-1 and its murine analogue, JE ( 25 ). The use of CCR2 −/− mice demonstrated that CCR2 was a dominant receptor for suppression of MPCs by MCP-1 and JE ( 25 ). The relevance of CCR2 as a receptor involved in negative regulation of MPCs was confirmed by the fact that the cycling status of MPCs in CCR2 −/− marrow is greater than that in CCR2 +/+ marrow ( 26 ). IL-8 and muMIP-2 bind CXCR2, and the use of CXCR2 −/− mice demonstrated that IL-8 and muMIP-2 did not inhibit proliferation of MPCs from CXCR2 −/− marrow, and that enhanced proliferation of MPCs was apparent in CXCR2 −/− compared with CXCR2 +/+ mice ( 27 ). In conclusion, our data identify CCR1 as the dominant receptor responsible for MIP-1α enhancement of growth factor–stimulated MPC proliferation and MIP-1α–induced MPC mobilization to peripheral blood, but rule out CCR1 as a dominant receptor for negative regulation of hematopoiesis by MIP-1α. In addition, the results identify an unexpected role for CCR1 as a negative regulator of G-CSF– dependent MPC mobilization to blood. Together with previous work, our results indicate that CCR1 is a highly versatile receptor able to mediate a broad range of MIP-1α actions, including specific steps in MPC proliferation, development, and distribution, as well as specific leukocyte trafficking. Delineation of specific downstream signaling events will be needed to understand the molecular basis for these diverse functional responses mediated by MIP-1α activation of CCR1. | Study | biomedical | en | 0.999998 |
10377196 | CCR2, CCR6, CCR7, and CCR9 were stably expressed in murine BaF/3 cells ( 9 ) using pME18S-neo (CCR2, 6, 7) or a murine retroviral system (CCR9 [reference 10 ]), whereas CCR3 ( 11 ), CCR8, and HCR/L-CCR ( 12 , 13 ) were stably expressed in rat Y3 cells using either pME18S-neo (CCR3, CCR8) or the murine retroviral system (HCR/L-CCR). CCR5, XCR1, GPR9-6, and STRL33 were stably expressed in human embryonic kidney (HEK) 293 cells using either pcDNA3.1 or pcDNA3.1/zeo(+) (Invitrogen). CXCR4 was analyzed as an endogenously expressed receptor present in the BaF/3 pre-B cell line. All lines were maintained in appropriate culture medium (RPMI or DMEM/10% FCS/10 ng/ml IL-3 for BaF/3 cells). Media for transfected cell lines also contained G418 (1 mg/ml) or zeocin (0.25 mg/ml; GIBCO BRL ) and were periodically tested for their ability to flux calcium in response to known ligands. BaF/3 cells were loaded with Fluo-3-AM ( Sigma Chemical Co. ) in appropriate culture medium (RPMI or DMEM/10% serum) for 1 h at 37°C, after which cells were washed three times in flux buffer (HBSS/20 mM Hepes/0.1% BSA) and aliquoted into a 96-well black-wall plates at a density of 10 5 cells/well. HEK 293 and Y3 cells were plated at a density of 5 × 10 4 cells/well 1 d in advance of assaying, loaded for 1 h in culture medium as above and washed four times. All plates were pre-coated with poly- l -lysine. Calcium flux was measured in all 96 wells simultaneously and in real time using a Fluorescent Imaging Plate Reader (FLIPR; Molecular Devices) and data was expressed as fluorescence units versus time. Chemokines were obtained commercially (R&D Systems or Peprotech ) or produced by DNAX/Schering-Plough. Chemotaxis was assayed in a 48-well microchamber (Neuroprobe) as previously described ( 14 ) using polycarbonate porous membranes (5-μm pore size). Assays were conducted over a 1-h period and cells were counted in an automated fashion on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/ ). Five high power (400×) fields were counted for each of the duplicate wells at a stated concentration. For HIV infection assays, pseudotyped virus was used in a single cycle infection assay and infectivity was monitored as a measure of luciferase activity according to previously published methods ( 15 ). Pseudotyped HIV-1 stock was prepared by cotransfecting HEK 293T cells with the envelope-deficient pNL4-3-luc-E − R − construct and a plasmid encoding the ADA envelope glycoprotein (pADAenv). 48–72 h after transfection, media was harvested, filtered (0.22 μm), aliquoted, and stored at −80°C. Pseudotyped virus was added to HEK 293T cells that were transiently transfected with CD4 alone or CD4 and CCR5. After a 4-d incubation at 37°C, cells were washed with PBS and lysed in reporter lysis buffer ( Promega ). Lysates were assayed for luciferase activity according to the instructions of the manufacturer. For inhibition, chemokines were added at 100 nM at the same time as virus. CCR8-Y3 cells (1D-21, described above) were resuspended in binding buffer (125 mM NaCl, 25 mM Hepes, 1 mM CaCl 2 , 5 mM MgCl 2 , and 0.5% BSA, pH 7.0; 200,000 cells in 200 μl) with 0.1 nM 125 I-labeled I-309 (100,000 cpm). Unlabeled vMip-I and I-309 were included as competitors where indicated. Reactions were incubated at room temperature for 3–5 h, harvested (Unifilter-96 Harvester; Packard Instrument Co. ) onto 96-well GF/C filter plates ( Packard Instrument Co. ), and washed with 4°C binding buffer containing 500 mM NaCl. The filter plates were dried at room temperature overnight, scintillation cocktail (Microscint-0; Packard Instrument Co. ) was added, and plates were counted (Topcount HTS; Packard Instrument Co. ). Data was analyzed by nonlinear regression (GraphPad Prism; GraphPad Software, Inc.) and is expressed as the average of triplicates (± SD). To identify a host-encoded receptor(s) for vMIP-I, we screened cell lines stably transfected with known or suspected chemokine receptors for calcium flux in response to vMIP-I and other chemokines. Among these cell lines we found only CCR8-Y3 cells to be highly responsive to vMIP-I . These same cells also responded to I-309 , a confirmed human ligand for CCR8 ( 16 – 19 ), but not to any of 39 other chemokines tested . The calcium response to vMIP-I was dose dependent and observable at picomolar concentrations . Prior incubation of CCR8-Y3 cells with vMIP-I also decreased subsequent signaling to I-309 in a dose-dependent manner . Similarly, I-309 stimulation reduced subsequent signaling to vMIP-I . At the lowest dose examined (0.01 nM first agonist), a slight enhancement of signaling was observed when the second agonist was added. 12 other receptors tested (CCR2, CCR3, CCR5, CCR6, CCR7, CCR9, CXCR3, CXCR4, XCR1, GPR9-6, STRL33, and HCR/L-CCR) failed to respond to either vMIP-I or I-309 with a calcium flux (data not shown). To characterize more fully the interaction between vMIP-I and CCR8, we examined the ability of vMIP-I to compete for 125 I-labeled I-309 binding to CCR8-Y3 cells. As shown in Fig. 2 , vMIP-I competed successfully for I-309 binding to CCR8-Y3 cells with a K i of 4.68 ± 0.44 nM, which was somewhat higher (sevenfold) than the K i observed for I-309 binding (0.65 ± 0.17 nM). In saturation binding experiments, I-309 bound to CCR8-Y3 cells with a K d of 0.40 ± 0.23 nM ( n = 5, data not shown). Interestingly the EC 50 for CCR8-Y3 cell calcium response was roughly equivalent for vMIP-I and I-309 stimulation (∼3.7 nM). Since previous reports have suggested that vMIP-I might interact with CCR5 ( 4 ), we examined CCR5–HEK 293 cells for a response to vMIP-I. These cells did not flux calcium in response to vMIP-I, SDF-1α, or eotaxin, but were responsive to monocyte chemoattractant protein (MCP)-2, MIP-1α, MIP-1β, and RANTES . vMIP-II has been suggested to act as an agonist for CCR3 ( 8 ), yet antagonizes other receptors, including CCR5 ( 20 ). Therefore, we examined whether vMIP-I could antagonize CCR5 signaling in response to its natural ligands. Prior incubation of HEK 293–CCR5 cells with vMIP-I was unable to antagonize subsequent responses to any of the CCR5 ligands tested, even when present at 100-fold excess amounts (data not shown). In addition, preincubation of the other available receptor cell lines (as above) with vMIP-I failed to inhibit subsequent signaling of these receptors in response to their known ligands (data not shown). These data suggest that, unlike vMIP-II, vMIP-I is not a broad-spectrum chemokine antagonist. To determine whether vMIP-I binding to CCR8-Y3 cells could mediate directed cell migration, we performed in vitro chemotaxis assays. CCR8-Y3 cells responded vigorously to both vMIP-I and I-309 . This response shows the typical bell-shaped curve previously observed in microchemotaxis assays with a maximal in the range of 1–10 nM for both vMIP-I and I-309. Background migration in this assay system was essentially zero, with fewer than five cells/five high power fields migrating in response to medium alone. These data demonstrate that vMIP-I acts as a CCR8 agonist for chemotaxis as well as calcium flux. Because our data indicate that vMIP-I does not interact with CCR5, and because reports of the ability of vMIP-I to inhibit HIV infection are contradictory, we examined whether recombinant vMIP-I would block CCR5-mediated HIV entry. To address this question we used ADA envelope pseudotyped HIV-1 in a luciferase-based viral entry assay. HEK 293 cells transiently transfected with CD4 alone or CD4 and CCR5 were used as target cells. As expected, 293 cells transfected with CD4 alone did not permit viral entry , whereas cells transfected with both CD4 and CCR5 were very efficient in allowing HIV entry. In contrast to the earlier results reported by Moore et al. ( 1 , 4 ), but in agreement with Boshoff et al. ( 8 ), we found vMIP-I to have no detectable effect on CCR5-mediated HIV infection . In contrast, RANTES was effective in blocking HIV infection of CD4/CCR5 double transfectants. This finding is consistent with our observation that vMIP-I is neither an agonist nor antagonist for CCR5 . Viruses have efficiently usurped various host genes in order to manipulate a variety of host-cell functions as well as to modify the host immune response. Through what amounts to in vivo recombinant genetics, virus-encoded molecules have been selected to retain and improve those functions that are advantageous to the virus, while abrogating those functions that are not (e.g., vIL-10). KSHV encodes several molecules that specifically target the chemokine subsystem of the immune response. We have provided the first functional characterization of one of these molecules, vMIP-I. The data presented here demonstrate that vMIP-I is a specific and potent agonist for the chemokine receptor CCR8, which is known to be preferentially expressed on Th2 cells. In addition we have addressed questions regarding the ability of vMIP-I to inhibit CCR5-mediated HIV infection. Understanding the functional role of KSHV-encoded chemokines may help elucidate the mechanisms underlying the pathology of Kaposi's sarcoma. One function of vMIP-I and vMIP-II may be to influence the balance of the immune response toward a Th2 phenotype. Several lines of evidence support this hypothesis. Recently, CCR8 has been shown to be preferentially expressed on human and mouse Th2 cells and its natural ligand, I-309, attracts Th2-polarized T cells in vitro with considerable vigor ( 21 , 22 ). Here, we have presented data that demonstrates KSHV- encoded vMIP-I is an agonist for CCR8 and that CCR8-transfected cells migrate vigorously in response to vMIP-I. In addition, KSHV-encoded vMIP-II has been reported recently to be chemotactic for CCR8-transfected Jurkat cells as well as Th2-polarized T cells ( 23 ). vMIP-II is also reported to interact with CCR3 ( 8 ), which is expressed on at least a subset of Th2 cells ( 24 – 26 ). Furthermore, vMIP-II is an antagonist for CCR5 and CXCR3 ( 20 ), which are preferentially expressed on Th1 cells ( 24 , 26 – 28 ). Finally, Sozzani et al. have reported that CD4 + and CD8 + T cell clones generated from the neoplastic skin of patients with Kaposi's sarcoma are more heavily skewed toward a type II cytokine profile than are clones obtained from patients with alopecia areata or atopic dermatitis ( 23 ). Another potential role for the vMIP-I–CCR8 interaction is in apoptosis. Van Snick et al. reported that I-309 and its murine homologue TCA-3 can block dexamethasone-mediated apoptosis of the BW5147 thymoma ( 29 ), suggesting a role for CCR8 in mediating this event. As a CCR8 agonist, vMIP-I might be used by KSHV to prevent apoptosis of a CCR8 + cell population. Alternatively, vMIP-I might be expressed in order to attract potential host cells for newly produced virus. An important aspect of KSHV pathology is the interaction of this virus with HIV. vMIP-II is reported to inhibit viral entry through CCR5, CXCR4, and CCR3 ( 8 , 20 ). The role of vMIP-I in this regard has been less clear. Using CD4 + /cat kidney cells transiently expressing both CCR5 and vMIP-I, Moore et al. demonstrated a reduction in the ability of R5 HIV-1 strains M23 and SF162 to enter these cells and express p24 versus cells expressing only CCR5 ( 4 ). In a subsequent report, Boshoff et al. reported no effect of vMIP-I on HIV-1 entry/p24 expression in U87/CD4 cells stably expressing CCR5, CCR3, or CXCR4, although vMIP-I did inhibit infection of PBMCs ( 8 ). In support of these later findings, we were unable to observe a calcium flux in response to vMIP-I in 293 cells stably expressing CCR5 . vMIP-I was also unable to antagonize RANTES, MIP-1α, or MIP-1β signaling in these cells. Furthermore, we did not observe any effect of vMIP-I on HIV infection of 293 cells transiently expressing both CCR5 and CD4, although RANTES was effective in abrogating viral entry . It is possible that differences in experimental systems can explain why neither our group nor Boshoff et al. was able to observe inhibition of CCR5-mediated HIV. Another possibility is that vMIP-I acts to inhibit HIV infection through some mechanism other than direct inhibition of binding. If this were true then a simple explanation would be that this mechanism simply does not operate in receptor-transfected 293 cells but is functional in cat kidney cells and PBMCs. Alternatively, it is possible that vMIP-I, when expressed endogenously by cat kidney cells, is posttranslationally processed in such a manner as to produce a chemokine that does interact with CCR5. However, this seems less likely, as Boshoff et al. used exogenously added recombinant vMIP-I to inhibit HIV entry into PBMCs ( 8 ). If this effect was mediated by CCR5, one would expect recombinant vMIP-I to be effective in transfected 293 cells as well. Given the results reported here, one obvious hypothesis is that the HIV infection of PBMCs in these experiments was mediated by CCR8, which has been reported to allow entry of some strains of HIV, including the ADA strain used for the experiments presented in this report ( 30 , 31 ). Indeed we investigated this possibility and have been consistently unable to observe infection of CCR8/CD4-transfected 293 cells by the ADA strain HIV, despite confirmed expression of both CCR8 and CD4. In any case, questions regarding the ability of vMIP-I to affect HIV pathology are clearly of interest and deserve further investigation. A great deal has been learned about KSHV in the past few years. This virus, which appears to be the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma ( 2 , 3 ), recently has also been linked to the development of multiple myeloma ( 32 ). Expression of the KSHV G-protein–coupled receptor in rodent fibroblasts leads to a proliferative phenotype, suggesting a role for this constitutively active chemokine receptor in cellular transformation ( 5 , 33 ). It has been reported that vMIP-I and vMIP-II are angiogenic ( 8 ), and that vMIP-II is a broad-spectrum chemokine antagonist ( 20 ). Understanding the role of vMIP-I in the context of these other molecules, particularly as a CCR8 agonist, should shed further light on our understanding of KSHV and HIV pathogenesis as well as on the role of chemokines in viral immunity. | Study | biomedical | en | 0.999996 |
10385516 | DG44 CHO cells with a double deletion for the dihydrofolate reductase (DHFR) locus were transfected with pSV2-DHFR-8.32, containing 256 copies of the lac operator sequence , and grown in selective media consisting of Ham's F12 media without thymidine and hypoxanthine (Specialty Media or GIBCO BRL ) and dialyzed FCS (Hyclone Laboratories, Inc.). The A03_1 cell line was subcloned from cells that underwent gene amplification in increasing concentrations of methotrexate (MTX) . The D11-1 clone was obtained in a similar way but no gene amplification was performed; the D11-1 clone was isolated after transfection with a modified pSV2-DHFR-8.32 vector containing scaffold-associated sequences flanking the DHFR gene. Transient transfection using Lipofectamine ( GIBCO BRL ) was done on cells plated 48 h earlier on 18-mm square glass coverslips in 35-mm petri dishes. Media were changed after 24 h and cells were stained and visualized 48–72 h after transfection. For initial experiments we used LAP348, a plasmid expressing the wild-type lac repressor fused with the AAD of VP16 and a nuclear localization signal (NLS) . Corresponding control experiments used p3′SS, an expression vector expressing the wild-type lac repressor fused with just an NLS . Later experiments used fusion proteins with a five amino acid COOH-terminal deletion of the lac repressor that forms dimers rather than tetramers . Construction of this p3′SS-dimer lac repressor plasmid and a derivative with an S65T green fluorescent protein (IGFP) fusion is described elsewhere . To make an analogous dimer construct containing the AAD of VP16, PCR was performed on LAP348 to use the BstII site in the lac repressor sequence and change the carboxyl end of the repressor sequence to have the five amino acid deletion and the linker with SauI. The BstEII–SauI PCR fragment containing the VP16 AAD was ligated into the BstEII-SauI sites of the p3′SS-IGFP-dimer lac repressor plasmid, to give the p3′SS-IGFP-dimer lac repressor-VP16 plasmid. From this plasmid, the BstEII–StuI fragment containing the VP16 AAD and truncated carboxyl end of lac repressor was ligated into the BstEII-StuI sites of the p3′SS-dimer lac repressor plasmid to make the p3′SS-dimer lac repressor-VP16 plasmid without the GFP. We modified all original vectors containing the fusion with the IGFP to have a new form, enhanced green fluorescent protein (EGFP) ( CLONTECH ), which contains an additional mutation (Phe64→ Leu) and changes in codon usage. To make the p3′SS-EGFP-dimer lac repressor construct, p3′SS-IGFP-dimer lac repressor was cut with XhoI and AseI (blunted) to include the IGFP-lac repressor-NLS regions. This fragment was ligated into the XhoI-SmaI sites of pCIneo ( Promega ) resulting in pCI neo IGFP-dimer lac repressor. Replacement of the IGFP sequence with EGFP was accomplished by using PCR to amplify the EGFP coding sequence, adding XhoI and EcoRI restriction sites at its ends. This was then inserted between the unique XhoI and EcoRI sites of pCI neo IGFP-dimer lac repressor, creating pCI neo EGFP-dimer lac repressor. To replace IGFP with EGFP in the p3′SS plasmids, the XhoI–EcoRV fragment from pCI neo EGFP-dimer lac repressor, containing EGFP linker and part of the lac repressor, was used to replace the corresponding Xho1– EcoRV fragment in the p3′SS IGFP-dimer lac repressor constructs, creating p3′SS-EGFP-dimer lac repressor and p3′SS-EGFP-dimer lac repressor-VP16 constructs. To make the pET28b EGFP-dimer lac repressor-VP16 construct, the p3′SS-EGFP-dimer lac repressor-VP16–containing vector was digested with DraI and XhoI. The pET28b plasmid (Novagen) was digested with NotI and nucleotides were filled in with Klenow to give blunt ends followed by digestion with SalI. The DraI-XhoI 2,385-kb DNA fragment was ligated in the sites created by the modification of pET 28b described above. The EGFP-dimer lac repressor-VP16 fusion protein was cloned into the pET28b vector next to a 6xHis tag. The fusion protein was purified from BL21 Escherichia coli cells 3 h after induction with isopropyl-1-thio-β- d -galactopyranoside at 32°C by passing the supernatant from a cell lysate over a nickel metal chelation resin ( CLONTECH ). The purity of the protein was assayed on a silver-stained SDS-PAGE and found to be ∼80–90%. The protein was stored in injection buffer (90 mM KCl, 10 mM NaH 2 PO 4 , pH 7.4) at −80°C and thawed immediately before microinjection. Microinjection was done following a standard procedure. Cells were plated in 35-mm ΔT3 dishes (Bioptics) 2 d before microinjection and were observed with an inverted light microscope (IMT-2; Olympus America, Inc.) equipped with a cooled, slow-scan CCD camera (Photometric). Microinjection was performed on cells kept at 37°C in a ΔT3 system (Bioptics). Micropipettes from borosilicate capillary tubes containing a filament were made using a micropipette puller (P-97; Sutter Instruments Co. ). Protein was prepared for microinjection at an appropriate dilution in injection buffer by microcentrifugation at 265,000 g for 10 min just before microinjection. Injections were performed with a micromanipulator (MO-204; Narishige) and a Nikon microinjector. After microinjection, the cells were either returned to the incubator or kept on the microscope for in vivo observations. In vivo observations were performed for a maximum of 6–7 h using conditioned media. The pH was maintained constant by hermetically sealing the ΔT3 dish. The media were replaced after 3 h. For the 19-h live observation, cells were returned to the incubator between image collection. Loading of the labeled nucleotide BrUTP was done by two methods: microinjection or lipid-mediated transfection. BrUTP sodium salt ( Sigma Chemical Co. ) was microinjected into normal or α-amanitin–treated A03_1 cells. Cells were grown as described above and microinjected using a solution of 35 mM BrUTP in injection buffer. Cells were returned to the incubator for 15 min before fixation. For lipid-mediated delivery, A03_1 cells were grown on coverslips for 1 d and transfected with the EGFP-dimer lac repressor-VP16 using the FuGENE6 ( Boehringer Mannheim ) transfection reagent. We followed the manufacturer's instructions, but used 1 μg of DNA and 5 μl FuGENE6 transfection reagent for a 35-mm petri dish. 2 d later, cells were transfected again with BrUTP using the DOTAP ( Boehringer Mannheim ) transfection reagent. 30 μl DOTAP was mixed with 70 μl Hepes, 20 mM FuGENE6, pH 7.4, and mixed with a 100-μl solution of 7.4 mM BrUTP in 20 mM Hepes, pH 7.4; the lipid–nucleotide complexes were allowed to form at room temperature for 15 min. Cells on 18-mm square coverslips were removed from the growth medium, washed in PBS, overlaid with 100 μl of the lipid–BrUTP mixture, and returned to the incubator for 15 min. Cells were washed in PBS, returned to the dishes containing the cell media, and further grown for 1 h. After either microinjection or transfection of the BrUTP, cells were fixed and stained as described below. Immunofluorescence staining and light microscopy were essentially performed as previously described . Double staining with primary antibodies against lac repressor (mouse) at a 1:1,000 dilution and acetylated histones (rabbit) (Serotec Inc., and a gift from David Allis, University of Virginia, HSC [H3 and H4]) at the following dilutions: 1:1,000 for acetylated H4, acetylated H4 at Lys 5, 8, or 16, acetylated H3, and acetylated H2A; and 1:500 for acetylated H4 at Lys 12 and acetylated H2B. A different double staining was done for the lac repressor (rabbit) at 1:1,000 to 1:100,000 dilution and anti–SM-100 (human) diluted 1:1,000, U2B diluted 1:10, nucleophosmin diluted 1:30, or fibrillarin (all three mouse) diluted 1:50. This was followed by incubation with anti–rabbit or anti–human antibodies conjugated with Texas red and anti–mouse antibody conjugated with FITC or Texas red (Jackson ImmunoResearch Laboratories, Inc.). We used 4′,6-diamidino-2-phenylindole (DAPI) as a DNA specific stain. We used the same conditions for bromouridine staining with the following modifications. Before staining, cells were fixed 20–45 min in 1.6% paraformaldehyde in PBS at room temperature. We used a 1:40 dilution of the mouse antibromouridine antibody ( Boehringer Mannheim ) and a 1:1,000 dilution of a Texas red–conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Primary antibodies for hGCN5 and hP/CAF (Santa-Cruz Biotechnology) were diluted 1:400 in PBS plus 3% donkey serum and 0.1% Triton X-100, and used on cells fixed either 10 min in 1.6% formaldehyde or first extracted in 0.1% Triton X-100 in PBS* (5 mM MgCl 2 and 0.1 mM EDTA in PBS) before fixation with 1.6% formaldehyde in PBS*. A blocking step in 10% donkey serum for 1 h at room temperature was used before application of the primary antibodies. We used 24 h incubation at 4°C for both the primary and secondary antibodies or incubation at room temperature, 6 h for the primary and 2 h for the secondary. The secondary antibody was donkey anti–goat IgG labeled with the red fluorophore (Alexa 594; Molecular Probes, Inc.). All other staining steps were identical to the staining procedure described before . Images were collected as optical sections using the Resolve3D data collection program (Applied Precision Inc.) with a fluorescence microscope essentially the same as the one built by Drs. Agard and Sedat at UCSF . The optical sections were deconvolved using an iterative constrained deconvolution algorithm . Details on the immunogold labeling and electron microscopy are found elsewhere . In brief, transfected cells were fixed without permeabilization for 2–4 h in 1.6% freshly prepared paraformaldehyde in calcium and magnesium-free PBS. To increase staining, we used long incubation times (21 h) at 4°C in a wet chamber for both primary and secondary antibodies. Similar staining conditions were used as for the immunofluorescence experiments, but the secondary antibody was coupled to a new, improved water soluble form of Nanoprobe, containing a 1.4-nm gold particle (a gift from Dr. Richard Powell, Nanoprobe). Silver enhancement, dehydration, and embedding, as well as lead citrate and uranyl acetate staining on freshly cut Epon sections were done as described , but a double silver enhancement was performed instead of a single procedure. Thick sections (0.4 mm) were examined using a transmission electron microscope (TEM) (Phillips CM200; Phillips Electronic Instruments, Inc.) at 200 kV. We displayed the images using the program NewVision on an SGI 4D/35 TG and assembled the selected images into figures using Adobe Photoshop. To examine the effects of targeting the VP16 AAD to specific chromosomal regions, we began by using the A03_1 CHO cell line containing an ∼90-Mbp amplified chromosome region ; because this heterochromatic amplified chromosome region is normally highly condensed, a decondensation in large-scale chromatin structure would be easier to detect. This cell line was created in two steps. CHO DG44 cells, with a double deletion of the DHFR gene, were transfected with a DHFR expression vector containing 256 direct repeats of the lac operator and stable transformants were selected. This was followed by gene amplification using stepwise increased concentrations of the DHFR inhibitor, MTX, to select cells that had formed chromosomal amplified regions, or HSRs . The cell clone A03_1, selected at 0.3 μM MTX, was found to contain a single, late replicating, heterochromatic HSR, stable in size and chromosome location . A careful cell cycle analysis revealed that the A03_1 HSR appears by light microscopy as a peripherally located, compact mass, 0.5–1.0 μm in diameter, throughout most of interphase. During a several hour period in middle to late S phase the HSR decondenses, increasing its diameter to ∼2 μm, and moves away from the nuclear periphery to the nuclear interior. This decondensation and movement is correlated with HSR DNA replication . We transfected A03_1 cells with either the vector LAP348 , expressing the lac repressor-VP16 AAD-NLS fusion protein, or p3′SS, expressing the lac repressor-NLS fusion protein . In these initial experiments, immunofluorescence staining with an anti–lac repressor primary antibody was used to visualize the HSR. Two types of novel decondensed structures were seen 24–72 h after transient transfection with the lac repressor-VP16 fusion protein but not with the lac repressor control: (1) a greatly enlarged HSR with fibrillar substructure occupying a large fraction of the nucleus ; (2) a ball-like structure, ∼1–3 μm in diameter, with peripheral repressor staining surrounding a spherical volume, devoid of internal DAPI staining . The extent of HSR decondensation in the first structure is well beyond that seen even during the HSRs decondensation during late S phase. For the purpose of statistical comparisons, we classified a given HSR in this first conformation if its projected area exceeded 2% of the total nuclear area. Over 40% of nuclei fell into this category (Table I ) after transfection with the lac repressor-VP16 fusion protein construct versus 0% (0/55) after the control transfection expressing lac repressor. The second ball structure is qualitatively distinct from any conformational stage seen during an extensive study of the HSR cell cycle dynamics. 72 h after transient transfection of the lac repressor-VP16 construct using Lipofectamine, 25% of nuclei contained decondensed HSRs in this category; none were observed in control experiments. Compact HSRs similar to those observed in control cells were observed in the remaining nuclei (32%). This compact morphology may be the result of a lower expression of lac repressor-VP16 in these cells and/or a cell cycle dependence for decondensation. The wild-type lac repressor forms a tetramer capable of binding two operators, raising the concern that bivalent lac repressor binding might influence the observed structures . However, a similar HSR decondensation was also observed after transient transfection with the dimer form of the lac repressor-VP16 fusion protein construct (Table I ). To verify that the decondensed HSR conformations were not altered by our fixation and immunostaining procedures, we performed control experiments using transient transfection with constructs coding for the EGFP-dimer repressor-VP16 AAD and EGFP-dimer repressor fusion proteins. Direct in vivo visualization revealed analogous decondensed HSR structures as seen after immunostaining . The observed decondensation is best described in terms of an unfolding and straightening of a large-scale chromatin fiber, forming enlarged HSR areas with extended fibers occupying up to 1/3–1/2 of the nuclear cross-sectional area . This fiberlike nature is more clearly demonstrated by serial optical sections and stereopairs (data not shown) generated from the deconvolved three-dimensional optical data sets. A small fraction of nuclei shows such extremely extended fibers that it is possible to estimate the total contour length from just a few optical sections , allowing calculation of the fiber compaction ratio. We measured the length for five such examples, obtaining values ranging from 25–40 μm with an ∼30-μm mean. The HSR size was previously estimated as 93 ± 10 Mbp, based on the ratio between the average HSR length relative to the total chromosome lengths within metaphase spreads . Together these measurements yield an ∼1,000:1 packing ratio for these extended fibers, well above the ∼40:1 ratio predicted for the 30-nm chromatin fiber. This measured value is in the hundreds to thousands compaction ratio range reported previously by fluorescence in situ hybridization studies for probes in the dystrophin gene . Higher resolution imaging was provided by TEM on preembedded immunogold stained specimens. Within 0.4-μm-thick sections, fiber segments with a length of 0.4–1.0 μm and diameter of ∼80–100 nm were visualized. The second ball-shaped decondensed structure, typically seen in 10–30% of cells after transient repressor-VP16 expression, is a qualitatively distinct conformation, different than anything that has been observed previously in many HSR-containing cell lines. The structure shows surprisingly little deviation from a spherical shape, and is distinguished as well by the peripheral localization of lac repressor staining. In midoptical sections, the repressor staining appears as a ring of punctate staining , whereas grazing optical sections show fibrillar staining , suggesting a wrapping of chromonema fibers over a spherical surface. This impression was supported by higher resolution TEM images of immunogold-stained sections . Serial semi-thick sections through an entire ball-shaped structure supported this interpretation as well as demonstrating the separation of this structure from the nuclear envelope (data not shown). One possibility we considered was that this ball-shaped HSR arises from the recruitment of splicing factors, generating an unusually large interchromatin granule cluster with the decondensed HSR wrapping around its periphery. However, staining A03_1 cells transiently expressing the lac repressor-VP16 fusion protein with antibodies to the U2B and SM-100 splicing factors instead showed a greatly increased but peripheral staining, colocalizing extensively with the lac repressor staining, but leaving the center unstained . This distribution is consistent with a VP16 AAD–induced, enhanced transcription of the intron-containing DHFR construct, with a concomitant recruitment of splicing factors. In a number of examples, interchromatin granule clusters adjacent to the ball-shaped structure appeared connected to the peripheral ring of repressor and splicing factor staining by a bridge of concentrated splicing factors . This is consistent with previous experimental work suggesting a dynamic redistribution of splicing factors and spatially organized targeting mechanisms . No accumulation of splicing factors near the A03_1 HSR was seen with the control lac repressor construct without the VP16 AAD fusion (data not shown). A second possibility was that the ball-shaped structure might represent a wrapping of the decondensed HSR around a nucleolus. A transient attachment of the A03_1 HSR adjacent to nucleoli has been seen during middle to late S phase during HSR replication . We stained A03_1 cells transiently transfected with lac repressor-VP16 with antibodies against fibrillarin or nucleophosmin, two different nucleolar-associated proteins. Fibrillarin immunostaining showed nucleolar localization, as expected, but no association with the ball structure in 29/30 nuclei ; moreover, the shape of the nucleolar staining is distinctly less round than the ball-shaped structure. TEM shows distinct textures of uranyl and lead staining in the center of the ball structure and immediately underlying the peripheral lac repressor staining , different from the nucleolar staining appearance. Based on these results, we conclude that these special ball-shaped structures cannot be attributed to a wrapping of a decondensed HSR around a preexisting nucleolus. Interestingly, nucleophosmin immunostaining showed a distinct ring, immediately concentric to the peripheral repressor staining, in 13/17 nuclei containing the ball-shaped structure. In some nuclei the nucleophosmin staining is concentrated inside the ball and is not present at any other sites, whereas in other nuclei both the ball-shaped HSR and nucleoli are stained ; an apparent decrease in nucleolar staining in cells showing bright staining within the ball-shaped structure suggested a recruitment of nucleophosmin away from nucleoli. Nucleophosmin is thought to be involved in ribosomal assembly and binds the 45S RNA in humans . This peculiar ball-shaped conformation was not specific to the A03_1 cell line but was also found in the D11-1 cells (see below) at comparable frequency. The frequency of the ball-shaped structures is dependent on the transfection method used for transient expression. The FuGENE6 reagent showed greatly reduced frequency of this structure over Lipofectamine (data not shown). We conclude that the ball-shaped conformation demonstrates a novel remodeling of a chromosome region, including polarized recruitment of splicing factors and nucleophosmin, which is induced by high concentrations of VP16, possibly as a result of increased transcriptional activity. Curiously, the closest analogous intranuclear structures previously described are the ball-shaped inclusions seen in nuclei after expression of the Huntingtin or ataxin-3 proteins with polyglutamate expansion . Whether there is any structural or functional relationship between these structures is currently being tested. The recruitment of locally high concentrations of transcription factors might lead to precipitation of wild-type proteins analogous to the aggregation seen with mutant proteins containing expanded polyglutamate repeats. Alternatively, these structures might represent exaggerated accumulations and self-assembly of nuclear proteins normally recruited at lower levels to active sites. The previous results were obtained 1–3 d after transient transfection. To establish the temporal sequence and identify the intermediates of the HSR decondensation, we directly observed the HSR conformation in living A03_1 cells after microinjection of purified EGFP-lac repressor-VP16. Roughly 10–15 min was required to visualize significant levels of microinjected protein transported into the nucleus. To allow visualization of the earliest stages of decondensation, we used A03_1 cells that stably expressed low levels of EGFP-lac repressor. Beginning within 15 min after injection, a noticeable increase in HSR size is observed . Further increases in size and appearance of internal fibrillar substructure take place over the next several hours. For technical reasons, these in vivo observations were made using a low NA dry lens. After fixing the sample, higher resolution data were acquired by optical sectioning using a high NA oil lens followed by deconvolution. Fibrillar substructure is clearly seen in these deconvolved images . In roughly one-third of the microinjected cells, the HSR assumes a ball-shaped structure. This appears to occur through a similar increase in HSR size, as described above, followed by the accumulation of an optically dense material in the middle of the decondensed HSR. The final form of the ball-like structure, showing fibers wrapped around a spherical structure, occurs within 10–19 h after microinjection of the EGFP-lac repressor-VP16 fusion protein . The mechanism of large-scale chromatin unfolding may involve one or more determinant factors. The simplest explanation would be that the observed A03_1 HSR decondensation is a consequence of transcription per se. Decondensation of 30-nm chromatin fibers and loss of nucleosome structure over the Balbiani ring genes in Chironimus polytene chromosome puffs requires maintenance of high levels of transcriptional activity . We first wanted to determine whether VP16 targeting increased transcriptional activity. If that was the case, we would next inhibit RNA polymerase II to determine whether changes in large-scale structure occurred in the absence of ongoing transcription. An indirect indication that the VP16 targeted chromosome site is transcriptionally active came from our observation of recruitment of splicing factors to the ball-shaped HSRs and to the fibrillar extended HSRs (data not shown). To directly verify whether VP16 increased transcription to the targeted chromosome site we used BrUTP incorporation followed by antibromodeoxyuridine immunostaining to detect newly synthesized RNA . Transient transfection of A03_1 cells with the EGFP-repressor-VP16 construct was followed 2 d later with introduction of BrUTP into cells by DOTAP for 15 min followed by a 1-h chase. Control experiments using α-amanitin to inhibit transcription verified that this nuclear staining was dependent on RNA pol 2 activity (see below). Only a low percentage of cells was exposed to BrUTP by this liposome introduction method, as determined by nuclear antibromouridine staining, and a much smaller number of cells both expressed the GFP-repressor-VP16 fusion protein and showed incorporation of BrUTP. 60% of cells (6/10) showing both EGFP-dimer lac repressor-VP16 expression and bromouridine incorporation also showed a bright bromouridine signal colocalizing with the A03_1 HSR . In these cells, the staining partially overlaps the actual lac repressor staining, with the remainder accumulated adjacent to the repressor staining. BrUTP labeling of control A03_1 cells expressing the GFP-lac repressor fusion protein did not show this bright BrUTP staining adjacent to the HSR in any of the cells (data not shown). To determine whether the large-scale decondensation observed for the A03_1 HSR was simply a consequence of increased transcriptional activity, we used α-amanitin to inhibit RNA Pol II. Previous results have shown that amanitin induces the degradation of RNA polymerase II large subunit . First, we tested whether the fibrillar structures formed after the VP16 targeting require active RNA Pol II to maintain their highly extended conformation. Cells were treated with α-amanitin 3 d after transient transfection with the EGFP-dimer lac repressor-VP16. Control experiments using BrUTP incorporation showed that this treatment eliminated nuclear BrUTP incorporation everywhere except the nucleoli (at the concentrations of α-amanitin we used, PolII but not PolI transcription is inhibited). These experiments showed stable, decondensed fibrillar structures after up to 24 h continuous exposure to α-amanitin . There did appear to be some degree of local refolding, with a reduction in the number of the most highly extended HSR conformations. Whether this was due to a partial role of ongoing transcription in maintaining a maximally decondensed HSR, or rather reflected a global effect on large-scale chromatin condensation because of long term α-amanitin treatment, was not clear. Second, we asked whether the initial opening of the HSR required active RNA Pol II. Therefore, we inhibited transcription of A03_1 cells for 4 h in 100 μg/ml α-amanitin before the microinjection of the lac repressor-EGFP-VP16. A similar degree of HSR decondensation was observed in the presence or absence of amanitin (five microinjected cells under same conditions that α-amanitin control was performed (see below), and >50 microinjected cells for several similar experimental conditions). To check the effectiveness of the α-amanitin inhibition, cells were microinjected with BrUTP and immunostained to assay BrUTP incorporation. A faint antibromouridine immunostaining signal appeared only over the cytoplasm and nucleoli, showing that Pol II but not Pol I was inhibited . These experiments clearly demonstrate that the decondensation of large-scale chromatin structure is not merely a consequence of ongoing transcription. Previous work has indicated that the A03_1 HSR contains ∼400 kb vector concatemers (with several minor populations from 500–1,000 kb) flanked by large regions of coamplified genomic DNA, averaging 1,000 kb in size . Our observation of extended structures, therefore, implies a straightening of a fiber that includes the 1,000-kb flanking regions. Light microscopy images of highly extended fibers within the HSR after VP16 targeting are suggestive of variations in repressor staining intensity on a size scale of a few tenths of a micrometer along the fiber length, but do not contain clearly visible gaps in repressor staining. This is expected given that the estimated 1,000:1 compaction ratio predicts a fiber length of 0.3 μm for these 1,000-kb flanking regions, which is small relative to the ∼0.25 μm halfwidth of the microscope point spread function. To demonstrate this propagation phenomenon more clearly, we used a different cell line cloned from an independent stable transformation of the DHFR expression vector containing the operator repeats. The D11-1 clone was isolated after transformation and before any MTX selection; it contains one large and one small chromosomal region containing lac operator repeats; pulse field gel analysis, however, indicates that these regions contain vector concatemers less than several hundred kilobases in size (Strukov, Y., and A. Belmont, unpublished data). Both regions appear normally as condensed, compact masses . Transient expression of the repressor-VP16 fusion protein leads to extensive large-scale chromatin decondensation of these chromosome regions, but in these D11-1 chromosome regions the vector concatemer insertion sites appear as spatially distinct staining segments, separated by clear gaps corresponding to flanking genomic DNA . The linear arrangement of these stained segments and gaps demonstrates that the extension and straightening of the large-scale chromatin fiber is not a local event, confined to regions of lac repressor-VP16 targeting, but instead involves the entire chromosome region including the flanking, coamplified DNA. If these flanking regions were packaged as extended 30 nm fibers, the ∼0.5-μm gaps would correspond to at least 60 kb of DNA. The fiber extension relative to the original size of the amplified chromosome region appears roughly comparable to that seen with the A03_1 HSR. In this case, if the flanking regions are packaged into similar large-scale chromatin fibers as observed for the A03_1 HSR, and for bulk chromatin , then this implies propagation over hundreds to thousands of kilobases. To further investigate the mechanism of HSR unfolding we examined the state of histone acetylation, which has long been linked with transcriptional activation. As mentioned in the introduction, genetic experiments in yeast combined with biochemical studies showed that the VP16 AAD can recruit the GCN5 histone acetyltransferase in a complex containing ADA proteins known as the SAGA HAT system . The recruitment of this protein complex at the VP16 targeted HSR would produce a specific pattern of histone hyperacetylation. To test if VP16 AAD targeting induces an increase in histone acetylation, A03_1 cells expressing the EGFP-dimer lac repressor-VP16 were stained with polyclonal antibodies reacting against all isoforms of acetylated histone H3, H4, and H2B, and against H2A acetylated at Lys5. To examine the acetylation pattern in even more detail, we used immunostaining with mAbs recognizing specifically each of the four different monoacetylated histone H4 isoforms (Lys 5, Lys 8, Lys 12, and Lys 16). The results of our immunostaining experiments were striking. An increase in histone acetylation signal, severalfold above the overall bulk chromatin staining was seen with all polyclonal antibodies used, suggesting that all core histones are hyperacetylated by the VP16 AAD targeting. This intense acetylation was observed in all HSR conformations including the condensed structures. The specific lysine residues of histone H4 that show an increase in acetylation at the HSR are lysines 5 and 12 and very weakly lysine 16. A qualitative summary of the data is found in Fig. 8 A. In control cells expressing the GFP-lac repressor, none of the antibodies showed an increased signal over the HSR with the exception of 10/45 (22%) cells stained with the antihistone H4 acetylated at Lys5 (3/45 well above background, 7/45 above background). We emphasize that these results should be regarded as qualitative rather than quantitative. Small variations in staining might reflect subtle differences between antibodies rather than the actual acetylation state. In support of these experimental concerns came our findings that different antibodies raised to tetraacetylated H3 and H4 peptides in David Allis's laboratory gave a slightly different result. Identical results were obtained with the two different polyclonal H3 antibodies, but a stronger signal in a larger percentage of cells (23 out of 25) was obtained for the second acetylated H4 antibody used. However, gross differences in immunostaining of bulk chromatin among various antibodies did not correlate with the degree of hyperacetylation at the A03_1 HSR . For example, the overall staining with the acetylated histone H3 antibody is poor in comparison with the acetylated H2A antibody, yet more cells showed a H3 hyperacetylated A03_1 HSR. Two major results emerge from this data. First, the VP16 AAD targeting induces a strong increase in histone acetylation signal at the targeted chromosome site. Second, a strong hyperacetylation at H2A (Lys 5) and H4 is observed. This is inconsistent with the acetylation patterns observed for the ADA and SAGA GCN5 containing HAT complexes, which primarily show histone H3 and H2B acetylation . Taken together, our results suggest significant activity of at least one additional HAT besides the ADA and SAGA complexes, which would be responsible for the strong H4 and H2A acetylation. Free GCN5 can acetylate H4 at lysines 8 and 16 but prefers H3 . Therefore, more specifically, at least one additional HAT complex is likely to rely on a catalytic domain other than GCN5, given the strong H2A acetylation and the strength and site specificity of the observed H4 acetylation. To directly test for the recruitment of different HATs by the VP16 AAD at the A03_1 HSR, we stained with commercially available antibodies raised against the following: 18– and 16–amino acid peptides from the amino and carboxyl termini of human GCN5; a 16–amino acid peptide from the carboxyl terminus of P/CAF, a HAT highly homologous to GCN5; and a large domain of CBP/P300 that has HAT activity for all core histones and interacts with P/CAF . An increased immunostaining coinciding with the HSR was seen in A03_1 cells expressing the EGFP-dimer lac repressor-VP16 for GCN5 (20/20 cells for the anti–COOH terminus antibody and 21/30 for anti–NH 2 terminus antibody), P/CAF (12/30 cells), and p300/CBP (27/49 cells) . A similar increase in signal was not observed in control cells expressing EGFP-dimer lac repressor (data not shown). The pattern of histone hyperacetylation observed in our system appears to be the result of the combined catalytic activity of several histone acetyltransferases, including GCN5, P/CAF, and p300/CBP, rather than the specific effect of a singular HAT. Recently, the VP16 activation domain has been shown to recruit the yeast NuA4 HAT complex that does not contain GCN5 but does acetylate both H4 and H2A. Interestingly, for both sets of antibodies against P/CAF and GCN5 we saw strong cytoplasmic staining in discrete spots in both CHO and HeLa cells. This pattern was seen with paraformaldehyde or methanol fixation, before or after detergent permeabilization, and is not observed with secondary antibody alone. We observed similar staining with two independent anti-GCN5 antibodies raised against different parts of the protein. The 16–amino acid peptide from the carboxyl region of GCN5 showed a dose-dependent blocking of the respective antibody staining, giving a pattern identical with the secondary alone. Considering these control experiments we believe that the observed cytoplasmic staining pattern is real and we are currently investigating its origins. Histone acetylation over large chromatin domains has been seen for the globin locus . One possible explanation for the observed large-scale chromatin unfolding is a direct modulation of chromatin packing by the acetylation of core histone tails within the extended fibers. Antiacetylated histone H4 staining (using the antibody from David Allis) of the D11-1 cell line after expression of GFP-lac repressor-VP16 showed an intense acetylation signal colocalizing with bound repressor-VP16, but this increased histone acetylation did not propagate across large gaps in the lac repressor staining . These results suggest that the observed propagation of large-scale chromatin decondensation is not simply a direct consequence of histone hyperacetylation. As summarized in the introduction, transcriptional activators can induce alterations of nucleosome structure but their effect on large-scale chromatin structure is poorly understood. We engineered a large heterochromatic chromosome amplified region containing repetitive DNA sequences that represented the target of an unusually strong viral transcriptional activator. The underlying rationale of this approach was to use this exaggerated, artificial system to amplify effects on large-scale chromatin structure induced by transcriptional activators to the extent that they could be easily analyzed in vivo at the single cell level by direct microscopy approaches. Observations of significant remodeling of large-scale chromatin structure induced by targeting of VP16 now provide an entry point into investigations of the relationship between transcriptional activation and large-scale chromatin architecture. Mitotic chromosomes are believed to form a radial loop– helical coil structure, a model based on a highly disruptive experimental approach involving histone extraction . An extension of this model for interphase chromosomes postulates that heterochromatic regions maintain their metaphase conformation whereas euchromatic regions unfold from the 700-nm structures to 200–240-nm fibers that themselves are organized as radial loops of 30-nm fibers. These loops can be more compact or more extended as a function of their transcriptional activity . More recently, light and electron microscopy visualization of chromatin at different stages during the mitotic and interphase cell cycle led us to propose a folded chromonema model based on ∼100-nm chromonema fibers formed by the compaction of 10-and 30-nm chromatin fibers . Because of the apparent tight folding, kinking, and supercoiling of these large-scale fibers, a clear, unambiguous demonstration of the distinct, fiberlike nature of these large-scale chromatin domains is provided only by a fraction of the total chromatin that exists as extended, spatially isolated fibers over ∼0.5–1.0-μm lengths. Our use of the lac operator-repressor system now supports the existence of these chromonema fibers in vivo . However, the difficulty of tracing chromonema fibers in the more compact folding patterns observed for the majority of chromosome regions raised questions about the generality of this folding motif. Because these fibers usually are tightly coiled, it is difficult to trace distinct fiber segments even for 0.5–2.0-μm lengths, particularly by light microscopy. The progressive unfolding of the A03_1 HSR and the very long, extended fibers observed in vivo after microinjection of the lac repressor-VP16 fusion protein provide additional strong support for the existence of chromonema fibers within the starting, condensed HSR conformation, and as a basic large-scale chromatin structural motif. Fibers arising from a 90-Mbp HSR could be visualized clearly for total lengths of 25–40 μm as distinct structures. These fibers had a compaction ratio of ∼1,000:1 consistent with the ∼80–100-nm diam visualized by immunogold EM staining. Our results provided a striking demonstration of the existence of distinct, large-scale chromonema fibers within supercoiled chromosomal regions in interphase nuclei. Dramatic modifications of large-scale chromatin structure by targeting large amounts of the VP16 transcriptional activator domain have been clearly demonstrated in our system. The molecular mechanism of these changes as well as the biological significance (see next subsection) are very complex questions that we are just beginning to address. Our demonstration that this large-scale chromatin decondensation does not require ongoing transcription rules out the possibility that the changes in structure are simply a result of chromatin modifications or DNA topological changes caused by RNA polymerase elongation. Instead, our results suggest that factors recruited by the VP16 AAD other than the polymerase itself lead to a significant reorganization of large-scale chromatin structure. Targeting of the VP16 AAD results in hyperacetylation of all core histones. Further results from immunostaining for histone acetyltransferases demonstrated the recruitment of several different HATs that probably act in concert to induce a complex pattern of histone acetylation. We have directly observed recruitment of GCN5, P/CAF, and CBP/P300 to the A03_1 HSR; other HATs such as mammalian homologues of NuA4 that bind VP16 and acetylate H4 and H2A are also candidates . Inhibition of histone deacetylases results in redistribution of certain heterochromatic proteins . The observed extension of large-scale chromatin fibers in our system might be explained at least in part by the release of heterochromatic proteins from the HSR as a consequence of histone hyperacetylation. A second possible mechanism for large-scale chromatin decondensation may involve chromatin remodeling complexes. Future work will aim at exploring the relationship between recruitment of components of chromatin remodeling complexes and the temporal sequence of large-scale chromatin decondensation. As discussed in the introduction, in vitro evidence suggests that the VP16 AAD recruits a number of different components of the transcriptional machinery. Therefore, dissecting the molecular mechanisms leading to large-scale chromatin decondensation will be a complex task, but one that can exploit the microscopy-based system we have developed, in conjunction with use of VP16 mutants, other lac repressor fusion proteins, and inhibition studies. An increased sensitivity to nuclease digestion extending for tens to hundreds of kilobases flanking transcriptionally active loci has been described in several well characterized examples . In the case of the β-globin locus, this generalized increased sensitivity has been linked to a cis-regulatory control element, the locus control region (LCR), containing multiple transcription factor binding sites . This chromatin opening activity has been proposed to facilitate long range activation of β-globin genes . A long standing question relating to genome organization has been the functional significance of the observed clustering of active, housekeeping genes within large, Mbp-sized chromosome R bands. Ultrastructural localization of bromouridine incorporation into newly synthesized RNA as well as older, tritiated uridine incorporation experiments have shown that most transcriptional activity localizes to the edge of condensed chromatin. This implies that differential compaction of large chromosome regions into condensed, heterochromatic, or extended euchromatic regions is functionally significant. Extending this further, there may be folding differences even within euchromatic chromosome regions that have significance in terms of overall permissiveness for transcriptional activity, leading to chromosome position effects. Experimentally, a differential, large-scale packing of chromatin over a scale of 0.1–1.5 Mbp has been reported recently for R versus G band chromosome regions; R bands are enriched in active housekeeping genes, and in the specific examples examined showed a more extended structure . The A03_1 HSR forms a compact, tightly folded interphase structure and condenses normally during mitosis. Targeting lac repressor-VP16 to this large, heterochromatic HSR induces a dramatic extension of large-scale chromatin fibers. Therefore, we speculate that highly active genomic regions form more extended large-scale chromonema fibers. This explains why we see the decondensed HSR occupying a much larger territory than observed typically for native chromosome regions of comparable size , since we have now created a 90-Mbp, highly active chromosome region. Normal chromosomes by comparison have smaller active regions dispersed among much larger transcriptionally inactive regions. The VP16-induced extended HSR structures thus may represent much larger, more exaggerated examples of similar changes in large-scale chromatin structure occurring in native chromosomes but confined to small regions. The assumption is that similar opening or extension in large-scale chromatin structure would occur normally over smaller regions, perhaps tens to hundreds of kilobases in size, flanking highly active individual gene loci. Our results demonstrate a specific long-range propagation of chromatin changes in the extension of large-scale chromatin fibers. Rather than a localized decondensation of the lac operator containing DNA segments , we instead saw a generalized straightening or uncoiling of large-scale chromonema fibers, spreading over coamplified genomic DNA flanking the vector repeats . Based on these observations we propose a model in which condensation is the default state for large-scale chromatin packing, with special opening sequences, capable of acting over large distances and perhaps related to the transcriptional machinery, required for euchromatic regions to maintain their extended structure. In our work with gene amplification, we have found that most HSRs assume a compact, chromosome territory-like structure in which highly folded fibrillar components are suggested at light microscopy but not easily delineated. Some HSRs, like the A03_1 HSR, are substantially more compact and do not show obvious fibrillar substructure at light microscopy resolution except when they decondense transiently during DNA replication. At the other extreme, we have isolated several cell lines in which highly extended fibrillar structures are seen for most cells. Our working hypothesis is that these extended HSRs are derived from gene amplification events in which euchromatic, highly active genomic regions containing these opening sequences are coamplified in the flanking DNA. We would predict that naturally occurring gene amplification events, as seen for oncogenes in tumor cells, or the endogenous, early replicating DHFR gene would tend to give rise to more open, extended HSRs than normal chromosomes, since they are derived from amplification of active genomic regions. The frequency of these opening sequences need not be very high if we consider the ability of the large-scale chromatin unfolding to propagate over large distances away from these sites. An example of such a unique sequence might be the β-globin LCR, which importantly is required for both the observed increased nuclease sensitivity surrounding the globin locus and the maintenance of normal, copy number–dependent expression in heterochromatic centromere regions . Transgene repeats and repetitive sequences in this model lead to condensed, large-scale chromatin structures at least in part because of the absence of opening sequences, which may be related to the transcriptional machinery. This model predicts the existence of special sequences, perhaps resembling the globin LCR, that would maintain a more open conformation and would be distributed throughout euchromatic regions. Interestingly, in vivo cross-linking experiments have indicated a wide distribution of binding sites for two homeodomain proteins . It is possible that distributed binding sites for different transcription factors may provide this opening function with LCRs corresponding to extreme examples of particularly active opening sequences. In the context of this model, the clustering of constitutively active, housekeeping genes would lead to a synergistic propagation of large-scale chromatin unfolding extending over the entire region . Gene regulatory sequences with opening activity would reinforce their neighbors in maintaining a larger, chromosome region in an open and transcriptionally permissive environment. This increased active gene frequency in certain chromosome regions would create an alliance against repressive effects of chromatin compaction. The extended structure produced would be important in increasing the general transcriptional potential of this chromosome region, while maintaining this potential during cell differentiation, particularly in cells that show significant global chromatin condensation. | Study | biomedical | en | 0.999996 |
10385517 | The template for the pre-mRNA substrate (T3-RNA1) has been described . Human antisense U2 snRNA was transcribed with T7 polymerase from a pGem-U2 construct digested with EcoRI . 2′- O -allyl oligoribonucleotides complementary to U2 snRNA were generous gifts from Angus Lamond (University of Dundee, Dundee, UK) and have been described by Barabino et al. . The Sm and C′ oligodeoxynucleotides are complementary to nucleotides 84–107 and 148–174 of U2 snRNA, respectively. Splicing factors and snRNPs were fractionated from HeLa cell nuclear extracts by DEAE-Sepharose, heparin-Sepharose, and Mono Q chromatography as described . SF3a and SF3b were further purified by spermine-agarose and Mono S chromatography . In addition, SF3b (Mono S), U1, and U2 snRNPs (Mono Q) were sedimented in glycerol gradients. Fractions were adjusted to 10% glycerol and layered onto 4-ml gradients of 15–40% glycerol in 100 mM KCl, 20 mM Hepes-KOH (pH 7.9), 1 mM MgCl 2 , 0.5 mM PMSF, 0.5 mM DTT, and 0.2 mM EDTA. Sedimentation was performed in a TST60.4 rotor (Kontron) for 8 h at 55,000 rpm. Fractions of 200 μl were collected from the top. U2 snRNPs complexes were assembled in the presence of SF3a and SF3b (Mono S fractions) and the 12S U2 snRNP (Mono Q) as described . The formation of presplicing complexes was performed in the presence of HeLa cell nuclear extract or partially purified splicing factors : SF1, ammonium sulfate–concentrated DS100 ; SF3a, Mono S ; U2AF, HS1000 , U1 snRNP purified by glycerol gradient centrifugation (see above), and the fractions indicated in the figure legends. IgG from monoclonal anti-SF3a66 antibody was bound to protein A–Sepharose (PAS) in NET-2 buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.5 mM DTT, 0.5% NP-40) for 1 h at 4°C. The material was washed with NET-2 to remove unbound IgG. PAS-coupled antibodies were incubated with combinations of SF3a, SF3b, and U2 snRNP for 90 min at 4°C. Unbound material was removed by washing with NET-2 and the samples were divided into three aliquots. Bound components were eluted with SDS sample solution from two aliquots each and separated by SDS-PAGE. Proteins were either stained with silver or blotted onto nitrocellulose membranes followed by detection of the U2 snRNP-specific protein B′′ with mAb4G3 (a gift from W. van Venrooij, University of Nijmegen, Nijmegen, The Netherlands) and Sm proteins with mAbY12 (a gift from J. Steitz, Yale University, New Haven, CT) as described . The third set of aliquots was treated with proteinase K (0.35 mg/ml proteinase K, 2% sarcosyl, 0.1 M Tris-HCl, pH 7.5, 20 mM EDTA) for 30 min at 60°C to release bound U2 snRNA, followed by phenol-chloroform extraction and ethanol precipitation. RNA was separated in a 14% polyacrylamide/8.3 M urea gel followed by Northern blotting and detection with radiolabeled antisense U2 snRNA as described below. U2 snRNP fractions isolated from HeLa cell nuclear extracts or reconstituted from individual purified components were digested with micrococcal nuclease for 10 min at 30°C . RNA was isolated by proteinase K digestion, separated in 14% polyacrylamide/8.3 M urea gels, and blotted onto GeneScreen membranes (Schleicher and Schüll) as described . RNA was cross-linked to the membranes by UV irradiation in a Stratalinker (Stratagene) and the membranes were prehybridized in 5× SSC, 5× Denhardt's solution, 1% SDS, 2.5% dextran sulfate, 50 mM sodium phosphate (pH 6.5), and 1 mg/ml salmon sperm DNA. The membranes were hybridized overnight in the same solution with uniformly labeled antisense U2 snRNA or 5′ endlabeled oligonucleotides at 25–40°C (depending on the oligonucleotides) followed by several washes in 2× SSC/1% SDS at 25°C. Glycerol gradient fractions containing purified U2 snRNPs or SF3b were negatively stained with 2.5% uranyl formate by the double carbon film method as described . The preparations were examined under a Zeiss EM109 electron microscope with an acceleration voltage of 80 kV and electron micrographs were taken with a magnification of 85,000. The majority of the U2 snRNP in HeLa cell nuclear extracts sediments with ∼17S in glycerol gradients (at 100 mM KCl) and most SF3a is found in the same gradient fractions . In contrast, after chromatography of nuclear extracts on DEAE-Sepharose, heparin-Sepharose, and Mono Q, SF3a and SF3b activity was detected in snRNP-free fractions eluting at ∼0.2–0.28 M KCl . In Western blots with antibodies directed against SF3a or SF3b subunits, the bulk of these proteins is found in the same fractions and only small amounts elute at higher salt concentrations (data not shown). U2 snRNP elutes from Mono Q in a highly reproducible heterogeneous pattern at salt concentrations >0.3 M . To test whether this heterogeneity resulted from a separation of the different forms of U2 snRNP, snRNPs of the Mono Q fractions were separated by nondenaturing PAGE . To visualize U2 snRNPs by autoradiography Mono Q fractions were incubated with a 5′ endlabeled oligoribonucleotide complementary to the 5′ end of U2 snRNA before gel electrophoresis. U2 snRNP eluting at ∼0.3 M KCl (fractions 34–38) migrated similar to U2 snRNP in nuclear extracts and a 17S U2 snRNP control obtained after glycerol gradient sedimentation. U2 snRNP eluting at 0.4 M KCl (fractions 48–50) corresponded in mobility to a particle formed in vitro with the 12S U2 snRNP and a SF3b-containing fraction , thus representing the 15S U2 snRNP. Most U2 snRNP eluted at high salt concentration (∼0.48 M KCl; fractions 54–60) and its migration corresponded to that of the 12S U2 particle. Thus, a large portion of SF3a and SF3b dissociates from the 17S U2 snRNP during the fractionation procedure, which is consistent with the observation that the 17S U2 snRNP is disrupted at salt concentrations >200 mM . As a first step in the identification of SF3b-associated polypeptides, Mono Q fractions containing the 15S U2 snRNP (which by definition should contain the Sm proteins, A′, B′′, and SF3b; see Introduction) were sedimented in a glycerol gradient. Individual gradient fractions were analyzed for their RNA and protein composition. The majority of U2 snRNA is detected in fractions 10–13 and the same fractions are highly enriched in polypeptides of ∼130–150 kD . In addition, a polypeptide of ∼50 kD cofractionates with U2 snRNA. We next tested whether the glycerol gradient fractions containing U2 snRNA were active in the assembly of presplicing complex A, i.e., could substitute for both, the 12S U2 snRNP and SF3b. Reactions were performed in the presence of SF1, SF3a, U2AF, and U1 snRNP which are required for presplicing complex formation in addition to SF3b and U2 snRNP . Complex A was efficiently assembled in the presence of partially purified U2 snRNP and SF3b or the Mono Q 15S U2 snRNP fraction used as the input for glycerol gradient sedimentation, but complex A was not formed in the absence of U2 snRNP and SF3b . Addition of glycerol gradient fractions 10–14 to the reconstitution reaction promoted the assembly of the complex. Complex formation was not observed when only SF3b was omitted from a spliceosome assembly reaction ; thus, we conclude that the 15S U2 snRNP fractions contain functional U2 snRNP and SF3b. We also sedimented partially purified SF3b in glycerol gradients. This step yielded fractions with extremely low activity in A complex formation (data not shown). Loss of activity could have been caused by dilution or inactivation of SF3b during centrifugation or by loss of another component(s) required for spliceosome assembly (see Discussion). As shown in Fig. 2 D, however, the protein content of glycerol gradient–purified SF3b with residual activity in complex formation and the purified 15S U2 snRNP was similar. Four proteins of ∼50, 130, 140, and 155 kD are present in both fractions. A polypeptide of 120 kD present in the 15S U2 snRNP is barely visible in SF3b. This protein is consistently coimmunoprecipitated with the 15S U2 snRNP, but is absent from the 17S U2 particle and does not represent SF3a120 (Nesic, D., and A. Krämer, unpublished observation). At present, it is unknown whether this protein represents a contamination or a 15S U2 snRNP-associated protein. A candidate for a specifically bound protein is Tat-SF1, the human orthologue of Cus2p which has been found associated with the U2 snRNP and interacts with SF3a . Two proteins of 29.5 and 28.5 kD present in the 15S U2 snRNP represent the U2 snRNP-specific A′ and B′′ proteins. These proteins are absent from SF3b fractions as confirmed by Western blotting with specific antibodies . From these results we conclude that SF3b comprises four subunits of 50, 130, 140, and 155 kD, which is further corroborated by immunoprecipitation experiments (see below). It is highly likely that the SF3b subunits correspond to the polypeptides of 53, 120, 150, and 160 kD found associated with the 17S U2 snRNP and to SAP49, 130, 145, and 155 detected by immunoprecipitation with anti-U2 snRNP antibodies . In accordance with the sizes determined for the U2 snRNP-associated SAPs , the SF3b subunits will be referred to as SF3b49, 130, 145, and 155. By nondenaturing PAGE we have shown previously that incorporation of SF3a into the U2 snRNP requires the presence of SF3b . In these experiments weak contacts between the 12S U2 snRNP and SF3a could have been disrupted during gel electrophoresis. Moreover, interactions between SF3a and SF3b could not be tested. We have addressed these issues in an immunoprecipitation experiment. A monoclonal antibody directed against SF3a66 was coupled to PAS and incubated with different combinations of partially purified SF3a, SF3b, and the 12S U2 snRNP . Immunoprecipitates were analyzed for the presence of SF3a and SF3b subunits by SDS PAGE . Other U2 snRNP-associated proteins were detected by Western blotting with antibodies directed against the U2 snRNP-specific protein B′′ and the Sm proteins . The presence of U2 snRNA was analyzed by Northern blotting with radioactively labeled antisense U2 snRNA . mAb66 specifically precipitated the proteins of 60, 66, and 120 kD from the SF3a fraction (lane 6), whereas no polypeptides were precipitated from control reactions containing SF3b and the 12S U2 snRNP (lanes 7 and 8). When SF3a, SF3b, and the 12S U2 snRNP were combined, the SF3a subunits, the proteins found in purified SF3b, B, B′, and B′′ and U2 snRNA were precipitated by mAb66, consistent with the formation of the 17S U2 snRNP (lane 12). In contrast, from reactions in which SF3a was incubated with either SF3b or the 12S U2 snRNP, only the SF3a subunits were precipitated (lanes 9 and 10). These results confirm that both the 12S U2 snRNP and SF3b are essential for the incorporation of SF3a into the particle . Furthermore, SF3a and SF3b do not detectably associate with one another in the absence of the 12S U2 snRNP. Thus, the SF3a interaction site is generated upon binding of SF3b to the 12S U2 snRNP. A requirement of U2 snRNA for the formation of the 17S U2 snRNP was tested by digestion of U2 snRNA with micrococcal nuclease at different stages of the assembly reaction. When the 12S U2 snRNP or a combination of SF3b and the 12S U2 snRNP (lane 14) was digested with micrococcal nuclease before the addition of the remaining components, neither the SF3b subunits nor the 12S U2 snRNP proteins were precipitated by mAb66. However, when the digestion was performed after preincubation of SF3a, SF3b, and the 12S U2 snRNP (lane 15), i.e., after the 17S U2 snRNP had been formed, SF3a, SF3b, and 12S U2 snRNP-associated proteins were found in the mAb66 precipitates and no intact U2 snRNA was detected. These results indicate that the U2 snRNA is required for the formation of the 15S and 17S U2 snRNP particles, but once the 17S U2 snRNP is formed, SF3a, SF3b, and the 12S U2 snRNP proteins remain associated with one another in the absence of intact U2 snRNA. In the Northern blot shown in Fig. 3 D the U2 snRNA was not completely digested, but distinct RNA fragments were protected from micrococcal nuclease digestion (not shown). We exploited this observation to analyze the portions in U2 snRNA that are inaccessible to micrococcal nuclease in the 12S, 15S, and 17S U2 snRNPs due to the binding of proteins. We first determined concentrations of SF3a, SF3b, and the 12S U2 snRNP that allow for the efficient assembly of the 15S and 17S U2 snRNPs without cross-contamination. Fig. 4 A shows that the 12S U2 snRNP was converted quantitatively into the 15S U2 snRNP at the highest concentration of SF3b. Efficient assembly of the 17S U2 snRNP was achieved at the highest concentration of SF3a. No 15S U2 snRNP was detected and only a minor contaminating complex that migrated slightly slower in the native gel than the 12S U2 snRNP was seen. This complex has not been analyzed further. For the protection experiment the 12S U2 snRNP (Mono Q) and 15S and 17S U2 snRNPs assembled under the conditions established above were subjected to micrococcal nuclease digestion. RNA was isolated from untreated and micrococcal nuclease-digested samples and processed for Northern blotting with radioactively labeled oligonucleotides complementary to different regions of U2 snRNA . Protected RNA fragments in the 12S U2 snRNP were only observed with oligonucleotides that are complementary to the 3′ half of U2 snRNA. The Sm oligonucleotide detected a smear of RNA at the bottom of the gel, possibly resulting from protection of the Sm-binding site by the Sm proteins . A smaller doublet and a larger fragment hybridized to oligonucleotide C′ (stem-loop IV); the larger fragment was also detected with oligonucleotide J (stem-loop III). The protection of stem-loop IV of U2 snRNA in the 12S U2 snRNP is consistent with the binding of the U2 snRNP-specific proteins A′ and B′′ to this region . A protection of stem-loop III was not necessarily expected. However, Hamm et al. found that deletion of stem-loop III of U2 snRNA resulted in a destabilization of the binding of the A′ and B′′ proteins in Xenopus oocytes, suggesting that stem-loop III influences the binding of these proteins. In addition, Reveillaud et al. observed a comparable protection even at higher micrococcal nuclease concentrations than those used here. In the 15S U2 snRNP several protected fragments were detected with oligonucleotides A (stem-loop I) and D (stem-loop IIa). The largest of these fragments also hybridized very weakly with oligonucleotide H (stem-loop IIb). (The weak hybridization of oligonucleotide H to this fragment as compared with full-length U2 snRNA could be explained if micrococcal nuclease cleaved within the region complementary to the oligonucleotide, thereby reducing the efficiency of hybridization to the remaining sequences.) Thus, binding of SF3b leads to a protection of the U2 snRNA in a region from stem-loop I to stem-loop IIa, which possibly extends into stem-loop IIb, indicating that the SF3b-binding site is located in the 5′ half of U2 snRNA. In addition, the protection in the 3′ half of U2 snRNA changed. The short doublet of fragments that hybridized to oligonucleotide C′ in the 12S U2 snRNP was no longer apparent and the fragment detected with C′ and J appeared more intense. Furthermore, oligonucleotides J and C′ hybridized to a larger doublet of fragments in the 15S U2 snRNP. A faint hybridization was also detected with oligonucleotide Sm, suggesting that the protection extended from stem-loop IV to the Sm-binding site. These changes could be caused either by direct contacts of SF3b with this region or a conformational change in the 3′ half of the U2 snRNP upon binding of SF3b that results in increased protection from micrococcal nuclease digestion by the U2A′ and B′′ and/or the Sm proteins. Binding of SF3a to the 15S U2 snRNP resulted in qualitative changes in the protection pattern of the 3′ half of U2 snRNA as apparent after digestion of the 17S U2 particle. Oligonucleotides Sm, J, and C′ efficiently hybridized to larger RNA fragments and the smaller fragment observed in the 15S U2 snRNP disappeared. Changes in the protection of the 5′ half of U2 snRNA (oligonucleotides A and D) were mainly quantitative in nature, with the exception of the smallest fragment present in the micrococcal nuclease digest of the 15S U2 snRNP but absent from the digest of the 17S U2 snRNP. Moreover, oligonucleotide H hybridized more efficiently to the two largest fragments; thus, the protection in the 5′ half of U2 snRNA extended into the region encompassing nucleotides 70–78 of U2 snRNA. These results suggest that the major site for interaction of SF3a is the 3′ portion of the U2 snRNP. The block of the micrococcal nuclease-sensitive site between the sequences complementary to oligonucleotides Sm and J may position SF3a relatively close to the Sm proteins. The binding of SF3b is affected as well. Because incorporation of SF3a into the U2 snRNP requires both SF3b and the 12S U2 snRNP (see above) it is possible that binding of SF3b to the 5′ half of U2 snRNA is stabilized by interaction with SF3a. Major micrococcal nuclease-sensitive sites were detected only in the region between oligonucleotides H and Sm, i.e., between stem-loop IIb and the Sm-binding site. Thus together, SF3a, SF3b, A′, B′′, and the Sm proteins appear to cover the U2 snRNA in the 17S U2 snRNP almost entirely. The morphology of the 12S and 17S U2 snRNPs has been studied previously by electron microscopy. The 12S U2 snRNP consists of two tightly attached domains . A core domain of ∼8 nm in diameter contains the Sm proteins and a slightly smaller head domain comprises the A′ and B′′ proteins. The 17S U2 snRNP displays a bipartite structure in which two globular domains of 10–12 nm each are connected by a thin RNase-sensitive filament . Based on the morphology and additional biochemical data, it was suggested that one of these domains comprises a large part of the 17S U2-specific proteins. The second domain should contain the 12S U2 domain; however, characteristic features of the 12S U2 snRNP were not apparent in either globular structure of the 17S U2 particle. Based on the results presented above, one might expect that one of the domains is built from the SF3b subunits whereas the second domain could represent SF3a in association with the 12S domain of the U2 snRNP. To test this assumption, glycerol gradient–purified SF3b and 15S U2 snRNP were adsorbed onto carbon film, negatively contrasted with uranyl formate and subjected to electron microscopy. Preparations of SF3b displayed globular structures of uniform size with a diameter of 10–12 nm . Close inspection of the shape of the particles and the accumulation of stain revealed structural details such as indentations and dot-like or elongated high intensity staining. Upon examination of ∼500 detailed views seven representative forms of SF3b that account for ∼80% of the images were distinguished. The images in Fig. 6 A (forms I and II) showed a typical dot-like high intensity stain, which was never located in the center but found in the periphery of the particle. The images are oriented such that the dot is visible in the upper third of the structure. The dot was relatively small (<2 nm) with intensive contrast (forms I and II) or larger (up to 5 nm) with an extended appearance that followed the contour of the particle (form III). A thin filamentous structure protruding from the left side was apparent in images of form III. Structures with a line of stain (oriented horizontally in the images shown) that bisects the particles were observed in forms IV–VI. The line either separated the SF3b particle into roughly two halves (form IV) or an upper third and a lower two-thirds (forms V and VI). Forms V and VI appeared as approximate mirror images with asymmetric bisecting lines that ended in an indentation on the left or right side of the particles, respectively. The last class of particles (from VII) was characterized by a single wedge-shaped indentation oriented upwards in the images shown. In summary, the SF3b protein complex has a globular appearance with a highly structured surface. The dot-like accumulation of stain most likely represents a deep hole or tunnel. The bisecting lines seen in other images could represent side views of such structure. On the other hand, the bisecting line could result from accumulation of stain in a surface furrow at the interface of two proteins. Purified 15S U2 snRNP displayed a bipartite structure in the electron microscope . A globular domain of ∼10–12 nm in diameter was connected by a thin filament to a second domain which was up to 12 nm long and 8 nm wide. The distance between the two domains measured up to 4 nm. The larger domain showed dot-like and elongated areas of high intensity staining, very similar to those observed with isolated SF3b . In addition, the contours of different images of this domain were comparable to those of SF3b, suggesting that the large domain represents the SF3b component of the 15S U2 snRNP. The smaller domain revealed many similarities to the structure of the 12S U2 snRNP determined by Kastner et al. . It consisted of a main body of ∼8 nm in diameter and a closely attached additional structure of 4 nm length and 6 nm width . In addition, a central dot, which is a characteristic feature of the snRNP core domain , was often visible in the main body. A very close similarity was observed between the structures of the isolated 12S U2 snRNP and SF3b and the two domains in the 15S U2 snRNP . Therefore, it is highly likely that the association between SF3b and the 12S U2 snRNP does not result in dramatic structural changes within either of the individual components. Considering the distance between the 12S U2 and SF3b domains visible in most images of the 15S U2 snRNP, direct communication between the individual domains is probably limited. In the 15S U2 particle a thin filament always connects SF3b to the 12S U2 snRNP core domain . In the images shown, the connecting filament protrudes from the left (forms I−IV) or right (forms V and VI) bottom part of the core domain. The smaller structure containing the A′ and B′′ proteins is more distantly located and found opposite of the SF3b domain in most projections. Thus, direct contacts between the SF3b and A′/B′′ proteins are unlikely. The connection to the 12S U2 domain with respect to SF3b can be described as follows. In the SF3b images that display dot-like staining the 12S U2 domain was either found close to or distant from (form II) the stained dot. A close location was usually apparent when the stained dot was large. The 12S U2 domain was located at a distance from the extended structure of high intensity stain in projections of form III. Similar to the corresponding images of isolated SF3b , the filamentous protrusion was visible on the left side of the particle. This protrusion was located in the lower part of the particle but was clearly separated from the filament that connects the 12S U2 to the SF3b domain. In forms IV–VI, which are characterized by a bisecting line, the 12S U2 domain was usually found close to the end of the line . Finally, in form VII the 12S U2 domain was located opposite to the wedge-like indentation. The distance between the SF3b and 12S U2 snRNP domains varies considerably between different forms. The connecting filament is clearly visible in those 15S U2 snRNP images that display a maximal distance between the two domains. In the example shown at higher magnification in Fig. 7 A the connecting filament (with a width of ∼2 nm) exits the core snRNP domain and appears to branch out before connecting to the SF3b domain. Compared with the 15S U2 snRNP the two domains of the 17S U2 particle are closer to one another and the connecting filament appears shorter and thicker . The typical features of the 15S U2 snRNP cannot be recognized in the 17S U2 domains, suggesting structural changes in both domains upon conversion of the 15S into the 17S U2 snRNP. In summary, the positioning of SF3a and SF3b inferred from the nuclease protection experiment agrees well with the morphology of the different particles visualized in the electron microscope. In the 12S U2 snRNP only the 3′ portion of U2 snRNA is protected, consistent with the binding of the Sm and A′/B′′ proteins to the Sm-binding site and stem-loop IV, respectively. Protection of the 5′ half upon binding of SF3b corresponds to the appearance of a novel structural domain in the 15S U2 snRNP that is very similar in size and shape to isolated SF3b . Binding of SF3a induces an extended protection in the 3′ half of U2 snRNA, which is reflected in a different appearance of the 12S domain in the 17S U2 snRNP. In addition, the shape of the SF3b portion of the U2 snRNP is changed in the 17S U2 snRNP, which correlates with a more efficient protection from micrococcal nuclease digestion in the 5′ half of the U2 snRNA . Comparison of polypeptides that are associated with the purified 17S U2 snRNP and SF3a or precipitated from spliceosomes with anti-U2 snRNP antibodies has previously suggested that SF3b consists of at least four subunits of 49, 130, 145, and 155 kD . Here we have demonstrated that SF3b purified from HeLa cell nuclear extracts comprises four subunits of the expected sizes. Polypeptides of identical apparent molecular mass are highly enriched in glycerol gradient fractions of the 15S U2 snRNP and these fractions substitute for the 12S U2 snRNP and SF3b activity in presplicing complex formation. Moreover, only the presumptive SF3b subunits are precipitated from purified or reconstituted 17S U2 snRNP with an antibody directed against SF3a66 in addition to the SF3a subunits, 12S U2 snRNP-associated proteins, and U2 snRNA. Together, the SF3a and SF3b subunits account for seven of the 17S U2 snRNP-specific proteins . Similarly, anti-U2 snRNP antibodies precipitated seven polypeptides in addition to the 12S U2 snRNP proteins from a fraction enriched in U2 snRNP . This raises questions about the 35 and 92-kD polypeptides found associated with the purified 17S U2 snRNP by Behrens et al. . It is possible that these proteins are even less tightly bound to the U2 snRNP than SF3a and SF3b and dissociate from the remainder of the particle during purification or immunoprecipitation procedures. The 35- and 92-kD proteins have not been detected in mAb66 immunoprecipitates of the 17S U2 snRNP, suggesting that they are not required for SF3a, SF3b, and the 12S U2 snRNP to remain stably associated with one another. However, this does not exclude the possibility that these proteins function in the assembly of the 17S U2 snRNP or the prespliceosome. Loss of the 35- and 92-kD proteins from the 15S U2 snRNP or SF3b fractions during glycerol-gradient sedimentation could, for example, explain the low activity of these fractions in presplicing complex formation. Brosi et al. previously established that the 17S U2 snRNP is formed in vitro by stepwise interactions of SF3b and SF3a with the U2 snRNP. The results presented here strongly suggest that the assembly pathway is initiated by contacts of SF3b with the 5′ half of U2 snRNA. First, in the 12S U2 snRNP only the 3′ terminal stem-loops and the Sm-binding site are protected from micrococcal nuclease digestion. In contrast, the region extending from stem-loop I to stem-loop IIa (and to a lesser extent stem-loop IIb) of U2 snRNA, which is fully accessible to micrococcal nuclease digestion in the 12S U2 snRNP, is protected in the reconstituted 15S U2 particle. Second, treatment of the 12S U2 snRNP with micrococcal nuclease before incubation with SF3a and SF3b prevents the assembly of the 17S U2 snRNP, indicating that at least part of the U2 snRNA is required for stable contacts. Moreover, visualization by electron microscopy demonstrated that the 15S U2 particle contains two structurally distinct domains. The smaller domain exhibits the typical features of the single domain of the 12S U2 snRNP, whereas the characteristic structure of SF3b is conserved in the larger domain. The domains are connected by a thin filament, which most likely represents U2 snRNA, in analogy to the 17S U2 snRNP where a corresponding connection is RNase-sensitive . The interpretation of these data is in agreement with and extends results of previous studies. Temsamani et al. reconstituted the U2 snRNP in a cytoplasmic S100 extract from synthetic U2 snRNA. Upon incubation in nuclear extract the particles (which exhibited a high mobility in native polyacrylamide gels) were converted to low mobility complexes that almost certainly correspond to the 17S U2 snRNP. Oligonucleotide-directed RNase H cleavage of the 5′ end of U2 snRNA and the branch site interaction sequence before incubation in the nuclear extract abolished 17S U2 snRNP formation. Behrens et al. reported that the 5′ end of the U2 snRNA was cleaved more efficiently by RNase H in the presence of a complementary oligonucleotide in the 17S than in the 12S U2 snRNP. In addition, nucleotides C8 and C10 were more accessible to chemical modification in the 17S than in the 12S U2 snRNP. Together these data suggested a structural change in the 5′ portion of U2 snRNA upon binding of the 17S U2-specific proteins that results in a (partial) melting of stem I and thus an increase in the potential for intermolecular base pairing. Furthermore, strengthening of stem-loop I by mutation inhibited splicing in a mammalian extract , which could reflect an interference with the binding of SF3b. U2 snRNA sequences on both sides of stem I engage in base pairing interactions with U6 snRNA . Therefore, it is intriguing to speculate that one or more SF3b subunits are directly involved in presenting this region of U2 to U6 snRNA for the formation of helix Ia/Ib which is an essential element of the catalytic center of the spliceosome both in mammals and yeast. The protection of the region encompassing stem-loop IIa in the 15S (and 17S) U2 snRNP is in good agreement with the result that chemical modification of nucleotides C40, G42, and C45 is less efficient in the 17S than the 12S U2 snRNP . Moreover, deletion of nucleotides 46–49 prevents the assembly of the 17S U2 snRNP . In addition, a mutation in Cus1p, the S . cerevisiae homologue of SF3b145 , suppresses mutations in yeast U2 snRNA that correspond to nucleotides 39–44, 52, and 61 of human U2 snRNA . Based on these results and the evolutionary conservation of SF3b145 and Cus1p we assume that the protection of stem-loop IIa reflects the binding of SF3b145. Preliminary evidence indicates that SF3b145, SF3b49, and one of the other large SF3b subunits cross-link to U2 snRNA (Gröning, K., and A. Krämer, unpublished observation). Moreover, Cus1p is apparently associated with an RNA-binding activity . Thus, it is highly likely that SF3b145 binds directly to stem-loop IIa of U2 snRNA. Whether or not the branch site interaction sequence of U2 snRNA is involved in interactions with the SF3b subunits is unclear. We have not detected any micrococcal nuclease-sensitive sites between stem-loops I and IIa in the 15S or 17S U2 snRNPs. However, this region is equally well accessible to oligonucleotide-targeted RNase H digestion and chemical modification in the 12S and 17S U2 snRNPs . This discrepancy could be explained by a different availability of the branch site interaction region to oligonucleotide-targeted RNase H or micrococcal nuclease digestion or to relatively mild conditions used for nuclease treatment in this report. The observation that U2 snRNAs carrying mutations within the branch site interaction region fail to assemble into the 17S U2 snRNP argues in favor of a contribution of these sequences to the assembly of the active U2 snRNP. Clearly, this issue needs further clarification. During the formation of the 15S U2 snRNP we also observed changes in the protection pattern in the 12S domain, in that the protection of stem-loops III and IV is more efficient and a weak protection of the Sm-binding site is apparent. Equivalent changes in the structure of the subdomains of the 15S U2 snRNP compared with isolated SF3b or the 12S U2 snRNP are not visible in the electron microscope. In fact, the domain containing the A′ and B′′ proteins appears to be located at a distance from the SF3b domain; therefore, we believe that direct contacts between these proteins are limited. Although we cannot rule out interactions between SF3b and the Sm proteins that may not be resolved in the electron microscope, we favor the notion that binding of SF3b to the 5′ half of U2 snRNA results in a subtle conformational change in the 12S domain. SF3a does not stably interact with either SF3b or the 12S U2 snRNP alone , indicating that both components contribute to the binding site for SF3a. Consistent with this, in the 17S U2 snRNP we observed an extended protection from micrococcal nuclease digestion that encompasses the Sm binding site and stem-loops III and IV of U2 snRNA as well as a more efficient protection of the 5′ half. In addition, we observed differences in the morphology of the 17S U2 snRNP in the electron microscope. Like the 15S U2 snRNP, the 17S particle is composed of two domains, both of which approximately correspond in size to the SF3b domain of the 15S U2 snRNP. However, structural details of neither the SF3b nor 12S domain are apparent in the two globular structures of the 17S U2 particle. Together, the results from micrococcal nuclease protection and electron microscopic analysis suggest that the main binding site for SF3a is the 12S domain of the U2 snRNP, thus contributing to the increase in size. The chemical modification pattern in the 3′ half of U2 snRNA was identical in the 12S and 17S U2 snRNP , which was interpreted to mean that most 17S U2 snRNP-specific proteins associate with the 5′ portion of the U2 snRNP. On the other hand, it is possible that binding of SF3a does not occur by tight protein–RNA interactions or induce substantial structural changes in the RNA, but may rely mainly on protein–protein interactions. Good candidates for interacting proteins are the A′ and/or B′′ proteins, because deletion of their binding site (nucleotides 154–167) prevents the assembly of the 17S U2 snRNP . Thus, the integrity of the 3′ end of U2 snRNA and/or the presence of the A′ and B′′ proteins may be necessary for binding of SF3a. An interaction of SF3a with these proteins (or this region of U2 snRNA) may also explain why U2 snRNAs carrying mutations in loop IV were inactive in mammalian splicing . Contacts with the Sm proteins are also possible, given that no micrococcal nuclease sensitive sites were detected between stem-loop IV and the Sm-binding site. In fact, the major micrococcal nuclease-sensitive site between sequences complementary to oligonucleotides Sm and J in the 15S U2 snRNP is completely protected in the 17S U2 particle, which locates SF3a close to the Sm proteins. The increased efficiency of micrococcal nuclease protection in the 5′ half of U2 snRNA observed upon binding of SF3a can be interpreted by stabilization of the interaction of SF3b with the U2 snRNA. This could either be the result of a structural change induced by binding of SF3a to the 3′ portion of U2 snRNP or by direct contacts between the SF3a and SF3b subunits. The idea of direct contacts between these proteins is supported by the result that the SF3a and SF3b subunits as well as the B′′, B, and B′ proteins are coprecipitated after micrococcal nuclease digestion of the reconstituted 17S U2 snRNP. Evidence for interactions between SF3a and SF3b or U2 snRNA comes from studies in S . cerevisiae . First, mutations in homologues of the SF3a subunits (Prp9p, Prp11p, and Prp21p) are synthetic lethal with a number of mutations in stem-loops IIa and IIb of U2 snRNA and the region between the branch site interaction sequence and stem-loop IIa . The same pattern of phenotype was observed with all three mutant proteins, which suggested that they interacted with the U2 snRNP as a functional unit. A candidate for interaction is Cus1p, the homologue of SF3b145. Mutations in Cus1p suppressed mutations in U2 snRNA downstream of the branch site interaction sequence and in stem-loop IIa . The synthetic lethality between mutations in the SF3a subunits and stem IIb may also explain the weak but apparent protection within this region. Second, extra copies of wild-type Cus1p partially suppressed the growth defect of prp11-1, but not prp9-1 or prp21-1, suggesting interactions between SF3b145 and SF3a66 . Third, in a yeast two-hybrid screen the gene product of Yml049c, which represents the yeast orthologue of SF3b130 , was found as a partner of Prp9p . Given these possible interactions, it may be surprising that the 17S U2 snRNP consists of two distinct globular domains in the electron microscope . On the one hand, this discrepancy could be explained if interactions between SF3a and SF3b were indirect and merely resulted in a structural change of the SF3b domain, which may be reflected in a loss of the typical features of this domain upon formation of the 17S U2 snRNP. On the other hand, interactions between SF3a and SF3b could be relatively weak and thus not be visible in the electron microscope. Another possibility is that protein–protein contacts involve structures that are beyond the detection limit of the electron microscope. In summary, the combined data from biochemical and electron microscopic analyses can be incorporated into a model in which SF3b initiates the assembly of the active U2 snRNP by interactions with the 5′ half of U2 snRNA, which positions this splicing factor close to U2 snRNA sequences that are essential for the catalysis of splicing. SF3a then associates with the 3′ portion of the U2 snRNP and, most likely, directly interacts with one or more of the SF3b subunits. | Study | biomedical | en | 0.999997 |
10385518 | Yeast genetic techniques and media were as described by Ausubel et al. . The wild-type strain YRW1 was MATa can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 ade2-1 tfc2::LEU2 pJA230 ( URA3 , TFC2 , and CEN3 ) . The YSC14 strain was derived from YRW1; it survived without the TFIIIA factor because of the presence of a multicopy plasmid (pRSC3) containing a 5S RNA gene under the control of the RNA polymerase III RPR1 promoter as described in Camier et al. . The RPR1-5S gene was mutagenized as described below to create a collection of pOK plasmids. Mutant cells carrying these new plasmids were obtained by transforming YRW1 and selecting the cells that survived without pJA230. The mutant strains contained the following plasmids: YOK69 contained pOK1 plasmid; YOK71 (pOK3); YOK72 (pOK4); YOK73 (pOK5); YOK 74 (pOK6); YOK76 (pOK7); and YOK77 (pOK8). The four nucleotides (GGCA) at the 5′ end of the mature 5S RNA in YSC14 strain were mutagenized according to the two-step PCR method described by Higuchi et al. . The sequence at the junction between the RPR1 promoter and the 5S DNA was GATT GGCA GGTT ; the italic sequence corresponds to the 5′ end of the wild-type 5S RNA and the underlined sequence corresponds to the sequence that was mutagenized. Two overlapping PCR fragments, one corresponding to the 5′ part of the RPR1-5S (from the 5′ end of the gene to downstream of the mutagenesis site), and the other to the 3′ part of the RPR1-5S (from upstream of the mutagenesis site to the 3′ end of the gene) were synthesized. These two PCR products were obtained using mutagenic primers and 5′ and 3′ primers containing a BamHI restriction site. Mutagenic PCR products were used as templates for a third PCR reaction to synthesize a complete RPR1-5S gene with mutations on both strands. The mutagenic primers for the synthesis of the 5′ fragments were: d(TGCGATT (T/C)(T/C)CT GGTTGCGGCCATAT ) for the preparation of plasmids pOK1, pOK3, pOK5, and pOK6, or d(TGCGATT GGTTGCGGCCATAT ) for the deletion of the GGCA sequence (plasmid pOK7). For the synthesis of the 3′ PCR fragments, the primers were d( GCAACC AG(A/G)(A/G) AATCGCAGCTCCC) for pOK1, 4, 5, and 6, or d( GCAACC AATCGCAGCTCCC) for the preparation of pOK7. The third PCR combined 1 pmol of each 5′ and 3′ PCR products and the 5′ and 3′ primers containing the BamHI restriction site. The resulting PCR fragments were digested with BamHI. The pOK plasmids derived from the pRSC3 plasmid by exchange of the BamHI fragment containing the wild-type RPR1-5S gene with the BamHI fragment containing a mutated RPR1-5S gene. The nature of the mutations was determined by sequencing. The pOK3 and pOK8 plasmids contained two repeats of the BamHI fragment from pOK4 and pOK1, respectively. RNAs were extracted as described by Schmitt et al. with some modifications. Cells corresponding to 10 ml of culture at an OD 600nm 0.4–0.6 were resuspended in 250 μl of 50 mM sodium acetate, pH 5.3, 10 mM EDTA, plus 25 μl of 10% SDS, mixed with 150 μl of acid-washed glass beads and 250 μl of phenol, vortexed vigorously, and incubated for 5 min at 65°C with intermittent vortexing. RNAs were precipitated with 0.3 M sodium acetate, pH 5.3, and 3 vol of ethanol at −20°C overnight. Small RNAs were separated on a denaturing 8% polyacrylamide gel and visualized by ethidium bromide staining. RNAs ranging from 1 to 5 μg were analyzed on 8% denaturing polyacrylamide gels and visualized by ethidium bromide staining. The gel picture was registered with an enhanced analysis system (E.A.S.Y.; Herolab) and the amount of each species was measured by densitometry using the NIH Image program. The curves for each RNA species were used to measure the average ratio between the amount of two RNA species (5S RNA and tRNA and 5.8S rRNA and 5S RNA). These experiments were reproduced at least three times for each strain. High molecular weight rRNAs were analyzed on 1.2% agarose gels after denaturation with glyoxal and dimethylsulfoxide as described in Ausubel et al. . After a run of 14 h at 55 V in 10 mM NaHPO 4 , pH 7.0, RNAs were transferred to a positively charged nylon membrane ( Boehringer Mannheim ) and cross-linked with UV. rRNAs were visualized by staining the membrane with 0.04% methylene blue in 0.5 M sodium acetate and 3 H-labeled rRNAs were detected by spraying the membrane with EN 3 HANCE ® ( DuPont ) and autoradiographed at −70°C. The 5′ end of 5S RNAs was identified by primer extension using the MMLV reverse transcriptase and the internal 5S RNA oligonucleotide GTCAGGCTCTTACCAGCTTAAC as described in Jacquet et al. . The identification of the 3′ end of the mutant 5S RNAs was carried out by sequencing using either chemical modifications or ribonuclease digestions as described below. The mutant 5S RNAs were isolated from denaturing gels and 3′ end was labeled with [ 32 P]pCp using T4 RNA ligase. The two labeled bands were separated on an 8% sequencing polyacrylamide gel, isolated, and analyzed separately. The chemical sequence was carried out using standard reactions as described in England et al. using diethylpyrocarbonate for the modification of A, dimethylsulfate for G, aqueous hydrazine for U, and 3 M NaCl in anhydrous hydrazine for C. After the modifications of the bases, the RNAs were digested with aniline and separated on a sequencing gel. The ribonuclease sequencing of the 3′ end–labeled 5S RNAs was performed as described in Donis-Keller et al. with modifications: 5S RNAs were partially digested with RNase T1 (that cleaves next to G residues) or RNase A (that cleaves after C and U). The products resulting from the two digestions were analyzed on a 6% denaturing polyacrylamide gel. The results obtained with the two methods were identical. The 5′ end of the 25S rRNA was analyzed by primer extension and the 3′ end of the 7S pre-rRNA precursor was analyzed by an RNase protection assay. Primer extension analysis was done according to the method described by Hong et al. . The 5′ end 32 P-labeled oligonucleotide for the analysis of the 5′ end of the 25S rRNA (cleavage at site C1) was TACTAAGGCAATCCCGGTTGG . The RNase protection assay was carried out using a 35 S-labeled transcript complementary to the ITS2 sequence, prepared with T7 RNA polymerase. The T7 template corresponds to a DNA fragment encoding the major part of the ITS2 sequence (nucleotides 1–215), amplified by PCR, using oligonucleotides CCTTCTCAAACATTCTGTTTGGTAG and GCCTAGACGCTCTCTTCTTA as primers and cloned under a T7 RNA polymerase promoter. Total yeast RNA was hybridized with T7 RNA transcripts and the resulting RNA duplexes were digested with a mixture of RNase A and RNase T1 and analyzed on a denaturing gel . Yeast polyribosomes were fractionated by sucrose gradients . Cells were grown in 100 ml of yeast extract/peptone/glucose (YPD) 1 medium at an OD 600nm 0.6–0.8. 50 μg/ml of cycloheximide was added to the culture, which was immediately transferred to ice. After 10 min, cells were collected by centrifugation and washed with lysis buffer containing 10 mM Tris-Cl, pH 7.4, 100 mM NaCl, 30 mM MgCl 2 , 50 μg/ml of cycloheximide, and 200 μg/ml of heparin. They were resuspended in two volumes of lysis buffer and one volume of acid-washed glass beads. The cells were disrupted by vortexing the suspension eight times for 15 s with a 30 s period of cooling on ice after each vortexing step. The samples were spun at 5,000 rpm for 5 min and the supernatants were centrifuged at 10,000 rpm for 10 min. A portion of the supernatant corresponding to 10 OD units at 260 nm was layered over an 11-ml linear sucrose gradient (7–47% wt/vol) containing 50 mM Tris-acetate, pH 7.6, 50 mM NH 4 Cl, 12 mM MgCl 2 , and 1 mM DTT. The sucrose gradients were centrifuged at 39,000 rpm for 2.5 h in an SW41 rotor (Beckman) and analyzed at 254 nm using a density gradient fractionator (model 640; Isco). Ribosomal subunit quantification was done according to the method described by Moritz et al. . Cycloheximide, heparin, and MgCl 2 were omitted from all buffers, and ribosomal subunits were separated on high salt (0.5 M KCl) sucrose velocity gradients. For pulse-chase labeling of pre-rRNA, 10 ml of cells growing in a synthetic medium without methionine at an OD 600nm 0.3–0.4 were labeled with 70 μCi/ml of [ methyl - 3 H]methionine for 1 min at 30°C. Unlabeled methionine was added to a final concentration of 5 mM and incubation was continued for various periods of time as indicated in the figures. Samples (2 ml) were centrifuged at room temperature. Cell pellets were immediately frozen in a dry ice/ethanol bath and stored at −20°C until RNA extraction and analysis as described above. The time of harvesting and freezing of the cells (1 min) was included in the chase times. Overproduction of the rpL5 protein was obtained by transforming the wild-type and mutant strains with the multicopy plasmid JW2601 carrying the RPL5 gene under the control of its own promoter. JW2601 was a gift of Dr. J.L. Woolford (Carnegie-Mellon University, Pittsburgh, PA), and it was derived from YEp352 ( URA3 , 2 μm). The open reading frame encoding the Lhp1 protein was a gift of Dr. S.G. Clarkson (Département de Génétique et de Microbiologie, Genève, Switzerland) . The LHP1 gene was cloned into the multicopy vector pFL44L ( URA3 , 2 μm) under the control of its own promoter (pFL44-LHP1) (Arrebola, R., personal communication). As a control, the wild-type and mutant strains were transformed with pFL44L ( URA3 , 2 μm) without insert. Strains were grown under selective conditions in a synthetic medium without uracil (−Ura) at an OD 600nm 0.2, diluted 10–10 3 -fold and spotted on −Ura plates. The plates were incubated at 30°C or 37°C for various periods of time as indicated in the figure legend. Photographs were scanned and arranged with Adobe Photoshop to have a homogenous black background. The preparation of cells for electron microscopy and freeze electron substitution was as described by Leger-Silvestre et al. . For all observations, the grids were contrasted with saturated aqueous uranyl acetate alone or combined with lead citrate (freeze substitution grids) and imaged in a JEOL-1200EX electron microscope operating at 80 kV. The grids for immunoelectron microscopy were pretreated for 15 min with PBS buffer, pH 7.6, containing 2% BSA and incubated for 2 h at room temperature with anti-Nop1 mAbs (1:10) provided by Dr. J. Aris (University of Florida, Gainesville, FL). The grids were washed for 30 min with PBS buffer containing 1% BSA and transferred for 1 h to colloidal gold-conjugate goat anti–mouse diluted 1:80 in the same buffer. After incubation, the grids were washed for 20 min in PBS buffer and 10 min in water, and air-dried. Controls were performed using gold-labeled antiserum alone. No labeling was detected on these grids. For in situ detection of ribosomal RNAs, a 35S pre-rRNA probe was synthesized by nick-translation in the presence of digoxigenin-11dUTP ( Boehringer Mannheim ) from a plasmid containing an rDNA unit. The probes for the 25S and 18S rRNA were synthesized by random priming using a DIG-High Prime kit ( Boehringer Mannheim ) from isolated restriction DNA fragments: the 3,533-bp KpnI-HindIII fragment for the 25S rRNA probe, or the 1,954-bp EcoRI-EcoRI fragment for the 18S rRNA probe. The probes were diluted (final, 2 ng/ml) in a hybridization medium containing 50% formamide and 10% dextran sulfate in 1× SSC. 20 μl of probe was added on the grids and denatured for 5 min at 75°C. Hybridization was carried out overnight in a wet chamber at 37°C. The grids were washed three times for 5 min in 50% formamide/2× SSC at 45°C, three times for 5 min in 0.1× SSC at 60°C, and a few seconds in 4× SSC at room temperature. Nonspecific immunological sites were blocked for 30 min with 5% BSA in 4× SSC. The probe was detected with an antidigoxigenin antibody gold-conjugate diluted 1:20 in 4× SSC. The grids were washed three times for 5 min each with 4× SSC and air-dried. For the specific detection of the ribosomal DNA, grids were pretreated before hybridization with DNase-free RNase at 100 μg/ml in 2× SSC for 1 h at 37°C. Grids were washed three times for 5 min in 2× SSC at room temperature and dehydrated through an ethanol series of 70, 90, and 100%. A collection of yeast strains surviving with mutated 5S RNA has been constructed using a genetic system described previously . In these strains, transcription of the endogenous 5S RNA genes was abolished by inactivating the gene coding for the transcription factor TFIIIA. The cells survive with 5S RNA expressed from a chimeric gene that does not require TFIIIA. The RPR1 promoter fused to the 5S RNA gene is transcribed by RNA polymerase III to give an RPR1-5S RNA precursor that is processed into 5S RNA molecules with extended 5′ and 3′ ends. The 5′ extremity contains four additional 5′ nucleotides GGCA (Table I , strain YSC14) and the 3′ extremity is heterogeneous, with 1–3 additional residues (data not shown). In wild-type 5S RNA, the 5′ and 3′ ends of the molecules are paired in a stem called helix I; the additional nucleotides could be part of an extended helix I and protected from degradation during RNA processing . To remove the extra nucleotides, mutations, which could potentially decrease interactions between the 5′ and 3′ ends of the 5S RNA, were introduced in the sequence upstream of the 5S RNA gene (Table I , strains YOK69, YOK73, YOK72, and YOK74). A deletion mutant was also constructed that removed the last four nucleotides of the RPR1 sequence at the junction with the 5S RNA gene (Table I , strain YOK76). All the mutations gave rise to functional 5S RNA, since the cells could grow in the absence of TFIIIA, at 30°C (Table I ). Small RNAs were prepared from all mutants and analyzed on a denaturing polyacrylamide gel . No mutation restored the normal size of the 5S RNA. The 5S RNA of all mutants migrated as two RNA bands of 125- and 127-nt long. The analysis of their 5′ and 3′ extremities by primer extension and RNA sequencing, respectively, showed that all the mutated 5S RNA still contained the 5′ and 3′ nucleotide extensions (data not shown). The cells surviving with mutant 5S RNA displayed very different growth rates both at 30°C and 37°C and presented defects in cell and colony morphology . The cells had a hyperpolarized growth and did not separate well after division, which resulted in filaments. These defects were particularly pronounced in slow growing mutants . The fact that the growth rate at 30°C and 37°C, and that the cell morphology could be improved by varying the mutations in the RPR1-5S gene indicated that these defects were due to the mutated 5S RNA rather than to the absence of TFIIIA. Duplication of the RPR1-5S insert in two different mutants resulted in a growth rate improvement both at 30°C and 37°C . This result suggested that the different growth rates reflected in part the amount of stable 5S RNA in the cell. The ratio between the amounts of tRNA and 5S RNA was determined by densitometry and found to be ∼2 in the wild-type cells and between 3 and >7 in the mutants (Table I ). The decrease in the amount of 5S RNA correlated well with the decrease in the cell growth rate. Apparently, mutated 5S RNA accumulated at different levels depending on the sequence located at the 5′ end of the molecule, and the different levels of 5S RNA determined the growth rate of the mutant cells. Woolford and co-workers have shown that free 5S RNA was not stable in vivo and that its stabilization required the binding of ribosomal protein rpL5 . One of the major binding sites of rpL5 to 5S RNA is helix I; the presence of extra nucleotides adjacent to that helix could perturb the binding of rpL5 and, therefore, result in decreased amount of stable 5S RNA. We found that in the slow growing mutants (YOK74, YOK76, YOK72, YOK73, and YOK71), the overexpression of the gene encoding rpL5 improved the growth rate at 30°C and 37°C . The amount of 5S RNA, analyzed in the most altered mutant (YOK74), was increased upon RPL5 overexpression, which is likely the cause of the improved growth rate (Table II ). Similar experiments were conducted with the LHP1 gene encoding the yeast homologue of the La protein, known to interact with the 3′ end of RNA polymerase III transcripts . La overexpression also led to an improved growth rate and resulted in an increased amount of stable 5S RNA . In this case, the nature of the mutant 5S RNA was modified, and the 5S RNA now migrates as a single band, corresponding to the slow migrating band of the doublet on denaturing polyacrylamide gels (data not shown). The 5′ end of 5S RNA, as determined by primer extension, was unchanged, suggesting that the increase in size was due to the protection of the extra 3′ nucleotides by bound Lhp1p. The increase in the amount of 5S RNA by overxpressing the LHP1 gene could reflect the existence of an in vivo pool of 5S RNA-Lhp1p. Alternatively, the slow migrating RNA band generated by LHP1 overexpression could have a higher affinity for rpL5 than the fastest migrating band and, therefore, be better stabilized. When analyzing the small RNA species present in 5S mutants, we noticed that whereas the ratio between tRNA and 5S RNA increased severalfold in slow growing cells, the ratio between 5.8S rRNA and 5S RNA remained practically constant, similar to the wild-type value (Table I ). This result indicated that the amount of 5.8S rRNA decreased concomitantly with the amount of 5S RNA. Since 5S RNA and 5.8S rRNA are part of the same large 60S ribosomal subunit, we analyzed the content in ribosomal subunits in the mutant strains. Polyribosomes and free ribosomal particles were separated on sucrose velocity gradients from wild-type cells and two different 5S mutants . Mutant ribosomal profiles showed marked alterations that were more accentuated in the slow growth mutant, YSC14, than in a better growing mutant, YOK77. First, the total amount of polyribosomes was significantly reduced in both mutants, suggesting a less efficient protein synthesis rate compared with wild-type cells. Second, the ratio between the amount of free 60S and free 40S ribosomal subunits was reversed in the mutants because of the accumulation of 40S subunits and a reduction in 60S particles. The imbalance between the 60S and 40S subunits was also evidenced by the presence of additional peaks sedimenting more quickly than the 80S monoribosomes or the polyribosomes. These peaks are likely to represent halfmers polyribosomes that contain mRNA associated with an integral number of ribosomes plus a stalled 48S preinitiation complex. The presence of halfmers can be an indication of a decreased amount of 60S subunits. To confirm the deficiency in 60S subunits, ribosomal profiles were run under conditions that dissociate the ribosomes. The ratio between the 60S and 40S subunits, which is ∼2 for wild-type cells, decreased to 1 in YSC14 cells (data not shown). Therefore, the decreased amount of 5S RNA observed in the mutant cells is accompanied by a decrease in the total amount of 60S subunits. This decrease very likely alters the protein synthesis rate that results in decreased growth rate. The shortage in mature 60S subunits could reflect the misincorporation of the mutant 5S RNA in the pre-60S particles, which in turn would lead to the accumulation of partially assembled 60S subunits. This possibility was particularly interesting since in all the mutant profiles analyzed, we reproducibly observed an additional peak sedimenting as a 35S particle, absent in the wild-type cells and whose intensity was particularly high in the YSC14 strain. However, the analysis of the RNA content of the peak showed that it did not contain any ribosomal RNA, but instead an RNA species of 2600 nt that corresponded to the 20S single-strand viral RNA (data not shown) . We did not identify any other peak that could represent the partially assembled 60S subunit. The analysis of the 5S RNA content in the 60S subunits showed that the different mutant 5S RNA species were all incorporated in the subunits, which shows that the decrease in 60S was not due to the selective incorporation of only some of these species. We explored the possibility that 5S RNA could play a role in the biogenesis of the 60S subunit and analyzed the preribosomal RNA processing steps in the mutants . The fate of the ribosomal precursors was followed in the WT strain and the YOK77 and YSC14 mutants by pulse-chase experiments after labeling with methyl- [ 3 H]methionine. The methylation patterns were quantified and the efficiencies of maturation of the different species were measured (Table III ). The efficiency of maturation of the 20S pre-RNA into 18S mature rRNA was similar in the mutants and the WT, ∼70% after a 2.5-min chase and 90% after a 5.5-min chase (Table III , ε 18 ). In sharp contrast, the accumulation of the mature 25S rRNA took much longer in the mutant cells compared with the WT, with a more pronounced defect in the YSC14 strain: after a 2.5-min chase no mature 25S rRNA was detected in YSC14 cells, only 15% in YOK77 cells, whereas the maturation was 70% complete in the WT cells (Table III , ε 25 ). The delay in the formation of the mature 25S rRNA was due to the delay of one specific step corresponding to the maturation of the 27SB precursor. The processing of the 27SA pre-RNA was not detectably altered since after a 2.5-min chase, most of the 27SA pre-RNA had been converted into 27SB pre-RNA in the three cell types (Table III , ε B ). No 32S pre-RNA or 35S pre-RNA was detected in the experiments, which suggests that the maturation of these species was not significantly altered in the mutants. Similar pulse-chase experiments performed in the presence of [ 3 H]uracil instead of [ 3 H]methionine also showed a specific delay in the processing of the 27SB precursor, which was again more pronounced in the YSC14 strain as compared with the YOK77 strain (data not shown). Therefore, the delay observed was not due to a delay in the methylation reaction. The processing of the 27SB precursor consists in the removal of a part of the internal transcribed spacer 2 (ITS2) between sites C1 and C2, through a still unknown mechanism . We analyzed the C1 and C2 cleavage sites by primer extension and RNase protection experiments, respectively. No difference was found between the 5S mutants and the wild-type cells (data not shown). Therefore, the 5S RNA mutations did not alter the cleavage sites in the precursor but had a strong effect on the processing reaction rate. The decreased processing rate of the RNA of the large subunit is likely at the origin of the decreased growth rate. There is indeed a good correlation between the processing rate and the growth rate, as shown for example in the YSC14 mutant, which grows four times more slowly than the WT, and processes the 27SB pre-RNA four times more slowly (half complete maturation of the 25S rRNA is observed after a 6-min chase compared with 1.5-min in the WT). As described above, one main consequence of the 5S mutations was to decrease the amount of stable 5S RNA in the cells. If the low processing rate in the mutants originated at least in part from the lower amount of stable 5S RNA, increasing the amount of 5S RNA by overexpression of the RPL5 or LHP1 genes should speed up the processing reaction. We analyzed the ribosomal processing in the strain YOK74 (the most affected mutant) transformed with either gene . When either rpL5 or Lhp1p was overproduced, the processing rate of the 27SB precursor was improved, since it was 67–90% complete after a 5.5-min chase compared with only half complete in the absence of overproduced protein. We conclude that the amount of stable 5S RNA in the cell influenced the rate of processing of the 27SB precursor, which suggests that the 5S RNA is recruited at this step of the processing pathway and that its recruitment is necessary for the processing. In S. cerevisiae , 5S RNA genes are present in the nucleolus. The absence of endogenous chromosomal 5S transcription and the defects in 60S subunit formation prompted us to analyze the structure of the nucleolus in these mutants. The small size of yeast and its tough cell wall considerably hindered precise structural analysis of its nucleolus by fluorescence and electron microscopy. Recently, by combining cryofixation and cryosubstitution, it has been possible to identify distinct substructures similar to the components of nucleoli of higher eukaryotes: the fibrillar centers, the dense fibrillar component, and the granular component . These techniques coupled with conventional in situ techniques at the electron microscopy level were applied to the analysis of some of the 5S mutants described here. In all mutants, a nucleolus could be morphologically identified. Its structure was similar for all the mutants but differed from a wild-type nucleolus since it appeared less dense, mostly fibrillar with less granular component . These characteristics were more pronounced in slow growing mutants. To appreciate the degree of disorganization of the nucleolus in the 5S mutants, we analyzed the localization of a nucleolar marker, the Nop1 protein, and the ribosomal RNA. The immunolocalization of Nop1p was normal and corresponded to the dense region of the nucleus referred to as the nucleolus . Therefore, the nucleolus of the mutants was not strongly disorganized. The ribosomal RNA was detected by in situ hybridization with digoxigenin-labeled probes complementary either to the 35S rRNA precursor or to the mature 25S rRNA . The results were identical for both probes but differed depending on the strain analyzed. For the wild-type strain, the nuclear labeling was, as expected, concentrated in the nucleolus. No significant labeling appeared in the nucleoplasm except for a few gold particles in the nuclear pores, which likely corresponded to preribosomal particles exported to the cytoplasm. For one of the most altered strains, YSC14, the precursor or the 25S rRNA was totally dispersed throughout the nucleus. For YOK69, one of the fast growing mutants, the situation was intermediate, with the nucleolus being mostly labeled plus regions at the periphery of the nucleoplasm. An accumulation of gold particles near the nuclear envelope was observed with both probes for all the 5S mutants analyzed. When similar experiments were conducted with a probe hybridizing to the 18S rRNA of the small ribosomal subunit, its localization was exclusively nucleolar for both wild-type and 5S mutant cells (data not shown). The ribosomal RNA mislocalized in the nucleus of 5S mutants corresponded, therefore, to the RNA of the large ribosomal subunit. The nuclear localization of the small subunit RNA remained unaffected. The novelty of this paper consists in the observation that 5S RNA plays a role in the processing of the large ribosomal subunit RNA. We show that there is a direct correlation between the rate of processing of the 27SB RNA precursor and the amount of 5S RNA present in the cells. We propose that 5S RNA binds to the preparticle containing the 27SB pre-rRNA and that this binding is necessary for the processing to proceed at a normal rate and give rise to the mature rRNA of the large subunit. This mechanism could participate in a quality control process ensuring that all newly formed mature 60S ribosomal subunits contain stoichiometric amounts of the three rRNA components. We have studied a collection of yeast strains surviving with mutant 5S RNA. The 5S mutations that correspond to nucleotide extensions at the 5′ and 3′ ends of 5S RNA result in decreased accumulation of 5S RNA in the cells, which is possibly due to the lower affinity of the ribosomal protein rpL5 for the mutant 5S RNA. In all the 5S mutants studied here, the decrease in the amount of 5S RNA was paralleled by a decrease in the amount of 60S subunits. No partially assembled 60S subunits devoid of 5S RNA were detected, suggesting that either they were degraded or their formation was impaired. The analysis of the preribosomal processing in the mutants favors the second hypothesis. We found that the rate of processing of 27SB pre-rRNA leading to mature 25S rRNA and 7S pre-rRNA (a precursor to the 5.8S rRNA) was considerably decreased in the 5S mutants. The slower rate was, at least in part, a consequence of the decrease in the amount of 5S RNA since it could be improved by the overproduction of rpL5 or Lhp1p that increased the amount of stable 5S RNA in the cells. We propose that 5S RNA is recruited by the preribosomal particles containing the 27SB precursor and that its binding allows processing to proceed at a normal rate. In the presence of limiting amounts of 5S RNA, 27SB pre-rRNA does not appear to accumulate, as judged by Northern analysis (data not shown) showing that it is eventually degraded. Our observation that 5S RNA plays a role in the formation of the mature 60S subunits is in agreement with previous observations made by other groups. Nazar and collaborators found that expressing mutant 5S RNA in otherwise wild-type cells (i.e. , also containing wild-type 5S RNA) led to a decrease in the production of 60S subunits suggesting that the 5S RNA was necessary for the formation or the stability of the subunit . Additional evidence came from the work of Woolford and collaborators who found that in the absence of rpL5 synthesis, no mature 60S subunits were produced, and of Lee and collaborators who observed a decreased production of 60S subunits in the presence of mutant rpL5 . Although these experiments altered rpL5 levels, they probably reflected the role of 5S RNA as well since rpL5 and 5S RNA are associated in an RNP that binds to the preribosomes. In yeast cells depleted of rpL5 no specific defect of the large ribosomal RNA processing was detected, but the whole processing pathway was slowed down. The delay of all the processing steps probably does not mean that rpL5 plays a role in all these steps. It may rather be due to a feedback mechanism, as proposed for two other yeast mutants (Nip7 and Spb4) altered in the processing of 27SB pre-rRNA and that displayed a delay in the processing of 35S and 32S pre-rRNAs . The maturation step affected by 5S RNA corresponds to two cleavage steps in the ITS2, at site C1 (the 5′ end of mature 25S rRNA) and site C2 (located within ITS2) that release the mature 25S rRNA and the 7S pre-rRNA, which is further processed to yield the mature 5.8S rRNA. No mutant has been isolated that presented a defect in only one of the cleavage steps, which suggests that the two cleavages are connected. The maturation of 27SB pre-rRNA is one of the slowest steps of the pathway, reflecting probably important structural rearrangements and the need to recruit several components , among which is probably the 5S–rpL5 complex. 5S RNA probably does not have a direct role in the cleavage reactions but could well be involved in the correct assembly of a maturation-competent preparticle. Other yeast mutants slowed down in the processing of 27SB pre-rRNA are assembly mutants altered in the ribosomal proteins rpL16 or rpL32 , mutants in putative RNA helicases , and mutants in a methylase . In parallel with the ribosomal processing defects, the 5S RNA mutants presented alterations of the nucleolar structure. Their nucleolus appeared less dense and with a less granular component than a wild-type nucleolus. The modification of the granular compartment is expected since this compartment corresponds to a concentration of preribosomal particles in the late stages of maturation , and the 5S mutants are altered in one of these stages. More unexpected was the delocalization of the large ribosomal subunit RNA throughout the nucleoplasm, which shows that the transport of the preparticles from the nucleolus to the cytoplasm is altered in these mutants. There is not a general defect in the nuclear transport since the localization of the small ribosomal subunit RNA was normal. Interestingly, in yeast mutants altered in a nucleoporin and, therefore, in the transport across the nuclear pores, a similar delocalization of 35S ribosomal RNA throughout the nucleus has been observed (Gas, N., unpublished observations). We propose that mutant preparticles not properly processed are not exported to the cytoplasm, which perturbs the whole trafficking of the presubunits in the nucleus. Very few data are available on the mechanism of transport of the preribosomal particles across the nucleoplasm and the nuclear envelope. There are some indications that transport and nucleolar organization could be linked, since mutants affected in proteins of the mRNA export pathway exhibit alterations of the nucleolus and defects in the preribosomal RNA processing . | Study | biomedical | en | 0.999998 |
10385519 | The yeast strains used in this study are listed in Table I . The diploid strain CBY1830-53 was constructed by a one-step gene disruption procedure ( 53 ), replacing one of the two SLI15 genes in DBY1830 with the sli15-Δ2:: HIS3 allele present on the ∼4.6-kb PvuII-ScaI fragment of pCC923. The sli15-3 strain CCY482-13D-1-1 was constructed by a recombination-mediated two-step gene replacement procedure ( 58 ), first by transforming CCY482-13D to Ura + with the integrating URA3 -plasmid pCC1147 that had been linearized at the unique SnaBI site, and then by screening for Ura − Ts − segregants of the Ura + Ts + transformant. This resulted in the replacement of the chromosomal copy of SLI15 gene in CCY482-13D with the sli15-3 mutant allele present on pCC1147. These gene replacements were confirmed by DNA hybridization. CCY598-49C and CCY598-52B, which contain a LEU2 marker integrated adjacent to the ipl1-2 locus, were constructed by transforming CCY405-10B with the integrating LEU2 -plasmid pCC512 that had been linearized at the unique NdeI site. The resulting Leu + transformant was backcrossed to yield CCY598-49C and CCY598-52B. CCY941-2C, which contains a URA3 marker integrated adjacent to the ipl1-2 locus, was similarly constructed, using the integrating URA3 -plasmid pCC821 that was linearized at the ClaI sites to transform CCY915-2B. The resulting Ura + transformant was backcrossed to yield CCY941-2C. CCY405-10B-2 and CCY1060-1D-4, which contain a LEU2 marker integrated adjacent to the SLI15 and sli15-3 locus, respectively, were constructed by transforming CCY405-10B and CCY1060-1D, respectively, to Leu + with the integrating LEU2 -plasmid pCC912 that had been linearized at the unique NheI site. The Escherichia coli strain DB1142 ( leu pro thr hsdR hsdM recA ) was used routinely as a host for plasmids, except in experiments involving recombinant protein expression, where the E. coli strains TOP10 , BL21 (F − ompT hsdS [ r B − m B − ] gal dcm ), and RR1 ( proA2 leuB6 galK2 xyl-5 mtl-1 ara-14 rpsL20 supE44 hsdS λ − ) were used. Yeast genetic manipulation as well as the preparation of rich medium (YEPD), synthetic complete medium (SC) lacking some amino acids, synthetic minimal medium (SD), and SD with necessary supplements were performed as described ( 52 ). 5-fluoroorotic acid (5-FOA; US Biological) was used at 1 mg/ml for plates to be incubated at 26°C and at 0.5 mg/ml for plates to be incubated at 37°C. Yeast cells were grown at 26°C unless otherwise specified. sli mutations that confer a lethal or very slow-growth phenotype only when combined with the ipl1-2 mutation were identified by a colony sectoring assay ( 2 , 35 ). The details of this genetic screen will be described elsewhere. In brief, an ade2 ade3 ura3 leu2 ipl1-2 haploid strain that contains IPL1 , URA3 , and ADE3 on a 2μ-plasmid (CCY396-8D) can grow well at 26°C even after spontaneous loss of the plasmid. Thus, it forms sectoring (red and white) colonies on medium containing adenine and uracil, and it is resistant to 5-FOA. This strain was mutagenized by treatment with ethyl methanesulfonate, and mutagenized colonies that were nonsectoring and 5-FOA–sensitive (i.e., could not grow well upon loss of the IPL1-URA3-ADE3 –plasmid) at 26°C were identified as potentially containing sli mutations. Standard backcrossing with ade2 ade3 ura3 leu2 ipl1-2 strains that were marked at the ipl1-2 locus with LEU2 (CCY598-49C and CCY598-52B) led to the identification of 23 sli mutant strains, including two that are relevant to this study. As expected, three ipl1 mutant strains were also identified. The cloned SLI15 gene complemented the mutant phenotypes of two of the sli mutants ( sli15-1 and sli15-13 ) (CCY757-1D and CCY822-6B), thus suggesting that these two sli mutants have mutations in the same gene. This was confirmed by linkage analysis. The SLI15 gene was cloned by complementation of the nonsectoring and 5-FOA–sensitive phenotypes of ade2 ade3 ura3 leu2 ipl1-2 sli15-1 cells that contained an IPL1-URA3-ADE3 –plasmid. Strain CCY822-6B was transformed with plasmid DNA from a yeast genomic library constructed in the LEU2 -CEN–plasmid p366 (gift of P. Hieter, University of British Columbia, Vancouver, Canada). Leu + transformants were selected on SC medium lacking leucine. Sectoring transformant colonies were identified and tested for their ability to grow on supplemented SD medium containing 5-FOA. Plasmids were recovered into E. coli from sectoring colonies that were 5-FOA–resistant. These plasmids were retested for their ability to complement the nonsectoring and 5-FOA–sensitive phenotypes of CCY822-6B. From ∼120,000 Leu + transformants screened, we obtained 20 plasmids that contained IPL1 and one plasmid (pCC879) that contained a different sequence. To confirm that pCC879 contained SLI15 , the integrating LEU2 -plasmid pCC912, which contained an ∼4.8-kb SacI-XhoI yeast genomic DNA fragment derived from the insert present within pCC879, was linearized at the unique NheI site present within this fragment and used to transform the ade2 ade3 leu2 ura3 ipl1-2 strain CCY405-10B. The resulting Leu + transformant (CCY405-10B-2) was mated with an ade2 ade3 leu2 ura3 ipl1-2 sli15-1 strain that contained an IPL1-URA3-ADE3 –plasmid (CCY822-7B). Sporulation and tetrad analysis of the resulting diploid revealed absolute linkage between the Leu + and 5-FOA– sensitive phenotypes. Thus, the insert DNA from pCC879 was derived from the SLI15 locus. Mutagenesis of SLI15 was carried out by in vitro error-prone PCR and in vivo gapped-repair as described ( 42 ). In brief, T3 and T7 primers ( Promega ) were used in a PCR reaction with Taq DNA polymerase ( Promega ) to amplify the SLI15 gene present on the low copy number LEU2 -plasmid pCC982. Approximately 0.5 μg of the ∼3.6-kb PCR product and ∼0.5 μg of the ∼7.5-kb NruI-SnaBI fragment of unmutagenized pCC982 were used to transform the yeast strain CCY1022-10C, which contained SLI15 on a URA3 -CEN–plasmid as the only source of SLI15. Leu + transformants were selected at 26°C on SC medium lacking leucine and were tested for their ability to grow at 26 and 37°C on supplemented SD medium lacking leucine but containing uracil and 5-FOA. Transformant colonies that could grow on 5-FOA plates at 26 but not 37°C were chosen. Such transformants were taken from the 5-FOA plates that had been incubated at 26°C and retested for their ability to grow on YEPD plates at 26 but not 37°C. The LEU2 -plasmids were recovered from such Ts − transformants into E. coli and retested for their ability to support growth of CCY1022-10C on 5-FOA plates at 26 but not 37°C. From ∼10,500 Leu + transformants screened by this method, 10 plasmids containing temperature-sensitive sli15 alleles ( sli15-3 to sli15-12 ) were isolated. One such plasmid, pCC1137, contains the sli15-3 mutant allele. Subcloning experiments were routinely carried out with the high copy number plasmids pSM217 and pSM218 (gift of P. Hieter), the low copy number plasmids pRS315 and pRS316 ( 64 ), and the integrating plasmids pRS305, pRS306, and YIp5 ( 58 , 64 ). Plasmids encoding epitope-tagged versions of Ipl1 or Sli15 were constructed by inserting a DNA fragment encoding three tandem copies of the HA- or Myc-epitope (from pMPY-3xHA or pMPY-3xMYC ) into the coding sequence of IPL1 (after the initiation codon) or SLI15 (before the stop codon). The HA-Ipl1 and Sli15-Myc fusion proteins are functional since they can complement the Ts − phenotype of ipl1-2 and sli15-3 cells, respectively (data not shown). Plasmids encoding the GST-Sli15 fusion protein were constructed in pEG(KT) ( 41 ) (for expression in yeast) and pGEX-2T ( 65 ) (for expression in E. coli ). The GST-Sli15 fusion protein is functional since it can complement the inviability phenotype of a sli15-Δ2::HIS3 mutant (data not shown). Plasmids encoding chimeric proteins containing the green fluorescent protein (GFP) ( 24 ) fused to Ipl1 (pCC959) or Sli15 were constructed with pRB2138 ( 11 ). The GFP-Ipl1 and GFP-Sli15 fusion proteins are functional since they can complement the Ts − phenotype of ipl1-2 and the inviability phenotype of sli15-Δ2::HIS3 cells, respectively (data not shown). The plasmid encoding His 6 -Ipl1 , which has six tandem histidine residues fused to the NH 2 terminus of Ipl1, was constructed with pTrcHis A (Invitrogen Corp.). The plasmid encoding the sli15-Δ2::HIS3 mutant allele (pCC923) was constructed by replacing the DNA sequence between the AvrII and NruI sites present in the low copy number URA3 -plasmid pCC883 with the ∼1.8-kb XbaI-SmaI fragment (containing HIS3 ) of pJJ217 ( 30 ). The plasmid encoding the TrpE-Ipl1 fusion protein (pCC134-16) was constructed by inserting the ∼1.5-kb EcoRI DNA fragment of pCC100 ( 13 ) into the EcoRI site of pATH10 ( 34 ), resulting in an in-frame fusion between trpE and codon 45 of IPL1. The TrpE-Ipl1 fusion protein was partially purified from E. coli cells (RR1) harboring pCC134-16 by a previously described method ( 78 ) and used as antigen in injections of guinea pigs. Anti-Ipl1 antibodies were affinity-purified with TrpE-Ipl1 that was immobilized on a nitrocellulose membrane ( 78 ). To prepare extracts from yeast cells that coexpressed HA-Ipl1 and GST or HA-Ipl1 and GST-Sli15, a saturated culture of TD4 that contained the plasmids pCC1128 (encoding HA-Ipl1) and pEG(KT) (encoding GST) or the plasmids pCC1128 and pCC1061 (encoding GST-Sli15) was diluted 20-fold into 50 ml of SC medium lacking uracil and leucine and with 2% raffinose (US Biological) instead of glucose as carbon source. After 6 h at 30°C, galactose (US Biological) was added to a final concentration of 4%, and the culture was incubated at 30°C for another 4 h. Cells were harvested and rinsed once with 10 ml of lysis buffer, which consists of 50 mM Hepes-KOH (pH 7.4), 200 mM KCl, 10% glycerol (vol/vol), 1% NP-40 (vol/vol), 1 mM EDTA, 1 mM dithiothreitol, 25 mM NaF, 1 mM NaVO 4 , and the following protease inhibitors ( Sigma Chemical Co. ): 2 μg/ml each of antipain, leupeptin, pepstatin A, chymostatin, and aprotinin; 10 μg/ml of phenanthroline; 16 μg/ml of benzamidine-HCl; and 1 mM PMSF. Cells were resuspended in 0.45 ml of lysis buffer and aliquoted into three 1.5-ml microcentrifuge tubes. Acid-washed glass beads (425–600-μm diam; Sigma Chemical Co. ) were added to each tube to give a final volume of ∼0.2 ml. After chilling on ice, the tubes were mixed by being vortexed for 1 min. This cycle of chilling and vortexing was repeated six more times. The lysates from the three tubes were combined, followed by a 10-min centrifugation at 20,800 g to remove cell debris. 140-μl aliquots of the resulting supernatant were distributed into three 1.5-ml microcentrifuge tubes, each containing 50 μl of a 50% slurry of glutathione-agarose beads ( Sigma Chemical Co. ) in equilibration buffer (50 mM Hepes-KOH, pH 7.4, 200 mM KCl, 10% glycerol [vol/vol], 1% NP-40 [vol/vol], 1 mM EDTA). The tubes were incubated at 4°C with constant agitation for 2 h. The glutathione-agarose beads were harvested by a 2-min centrifugation at 960 g , followed by one, three, or five 5-min washes with 250 μl of lysis buffer. The proteins bound on the glutathione-agarose beads in each tube were eluted by the addition of 60 μl of sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecyl sulfate [wt/vol], 0.1% bromophenol blue [wt/vol], 10% glycerol [vol/vol]). To prepare extracts from yeast cells that coexpressed HA-Ipl1 and Sli15 or HA-Ipl1 and Sli15-Myc, a saturated culture of TD4 that contained the plasmids pCC1128 (encoding HA-Ipl1) and pCC1192 (encoding Sli15) or the plasmids pCC1128 and pCC1193 (encoding Sli15-Myc) was diluted into 50 ml of SC medium lacking leucine and uracil to give a cell density of ∼2 × 10 6 /ml. After 5 h at 30°C (when cultures had reached a cell density of ∼1.5 × 10 7 /ml), cells were harvested and cell extracts were prepared as described above for GST pulldown experiments, except that lysis buffer with protease inhibitors was replaced by buffer B (50 mM MOPS, pH 7.0, 200 mM KCl, 5 mM dithiothreitol, 10% glycerol [vol/vol], 1% NP-40 [vol/ vol], 25 mM NaF, 0.5 mM NaVO 4 ) with protease inhibitors. After clearing of cell debris, 140 μl of cell lysate was added to a 1.5-ml microcentrifuge tube that contained 50 μl of a 50% slurry of BSA-coated protein A–agarose beads ( Pharmacia ) in buffer A, which consists of 50 mM MOPS, pH 7.0, 200 mM KCl, 3 mM Na azide, 5% glycerol (vol/vol). The tube was incubated at 4°C with constant agitation for 30 min. After a 2-min centrifugation at 960 g , the supernatant was transferred to a 1.5-ml microcentrifuge tube that contained 1 μl of mouse anti-Myc ascites fluid (gift of B. Haarer, University of Texas at Austin, Austin, TX). After a 1-h incubation at 4°C with constant agitation, 50 μl of a 50% slurry of BSA-coated protein A–agarose beads (in buffer A) was added, followed by a 30-min incubation at 4°C with constant agitation. The protein A–agarose beads were harvested by a 2-min centrifugation at 960 g , followed by two 5-min washes with 250 μl of buffer B. The proteins bound on the protein A–agarose beads in each tube were eluted by the addition of 50 μl of sample buffer. To prepare extracts from E. coli that expressed GST, GST-Sli15, His 6 -Ipl1, or His 6 -NonO-C protein, a saturated culture of BL21 that contained the plasmid pGEX-2T (encoding GST) or pCC1062 (encoding GST-Sli15) or a saturated culture of TOP10 that contained the plasmid pCC1167 (encoding His 6 -Ipl1) or pRBD2-nonO (encoding His 6 -NonO-C; gift of C. Huang and P. Tucker, University of Texas at Austin, Austin, TX) was diluted 100-fold into 50 ml of Luria broth with ampicillin. After 3 h at 37°C, IPTG (isopropyl-β- d -thiogalactopyranoside) was added to a final concentration of 1 mM, followed by a 4-h incubation at 26°C. Cells were harvested, rinsed once in 10 ml of ice cold lysis buffer (see above), and resuspended in 2.5 ml of the same buffer. Cells were lysed in a French press at 16,000 psi, and cell debris was removed by a 10-min centrifugation at 20,800 g , thus generating the supernatant fraction. 0.4 ml of supernatant containing GST or GST-Sli15 was added to a 1.5-ml microcentrifuge tube that contained 0.1 ml of a 50% slurry of BSA-coated glutathione-agarose beads in equilibration buffer (see above). After a 0.5-h incubation at 4°C with constant agitation, the supernatant was removed and replaced by 0.8 ml of supernatant that contained His 6 -Ipl1 or His 6 -NonO-C. After a 1.5-h incubation at 4°C with constant agitation, the supernatant was removed and the glutathione-agarose beads were rinsed twice with 1 ml of lysis buffer. The proteins bound on the glutathione-agarose beads were eluted by the addition of 60 μl of sample buffer. Protein samples were electrophoretically separated in SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with 2,000-fold diluted rabbit anti-GST antibodies (Molecular Probes, Inc.), 1,000-fold diluted mouse anti-HA ascites fluid (BABCO), 1,000-fold diluted mouse anti-Myc ascites fluid (gift of B. Haarer, University of Texas at Austin), 200-fold diluted affinity-purified guinea pig anti-Ipl1 antibodies, or 2,500-fold diluted rabbit anti-NonO antibodies (gift of C. Huang and P. Tucker, University of Texas at Austin). Proteins recognized by primary antibodies were visualized by the ECL chemiluminescent system ( Amersham Corp. ). Immunofluorescence staining of yeast cells was carried out as described ( 47 ). In experiments that involved immunostaining of microtubules in yeast cells that expressed GFP fusion proteins, cells were fixed in 3.7% formaldehyde at room temperature for 30 min, followed by standard procedures for immunofluorescence staining ( 47 ), except that the methanol- and acetone-fixation steps were omitted. In experiments that did not involve immunostaining, GFP fusion proteins were observed either in live yeast cells or in cells that had been fixed for 10 min at room temperature in 3.7% formaldehyde. In experiments in which visualization of DNA as well as GFP fusion proteins was desired, 4′,6-diamidino-2-phenylindole (DAPI; Accurate Chemical Co.) was added to the growth medium to a final concentration of 2.5 μg/ml 15 min before preparation of cells for observation. We have shown previously that ipl1-2 mutant cells do not have a uniform arrest phenotype at the restrictive temperature of 37°C ( 13 ). Instead, they go through the cell cycle, missegregate chromosomes severely, undergo cytokinesis, and become inviable. In a temperature-shift experiment, over 20% of the large-budded cells in an asynchronous culture of ipl1-2 cells that had been incubated at 37°C for 3–4 h had clearly failed to segregate chromosomal DNA evenly to the opposite poles of apparently normal looking mitotic spindles ( 13 ). The percentage of ipl1-2 cells that had missegregated chromosomes was probably much higher (see below and reference 8 ), since we were conservative in our scoring of uneven chromosomal DNA masses. To find out whether this failure in chromosome segregation was caused by a failure in sister chromatid separation, we examined the distribution of chromosome V that was marked by the binding of Tet repressor-green fluorescent protein (TetR-GFP) to Tet operator sites located adjacent to the centromere of chromosome V ( 40 ). At both 26°C and after a 2-h incubation at 37°C, ≥90% of large-budded wild-type haploid cells that were in early anaphase had clearly separated sister chromatids, as indicated by the presence of two dots of TetR-GFP signal . As these cells reached late anaphase or early G1, essentially all cells had separated and properly segregated sister chromatids, as indicated by the presence of a single dot of TetR-GFP signal within each of the two evenly segregated chromosomal DNA masses . In contrast, ipl1-2 cells had properly separated and segregated sister chromatids at 26 but not 37°C. After a 2-h incubation at 37°C, among ipl1-2 cells that were in early anaphase, ∼60% had only a single dot of TetR-GFP signal , and ∼22% had two dots that were located unusually close to each other . As ipl1-2 cells reached late anaphase or early G1, only ∼30% of them appeared to have properly separated and segregated the sister chromatids of chromosome V. Approximately 43% had a single dot of TetR-GFP signal , and ∼27% had two dots that were located within only one of the two (often unevenly segregated) chromosomal DNA masses . The presence of a single dot of TetR-GFP signal in ipl1-2 cells suggested that sister chromatids had failed to separate, or that sister chromatids had separated but had failed to be properly segregated to opposite poles, thus resulting in the juxtaposition of two dots of TetR-GFP signal that were scored mistakenly as a single dot ( 70 ). While we cannot distinguish between these two possibilities, the presence of a large fraction of ipl1-2 cells with two dots of TetR-GFP signal within only one of the two chromosomal DNA masses indicated that sister chromatids had separated in these cells, but these separated sister chromatids had failed to segregate away from each other towards the two opposite poles. Thus, if a defect in sister chromatid separation exists in ipl1-2 cells, it cannot be the only cause of chromosome missegregation in these cells, as sister chromatids that have separated also fail to be properly segregated. In addition to the most prominent phenotype of uneven chromosome segregation described in the last section, a small fraction of ipl1-2 cells exhibits some other cytological defects. After 3–4 h at 37°C, ∼10% of the large-budded cells in an asynchronous culture of ipl1-2 cells exhibited defects in nuclear migration and/or mitotic spindle orientation . A smaller fraction of ipl1-2 cells appeared to have monopolar spindles , which are suggestive of defects in spindle pole body duplication or separation. This latter phenotype is more noticeable in ipl1-2 cells that had been presynchronized at G1 before being released into the cell cycle at 37°C (data not shown). To find out whether ipl1-2 cells have normal spindle poles, we examined the localization of Nuf2-GFP in ipl1-2 cells. Previous immunofluorescence and biochemical studies have shown that Nuf2 is associated with the intranuclear region of the spindle pole body ( 45 , 77 ), and Nuf2-GFP is a marker commonly used for observing spindle pole dynamics ( 31 ). Consistent with previous reports, our immunofluorescence study with wild-type cells showed that at 26°C and after a 2–3 h incubation at 37°C, Nuf2-GFP was found concentrated at the spindle poles. In cells that had a mitotic spindle, the intensities of the Nuf2-GFP signal were even at the two poles . In contrast, the localization of Nuf2-GFP appeared abnormal in ipl1-2 cells, especially after a 2–3-h incubation at 37°C. First, Nuf2-GFP was no longer restricted to the spindle poles. Instead, it was also found in many ipl1-2 cells as extra dots that colocalized with mitotic spindles that were short to medium in length . This pattern of Nuf2-GFP was found at much lower frequencies in wild-type cells. A similarly abnormal localization of Nuf2 has been reported in the nuf2-61 mutant ( 45 ), thus suggesting that Nuf2 function may be compromised in ipl1-2 cells. However, we have so far not detected genetic interaction between IPL1 and NUF2. The Ts − phenotype of ipl1-2 or nuf2-61 cells is not complemented by a high copy number plasmid carrying NUF2 or IPL1 , respectively, and ipl1-2 nuf2-61 double mutant cells are no more defective in growth at elevated temperatures than ipl1-2 and nuf2-61 single mutant cells. Furthermore, the abundance and electrophoretic mobility of Nuf2 are not affected in ipl1-2 cells (data not shown). Second, for a large fraction of ipl1-2 cells with mitotic spindles, the intensities of Nuf2-GFP signal were no longer even at the two poles . In extreme cases, the Nuf2-GFP signal was barely or not detectable from one pole . There was a general correlation between the pattern of uneven Nuf2-GFP distribution and the pattern of uneven chromosome segregation. Examination of ipl1-2 cells that were in late anaphase or early G1 revealed that the pole with the greater intensity of Nuf2-GFP signal was most often associated with a greater-than-normal amount of chromosomal DNA, and in no case was stronger Nuf2-GFP signal associated with a smaller-than-normal amount of chromosomal DNA . The presence of ipl1-2 cells with uneven distribution of Nuf2-GFP but apparently even distribution of chromosomal DNA masses, and cells with uneven distribution of chromosomal DNA masses but apparently even distribution of Nuf2-GFP, may simply reflect the intrinsic difficulty in scoring (smaller) differences in the intensity of Nuf2-GFP signal and chromosomal DNA masses. Furthermore, since yeast chromosomes vary greatly in size, chromosomal DNA mass may not be strictly correlated with chromosome number. To find out whether other spindle pole–associated components may also be mislocalized in ipl1-2 cells, we examined the localization of the spindle pole body central plaque component Spc42 ( 55 ). At 26°C, a functional Spc42-GFP fusion protein (gift of J. Kilmartin, Medical Research Council, Cambridge, England) was evenly distributed at opposite spindle poles in 100% of wild-type and ipl1-2 cells. After a 3-h incubation at 37°C, the Spc42-GFP signal at opposite spindle poles was slightly uneven in <3% of wild-type and ipl1-2 cells. Furthermore, Spc42-GFP was never detected along the mitotic spindle in either cell type (data not shown). Thus, Nuf2-GFP, but not Spc42-GFP, becomes distributed abnormally in ipl1-2 cells. This abnormality suggests that potential defects in the spindle poles may contribute to chromosome missegregation in ipl1-2 cells. This subject will be taken up further in the Discussion. To determine the subcellular localization of Ipl1, we generated a fusion gene encoding the GFP and full-length Ipl1. The GFP-IPL1 fusion gene, which was under the control of the ACT1 promoter, was functional (see Materials and Methods). In wild-type yeast cells that carried pCC959, a low level of GFP-Ipl1 signal could be detected in the cytoplasm . The intensity of this cytoplasmic signal varied somewhat in different cells, perhaps due to the small variations in the copy number of pCC959. GFP-Ipl1 was also found enriched in the nucleus, with special concentration on the mitotic spindle apparatus. In unbudded and small-budded cells, GFP-Ipl1 was sometimes found in a dot-like structure that typically colocalized with the edge of the nucleus . Immunofluorescent staining of microtubules in these cells indicated that this dot-like structure represented the spindle pole body (data not shown). In large-budded cells that had completed chromosome segregation, GFP-Ipl1 was often found concentrated on elongated or disassembling spindles . At a lower frequency, GFP-Ipl1 was also found concentrated on mitotic spindles that were short to medium in length. We do not know whether the apparent differences in our ability to detect GFP-Ipl1 on spindles of different lengths were due to cell cycle-specific changes in the localization pattern of GFP-Ipl1, or the possibility that GFP-Ipl1 signal on short/medium-length spindles might be more readily obscured by the overall nuclear signal. We have so far not detected GFP-Ipl1 signal on cytoplasmic microtubules. However, we cannot rule out the possibility that GFP-Ipl1 might be present at low levels on cytoplasmic microtubules. To identify other proteins that may play a role in the Ipl1-mediated chromosome segregation process, we reasoned that nonlethal mutations that lower or abolish the function of proteins that play a positive role in this process (e.g., as substrates or positive regulators of Ipl1) may be tolerated in wild-type cells but not in mutant cells with reduced Ipl1 function ( 21 ). ipl1-2 mutant cells have a normal growth rate at 26°C but they do not have normal Ipl1 protein kinase function, since ipl1-2 cells exhibit an ∼10-fold increase in the frequency of chromosome gain at this temperature ( 8 ). Thus, we carried out a genetic screen at 26°C for nonlethal sli mutations that confer a lethal or very slow-growth phenotype only when combined with the ipl1-2 mutation (see Materials and Methods). Among 23 sli mutants isolated, two contain recessive mutations in the SLI15 gene. The wild-type SLI15 gene was cloned by complementation of the mutant phenotypes of sli15-1 ipl1-2 cells (see Materials and Methods). Subcloning and partial sequencing revealed that SLI15 is identical to YBR156C , which potentially encodes a protein of 698 residues, with a predicted molecular mass of ∼79 kD and a predicted pI of ∼10. The predicted Sli15 protein sequence is not highly similar to that of any protein listed in the sequence databases at the National Center for Biotechnology Information. A putative nuclear localization signal is present at residues 530– 546 ( 48 ), and the region (residues 517–565) surrounding this putative localization signal is predicted to have a high probability of adopting a coiled-coil conformation ( 38 ). It has been reported previously that disruption of YBR156C ( SLI15 ) leads to loss of cell viability ( 51 ). We have confirmed this result by creating a diploid yeast strain that is heterozygous for the sli15-Δ2:: HIS3 null mutation. Tetrad analysis of this diploid strain revealed that sli15-Δ2::HIS3 haploid cells are inviable. Thus, Sli15, like Ipl1, is essential for yeast cell viability. To determine the subcellular localization of Sli15, we generated a fusion gene encoding GFP and full-length Sli15. The GFP-SLI15 fusion gene, which was under the control of the ACT1 promoter, was functional (see Materials and Methods). In wild-type yeast cells, GFP-Sli15 was found to be present at a low level throughout the nucleoplasm . Furthermore, GFP-Sli15 was highly concentrated on mitotic spindles. In unbudded cells, GFP-Sli15 was present in a dot-like structure that represented the spindle pole body, as indicated by its colocalization with the vertex of microtubule staining . In a small fraction of unbudded cells, GFP-Sli15 was also found on what appeared to be intranuclear microtubules. In budded cells, GFP-Sli15 was concentrated on mitotic spindles of all different lengths, including those that were in the process of disassembly . We did not detect GFP-Sli15 signal on cytoplasmic microtubules, but we cannot exclude the possibility that GFP-Sli15 might be present at low levels on cytoplasmic microtubules. The sli15-1 and sli15-13 mutants that we identified originally in the synthetic lethal screen do not have major growth phenotypes at 13–37°C. Thus, we carried out in vitro mutagenesis of SLI15 and screened for sli15 mutant alleles that confer a Ts − growth phenotype at 37°C (see Materials and Methods). One such allele, sli15-3 , was used to replace the chromosomal copy of the wild-type SLI15 gene in a haploid IPL1 strain (see Materials and Methods). The resulting sli15-3 strain is Ts − for growth at ≥33°C . The sli15-3 mutation, like the sli15-1 and sli15-13 mutations, causes a synthetic lethal phenotype at 26°C when combined with the ipl1-2 mutation. Tetrad analysis of a diploid heterozygous for ipl1-2 and sli15-3 revealed that 0 of 7 ipl1-2 sli15-3 haploid meiotic products were viable at 26°C, whereas 12 of 13 ipl1-2 SLI15 haploids and 13 of 13 IPL1 sli15-3 haploids were viable at this temperature. To examine the phenotype of sli15-3 cells, we have carried out temperature-shift experiments with asynchronous cultures of sli15-3 cells. After a 4-h shift to 37°C, only 15– 20% of sli15-3 cells remained viable, and they did not arrest with a uniform cell morphology. Immunofluorescence microscopy showed that >20% of large-budded sli15-3 cells had clearly failed to segregate chromosomal DNA evenly to the opposite poles of apparently normal looking mitotic spindles ; ∼10% of large-budded cells seemed to be defective in nuclear migration and/or mitotic spindle orientation ; and a smaller fraction of large-budded cells appeared to have monopolar spindles . These phenotypes of sli15-3 cells are very similar to those of ipl1-2 cells. We have also examined the separation and segregation of sister chromatids in sli15-3 cells, using the TetR-GFP assay described above. After a 2-h shift to 37°C, among sli15-3 cells that were in early anaphase, ∼53% had only a single dot of TetR-GFP signal , and ∼25% had two dots that were located unusually close to each other . As sli15-3 cells reached late anaphase or early G1, only ∼39% of them appeared to have separated properly and segregated the sister chromatids of chromosome V. Approximately 32% had a single dot of TetR-GFP signal , and ∼29% had two dots that were located within only one of the two (often unevenly segregated) chromosomal DNA masses . Thus sli15-3 cells, like ipl1-2 cells, are defective in sister chromatid segregation (and possibly sister chromatid separation). Finally, we also examined the distribution of Nuf2-GFP in sli15-3 cells. After 2–3 h at 37°C, Nuf2-GFP was no longer restricted to the spindle poles. Instead, it was also found in some sli15-3 cells as extra dots that colocalized with mitotic spindles that were short to medium in length . Furthermore, the intensities of Nuf2-GFP signal were no longer even at the two poles of a large fraction of mitotic spindles . Examination of sli15-3 cells that were in late anaphase or early G1 revealed that the pole with greater intensity of Nuf2-GFP signal was most often associated with a greater-than-normal amount of chromosomal DNA, and in no case was stronger Nuf2-GFP signal associated with a smaller-than-normal amount of chromosomal DNA . These abnormal patterns of Nuf2-GFP distribution are very similar to those observed in ipl1-2 cells. Furthermore, this abnormality is limited to Nuf2-GFP, as a functional Spc42-GFP fusion protein is evenly distributed to the spindle poles in sli15-3 cells (data not shown). The genetic interaction between ipl1-2 and sli15 mutations, the similar phenotypes of ipl1-2 and sli15-3 cells, and the similar subcellular localization of GFP-Ipl1 and GFP-Sli15 suggest that these two proteins may associate with each other. To examine this possibility, we used a high copy number plasmid to express in yeast a functional version of Ipl1 that was fused to the HA-epitope (HA-Ipl1). We coexpressed in the same cells GST or a functional version of Sli15 that was fused to GST (GST-Sli15). The latter proteins were expressed under the control of the GAL1 / 10 promoter and were thus present in great excess. As shown in Fig. 7 A, affinity purification of GST-Sli15, but not the more abundant GST, led to the copurification of HA-Ipl1. The association between HA-Ipl1 and GST-Sli15 was moderately stable, as repeated rounds of washing led to only partial disruption of this association. Comparison of the level and electrophoretic mobility of HA-Ipl1 in cells that coexpressed GST or GST-Sli15 revealed two interesting features. First, the level of HA-Ipl1 was much higher in cells that coexpressed GST-Sli15, thus suggesting that Sli15 may serve to stabilize Ipl1. Second, in cells that coexpressed GST, HA-Ipl1 could be detected as two forms that differed in electrophoretic mobility, with the slower-migrating form being much less abundant . In cells that coexpressed GST-Sli15, the relative abundance of the slower-migrating form was increased and this form of HA-Ipl1 appeared to be more readily copurified with GST-Sli15. The slower-migrating form could be converted to the faster-migrating form by endogenous phosphatases that were present in the yeast extract, as the slower-migrating form was detected only when the yeast extract was prepared in the presence of phosphatase inhibitors . These results indicated that the slower-migrating form of HA-Ipl1 is phosphorylated and that GST-Sli15 (and probably Sli15) may promote the phosphorylation of HA-Ipl1. We have also used high copy number plasmids to coexpress in yeast HA-Ipl1 and Sli15 or a functional version of Sli15 that was fused to the Myc-epitope (Sli15-Myc). As shown in Fig. 7 C, immunoprecipitation of Sli15-Myc from a crude yeast lysate with anti-Myc antibodies led to the coprecipitation of HA-Ipl1. Interestingly, Sli15-Myc appeared as multiple electrophoretic forms, thus suggesting that Sli15 may also be a phosphoprotein. To find out whether Sli15 binds Ipl1 directly, we expressed GST-Sli15 and His 6 -Ipl1 in E. coli. As shown in Fig. 7 D, His 6 -Ipl1 that was present in an E. coli crude extract associated with GST-Sli15, but not GST, that was immobilized on glutathione-agarose beads. The association between GST-Sli15 and His 6 -Ipl1 was specific, as the control protein His 6 -NonO-C failed to associate with GST-Sli15. These results together indicate that Sli15 is a binding partner of Ipl1 in vivo and that these two proteins most probably bind to each other directly. The results described so far strongly suggest that Ipl1 and Sli15 function in a complex to promote proper chromosome segregation. Mutations that affect the function of one component of a protein complex can sometimes be suppressed by overproduction of other components of the complex ( 22 ). Thus, we examined whether overproduction of Ipl1 or Sli15 can suppress the sli15-3 or ipl1-2 mutation, respectively. Our results showed that a high copy number SLI15 -plasmid had no effect on the Ts − growth phenotype of ipl1-2 cells (data not shown). In contrast, a low copy number IPL1 -plasmid could suppress partially and a high copy number IPL1 -plasmid could suppress almost completely (data not shown) the Ts − phenotype of sli15-3 cells at 37°C. Suppression requires residual Sli15 function, as a high copy number IPL1 -plasmid could not suppress the inviability of sli15-Δ2::HIS3 cells (data not shown). One possible interpretation of these results is that the sli15-3 mutation compromises the ability of mutant Sli15 to associate with Ipl1, whereas the ipl1-2 mutation compromises the catalytic activity of the Ipl1 protein kinase. In fact, the ipl1-2 mutation is known to alter a residue located in the COOH-terminal catalytic domain of Ipl1 ( 13 ). We have shown previously that the Ts − phenotype of some ipl1 mutants can be suppressed partially by overproduction of a truncated and dominant negative form (Glc7-Δ186-312) of PP1 or by overproduction of Glc8, an inhibitor of PP1, thus suggesting that PP1 acts in opposition to the Ipl1 protein kinase in regulating chromosome segregation ( 13 , 74 ). If the sli15-3 mutation indeed leads to a reduction in Ipl1 protein kinase function, we might expect the perturbations described above also to result in suppression of sli15-3 mutant phenotype. We found this to be true, as high copy number plasmids containing either the glc7-Δ186-312 dominant negative allele or the wild-type GLC8 gene could suppress the Ts − phenotype of sli15-3 cells at 33°C . Thus, SLI15 behaves genetically as a positive regulator of IPL1. The most prominent cytological phenotype of ipl1-2 mutant cells is the uneven segregation of chromosomes to opposite poles of apparently normal looking mitotic spindles that are capable of undergoing elongation and disassembly ( 13 ). At the restrictive temperature, sister chromatids of chromosome V often appear unseparated in ipl1-2 cells , suggesting that sister chromatid separation has failed, or that sister chromatids that have separated are not properly segregated away from each other to opposite poles of the mitotic spindle. In a separate study, it has been shown that the sister chromatid cohesion protein Mcd1/Scc1 ( 20 , 40 ) dissociates with wild-type kinetics from the chromosomes of ipl1 cells, thus suggesting that sister chromatids probably are separated normally in ipl1 cells ( 3 ). Consistent with the idea that ipl1-2 cells are defective in sister chromatid segregation but not separation, sister chromatids of chromosome V are clearly separated in the majority of ipl1-2 cells, although such separated sister chromatids very often are not segregated to opposite poles . However, sister chromatid segregation clearly does not fail in all ipl1-2 cells. This observation is consistent with our previous finding that at least some sister chromatids segregate away from each other in ipl1-2 cells, since the mitotic spindle-mediated poleward forces (acting on sister kinetochores) that cause the breakage of topologically intertwined sister chromatids in top2-4 mutant cells ( 26 , 67 ) also cause chromosome breakage in top2-4 ipl1-2 cells ( 12 ). Defects in the spindle pole bodies, the kinetochore microtubules, or the kinetochores themselves can all lead to a failure in sister chromatid segregation. Nuf2 is a spindle pole–associated protein that can be copurified with yeast spindle poles ( 77 ), and immunofluorescence microscopy has shown that Nuf2 colocalizes with Ndc80 to the intranuclear region of spindle poles ( 45 ). Interestingly, the phenotype of ndc80 mutant cells is similar, although not identical, to that of ipl1-2 mutant cells. During mitosis, most of the chromosomal DNA remains at one pole of the elongated mitotic spindle in ndc80-1 cells ( 77 ). Immunoelectron microscopy has shown that Ndc80 is associated with spindle microtubules, particularly at regions close to the spindle pole body ( 54 ). It was proposed that Ndc80 may be associated specifically with kinetochore microtubules ( 54 , 77 ). If Nuf2 is localized similarly to the kinetochore microtubules, the observation that uneven amounts of Nuf2-GFP are often found at the spindle poles of ipl1-2 cells would suggest quantitative or qualitative differences between the kinetochore microtubules that emanate from opposite spindle poles, possibly as consequences of defects in spindle pole body. For example, the number of kinetochore microtubules emanating from opposite spindle poles may differ greatly in ipl1-2 cells. We do not favor this idea because kinetochore microtubules make up most of the spindle microtubules ( 79 ), and immunofluorescent staining of microtubules has not revealed major differences in the intensity of half spindles. Alternatively, the properties of the kinetochore microtubules that emanate from the opposite poles may differ, with those from one pole being less proficient in bringing about sister chromatid segregation to that pole. Such properties may include the ability of kinetochore microtubules to attach to kinetochores and undergo polymerization or depolymerization. In addition to ndc80 mutants, some ndc10 mutants also have phenotypes that are similar, but not identical, to those of ipl1-2 cells. During mitosis, essentially all chromosomes remain at one pole of the elongated mitotic spindle in ndc10-1 and ndc10-2 cells, and chromosome missegregation is not associated with cell cycle arrest ( 16 , 66 ). NDC10 encodes an essential component of yeast kinetochores ( 10 , 29 ). We have examined possible genetic interaction between ipl1 and ndc10 mutations and found that some, but not all, ipl1-2 ndc10-2 double mutants have a restrictive growth temperature lower than that of either single mutant (our unpublished results). Immunofluorescence microscopy has shown that Ndc10 localizes to the spindle pole body region of nearly all cells and also along some short mitotic spindles ( 16 ). This observation raises the question whether Nuf2 may actually be associated with yeast kinetochores. We do not favor this idea since Nuf2, but not Ndc10 (or other kinetochore components), is known to be copurified with spindle poles ( 54 , 77 ). Furthermore, our preliminary results suggest that whereas Nuf2-GFP is concentrated in a single dot-like structure at the spindle poles of wild-type cells, Ndc10-GFP (gift of J. Kahana, Harvard University Medical School, Boston, MA) is more often found in multiple dots that cluster around the spindle poles and also less frequently along mitotic spindles, including those that are elongated. If Nuf2 is associated with kinetochores, the uneven amounts of Nuf2-GFP found at opposite spindle poles in some ipl1-2 mutant cells may simply reflect unequal numbers of chromosomes (and their kinetochores) that are segregated to the two poles. Furthermore, the presence of Nuf2-GFP in dot-like structures along some mitotic spindles in ipl1-2 cells may reflect a failure of kinetochores to move to opposite spindle poles , and it would also suggest that the kinetochores are attached to the kinetochore microtubules in ipl1-2 cells. In a separate study, it has been shown that Ipl1 can phosphorylate Ndc10 in vitro, and that the kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules, thus suggesting that Ipl1 may affect kinetochore functions ( 3 ). A better understanding of the actual site of Nuf2 localization and the quantity and quality of kinetochore microtubules present in ipl1 cells will help us understand to what degree the chromosome missegregation observed in ipl1 cells is due to defects in spindle pole or kinetochore function. A functional GFP-Ipl1 fusion protein is localized to the entire mitotic spindle , thus suggesting that Ipl1 may play important roles not only at spindle poles, kinetochore microtubules, or kinetochores. Furthermore, instead of the most prominent phenotype of uneven chromosome segregation, a small fraction of ipl1-2 cells exhibits one of two other phenotypes. First, some ipl1-2 cells exhibit a nuclear migration defect , which has also been reported for the pac15-1 / ipl1 mutant ( 14 ). Nuclear migration in yeast is dependent on cytoplasmic microtubules ( 46 , 71 ) and microtubule-based motor proteins (for review see reference 69 ). We have not detected GFP-Ipl1 on cytoplasmic microtubules. However, it is possible that spindle pole–associated Ipl1 acts on (motor proteins present at) the minus ends of cytoplasmic microtubules to influence their functions. Second, some ipl1-2 cells appear to have monopolar spindles , which are suggestive of defects in spindle pole body duplication or separation. This phenotype of ipl1 cells is reminiscent of that of Drosophila aurora mutants, which are defective in centrosome separation and form monopolar spindles due to mutations in the gene encoding a homologue of Ipl1 ( 15 ). In budding yeast, spindle pole body separation requires the function of the kinesin-related Cin8 and Kip1 motor proteins ( 28 , 50 , 57 ). Interestingly, the nonessential Cin8 motor protein becomes indispensable in ipl1 mutant cells (14 and our unpublished results), thus suggesting that Ipl1 may act on kinesin-related motor proteins. In this regard, it is interesting to note that microinjection of antibodies against HsEg5, a human homologue of Cin8 and Kip1, leads to the abnormal distribution of some centrosome-associated proteins in HeLa cells ( 76 ). Furthermore, C. elegans embryonic cells lacking the centrosome-associated Ipl1-homologue AIR-1 kinase are also defective in the localization of the centrosome-associated protein PIE-1 ( 60 ). Several lines of evidence indicate that there is a very close functional relationship between Ipl1 and Sli15. First, nonlethal mutations in SLI15 exacerbate the Ts − growth phenotype of ipl1-2 cells, leading to cell inviability at 26°C. Second, sli15-3 and ipl1-2 cells have very similar mutant phenotypes, including failure of separated sister chromatids to be properly segregated , abnormal distribution of Nuf2-GFP , and minor defects in nuclear migration and bipolar spindle formation . Third, GFP-Ipl1 and GFP-Sli15 are both localized on the mitotic spindle . Fourth, Ipl1 and Sli15 associate with each other in vivo, most probably through direct binding . These results suggest that Sli15 may function as a major physiological substrate and/or as a positive regulatory binding partner of Ipl1. The following observations support the idea that Sli15 functions as a positive regulatory binding partner of Ipl1, although they by no means preclude the possibility that Sli15 may also function as a major physiological substrate of Ipl1. First, the Ts − phenotype of sli15-3 cells can be suppressed by a small increase in the gene dosage of IPL1 or perturbations that lower the in vivo function of PP1 . Such perturbations also suppress the Ts − phenotype of ipl1 mutant cells ( 13 , 74 ). Second, the sli15-3 mutation exhibits synthetic lethal genetic interaction with the same spectrum of mutations that are synthetic lethal with ipl1-2 (our unpublished results). Third, the abundance of HA-Ipl1 is increased in cells that overexpress GST-Sli15, and the phosphorylation state of HA-Ipl1 is also altered in such cells . Furthermore, the phosphorylated form of HA-Ipl1 appears to be copurified preferentially with GST-Sli15, thus suggesting that GST-Sli15 may promote the phosphorylation of HA-Ipl1. As a binding partner of Ipl1, Sli15 potentially may stimulate the protein kinase activity of Ipl1 or it may stabilize Ipl1 and target it to its sites of action. We are currently testing the ability of Sli15 to stimulate the in vitro kinase activity of Ipl1. We do not know whether the mitotic spindle association of Ipl1 is dependent on Sli15 function, since we have not been able to localize Ipl1 when it is expressed from the chromosomal copy of IPL1 , and a small increase in the dosage of IPL1 suppresses the Ts − phenotype of sli15-3 cells . It is known that the transcript level of IPL1 varies through the cell cycle, peaking in late G1 ( 9 , 68 ), presumably due to the presence of a putative MluI cell cycle box ( 39 ) in the promoter of IPL1. Interestingly, the transcript level of SLI15 fluctuates less so through the cell cycle ( 9 ). This raises the possibility of the existence of two populations of Ipl1, one of which is in association with Sli15. Consistent with this possibility, GFP-Sli15 appears to be mostly restricted to the mitotic spindle whereas GFP-Ipl1 appears to be more broadly distributed . The cells from a majority of human colorectal tumors are aneuploid ( 5 ), probably due to increased rates of chromosome missegregation in such cells ( 37 ). The gene encoding the Ipl1-related Aurora2 kinase is amplified and/or overexpressed in a variety of human tumors, including a significant fraction of colorectal and breast tumors ( 4 , 62 , 81 ). Ectopic expression of Aurora2 in cultured fibroblasts leads to chromosome missegregation, centrosome amplification, and cellular transformation. Since overexpression of GST-Sli15 leads to an increase in the level of HA-Ipl1, overexpression of a Sli15-related binding partner of human Aurora2 may also lead to an increase in the level of Aurora2. A human homologue of Sli15 has not yet been identified. If such a homologue exists, it will be important to find out whether amplification and/or overexpression of the gene encoding this Sli15-homologue may also be correlated with diverse forms of human cancer. | Study | biomedical | en | 0.999997 |
10385520 | MLH1 null animals were produced from matings of male and female animals heterozygous for a targeted disruption of the Mlh1 gene . The Mlh1 genotype of offspring was determined by PCR from ear punch tissue using conditions and primers as described . To analyze homologue synapsis, air-dried preparations of oocyte nuclei were made from 16–17-d fetal ovaries using the technique described . The Mlh1 genotype of individual fetuses was determined by PCR from tail tissue using conditions and primers as described . Immunostaining was performed as described using a goat monoclonal antibody to rat SCP3 (a component of the lateral elements of the synaptonemal complex) generously supplied by T. Ashley (Department of Genetics, Yale University School of Medicine, New Haven, CT), and an FITC-conjugated donkey anti–goat secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Immunoreacted slides were scored on a Zeiss Axioplan fluorescent microscope. For chiasma counts, air-dried chromosome preparations were made from diakinesis stage oocytes using the technique described . Preparations were stained with Giemsa (Harleco) and the number and position of chiasmata was scored by two independent observers. Air-dried testis preparations were made according to the method described , the preparations were stained with Giemsa (Harleco), and the configuration of the sex chromosomes was scored by two independent observers. For MLH1 null males, the total number of chiasmate bivalents in each cell was also recorded. To obtain ovulated oocytes for first polar body extrusion experiments, females were injected with 2.5 IU of pregnant mare serum gonadotropin ( Sigma ) followed 42–44 h later by an injection of 5 IU human chorionic gonodotropin ( Sigma ). Ovulated oocytes were collected from the oviducts 15 h after the human chorionic gonodotropin injection, freed of adherent cumulus cells by a brief exposure to hyaluronidase (200 μg/ml; Sigma ), washed, and fixed as described below. For all studies of MI, prophase arrested oocytes were obtained from 3.5–4-wk-old females. Ovaries were removed and placed in Weymouth's MB752/1 medium ( GIBCO BRL ) supplemented with 10% fetal calf serum and 0.23 mM sodium pyruvate. Antral follicles were punctured with 26 gauge needles to obtain immature oocytes at the germinal vesicle stage. Germinal vesicle stage oocytes were cultured in microdrops of medium under oil at 37°C in 5% CO 2 in air. After 2 h in culture, oocytes were scored for germinal vesicle breakdown, indicating resumption of MI, and any oocytes remaining at the germinal vesicle stage were excluded from the experiment. For studies of MI spindle pole formation, donor females were injected with 2.5 IU pregnant mare serum gonadotropin 42–44 h before oocyte collection. For analyses of chromosome behavior and meiotic spindle formation during MI, oocytes were cultured for a total of 2, 4, 6, 8, 10, 12, or 18 h before fixation. At the end of the culture period, oocytes were embedded in a fibrin clot attached to a microscope and fixed as described previously . Fixed oocytes were incubated with an antibody to β-tubulin ( Sigma ) and detected with an FITC-conjugated secondary antibody according to the technique described . Meiotic staging, analysis of chromosome behavior, and spindle characteristics and length were scored by two independent observers using a Zeiss Axioplan microscope fitted with a micrometer in one eyepiece. Following immunofluorescence staining, a subset of the oocytes fixed after 6 h in culture was hybridized as described previously with the X-specific probe, DXWas 70 (American Tissue Type Culture Collection). The X chromosome probe was labeled with digoxigenin ( Boehringer Mannheim Biochemicals ) and detected with FITC-conjugated anti-digoxigenin ( Boehringer Mannheim Biochemicals ). Following hybridization and detection, oocytes were analyzed on a confocal microscope (Bio-Rad Laboratories) to characterize the location of the X chromosome homologues on the MI spindle. To determine if the process of homologue synapsis occurs normally in oocytes from MLH1 null females, we analyzed air-dried preparations of pachytene stage nuclei from 16– 17-d MLH1 null fetuses and control siblings. Normal pachytene stage oocytes exhibiting fully synapsed homologues were observed , and no differences in the morphology of the complexes in control and MLH1 null siblings were evident. To determine the consequences of MLH1 deficiency on recombination, we analyzed air-dried preparations of oocytes at the diakinesis stage. The number and distribution of chiasmata were virtually identical for wild-type and heterozygous control females (Table I ). In contrast, we observed an ∼10-fold reduction in chiasmata in oocytes from MLH1 null females . The average number of chiasmata in null females was only 1.9 per oocyte, with a maximum of 6 observed in a single cell and zero exchanges observed in 15% of cells. Moreover, the placement of chiasmata was slightly skewed by comparison with cells from controls, with a preponderance of terminal exchanges and very few interstitial exchanges (Table I ). The conclusion that recombination is greatly reduced in MLH1 null males and females is based solely on cytological evidence. Thus, the possibility that recombination occurs normally but functional chiasmata are not produced cannot be excluded. To test this hypothesis, we introduced a structurally abnormal Y chromosome that would allow detection of recombination in the absence of chiasmata. As shown in Fig. 3 a, recombination between the X and Y chromosomes in males carrying this structurally rearranged Y chromosome significantly alters the length of the recombinant X and Y chromatids. Thus, premature resolution of an exchange event would be cytologically detectable at diakinesis as univalent X and Y chromosomes with chromatids of different lengths. In MLH1 null males , as in null females (Table I ), the vast majority of chromosomes were present as unpaired univalents at diakinesis. An average of 2.7 chiasmata per cell was observed, with a maximum of 8 exchanges observed in a single cell and zero exchanges observed in 14% of cells (data not shown). The X chromosome, however, displayed a unique morphology, presumably reflecting its unique chromatin configuration and sequestration in the sex vesicle , which allowed us to unambiguously distinguish it from univalent autosomal chromosomes. Only 3 cells with recombinant X and Y chromosomes were observed among the 400 diakinesis cells scored . In addition, in all 397 cells in which the X and Y chromosomes were present as univalents, both X chromatids were of equal size , indicating failure of recombination rather than precocious resolution of an exchange event. Previous studies of oocytes from MLH1 null females suggested a variety of aberrations, including failure to extrude a second polar body and a reduced rate of fertilization and cleavage . Our initial attempts to obtain oocytes arrested at the second meiotic division (MII) from superovulated females suggested that the first polar body extrusion rate is also extremely low. Ovulated oocytes are enclosed in a sticky mass of cumulus cells which are routinely dispersed by brief exposure of the oocyte/cumulus cell mass to a hyaluronidase solution. In initial studies, we noted that hyaluronidase treatment induced polar body extrusion in a small proportion of oocytes from MLH1 null females. Hence, to avoid artificial induction of polar body extrusion, we studied oocytes meiotically matured in vitro. A large cohort of follicles initiates growth in the neonatal murine ovary. This cohort develops almost synchronously and by 3–4 wk after birth a large population of oocytes can be obtained from the ovary, most of which are meiotically competent and will spontaneously resume and complete MI in vitro . When oocytes from control females were collected and cultured in this fashion, 65% had completed MI and extruded a first polar body after 15 h in culture; the remaining oocytes were meiotically incompetent and arrested at metaphase I. In contrast, after 15 h in culture, only 7% of the oocytes collected from MLH1 null females had extruded a polar body and, after extended time in culture (18–20 h), only 18% of the oocytes had completed MI (Table II ). Further, immunostaining with an antibody to β-tubulin demonstrated a variety of abnormalities in the vast majority of oocytes that extruded a polar body, including incomplete cytokinesis, lack of chromatin in the polar body, and abnormalities in MII spindle formation (data not shown). To determine why the majority of oocytes failed to extrude a polar body, we analyzed MI in oocytes from MLH1 null females. Oocytes were collected and cultured as described for the polar body experiments and fixed after 2, 4, 6, 8, 10, or 12 h in culture to study successive stages of MI (Table III ). After 4 h in culture, oocytes from control females were at prometaphase of MI and exhibited either a very early prometaphase configuration, with condensed chromosomes surrounded by a mass of microtubules , or early spindle formation with chromosomes congressing to the spindle equator . Aberrations in chromosome orientation were evident at both stages of prometaphase in oocytes from MLH1 null females: in early prometaphase cells, the majority of the chromosomes were arranged at the periphery of the microtubule mass, with the chromosome arms radiating outward like the petals of a flower . The few centrally located chromosomes in these cells were almost exclusively bivalents, suggesting that the aberrant chromosome behavior is limited to univalent chromosomes. In cells with evidence of spindle pole formation, there was no evidence of orderly chromosome congression . With increasing time in culture the aberrations in oocytes from MLH1 null females became more pronounced. In control oocytes, organization of the MI spindle and the congression of the chromosomes to the spindle equator occurred within the first few hours of culture and, by 8 h, the majority of oocytes had attained a metaphase configuration . In contrast, the gross disturbances in chromosome congression evident at the early stages of bipolar spindle formation in oocytes from MLH1 null females persisted over time, and a normal metaphase configuration was never observed . In addition, defects in the organization of the midzone microtubules were a characteristic feature . Anaphase figures were first observed in control oocytes after 8 h and, by 12 h, the majority of oocytes had initiated anaphase or completed the division and arrested at second meiotic metaphase. In oocytes from null females, however, the congression failure phenotype persisted over time, with the only difference being an increase in the frequency of oocytes with gross spindle aberrations, e.g., grossly unequal poles, collapse of the spindle, and extremely elongated spindles, some of which were tripolar . The aberrations evident at early prometaphase and the increase in spindle abnormalities with increasing time in culture suggest that both the organization and maintenance of the MI spindle is aberrant in oocytes from MLH1 null females. To perform a temporal analysis of spindle pole formation and spindle elongation, females were injected with pregnant mare serum gonadotropin 44–48 h before oocyte collection. Priming with exogenous hormones results in a more synchronous population of oocytes and also slightly accelerates progression through MI. Pole-to-pole measurements were obtained for oocytes from MLH1 null females and controls collected after 2–12 h in culture. As shown in Fig. 6 , the appearance of organized spindle poles was not delayed, indeed, it was slightly accelerated in oocytes from null females. Although pole formation was not observed in any of the 33 oocytes from control oocytes analyzed after 2 h in culture, 3/43 (7%) of oocytes from null females exhibited organized poles. After 4 h in culture, pole formation was evident in the majority (19/29) of oocytes from null females and, although poles were evident in a significant minority (21/76) of oocytes from control females, the average pole-to-pole distance was significantly greater in oocytes from null females ( t = 2.9; P < 0.01). Moreover, the average interpolar distance continued to increase over time in culture and remained significantly greater than the average for controls at all time points . In mammalian cells, several of the proteins that associate with metaphase chromosomes relocalize to the spindle midzone at anaphase and remain associated with the interzonal microtubules as the chromosomes move to the poles. The interzonal microtubules and associated proteins are thought to be important in positioning of the cleavage furrow . Given the aberrations in both chromosome alignment and midzone microtubule organization in oocytes from MLH1 null females, it remained possible that the extremely elongated spindles observed after extended time in culture represented an “abortive” anaphase. To test this hypothesis, we analyzed extremely elongated spindles for evidence of the chiasma resolution that occurs at the onset of anaphase. From a group of oocytes cultured for 12 h, a subset of 22 oocytes with the longest spindles (i.e., with a pole-to-pole measurement exceeding 80 μm) was selected. In 9 of the 22 oocytes (41%) chiasmate bivalents were obvious, in an additional 9 oocytes a group of chromosomes was present at the spindle equator but it was impossible to distinguish a chiasmate bivalent, and in 4 oocytes (18%) it was possible to conclude that no exchange bivalents were present (e.g., either the chromosomes were randomly dispersed over the length of the spindle and all visible or were present as 2 distinct groups at opposite spindle poles). These observations provide evidence that the elongated spindle phenotype does not represent an attempt to initiate anaphase, a conclusion supported by the results of histone H1 kinase assays to determine if spindle pole elongation was associated with the characteristic decline in cyclin B levels that accompanies anaphase onset (data not shown). Interestingly, the proportion of cells with no exchange bivalents among the oocytes with extremely elongated spindles (18% of the total) was virtually identical to the frequency of achiasmate cells scored at diakinesis (15% of the total). This suggests that the fate of oocytes with zero chiasmata is no different from that of cells with one or more exchange bivalents. Despite the disturbance in congression, the number and distribution of chromosomes on opposite sides of the bipolar spindle was nearly always equivalent in oocytes from MLH1 null females . The balanced nature of these MI spindles was particularly striking in oocytes fixed during the early stages of MI spindle formation (i.e., oocytes fixed after 4–6 h in culture). To determine if this was due to the fact that homologous chromosomes maintained a spatial orientation during the early stages of spindle formation despite their univalent status, a subset of oocytes fixed after 6 h in culture was hybridized with an X chromosome–specific probe. The signals for the 2 X homologues were in close proximity and located at the spindle equator in 4 of the 44 cells analyzed. This is the expected configuration for an exchange bivalent. In the remaining 40 cells, the 2 X chromosomes were present as univalents with widely separated signals. In these cells the location of the homologues with respect to each other appeared to be random: the two X chromosome signals were located on opposite sides of the spindle equator in 17/40 (43%) cells and on the same side of the spindle equator in 23/40 (58%). In MLH1 null male mice, meiotic recombination is reportedly reduced 10–100-fold . Synapsis occurs normally, but homologues separate prematurely due to the absence of functional chiasmata and the cells arrest between pachytene and MI . Because a reduction in functional chiasmata might reflect the premature separation of exchanged bivalents rather than a primary defect in recombination, we created MLH1 null mice carrying a structurally rearranged Y chromosome that would make it possible to detect recombination even if the resultant chiasma could not be maintained . The analysis of meiotic cells from these males provides compelling genetic evidence that lack of the MLH1 protein reduces the level of recombination during mammalian meiosis. Our studies of oocytes from MLH1 null females suggest that lack of the MLH1 protein has a comparable effect on oogenesis. As in the null male, we observed no defects in homologue synapsis in studies of pachytene stage oocytes from MLH1 null females, and analysis at the diakinesis stage demonstrated a 10–100-fold reduction in functional chiasmata. In yeast, the Mlh1 protein plays a unique role in promoting recombination and is involved in both crossover and gene conversion pathways ; Mlh1-deficient yeast show a high level of postmeiotic segregation, indicating failure to correct mismatches in DNA heteroduplexes formed during recombination . Unlike the situation in yeast where the mechanism of resolution of recombination intermediates can be inferred from the analysis of the products of meiosis, failure to complete MI in the MLH1 null female mouse precludes this type of analysis. Nevertheless, our results are consistent with the interpretation that—as is the case in yeast and in the male mouse—absence of the MLH1 protein disrupts the normal recombination pathway. Interestingly, our studies demonstrate that oocytes with few or no functional exchanges can initiate but not complete MI. The ensuing discussion of this finding is predicated on the assumption that defects in MI result from the reduction in recombination levels. In the absence of chiasmata, the majority of chromosomes were present as univalents at prometaphase of MI . Although virtually all oocytes from control animals had reached metaphase after 8 h in culture, a normal metaphase configuration was never observed in oocytes from MLH1 null females. Even after an extensive period of time (as long as 18 h after nuclear envelop breakdown) the orientation of the majority of univalent chromosomes on the MI spindle was random, suggesting that most were unable to form stable bipolar attachments necessary for congression to the spindle equator. Proper segregation at MI requires that the kinetochores on homologous chromosomes—not sister chromatids— attach to opposite spindle poles. Chromosome transfer experiments in grasshopper spermatocytes in the mid-1970s suggested that the MI segregation pattern is a specialized property of MI chromosomes . This conclusion has been supported by subsequent morphological evaluations of meiotic kinetochores that indicate that sister kinetochores are not present as physically separate domains until anaphase I . Studies in rat kangaroo kidney cells have demonstrated that a chromosome fragment created by laser microsurgery that contains a single kinetochore can form stable attachments to opposite spindle poles and congress to the spindle equator during mitotic cell division . In contrast, in our meiotic studies, univalent chromosomes with two kinetochores were only rarely oriented at the equator in a manner that suggested attachment to opposite spindle poles. This suggests that the constraint on sister kinetochores at MI is strong. It is not, however, absolute. Equational segregation of univalent chromosomes at MI has been reported in a variety of species, including mammals . Further, previous studies of a murine univalent X chromosome have demonstrated that bipolar attachment and resultant equational segregation at MI occurs during female meiosis but that it is not the favored mechanism of segregation . The present results extend these observations and suggest that, even after extended time, the majority of murine univalent chromosomes are incapable of differentiating separate functional sister kinetochore domains before anaphase I. In yeast mutants with a single-division meiotic phenotype, the ability of sister kinetochores to act independently and direct equational segregation of sister chromatids at MI varies for individual centromeres . This suggests that the degree to which sister kinetochores are physically constrained at MI may be at least partially dependent upon centromere and centromere-associated sequences. In the human, the inappropriate segregation of sister chromatids at MI has been postulated to be a major mechanism of meiotic nondisjunction . Further study of the MLH1 null mouse to identify chromosomes with a propensity to differentiate separate kinetochore domains at MI may provide a means of understanding the structural features that facilitate the coordinated behavior of sister chromatids during MI. The meiotic spindle in oocytes from a variety of evolutionarily diverse animal species, including mouse , Drosophila , Xenopus , Ascidians , and Caenorhabditus elegans , forms through the action of multiple microtubule organizing centers rather than from a pair of centrosomes. The fact that chromatin promotes microtubule organization and the observation that the presence of several individual groups of chromosomes can result in the formation of multiple independent spindles suggest that, in these meiotic systems, chromosomes play a fundamental role in the early events of spindle organization. In the present study of meiosis in MLH1 null female mice, the aberrant situation created by the presence of multiple univalent chromosomes has made it possible to discern an additional role of the chromosomes in meiotic spindle formation. At very early prometaphase, we observed an abnormal rosette configuration, with univalent chromosomes located at the periphery of the microtubule mass . The unusual positioning of univalent chromosomes in comparison with bivalents during early prometaphase suggests that, in normal oocytes, bivalent chromosomes must be establishing bipolar attachments as the antiparallel arrays of microtubules form. Indeed, consistent with this suggestion, the chromosomes on very early bipolar spindles in oocytes from normal females are frequently nearly aligned at the spindle equator . If bipolar attachment of most bivalents is, in fact, coincident with spindle pole formation, then the attachment and congression of the chromosomes is a very different process during female meiosis than during mitotic cell division. The simultaneous formation of spindle poles and the establishment of bipolar attachments may be a characteristic feature of meiotic systems in which the MI spindle forms through the action of multiple microtubule organizing centers. In the Xenopus oocyte, each bivalent appears to form a mini spindle and the MI spindle is established from the coalescence of the individual mini spindles (Duesbery, N., personal communication). In the mouse, detailed studies of very early stages of meiotic spindle formation have been possible in an extremely protracted MI division resulting from the microinjection of Mos into oocytes held in meiotic arrest with the drug IBMX . Interestingly, immunostaining with a centrosomal antibody indicates that multiple foci become regionalized to opposite sides of the condensed chromosome mass, as though one or several bivalents organize individual bipolar spindles that subsequently coalesce into the two discrete poles of a bipolar spindle. These observations support our conclusion that the bipolar attachment of most bivalents occurs concurrently with spindle pole organization. Our results also suggest that the chromosomes play a role in the organization and stabilization of the meiotic spindle. In both meiotic progression studies (Table III ) and subsequent studies of pole formation and spindle elongation precocious appearance of the spindle poles was observed in oocytes from MLH1 null females. In view of the compromised spindle integrity apparent at late prometaphase , the apparent acceleration in the organization of the MI spindle is puzzling. It is possible that under normal conditions the process of bivalent attachment, detachment, and reorientation necessary to achieve stable bipolar attachments influences the process of pole formation, slowing the process. However, we think it more likely that the early appearance of spindle poles in oocytes from MLH1 null females reflects an inability to tether the newly forming spindle poles. This is partially analogous to the situation in mitotic cells where the bipolar attachment of at least one chromosome is necessary to tether the centrosomes . In contrast to mitotic cell division, however, our studies suggest that a critical mass is required to tether the forming spindle poles during mammalian female meiosis. That is, both precocious pole formation among cells scored after several hours in culture and spindle defects (i.e., spindles with unequal poles or elongated, collapsed, and tripolar spindles) among cells scored after extended time in culture were a feature of cells with zero, one, or several bivalent chromosomes present at the spindle equator . Thus, we propose that the chromosomes not only play a role in organizing the MI spindle, but that the formation of stable bipolar attachments is necessary both to stabilize the interzonal microtubules and to control the movement of the spindle poles. This implies that tension is used differently in female meiotic systems that utilize multiple microtubule organizing centers, and is consistent with recent studies of the cell cycle checkpoint protein MAD2 in maize, where the meiotic spindle also forms by an inside-out mechanism. In contrast to mitotic cells where loss of MAD2 staining was correlated with initial microtubule attachment, loss of MAD2 staining in meiotic cells appeared to be tension-dependent . According to our hypothesis, the extremely long spindles that are characteristic of oocytes from MLH1 null females are not the result of congression failure per se, but rather of the failure of the vast majority of univalent chromosomes to form stable bipolar attachments. Several lines of evidence support this conclusion: first, when normal meiotic bivalents or replicated mitotic chromosomes are present, defects in chromosome congression induced by the microinjection of kinetochore antibodies or immunodepletion of the kinetochore-associated motor protein, CENP-E , apparently are not associated with spindle pole elongation; second, in meiotic studies of human oocytes and in oocytes from a mouse mutant (Hunt, P.A., manuscript in preparation) we have observed defects in meiotic chromosome congression without corresponding spindle pole elongation. In all of these situations normal MI bivalents are present and the ability of these bivalents to form bipolar attachments is presumably normal. Importantly, in the mouse mutant, although congression to the spindle equator was disrupted, anaphase onset occurred normally (Hunt, P.A., manuscript in preparation). The suggestion that the tension created by the bipolar attachment of chromosomes plays an essential role in the formation of a stable MI spindle appears to be at odds with several previous observations that suggest that the chromosomes are passive players in the process. First, stable bipolar spindles can be assembled around chromatin-coated beads in Xenopus extracts and, in the absence of kinetochores, the beads can align at the spindle equator in a metaphase-like configuration . Second, studies of mechanically bisected MI and MII stage mouse oocytes suggest that normal-appearing, stable bipolar spindles can form in chromosome-free oocyte fragments . Finally, in grasshopper spermatocytes, the removal of the chromosomes from bipolar spindles does not interfere with the onset of anaphase . These results clearly demonstrate that microtubule self-assembly of a bipolar spindle is possible and that anaphase onset can occur in the absence of cues from the chromosomes. They do not, however, provide any indication of the complexity that may be imposed on the process by the presence of chromosomes. For example, in the grasshopper spermatocyte, although a stable MI spindle persists and can initiate anaphase after the removal of the chromosomes , the presence of a single chromosome that is not removed but merely dragged off the spindle and left in the cytoplasm results in spindle depolymerization . Thus, in the MLH1 null mouse, the presence of multiple univalent chromosomes creates an aberrant situation that provides a means of unraveling the complexity of the normal process of MI spindle formation in a centrosome-free system. Previous attention has focused on the role of tension in the alignment of the chromosomes at the spindle equator. The formation of a bipolar attachment places opposing kinetochores of a bivalent (or sister kinetochores at MII or mitosis) under tension, stabilizing the microtubule connections to the kinetochores . However, to our knowledge an effect of tension on pole formation and spindle integrity has not been described previously. Is the role of the chromosomes in the stabilization of the MI spindle unique to mammalian female meiosis? Tension is used in a very different way in Drosophila oogenesis: metaphase I is a normal cell cycle arrest point and the tension resulting from the bipolar attachment of one or more exchange bivalents is necessary to achieve this arrest . However, when tension is lacking, not only is the MI arrest voided but the MI spindle becomes significantly elongated (Hawley, R.S., personal communication). Thus, the role of tension in stabilizing the MI spindle may be a generalized feature of oogenesis in many animal species that reflects the manner in which the spindle is organized. Several lines of evidence suggest that congression of all chromosomes to the spindle equator is not a prerequisite for anaphase onset in murine oocytes, and mammalian female meiosis has been hypothesized to lack an important cell cycle control mechanism to detect misaligned chromosomes . Our current studies demonstrate that if the majority of chromosomes fail to form a bipolar attachment, a stable metaphase spindle cannot be assembled and anaphase onset is prevented. These observations are consistent with a number of previous studies which demonstrate that exposure of MI oocytes to spindle disrupting drugs prevents or significantly delays anaphase onset . Taken together, these results suggest that the cell cycle checkpoint that monitors both spindle assembly and chromosome alignment in mitotic cells functions only to detect spindle aberrations during female meiosis. We believe that this important difference in cell cycle control reflects differences in spindle assembly: in mitosis, where specialized structures organize the spindle and the attachment and congression of each chromosome occurs independently, stringent mechanisms have evolved to ensure that all chromosomes have congressed to the spindle equator before anaphase onset is initiated. In contrast, in female meiosis where the spindle is formed from multiple microtubule organizing centers and the bipolar attachment of most bivalents appears to be coincident with the formation of the spindle poles, control mechanisms emphasizing spindle stabilization rather than chromosome head counting have evolved. Although mechanisms for the segregation of nonexchange homologues have been described in a variety of species , it remains unclear whether mammalian female meiosis has a backup mechanism to ensure the segregation of homologues that fail to recombine. Interestingly, despite the lack of chromosome congression, in the majority of oocytes from MLH1 null females fixed during the early phases of meiotic spindle formation the chromosomes appeared to be spatially balanced on the spindle . Although we made no attempt to quantify the amount of chromosomal balancing, hybridization with an X chromosome–specific probe suggested that the placement of the two X homologues with respect to each other in these cells was random. Thus, we believe that the balanced orientation of the univalent chromosomes in oocytes from MLH1 null females is a property of the meiotic spindle. In Drosophila , the crowded spindle model has been proposed to explain the segregation of chromosomes in the absence of physical attachments . According to this model, a given nonexchange chromosome is more likely to make an attachment to the spindle pole that is not already occupied by a univalent. The situation is more complex in the mouse with its 20 pairs of chromosomes than in Drosophila with its 4 pairs. Nevertheless, the behavior of the chromosomes in MLH1 null females suggests that some mechanism of balancing the number of chromosomal bodies on the MI spindle exists, although in MLH1-deficient females it appears to be unrelated to homology. | Study | biomedical | en | 0.999996 |
10385521 | All yeast strains were derived from DF5 and the procedures for yeast manipulation were as described . KAP114 was deleted by integrative transformation of HIS3 . Heterozygous diploids were sporulated and tetrads dissected to generate kap114 haploid strains . kap114/kap123 deletion strains were constructed by mating, sporulating, and dissecting the relevant strains. Diploid strains expressing the Kap114–protein A (PrA) and TBP-PrA fusion proteins were constructed by integrative transformation of the coding sequence of four and a half IgG binding repeats of Staphylococcus aureus PrA immediately upstream of the relevant stop codon as described . Haploid strains were generated by sporulation and dissection. Other strains were constructed by PrA integration downstream of TBP in the relevant haploid deletion strains. After fixation in 3.7% formaldehyde for 20 min, immunofluorescence microscopy on yeast spheroplasts was done as previously described . PrA tags were visualized using rabbit anti–mouse IgG (preadsorbed against formaldehyde-fixed wild-type yeast cells) followed by Cy3-conjugated donkey anti–rabbit IgG ( Jackson Labs ). GFP-expressing cells were applied to slides and briefly permeabilized in methanol, followed by acetone. All cells were mounted in a 4′,6-diamidino-2-phenylindole (DAPI)–containing medium. All images were viewed under the 63× oil objective on a Zeiss Axiophot microscope, images were collected with a video imaging system, and manipulated in the computer program Adobe Photoshop. Postnuclear, postribosomal cytosol was prepared from 4 liters of the TBP-PrA and Kap114-PrA strains and 1 liter of TBP-PrA/ kap114 and 160 ml of TBP-PrA /kap114/kap123 strains grown to an OD 600 of 1.6 as described . Kap114-PrA or TBP-PrA and associated proteins were immunoisolated by overnight incubation of cytosol with rabbit IgG Sepharose (Cappel Laboratories) as described. After washing in TB (20 mM Hepes, pH 7.5, 110 mM KOAc, 2 mM MgCl 2 , 1 mM DTT, 0.1% Tween 20), proteins were eluted from the Sepharose with a step gradient of MgCl 2 and precipitated with methanol before analysis by SDS-PAGE. Coomassie staining bands were excised and prepared for analysis by MALDI-TOF mass spectrometry (MS) and by peptide sequencing . The entire coding region of KAP114 was amplified by PCR and ligated into the vector pGEX5X-1 ( Pharmacia Biotech ). The resulting GST-Kap114 fusion protein was purified from induced bacteria following the manufacturer's instructions. Purified GST was obtained from the manufacturer ( Pharmacia ) and recombinant TBP expressed in bacteria as a His 6 fusion protein was a gift of Dr. P.A. Weill (Vanderbilt University, Nashville, TN). Approximately 0.5 μg or 1 μg of GST-Kap114 or 2 μg of GST was bound to 20 μl of a 50% slurry of glutathione-Sepharose in TB/ 0.1% casamino acids. After washing, Sepharose was incubated with ∼1 μg of TBP for 30 min in the above buffer. The supernatant was collected and constituted the unbound fraction, the Sepharose was washed six times in TB, and the bound fraction was collected by boiling in the sample buffer. Ran (Gsp1p) was expressed in bacteria and purified and loaded with GTP or GDP as described . Oligonucleotides (TATA, 5′-CTGTATGTATATAAAACG-3′; M1, 5′-CTGTATGTAGAGAAAACG-3′) were annealed by standard procedures and diluted in TB. Purified TFIIA was a gift from Dr. S. Hahn (Fred Hutchison Cancer Research Center, Seattle, WA) . Kap114-PrA and TBP were isolated in batch by incubation with IgG Sepharose overnight, ∼1–1.5 ml of cytosol (equivalent to 33–50 ml of yeast grown to an OD 600 of 1.6) per dissociation was used. After washing in TB, the Sepharose was divided into separate microfuge tubes for each experiment, resulting in ∼10–20 μl of Sepharose per tube. Ran, DNA, and TFIIA were added as indicated to give a final volume of 100 μl. 0.1 mM GTP was included, except when using RanGDP. Tubes were rotated for 50 min at room temperature. The entire contents of the tube were transferred to a 2-ml disposable column and the liquid was drained and collected. The Sepharose was washed with a further 100 μl of TB, together these fractions constituted the eluted fraction. The columns were washed with 10 ml of TB and drained. Proteins bound to the Sepharose were eluted either with 200 μl of 1 M MgCl 2 followed by 200 μl of 4.5 M MgCl 2 , or directly with 4.5 M MgCl 2 to elute the Kap114-PrA and bound TBP from the Sepharose, this constituted the bound fraction. All fractions were precipitated and separated by SDS-PAGE and immunoblotted. Amido black–stained blots were observed to see the excess unbound Ran in the eluted fraction that acted as an internal standard for the protein loading in this fraction. Procedures for Western blotting and subsequent detection by enhanced chemiluminescence were as described ( Amersham ). An affinity-purified rabbit polyclonal antibody was used to detect TBP. This antibody also interacts with PrA, so the levels of Kap114-PrA were compared as an internal standard for the bound fractions. All dissociations were carried out several times and representative experiments are shown. In yeast, TBP is encoded by the gene SPT15 . Analysis of the TBP amino acid sequence revealed no known NLSs. To find the Kaps that are responsible for the import of TBP, we sought to isolate proteins that interacted with the low abundance, cytosolic pool of TBP. This pool most likely represents newly synthesized TBP en route to the nucleus. We genomically tagged TBP with an in frame carboxy-terminal fusion of the IgG-binding domain of PrA. Therefore, haploid cells expressed a single PrA-tagged copy of TBP (TBP-PrA) from its endogenous promoter. The tagged version of TBP was functional as it complemented growth and was localized to the nucleus (see below). TBP-PrA and associated proteins were isolated by incubation of a postribosomal cytosol with IgG-Sepharose and elution with a step gradient ranging from 0.05 to 4.5 M MgCl 2 . The eluted fractions were analyzed by SDS-PAGE and Coomassie blue staining . As expected, the TBP-PrA, which binds IgG with high affinity because of the PrA moiety, was eluted at high concentrations of MgCl 2 . A few faint bands were visible in every fraction suggesting that they may be nonspecific contaminants or keratin. A band of ∼116 kD, visible mainly in the 0.25- and 1-M MgCl 2 eluates, was in the expected size range for an associated Kap . The band was excised and analyzed by MS. The band represented a protein encoded by the uncharacterized open reading frame YGL241W . YGL241W is predicted to encode a protein of 1,004 amino acids with an expected molecular mass of 113.9 kD. Comparison of this protein sequence with the database showed strongest homology with Kap family members (15–17% identity), suggesting that this protein was, indeed, a member of the Kap superfamily . Consistent with its role in nuclear transport (described below) and the standard nomenclature for other yeast Kaps, we will refer to this protein as Kap114p. To verify that the interaction of Kap114p with TBP was specific, we tagged Kap114p as before with PrA (Kap114-PrA). Using cytosol prepared from the Kap114-PrA strain we purified by affinity chromatography those proteins that interacted with Kap114p. Kap114-PrA eluted at the high concentrations of MgCl 2 , whereas several bands, including one of ∼27 kD, eluted at 0.25–1 M MgCl 2 . The 27-kD band was excised and subjected to MS analysis and peptide sequencing which showed that this protein was indeed TBP. These data confirm that Kap114p and TBP interact in the cytosol, suggesting that TBP might represent an import substrate for Kap114p. Other bands detected in this experiment may also include Kap114p interacting proteins and, hence, potential import substrates for Kap114p. To determine whether the interaction between Kap114p and TBP was direct, a complex was formed between bacterially expressed, purified GST-Kap114 and bacterially expressed, purified His 6 -tagged TBP . In control experiments very little TBP could be detected bound to glutathione-Sepharose alone or to GST bound–Sepharose . However, a much greater amount of TBP bound to GST-Kap114 bound– Sepharose, suggesting that the binding was due to the presence of Kap114p . TBP binding was also seen to increase with increasing amounts of GST-Kap114 immobilized on the Sepharose . These results suggested that TBP and Kap114p interacted directly. Using the Kap114-PrA–tagged strain it was possible to determine the localization of the Kap114p by visualization of the PrA tag. The Kap114-PrA was localized to the cytoplasm and nucleus, which would be consistent with its function as a nucleocytoplasmic shuttling protein . To determine whether the gene encoding Kap114p was essential for cell growth the gene was replaced by genomic integration with HIS3 . Viable haploid HIS3 yeast strains deleted for KAP114 (Δ kap114 ) were obtained (data not shown), suggesting that the KAP114 gene was not essential for cell growth. Deletion strains were also grown on rich medium at 37°C and 17°C, however, no growth defect was detected under these conditions (data not shown). To ascertain whether TBP was imported into the nucleus by Kap114p in vivo, the localization of TBP-PrA in the KAP114 deletion strain (Δ kap114 ) was determined. As expected TBP-PrA appeared nuclear in wild-type cells, whereas in Δ kap114 cells a large proportion of the TBP-PrA was now localized to the cytoplasm . These results were confirmed using an antibody to detect endogenous TBP in the Δ kap114 strain demonstrating that the mislocalization was not dependent on the PrA moiety (data not shown). Thus the nuclear import of TBP appears to be partially abrogated in the absence of Kap114p, confirming that Kap114p imports TBP in vivo. The localization of Lhp1p-GFP and a human La–GFP reporter, which have been previously shown to be substrates for other Kap-mediated import pathways , were also analyzed in the Δ kap114 strain. These proteins were correctly localized to the nucleus in this strain , suggesting that we were not observing a general defect in nuclear transport and the effect on TBP localization was specific to deletion of KAP114 . As strains deleted for KAP114 appeared to have no growth defect under the described conditions and some of the TBP was nuclear in the absence of Kap114p , it seemed likely that a fraction of the TBP was imported into the nucleus via an alternative pathway. Cytosol was made from the TBP-PrA/Δ kap114 strain to determine which Kaps interacted with TBP in the absence of Kap114p. Coomassie blue staining of TBP-PrA–copurifying proteins revealed one in the correct molecular weight range for a Kap . MS analysis revealed that this Kap was Kap123p, which has been previously shown to be involved in the import of ribosomal proteins . Kap123p is the most abundant Kap and is not encoded by an essential gene, although deletion strains grow very slowly at 37°C . TBP was PrA-tagged in the Δ kap123 strain and the localization of TBP-PrA was determined by immunofluorescence. TBP-PrA did not appear to be mislocalized in this strain at any temperature . As Kap114p was still present in this strain we made a double deletion of KAP114 and KAP123 (to generate Δ kap114 /Δ kap123 ), to determine whether they were synthetically lethal. This strain was also viable and phenotypically appeared similar to the single mutants, in that TBP was mislocalized to a similar degree as in Δ kap114 cells and that the cells grew poorly at 37°C as observed in the Δ kap123 strain . These results suggested that the essential protein TBP may in fact have several routes into the nucleus and to find further import pathways for TBP, TBP-PrA–copurifying proteins were isolated from the Δ kap114 /Δ kap123 strain as before. A very faint band in the correct molecular weight range for a Kap was detected by Coomassie blue staining of the proteins after separation by SDS-PAGE (data not shown), however, there was insufficient material for MS analysis. Another Kap, Kap121p, has been previously shown to have overlapping functions with Kap123p and to compensate for a lack of Kap123p in the import of ribosomal proteins . We determined whether the TBP-PrA–interacting band of the Δ kap114 /Δ kap123 strain represented Kap121p by probing the eluted fractions, after transfer to nitrocellulose, with a polyclonal antibody to Kap121p . This analysis showed that this band indeed contained Kap121p . Kap121p is encoded by an essential gene and a temperature sensitive strain, pse1-1 , has been described . A pse1-1 strain expressing TBP-PrA was grown at the restrictive temperature for 3 h to determine the effect on TBP localization. After this treatment, TBP-PrA was still localized to the nucleus . This suggests that although Kap121p and Kap123p may play a role in the import of TBP, this role is likely to be redundant with Kap114p. Interestingly, Kap121p could also be detected by immunoblot in TBP-PrA isolation experiments in the presence of Kap114p (data not shown). These data suggest that Kap114p mediates the bulk of TBP nuclear import, as evidenced by the fact that only a kap114 deletion leads to mislocalization of TBP, even in the presence of an otherwise wild-type complement of Kaps. In the nucleus, the dissociation of Kaps from their cognate import substrates is thought to be achieved by RanGTP and not RanGDP . To determine whether this is also the case for the Kap114p–TBP complex, we incubated the IgG-Sepharose bound Kap114-PrA-TBP complex, isolated from yeast cytosol, with RanGTP or RanGDP. The material eluted after the incubation with Ran was collected (constituting the unbound fraction) and the IgG-Sepharose bound material was eluted with 1 M MgCl 2 to dissociate the remaining TBP, followed by 4.5 M MgCl 2 to dissociate the Kap114-PrA (both fractions constituting the bound fraction). After separation by SDS-PAGE and transfer to nitrocellulose, TBP and Kap114-PrA were detected by immunoblotting with an anti-TBP polyclonal antibody. This antibody not only recognizes TBP but also Kap114-PrA, by virtue of its PrA tag. TBP was not eluted at high or low concentrations of RanGDP (data not shown). A high concentration of RanGTP (9.5 μM) led to dissociation of more than half the TBP from the complex . However at lower concentrations of RanGTP (480 nM) only a small amount of TBP was dissociated . These results suggested that RanGTP rather than RanGDP was required to effect dissociation of TBP from Kap114p and the extent of dissociation depended on the concentration of RanGTP. Many previously published Kap import substrate dissociation experiments have been carried out at RanGTP concentrations in the 5–10-μM range and even at these concentrations many of the dissociation experiments appeared to be fairly inefficient. The concentration of unbound RanGTP in the nucleus is unknown, however, a number of RanGTP-binding proteins such as the 14 Kaps, RanBP1, and the RanGEF, present in the nucleus, will compete for binding to RanGTP. Additional factors might, therefore, be necessary to stimulate RanGTP-dependent dissociation of import substrates from their cognate Kaps. To investigate this possibility we reduced the RanGTP in our experiments to a level where we saw virtually no dissociation of TBP from Kap114p. As TBP functions by binding the TATA box at the core promoter, we first determined whether an 18-bp ds oligonucleotide containing the CYC1 TATA box might stimulate the dissociation of TBP from Kap114p. This particular ds oligonucleotide was also used in the cocrystallization of TBP bound to DNA . Isolated Kap114-PrA-TBP complex was incubated with 380 nM RanGTP, and very little dissociation of TBP from the Kap was observed . In these experiments the dissociated TBP fraction was collected and the bound fraction containing Kap114-PrA and bound TBP was eluted as one fraction with 4.5 M MgCl 2 . Surprisingly, the addition of increasing concentrations of TATA-containing DNA, in the presence of RanGTP, led to the dissociation of most of the TBP from Kap114p . Even with a low concentration of DNA (5 ng/μl or 430 nM) at least half the TBP was released from Kap114p . In contrast, when the complex was incubated with a high concentration of DNA but no RanGTP, no dissociation was observed, demonstrating that DNA alone could not release TBP from Kap114p . The DNA-mediated dissociation was dependent on RanGTP rather than RanGDP, as high concentrations of RanGDP (4.8 μM) and DNA (10 ng/μl) together caused no dissociation . Using biotinylated ds TATA oligonucleotides captured on streptavidin beads, it was also possible to show that the released TBP was bound to DNA (data not shown). These experiments show that at this concentration of RanGTP, TBP is released from Kap114p only in the presence of DNA and suggests a possible mechanism for the specific release of TBP at its DNA target. To determine whether this effect was specific for ds TATA-containing DNA, several controls were carried out using mutant TATA-containing ds DNA, single stranded DNA, and total yeast RNA in the presence of 290 nM RanGTP . Total yeast RNA did not stimulate dissociation, suggesting that we were not observing a nonspecific effect of large negatively charged molecules . We saw a spectrum of activity using other DNA controls, some double and single stranded templates led to low levels of dissociation (data not shown), suggesting that they would not stimulate the release of TBP from the Kap. Other DNA controls including the ds oligonucleotide, M1, that was identical to the TATA oligonucleotide but included two mutations in the TATA box stimulated dissociation quite efficiently. Single stranded TATA DNA and single stranded M1 DNA stimulated some dissociation of the TBP from the Kap. However, most of the controls did not stimulate dissociation as well as the original TATA-containing ds oligonucleotides . As DNA stimulated the dissociation of TBP in the presence of RanGTP, it was possible that a TBP-interacting protein could also function in this way. We examined TFIIA as it has been shown to bind TBP alone and complexed with DNA . At the RanGTP concentrations (290 nM) used in this experiment, almost no dissociation of TBP from Kap114p was observed and the addition of ds TATA-containing DNA (2 ng/μl) with RanGTP resulted in the dissociation of more than half of the TBP . The addition of TFIIA (65 nM) together with RanGTP, however, did not result in the dissociation of any TBP . At this concentration, analysis of amido black–stained blots suggested that TFIIA was in excess of TBP (data not shown). RanGTP-dependent dissociation was also not observed when a higher concentration of TFIIA (200 nM) was used (data not shown). This suggested that TFIIA could not stimulate the RanGTP-dependent dissociation of TBP from the Kap in the absence of ds DNA, raising the possibility that Kap114p may prevent TBP from associating with TFIIA before it has bound DNA. When TFIIA was added together with RanGTP and DNA, as expected most of the TBP was dissociated from Kap114p in a similar manner to that seen with RanGTP and DNA alone . However, the addition of TFIIA appeared to stimulate the dissociation seen with RanGTP and DNA. This may be due to the fact that TFIIA stabilizes the TBP–DNA interaction . Here we show that the major import pathway for TBP is mediated by the newly characterized Kap, Kap114p. TBP is also imported into the nucleus by Kap114p-independent pathways and we suggest that these pathways may be mediated by Kap123p and Kap121p. As expected for a Kap family member, the dissociation of TBP and Kap114p is RanGTP-dependent. However, we show here that this dissociation is greatly stimulated by the addition of TATA-containing ds DNA. This suggests a mechanism for the targeted dissociation of TBP at the promoter of genes to be transcribed. The isolation of a cytosolic Kap114p–TBP complex suggested that Kap114p mediated the import of TBP into the nucleus and analysis of the localization of TBP in the absence of Kap114p confirmed this. Our ability to reconstitute the Kap114p–TBP complex using recombinant proteins suggested that they interact directly. In addition, the Kap114p–TBP complex, isolated from yeast cytosol, was resistant to incubation with S7 nuclease and DNase, suggesting that the complex was not mediated by nucleic acid (data not shown). Strains lacking Kap114p are viable, in contrast to strains lacking TBP, which is encoded by an essential gene. In common with the Pdr6p/Kap122p–mediated import pathway for TFIIA (Titov, A., and G. Blobel, manuscript submitted for publication) and the import of ribosomal proteins by Kap123p proteins , this represents another example of a nonessential Kap importing an essential substrate. This led us to search for additional pathways into the nucleus for TBP. The isolation of TBP-interacting Kaps, Kap123p and Kap121p, suggested that they too might mediate import of TBP. In both a Kap123p deletion strain and in a Kap121p temperature sensitive strain, however, we saw no mislocalization of TBP. The fact that we could detect no other Kaps in a complex with TBP-PrA, and that these two Kaps were not isolated in analogous coprecipitation experiments with Lhp1p-PrA (Rosenblum, S., and G. Blobel, unpublished data) were indicative of a potential role for Kap123p and Kap121p in TBP import. The demonstration of direct binding and of an effect on nuclear import will be needed, however, to conclusively demonstrate the role of Kap123p and Kap121p in TBP import. It is likely that Kap114p mediates the bulk of TBP nuclear import, as evidenced by the fact that among the TBP-interacting Kaps, only a kap114 deletion leads to mislocalization of TBP, even in the presence of an otherwise wild-type complement of Kaps. We do not know if these are the only Kaps responsible for the import of TBP into the nucleus. Kap121p is encoded by an essential gene , and we were, therefore, not able to test for synthetic lethality by deletion of all three genes in one strain. It is possible that there are additional Kap-mediated pathways for the import of TBP as Kap121p was only detected by immunoblotting, indicating that other Kaps could be interacting below the limits of detection by Coomassie blue staining. In strains lacking both Kap114p and Kap123p only very small amounts of TBP–copurifying Kap were detected by our assay. As some TBP was being imported into the nucleus to support normal transcription and cell growth (discussed below), it is possible that we are not able to detect very nonabundant Kaps in this way. The fact that we could not detect them may suggest that these Kaps interact with their substrates with lower affinity or the interaction is less stable. It is also possible that some TBP is entering the nucleus by a Kap-independent pathway or by diffusion. Diffusion is unlikely, however, as TBP is believed to dimerize in the cytoplasm, increasing its molecular weight to beyond the exclusion limit of the NPC . Like mammalian ribosomal proteins, this is an example of a single substrate that appears to have multiple secondary pathways into the nucleus . It may prove to be a general phenomenon, whereas one Kap plays a major role in the import of a given substrate, this function is redundant with several other Kaps. Such a multiply redundant, hierarchical import pathway also suggests that the coordinate regulation of the Kap family is likely to be very complex. This may help explain why there are so many nonessential or redundant Kaps. Of the fourteen β Kaps present in the yeast genome, only four are encoded by essential genes (Kap95p, Kap121p, Crm1p, and Cse1p), whereas deletion of the others lead to varying effects on cell growth . It is possible that the essential Kaps transport essential proteins, or they may play a more general role and can backup many pathways. Alternatively, they may carry out the bulk of nuclear transport, although their relative abundance would suggest that this is not the case . Kap121p has been shown to functionally overlap with Kap123p and can participate in the import of ribosomal proteins . If Kap123p and Kap121p also play a role in the import of TBP, it raises the possibility that the Kap family may be divided into functional subgroups. Future identification of additional substrates for these three Kaps will determine whether this subgroup coordinates the import of other proteins. Kap123p copurified with TBP-PrA in the absence of Kap114p, whereas Kap121p was detected in small quantities in both the presence and absence of Kap114p. As Kap114p would be present in a wild-type yeast cell, it is possible that some TBP import is constitutively mediated by other Kaps or that other Kaps participate in TBP import only under specific conditions. These conditions could be dictated by the available carbon source, stress, or phase of the cell cycle amongst others. Several Kaps have been shown to have more than one substrate, and the fact that there are many more nuclear proteins than Kaps suggests that this will be a general phenomenon . We observed several Kap114- PrA copurifying bands in addition to TBP, suggesting that it also may have other import substrates and future characterization should determine the identity of these proteins. The Kaps may be coordinated in a complex network of overlapping substrate specificities, raising the problem of how several Kaps can recognize the same substrates while distinguishing between others. The recognition of a substrate by a Kap is determined by the NLS contained in the substrate. The crystal structure of TBP has been elucidated both alone and complexed with DNA, TFIIA, and TFIIB . This has allowed the precise binding sites of these molecules within TBP to be determined. It is not yet known whether the TBP NLS recognized by Kap114p overlaps with one of these sites, such as the DNA-binding domain, or whether the Kap recognizes another domain of TBP. It is possible that other Kaps, for example Kap121p and Kap123p, may recognize the same NLS in TBP as Kap114p. However, comparison of TBP with known Kap123p and Kap121p substrates has not yet revealed any similarities at the amino acid level that may represent a consensus NLS. To function in transcription, TBP in the nucleus must be first dissociated from Kap114p and then interact with DNA and other components of the transcriptional machinery. All the Kaps are believed to bind RanGTP with their homologous amino-terminal domain as shown in the structure of mammalian Kap β2 complexed with RanGTP . As expected RanGTP, which is believed to be the predominant form of Ran in the nucleus, is necessary for the dissociation of TBP from Kap114p. RanGDP does not appear to be able to function in this process. However, we observed in vitro that not all the TBP was dissociated from Kap114p, even at high (10 μM) RanGTP concentrations. Previously published Kap import substrate dissociations have been carried out at RanGTP concentrations in the 5–10-μM range, however, many of these dissociation experiments appeared similarly inefficient . Although the total Ran concentration in the whole cell has been suggested to be as high as 5–10 μM, it is difficult to predict the concentration of unbound RanGTP in the nucleus . As there are at least 16 RanGTP binding partners present in the nucleus, which bind RanGTP with dissociation constants in the micromolar to nanomolar range, the concentration of unbound RanGTP may be much lower than this . At lower (190–380 nM) concentrations of RanGTP, we observed almost no dissociation of TBP from Kap114p that led us to search for stimulatory factors. We identified ds TATA–containing DNA as one such factor. As nonnucleosomal ds TATA–containing DNA would be the available form of DNA at genes about to undergo transcription, this suggests a mechanism whereby TBP may only be dissociated from the Kap at its point of function. The dissociation caused by TATA-containing DNA was most efficient, however, other DNAs could also stimulate dissociation of TBP from Kap114p to varying extents. This lack of specificity in vitro may be because TBP can bind all DNA with some affinity . In vivo, other GTFs such as TFIIA and TFIIB would be present and there is evidence to suggest that they increase the specificity of TBP for TATA sequences . Alternatively, in vivo the initial dissociation at the promoter may be TATA-independent; once dissociated, TBP could then move to the TATA box itself to form a high affinity interaction . In this way, the dissociation would still most likely occur at the promoter as only a small proportion of DNA in the nucleus is actually available for TBP binding. Most DNA is incorporated into nucleosomes, and naked, nonnucleosomal DNA is only likely to be found at promoters of genes that are about to be transcribed or after the replication fork . The TBP-interacting protein TFIIA did not stimulate the dissociation of TBP in the absence of TATA-containing DNA. It is possible that association of TBP with Kap114p prevents TBP from prematurely interacting with other PIC components before reaching the promoter. TFIIA did stimulate dissociation in the presence of DNA, which may be due to its ability to stabilize TBP–TATA interactions . It is not yet known whether other TBP-interacting proteins such as TBP-associated factors, TFIIB, negative regulators of TBP, and components of the SAGA complex can stimulate the dissociation of TBP from Kap114p, either with or without DNA. Future experiments will be needed to determine the sequence of events of PIC complex formation in vivo. There is believed to be enough TBP present in the cell to participate in transcription from all 6,000 plus promoters at one time . There are at least three mechanisms by which the availability of TBP and its ability to function at specific promoters is negatively regulated. These include the proteins or protein complexes Mot1p, NC2, and Nots . It is likely that much of the cellular TBP is negatively regulated by these factors, which may act by sequestering TBP . Our in vitro dissociation experiments would argue that the Kap participates in this regulation, by only releasing the TBP it has imported, at its point of function. By reducing the amount of RanGTP we observed a dissociation requirement for further factors that may prove to be a general phenomenon in nuclear transport. Previous studies using Kap111p/Mtr10p have shown that the addition of total cellular RNA also stimulated the RanGTP-dependent dissociation of the mRNA-binding protein Npl3p, from Kap111p . The DNA-stimulated, RanGTP-mediated targeted dissociation of TBP from Kap114p we observe in vitro suggests that in vivo dissociation might be effected at much lower concentrations of RanGTP than previously thought. For targeted dissociation to occur, Kap114p would be predicted to travel with TBP from the nucleoplasmic face of the NPC to the promoter, and this raises the possibility that this Kap may also be functioning in intranuclear delivery of TBP. Very little is known about the movement of molecules within the nuclear interior and it is possible that Kaps function not only in nucleocytoplasmic transport but may also mediate the efficient transport of proteins within the nucleus. It was surprising that although much of the TBP was mislocalized in the Δ kap114 deletion strain, we could detect no growth defect. It is possible that the small amount of TBP that was imported into the nucleus was sufficient to support transcription. Whether this small amount of TBP is now no longer subject to the negative regulation discussed above, and is all functioning in transcription, remains to be determined. In the absence of Kap114p, TBP imported by other Kaps such as Kap121p and Kap123p, may also be dissociated in a DNA-stimulated, RanGTP-dependent fashion at its point of function and it will be interesting to determine whether this will be a generalized theme in nuclear transport. The experiments presented here for TBP and previously published data for TFIIA suggest that at least two components of the PIC are imported by separate pathways (Titov, A., and G. Blobel, manuscript submitted for publication). It remains unclear how other components of the PIC are imported into the nucleus and whether their import by separate pathways plays a role in their regulation. In summary, we have identified a new pathway for protein import into the nucleus, mediated by the novel transport factor Kap114p. This pathway constitutes the major import pathway for TBP, however, other Kaps also appear to participate in TBP import. The import of TBP is one of the first examples in yeast of a substrate having multiply redundant, hierarchical import pathways and suggests that the Kaps may be coordinated in a complex network of overlapping substrate specificities. We also present data suggesting that TBP is only released by the Kap at its point of function, raising the possibility that the Kaps may play a role in the intranuclear targeting of their substrates as well as nucleocytoplasmic transport. | Study | biomedical | en | 0.999997 |
10385522 | Mouse monoclonal (clone 2H11) and rabbit polyclonal anti-HRPs were purchased from Advanced ImmunoChemical Inc. and from DAKO, respectively. Polyclonal antibodies against secretogranin II and synaptophysin/p38 have been described previously . Immobilon-P membrane was obtained from Millipore ; 3 H-Dopamine was from Amersham International ; NHS-SS-biotin was from Pierce; Express 35 S-labeling mix of methionine and cysteine was from NEN Life Science Products; and Amplify™ was from Amersham . Other chemicals were purchased from Sigma Chemical Co. Mouse receptor grade epidermal growth factor (EGF), human iron saturated transferrin (Trn), protein A, and 2H11 were iodinated using the modified IODO-GEN procedure as described elsewhere . A chimeric cDNA comprising the human growth hormone signal sequence followed by HRP, the transmembrane domain, and cytoplasmic tail of P-selectin was generated as previously described , as were chimeras with deletions of the cytoplasmic tail. The tetra-alanine substitutions and point mutations were made as described . Two additional mutants were constructed in the same way using the following primers: Y777A: AGCCACCTAGGAACAGCTGGAGTTTTTACAAA and Y777F: AGCCACCTAGGAACATTTGGAGTTTTTACAAA. Only the sense primer is shown, the anti-sense primer used is the exact complement. The constructs obtained were confirmed by sequencing. The rat pheochromocytoma cell line PC12 (CCL23; American Type Culture Collection) was maintained and transfected as described previously . Where necessary, cells were stimulated with 10 mM Carbachol for 30 min at 37°C. Cells were labeled with 125 I-Trn or 125 I-EGF as described previously . Removal of cell surface–bound ligands was as described in Blagoveshchenskaya et al. . 125 I-2H11 (1 μg/ml) was internalized for 1 h at 37°C in growth medium and then cells were placed on ice. Antibody present on the plasma membrane was removed by treatment of cells with acidic buffer (100 mM sodium acetate, pH 4.0, and 500 mM NaCl) on ice twice for 10 min each. DCG were labeled with 3 H-Dopamine as previously described . PC12 cells grown on 150-mm dishes to 70% confluency were rinsed twice with methionine/cysteine-free DME supplemented with 0.5% FCS and cultivated in this medium for 30 min in the CO 2 -incubator. Cells were pulsed in 15 ml of methionine/cysteine-free DME containing 2 mCi of Express 35 S-labeling mix and 1% FCS for 10 min at 37°C, washed with growth medium, and chased for either 20 min at 37°C or 16 h at 37°C in fresh growth medium. 35 S-labeled cells were washed three times with ice-cold HSE buffer (0.25 M sucrose, 10 mM Hepes-KOH, pH 7.2, 1 mM EDTA, 2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ ml pepstatin A) and subjected to subcellular fractionation for isolation of immature and mature DCG (see below). Subcellular fractionation of PNS on the initial 1–16% Ficoll velocity gradients, secondary 5–25% Ficoll velocity gradients for lysosome isolation and 5–25% Glycerol velocity gradients for SLMV isolation were carried out as described Cells were fed with 125 I-2H11 as described above, homogenized, and then subjected to a two-stage fractionation procedure for lysosomal isolation. Targeting data were presented as a lysosomal targeting index (LTI), i.e., the amount of 125 I-2H11 radioactivity present in the lysosomal peak for each chimera normalized to that for wild-type ssHRP P-selectin . In all experiments, the LTI for wild-type ssHRP P-selectin was set at 1. To take into account variations of expression level and lysosomal yield, the amount of 125 I-2H11 radioactivity present in the lysosomal peak ( 125 I-2H11 peak) has been corrected for the amount of NAGA activity (NAGA peak) within the lysosomal fractions and for total 125 I-2H11 radioactivity in the homogenate ( 125 I-2H11 hmg). After simplifying the original equation, the LTI was defined as follows: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}LTI=\frac{\frac{mutant\;\hspace{.167em}^{125}I-2H11\;peak}{mutant\;\hspace{.167em}^{125}I-2H11\;hmg}:\frac{\hspace{.167em}^{}mutant\;NAGApeak}{\hspace{.167em}^{}mutant\;NAGA\;hmg}}{\frac{WT\;\hspace{.167em}^{125}I-2H11\;peak}{WT\;\hspace{.167em}^{125}I-2H11\;hmg}:\frac{WT\;NAGApeak}{\hspace{.167em}^{}WT\;NAGAhmg}}.\end{equation*}\end{document} Typically, the LTI for tail-less ssHRP P-selectin763 was subtracted from those for the other chimeras in each experiment to provide a baseline, i.e., the LTI for ssHRP P-selectin763 was considered as 0. The LTIs of the mutants were therefore described on a scale within a range set by ssHRP P-selectin (1) and ssHRP P-selectin763 (0). SLMV targeting indexes were calculated by normalizing the amount of HRP activity in the peak for synaptophysin (p38) recovery and chimera expression level as indicated below. Amounts of p38 in the SLMV peaks and in the homogenate were determined by quantitative Western blotting as described . 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}SLMV-TI=\frac{\frac{mutant\;HRP\;peak}{mutant\;HRP\;hmg}:\frac{mutant\;p38\;peak}{mutant\;p38\;hmg}}{\frac{WT\;HRP\;peak}{WT\;HRP\;hmg}:\frac{WT\;p38\;peak}{WT\;p38\;hmg}}.\end{equation*}\end{document} Those fractions (14–20) from initial 1–16% Ficoll gradients containing most 3 H-Dopamine radioactivity were pooled, diluted with HB (320 mM sucrose, 10 mM Hepes-NaOH, pH 7.3), and 4 ml of this material was then layered on top of 9 ml preformed 0.9–1.85 M sucrose gradients. The gradients were centrifuged at 35,000 rpm for 20 h in a SW40Ti rotor and fractionated. The lighter peak of HRP activity containing NAGA and internalized 125 I-EGF corresponds to late endosomes, while the denser peak, which overlaps with 3 H-Dopamine distribution, contains the DCG . To quantitate targeting to DCG, granule targeting indexes (GTI) were calculated essentially as described above, except 3 H-Dopamine was used to normalize for DCG recovery as follows: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}GTI=\frac{\frac{\hspace{.167em}^{}mutant\;HRP\;peak}{\hspace{.167em}^{}mutant\;HRP\;hmg}:\frac{mutant\;\hspace{.167em}^{3}H\;peak}{mutant\;\hspace{.167em}^{3}H\;hmg}}{\frac{\hspace{.167em}^{}WT\;HRP\;peak}{\hspace{.167em}^{}WT\;HRP\;hmg}:\frac{WT\;\hspace{.167em}^{3}H\;peak}{WT\;\hspace{.167em}^{3}H\;hmg}}.\end{equation*}\end{document} The gradient system used was that established earlier by Tooze and Huttner and then modified . In brief, after three washes with ice-cold HSE buffer, cells from one 150-mm dish were scraped in this buffer and pelleted by low-speed centrifugation at 1,000 g for 3 min. The supernatant was discarded and cells resuspended in 2 ml of fresh HSE buffer were then passed 10 times through a ball bearing homogenizer with 0.01-mm clearance. After centrifugation of the homogenate at 1,000 g for 10 min, 1.3 ml of PNS was layered on top of an 11-ml preformed 0.3–1.2 M sucrose gradient made in 10 mM Hepes-KOH, pH 7.2, and centrifuged in a SW40Ti rotor for 19 min with slow acceleration and deceleration. The gradients were then collected in 1-ml fractions from the top of the tube. After fractionation of the initial 0.3–1.2 M sucrose gradients, the fractions containing iDCG (fractions 2–4) or mDCG (fractions 4–6) were pooled, diluted with 10 mM Hepes-KOH, pH 7.2, to a final volume of 4 ml, and then layered on top of 9-ml preformed 0.9–1.7 M sucrose gradients made in 10 mM Hepes-KOH, pH 7.2, and recentrifuged to equilibrium for 21 h in a SW40Ti rotor. 1-ml fractions were collected from the top of the equilibrium gradients. 200 μl of each fraction was diluted in NDET buffer to 10 ml (final concentration: 1% NP-40, 0.4% sodium deoxycholate, 66 mM EDTA, 10 mM Tris-HCl, pH 7.4, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin A) and used for immunoprecipitation of 35 S-labeled SgII. The rest was aliquoted and used for measurement of HRP activity or for Western blotting with anti-SgII. Immunoprecipitation using polyclonal anti-SgII followed by SDS-PAGE was carried out as described previously . The gels were exposed to a PhosphorImager screen for weak β emission (Bio-Rad Laboratories) and then to x-ray film for 2 d. Quantitation of data was performed using Molecular Analyst software (Bio-Rad Laboratories). The distribution of unlabeled SgII across the gradients was analyzed by Western blots of fractions followed by detection with polyclonal anti-SgII, then with 125 I–protein A and PhosphorImager exposure. N -acetyl-β- d -glucosaminidase activity was determined as described . Protein concentration was measured using the Micro BCA protein assay kit (Pierce) according to the manufacturer's instructions. HRP activity in the samples was determined in triplicate as previously described . HRP proteolysis, internalization assays, and the DAB cytochemistry were carried out as described . We set out to reexamine the determinants needed for DCG targeting of P-selectin in PC12 cells where parallel analyses of SLMV and lysosomal targeting were being carried out. To quantitatively evaluate targeting of HRP– P-selectin chimeras to DCG at steady state conditions, we have introduced an improved subcellular fractionation protocol for DCG isolation, as illustrated in Fig. 1 , because of the contamination of DCG enriched fractions by late endosomes. The first stage of DCG isolation is a velocity centrifugation of PNS on preformed linear 1–16% Ficoll gradients as described earlier . This procedure was originally developed to separate SLMV and DCG, but has also proved successful for the separation of plasma membrane, multiple endosomal compartments, and lysosomes . To determine the position of intracellular compartments on this gradient, the distributions of 125 I-Trn (early recycling endosomes), 125 I-EGF (late endosomes), 3 H-Dopamine (DCG), and NAGA activity (lysosomes) were monitored. PC12 cells transfected so as to transiently express ssHRP P-selectin were loaded either with 3 H-Dopamine, 125 I-Trn, or 125 I-EGF, homogenized, and a PNS was then centrifuged on linear 1–16% Ficoll gradients. The early endosomes containing 125 I-Trn sedimented in the low density fractions 5–10 . After binding to the plasma membrane at 4°C, 125 I-EGF was allowed to internalize for 20 min at 37°C to reach late endosomal compartments . The distribution of 3 H-Dopamine overlaps both with 125 I-EGF and partially with NAGA in fractions 14–20 , indicating that DCG and late endosomes have similar sedimentation characteristics under these centrifugation conditions. To separate late endosomes from DCG, fractions containing the majority of 3 H-Dopamine radioactivity (14–20) were pooled and recentrifuged on 0.9–1.85 M sucrose gradients. The single peak of HRP activity seen on the initial Ficoll gradient splits into two peaks on the sucrose gradient . The lighter peak of HRP activity corresponds to late endosomes since it cosediments with NAGA and 125 I-EGF internalized for 20 min at 37°C, but is devoid of 125 I-Trn. Although we have no direct evidence from these data as to whether the HRP activity is within the same organelles which contain NAGA and 125 I-EGF, we have used a diaminobenzidine density shift procedure to show that this is the case in H.Ep.2 cells . The second, denser peak of HRP activity codistributes with the single peak of 3 H-Dopamine, thereby indicating the position of DCG on this gradient . Therefore, to quantitate targeting to DCG for each chimera, we measured the amount of HRP activity within the latter peak. We have carried out a screen for short discrete determinants in the cytoplasmic domain that might mediate trafficking to the DCG in PC12 cells, using a series of HRP– P-selectin chimeras . Inclusion of the transmembrane domain, as in these chimeras, has been recently shown to increase the efficiency of targeting to DCG in neuroendocrine RIN5 cells, presumably by providing a more native context in which the cytoplasmic domain operates . Cells expressing HRP–P-selectin chimeras were loaded with 3 H-Dopamine, homogenized, and then the PNS was fractionated as described above. We have monitored the targeting efficiency for each chimera by calculating a granule targeting index (GTI). Most of the chimeras with tetrapeptide substitutions show GTI similar to that for ssHRP P-selectin (GTI = 1) . However, targeting to granules of ssHRP P-selectinYGVF and ssHRP P-selectinTNAAF was reduced by ∼85%: 0.12 ± 0.06 (± SE) and 0.172 ± 0.1, correspondingly . YGVF resembles a typical Tyr-based sorting signal conforming to the consensus YXXø (where X is any amino acid and ø is a bulky hydrophobic amino acid). Since Tyr is often a critical residue within such signals , we examined the contribution of Tyr777 to DCG targeting. As shown in Fig. 3 C, substitution of Tyr777 by Ala, but not by Phe, reduced the GTI by 79%, i.e., to the level of ssHRP P-selectinYGVF , while substitution of Phe780 by Ala did not cause a significant fall of the GTI. Therefore, Tyr777 makes a major contribution to the promotion of granule targeting but can be substituted by another aromatic amino acid without loss of function. Substitution of the sequences surrounding YGVFTNAAF does not have a pronounced effect on DCG targeting. However, we have also tested the effects of grosser alterations within the C2 domain . In agreement with the effect of tetrapeptide alanine substitutions , removal of four carboxy-terminal amino acids (DPSP) did not affect the GTI , whereas larger truncations including removal of TNAAF or TYGVFTNAAF reduced the GTI to levels similar to that of ssHRP P-selectinY777A . Deleting the entire C1 domain also caused the GTI to fall, implying that the spacing between YGVFTNAAF and the lipid bilayer may be important for granule targeting in PC12 cells. Although we and others have shown that when expressed in neuroendocrine cells, P-selectin is targeted to DCG , the distribution of this protein between iDCG and mDCG has not been determined, although current models for granule biogenesis imply that any protein found within mDCG will first enter the iDCG from the TGN. It is of interest to determine whether a mutant that fails to accumulate in mDCG also fails to enter the iDCG (i.e., sorting occurs at the level of the TGN) or is found only in the precursor iDCG population (i.e., signal-mediated sorting is occurring during maturation of iDCG to mDCG). To address this issue, we have adopted a well-established subcellular fractionation procedure for separation of iDCG and mDCG . PC12 cells transiently expressing wild-type HRP P-selectin were pulsed and then chased to label iDCG or mDCG followed by fractionation as described (Materials and Methods). Fractions rich in iDCG or mDCG were collected and recentrifuged on 0.9–1.7 M sucrose equilibrium gradients followed by fractionation and immunoprecipitation with anti-secretogranin II (SgII, a soluble marker protein of DCG) and SDS-PAGE. The distribution on the secondary equilibrium gradient of 35 S-labeled SgII shows a clear separation of iDCG from mDCG , with iDCG peaking in fractions 7–10 and mDCG in fractions 10–13. We wished to determine whether the HRP activity of wild-type ssHRP P-selectin is present within these peaks. Since, this analysis is performed at steady state conditions rather than in pulse–chase experiments, we first examined the steady state distribution along the gradients of the defining marker, SgII. Western blotting shows that even at steady state, SgII is found within those same peaks corresponding to iDCG and mDCG as revealed by pulse–chase experiments . To confirm that SgII is indeed present within functional secretory granules, we examined the effects of secretagogue stimulation on both peaks. In agreement with previous studies , the peaks corresponding to both iDCG and mDCG diminished after carbachol treatment . Having established the distribution of the secretagogue-responsive peaks of SgII corresponding to iDCG and mDCG, we determined the distribution of HRP–P-selectin chimeras on the same gradients. Accordingly, PC12 cells expressing ssHRP P-selectin were fractionated as above followed by measurement of HRP activity across the secondary equilibrium gradients. We have also analyzed the effect of secretagogue stimulation on distribution of HRP activity across these gradients. Fig. 4 , G and H, show that ssHRP P-selectin is present within both iDCG (fractions 7–10) and mDCG (fractions 10–13) and that both peaks of HRP activity are responsive to secretagogue. Since P-selectin has a more complex intracellular distribution than SgII, we expected that in addition to the DCG peaks, there would also be HRP activity corresponding to other organelles. Indeed, in addition to the peaks representing iDCG and mDCG, there is substantial HRP activity within a lighter peak . However, unlike the fall of HRP activity within iDCG and mDCG, HRP activity in the lighter peak increases after secretagogue stimulation. Since secretagogue action transfers ssHRP P-selectin from the DCG to the plasma membrane and thence via endosomes to SLMV , we presume that the material in fractions 5 and 6 represents a mixture of SLMV, plasma membrane, and endosomes. Quantitation of HRP activity revealed that 7.2% of the ssHRP P-selectin in the homogenate was recovered within the iDCG, whereas 14% was present in the mDCG. Importantly, this ratio is comparable to that for SgII recovered at steady state conditions within the iDCG and mDCG, as judged by quantitative Western blotting, 10 and 28%, respectively. Thus, when expressed in PC12 cells, ssHRP P-selectin is found in the iDCG and mDCG in a ratio similar to that of the DCG marker protein SgII. Having established that wild-type ssHRP P-selectin is sorted to both iDCG and mDCG, we determined whether a DCG targeting mutant is present in iDCG but not in mDCG, or whether it is found in neither population. Therefore, we have examined the targeting of ssHRP P-selectinY777A to both populations of DCG. After the fractionation of PNS obtained from cells expressing ssHRP P-selectinY777A , no peaks of HRP activity within the fractions corresponding either to iDCG (fractions 7–10) or to mDCG (fractions 10–13) was detected , despite the presence of HRP activity in the lighter peak (fractions 5–6) as for ssHRP P-selectin . Moreover, secretagogue stimulation did not cause an increase of HRP activity within the light membrane peak, confirming that ssHRP P-selectinY777A is not sorted to either population of DCG. These data strongly suggest that Tyr777-dependent sorting of HRP–P-selectin chimeras to iDCG occurs at the level of the TGN. Previously, we have demonstrated that HRP–P-selectin chimeras are efficiently transported to SLMV in PC12 cells . We have now determined in detail the sequence(s) within the cytoplasmic domain of P-selectin that are needed for trafficking to SLMV, exploiting a glycerol gradient fractionation scheme . We have previously determined that >90% of ssHRP P-selectin from the SLMV peak on glycerol gradients could be immunoisolated using an antibody to the cytoplasmic domain of p38/synaptophysin . Whereas both p38 and ssHRP P-selectin are present within the SLMV peak on this type of gradient, these fractions are completely devoid of 125 I-Trn, thus confirming that the SLMV peak is free from endosomal recycling vesicles . After expression of the HRP–P-selectin chimeras in PC12 cells and subcellular fractionation on glycerol gradients, a SLMV targeting index (SLMV-TI) was calculated for each chimera . Within the C1 domain, substitution of KCPL reduced SLMV targeting by 80% (0.204 ± 0.05) compared with the SLMV-TI of ssHRP P-selectin . Analysis of single amino acid substitutions across KCPL reveals that replacement of Leu768 with Ala resulted in a similar reduction of SLMV targeting (0.23 ± 0.01) , suggesting that Leu768 within KCPL is required for SLMV targeting. In contrast to the C1 domain, most tetrapeptide substitutions within the C2 domain reduced targeting to SLMV to some degree. In the most dramatic of these, the SLMV-TI for ssHRP P-selectinYGVF was as low as that for tail-less ssHRP P-selectin763 (SLMV-TI = 0), which was found to accumulate at the plasma membrane . Analysis of point mutations within YGVF indicates that Tyr777 makes a major contribution to SLMV targeting and that this residue is tolerant of substitution by another aromatic amino acid suggesting that, in at least this respect, targeting to both RSO is mediated by the same determinant. Substitution or deletion of DPSP (ssHRP P-selectinDPSP and ssHRP P-selectin786 ) reduced SLMV targeting by ∼75% (0.25 ± 0.05 and 0.27 ± 0.055, respectively). Altering the sequence between DPSP and YGVF, as seen with ssHRP P-selectinTNAAF , revealed a less severe phenotype (0.43 ± 0.108). Deletions of the C1 or C2 domains, or of the nine carboxy-terminal amino acids (ssHRP P-selectinΔC1 , ssHRP P-selectin776 , and ssHRP P-selectin782 , respectively) also dramatically inhibited SLMV targeting . When expressed in several cell lines, including PC12 cells, a substantial proportion of newly synthesized P-selectin is constitutively transported to the plasma membrane and then efficiently endocytosed to lysosomes for degradation . Findings of cell type–specific variations encouraged us to analyze lysosomal targeting in PC12 cells. We have previously established two assays that allow for quantification of lysosomal targeting of HRP chimeras. The first, an HRP proteolysis assay, reveals exposure of chimeras to intracellular proteolytic activity under steady state conditions in living cells. The second assay uses sequential Ficoll velocity gradients for the isolation of lysosomal compartments allowing for calculation of lysosomal targeting indexes . As measured by HRP proteolysis, substitution of KCPL within the C1 domain dramatically reduced HRP clipping to 1.43 ± 1.08%, as compared with the average for ssHRP P-selectin which was 16 ± 2% . Chimeras in which other sequences within the C1 domain have been substituted, i.e., ssHRP P-selectinKDDG and ssHRP P-selectinNPHS , were proteolyzed as efficiently as ssHRP P-selectin . Analysis of point mutations across KCPL revealed that as for SLMV targeting, Leu768 also operates to promote lysosomal targeting . In contrast, two mutations of the C2 domain, ssHRP P-selectinYGVF and ssHRP P-selectinDPSP , exhibited a significant increase in HRP proteolysis, up to 45 ± 5% and 35 ± 6% compared with 16 ± 2% for the wild-type chimera. Tyr777, a critical residue within YGVF for targeting to DCG and SLMV, was also found to be an important residue in preventing lysosomal targeting, since when substituted by Ala but not by Phe, it caused increased HRP clipping of 32 ± 4.4% . The analysis of targeting to lysosomes described above was performed under steady state conditions and does not answer the question of whether chimeras pass exclusively via the plasma membrane to lysosomes or are transported directly from the TGN. This is an important point to establish, since a direct route from the TGN to the lysosomes might bypass the compartment from which SLMV bud, thus leading to a misinterpretation of the SLMV targeting data. Therefore, we determined the efficiency of delivery of internalized 125 I-2H11 from the plasma membrane to lysosomes using the fractionation procedure designed for isolation of lysosomal compartments. After a 1-h uptake of 125 I-2H11 at 37°C, 11% of 125 I-2H11 radioactivity was present in the lysosomal peak of cells expressing ssHRP P-selectin . However, only 2.8% of the 125 I-2H11 intracellular radioactivity was detected in the lysosomal peak from cells expressing ssHRP P-selectin763 . If most ssHRP P-selectin transfers to lysosomes directly from the TGN, then the amount of 125 I-2H11 recovered in the lysosomal peak should have been as low as that for ssHRP P-selectin763 which is clearly not the case. This suggests that the ssHRP P-selectin that is not sorted to DCG and SLMV is delivered to lysosomes via the plasma membrane. To ascertain whether those chimeras that exhibit the greatest differences in lysosomal targeting also travel via the plasma membrane, we have calculated LTI by 125 I-2H11 uptake for each chimera. The LTI for ssHRP P-selectinYGVF and ssHRP P-selectinDPSP exceeded those for ssHRP P-selectin implying that these mutant chimeras are indeed targeted to lysosomes via the plasma membrane . ssHRP P-selectinL768A , which was incapable of targeting to lysosomes, as measured by HRP proteolysis , is not detected in lysosomal fractions as seen by 125 I-2H11 uptake either, indicating that the results obtained by either assay are consistent. As shown above, Leu768 was found to promote lysosomal targeting of HRP–P-selectin chimeras in PC12 cells. Mutation of this amino acid might act either by blocking exit from the endosomes or by affecting internalization from the plasma membrane. Since the internalization rate for ssHRP P-selectinL768A is 75% of that of wild-type ssHRP P-selectin (see Table II ), the latter was not the case. To determine whether ssHRP P-selectinL768A accumulates within Trn-containing endosomes, we have exploited a DAB density shift procedure. DAB cross-linking in the presence of H 2 O 2 results in an increase in density of the vulcanized HRP-containing organelles, as seen by subcellular fractionation. PC12 cells transfected so as to transiently express ssHRP TrnR(tail−) , ssHRP P-selectin , or ssHRP P-selectinL768A were analyzed for their ability to shift 125 I-Trn using a DAB-reaction protocol. As shown in Fig. 9 and quantitated in Table I , in control cells expressing ssHRP TrnR(tail−) only 8% of the 125 I-Trn was shifted . We consider this value to be a basal level, representing signal-independent traffic through the endosomes. It should be noted that DAB shift percentages (Table I ) do not measure absolute amounts of chimera within the Trn-containing endosomes since the procedure was performed on transiently expressing cells. They provide comparative data showing differences in the levels of colocalization between different HRP chimeras and 125 I-Trn. ssHRP P-selectin demonstrated higher colocalization with 125 I-Trn than ssHRP TrnR(tail−) , shifting 13% of the original radioactivity. However, in cells expressing ssHRP P-selectinL768A , the shift was 27%, suggesting that this chimera is concentrated within early Trn-containing endosomes and indicating that Leu768 operates at the level of Trn-positive endosomes to mediate transfer to SLMV and to lysosomes. Some of the variations in targeting to post-Golgi destinations might be attributable to alterations in internalization rates, as demonstrated for VAMPII . Therefore, we have measured internalization rates for ssHRP P-selectin , ssHRP P-selectin763 , ssHRP P-selectinYGVF , ssHRP P-selectinTNAAF , ssHRP P-selectinDPSP , and ssHRP P-selectinL768A using a cell surface biotinylation procedure . First order internalization rates were calculated from data obtained during the first 4 min of endocytosis at 37°C and expressed as %/min (Table II ). In agreement with previous studies , ssHRP P-selectin763 was internalized ∼10-fold less efficiently than ssHRP P-selectin . Partial inhibition of endocytosis (by 40%) was observed for ssHRP P-selectinDPSP , while the other chimeras revealed smaller reductions. These data strongly suggest that RSO and lysosomal targeting signals operate independently of internalization from plasma membrane. Recent data from Shi et al. have indicated that targeting of VAMPII to SLMV in PC12 cells can occur by two pathways: either directly from the plasma membrane or from endosomes. The former route is thought to be AP2, clathrin, and dynamin dependent and is BFA insensitive, whereas the latter is AP3 and adenosine ribosylation factor (ARF) 1 mediated and is BFA sensitive . Our data presented above suggest the involvement of an endosomal intermediate en route to SLMV, since both SLMV and lysosomal targeting are dependent on Leu768. To provide further support for this hypothesis, we have examined the BFA sensitivity of SLMV targeting for wild-type HRP P-selectin . Fig. 10 A shows that BFA treatment resulted in an 88% reduction of the HRP activity recovered within the SLMV peak. The magnitude of this effect is similar to the 77% reduction in SLMV targeting found for ssHRP P-selectinL768A . This strongly suggests that HRP–P-selectin chimeras are passing to SLMV via an endosomal intermediate. As a control, we analyzed levels of the endogenous SLMV membrane protein synaptophysin/p38 in those same SLMV peaks in which HRP activity was measured . As shown in Fig. 10 B, levels of p38 in the SLMV peaks from BFA-treated and untreated cells were identical, confirming the observation of Schmidt et al. that p38 is sorted to the SLMV from a plasma membrane– derived subcompartment and that this process is therefore BFA insensitive. In this work, we have explored the relationships between trafficking to SLMV, DCG, and lysosomes in PC12 cells, by characterizing the targeting signals of P-selectin, which is localized to all three organelles in this cell line. We have used quantitative measures of the targeting of a series of HRP–P-selectin chimeras to identify the sequences needed for delivery to a variety of destinations. In summary of our results , we conclude that: (a) While a Tyr-based motif within the cytoplasmic domain is critical for trafficking of HRP–P-selectin to both DCG and SLMV, additional determinants are required to direct this protein to SLMV. (b) There is an overlap between trafficking routes to SLMV and to lysosomes, since Leu768 is essential in exiting from the Trn-positive endosomes for subsequent transfer to either of these organelles. (c) There are complex relationships between targeting to RSO and to lysosomes, since reduction of SLMV or DCG targeting results in a corresponding rise in lysosomal delivery but does not affect delivery to the alternative RSO. We have discovered that alanine substitution of two short colinear determinants located within the C2 domain of the cytoplasmic tail, YGVF and TNAAF, causes a considerable reduction in targeting to the DCG in PC12 cells. HRP–P-selectin chimeras in which these sequences are substituted by Ala were targeted to DCG at levels only 10% above that found for the tail-less ssHRP P-selectin763 . Analysis of chimeras in which sequences on either side of YGVFTNAAF are deleted shows that removal of the four–amino acid sequence at the carboxy side did not affect DCG targeting. Deleting the C1 domain reduced DCG targeting to basal levels, suggesting that the spacing of YGVF and TNAAF from a lipid bilayer is important for the proper functioning of DCG targeting determinants. When carboxy-terminal deletions are extended to remove first TNAAF and then both TNAAF and YGVF, we do not find an additive inhibition of DCG targeting, since removal of TNAAF alone (ssHRP P-selectin782 ) caused the same reduction as removal of YGVFTNAAF (ssHRP P-selectin776 ). In addition, while TNAAF does not reveal homology to other sorting signals known to promote targeting to different post-Golgi destinations, YGVF is similar to other Tyr-based signals . We have found that Tyr777 is important for YGVF to function and is tolerant of conservative replacement with another aromatic amino acid . Therefore, we speculate that Tyr777 plays a major role within the targeting determinant, while TNAAF may make a local structural contribution to its function. Our results on identification of DCG targeting determinants within P-selectin partly agree with those obtained in the anterior pituitary line AtT-20 by Modderman et al. . These authors found that Leu768/Asn769 within the C1 domain, as well as Tyr777, Gly778, and Phe780 within the C2 domain, are needed for DCG targeting in AtT-20 cells. Interestingly, the Leu768/Asn769 DCG determinant in AtT-20 cells overlaps with that (Leu768) which functions as a lysosomal targeting signal in both PC12 and NRK cells , but has no effect on DCG targeting in PC12 cells. One plausible explanation for this apparent discrepancy is that a recycling route leading to DCG via plasma membrane and endosomal intermediates is more important in AtT-20 than in PC12 cells for maintaining steady state levels of proteins within the granule membrane. If so, then this would explain why mutation of Leu768, which leads to an accumulation of P-selectin within the early Trn-containing endosomes, reduces levels of the protein in the DCG within AtT-20 cells, but not in PC12 cells. The DCG targeting determinants found in P-selectin do not reveal any homology to the DCG targeting signal identified in VAMPII . Our finding that a Tyr-based sequence promotes DCG targeting was unexpected; Tyr-based motifs are generally thought to be involved in the recruitment of adaptors followed by the binding of clathrin and are therefore concerned with selection for entry into small vesicles. This is difficult to reconcile with a simple model for entry of membrane proteins into forming granules in which sorting in the plane of the membrane precedes budding of an iDCG from the TGN. The role of clathrin and AP1 during subsequent maturation of forming granules is thought to be in the removal via small vesicles of proteins like furin and M6PR that are present in immature but not mature granules . Our data show that the ratio of relative levels of HRP–P-selectin within iDCG (7.2%) and mDCG (14%) is similar to that found for SgII (10 and 28%, respectively). Therefore, we conclude that Tyr 777 promotes sorting to DCG at the level of the TGN since ssHRP P-selectinY777A was not found in either population of DCG . These data lead us to suggest an alternative mechanism used in delivery of membrane proteins to this RSO. We speculate that coats might be involved as planar matrices in concentrating proteins within the TGN into “rafts” that could interact with the forming dense core, thus driving granule formation in a process analogous to virus budding. However, until now, this mechanism of sorting at the level of the TGN has only been demonstrated for transmembrane proteins that are involved in apical transport in epithelial cells . A contribution of the transmembrane domain of P-selectin to the formation of DCG would be consistent with the raft model, since the transmembrane domain might facilitate the inclusion of protein within such a raft. Although AP1 and clathrin have not yet been shown to be directly involved in granule formation in the TGN in vivo, in vitro granule budding assays have documented a role for ARF1 (which is implicated in recruitment of AP1 and clathrin to the TGN) in promoting the formation of DCG . Currently, granule formation is very poorly understood, but a role for cytoplasmic factors, as well as interactions between the core and the lumenal domains of membrane proteins, clearly must be included in models of granule biogenesis. In this study, we show that KCPL mediates sorting from early Trn-containing endosomes to late endosomes and lysosomes in PC12 cells. Detailed site-directed mutagenesis revealed that Leu768 within this tetrapeptide promotes lysosomal targeting, i.e., operates as a positive lysosomal targeting signal in PC12 cells . Substitution of Leu768 caused the retention of this mutant chimera within Trn-positive endosomes as seen by colocalization with 125 I-Trn using a DAB-shift procedure . In addition, some tetra-alanine substitutions within the C2 domain, most notably, YGVF and DPSP, have been found to increase delivery to protease-rich compartments in PC12 cells. This strongly suggests that YGVF and DPSP operate as lysosome avoidance signals at late stages of endocytosis , in agreement with results that we obtained previously in H.Ep.2 cells using double mutants . To date, very little is known about the sequences needed for targeting to SLMV. The only protein within which such signals have been characterized in detail is VAMPII, in which an amphipathic α-helix was found to be a necessary requirement for SLMV targeting in PC12 cells . However, the SLMV targeting signal in VAMPII was later found to be required for endocytosis from plasma membrane implying that internalization and SLMV targeting are controlled by the same signal . By contrast, our results show that in HRP–P-selectin chimeras, determinants located in both the C1 and C2 domains are implicated in targeting to SLMV regardless of internalization . Within the C1 domain, KCPL and, in particular, Leu768 are necessary requirements for SLMV targeting . In addition, most sequences within the C2 domain; YGVF (in particular, Tyr777), as well as DPSP and, to a lesser extent, TNAAF, are needed for SLMV targeting . Thus, the trafficking of HRP–P-selectin chimeras to SLMV is controlled by multiple signals located in different portions of the cytoplasmic tail. An important consideration in understanding SLMV biogenesis is how the signal-mediated trafficking steps en route to SLMV operate with respect to those en route to other post-Golgi destinations, such as lysosomes and DCG. We have discovered that Leu768 is needed for both SLMV and lysosomal targeting. Since substitution of Leu768 leads to an accumulation within early Trn-positive endosomes, exit from this endosomal compartment is not only required for lysosomal targeting but also for delivery to SLMV, implying that SLMV and lysosomal trafficking involve the same endosomal intermediate. In contrast, mutations of sequences within the C2 domain, i.e., YGVF, TNAAF, and DPSP, block SLMV targeting, but also cause an increase in targeting to lysosomal compartments, as judged by subcellular fractionation and HRP proteolysis . Thus there is clearly a signal-dependent trafficking choice between endosomes, lysosomes and SLMV. Currently, there is evidence for two possible routes taken by membrane proteins targeted to SLMV: (a) direct budding from the plasma membrane (or an elaboration thereof) and (b) via an endosomal intermediate . The former route was found to be adaptor complex AP2, clathrin, and dynamin dependent , while the latter is temperature sensitive and ARF1 and adaptor complex AP3 dependent . However, it may well be that the two pathways can coexist in the same cell . The results described in this paper support the view that the delivery of P-selectin to the SLMV proceeds via an endosomal intermediate which is Trn-positive and is most likely analogous to that observed by Lichtenstein et al. , since failure to exit from this compartment, as exemplified by ssHRP P-selectinL768A , also impairs delivery to the SLMV. Studies from Faundez et al. and Shi et al. suggest that generation of SLMV from endosomal intermediates is BFA sensitive, whereas the direct formation of SLMV from the plasma membrane is not . We have now demonstrated that SLMV targeting of HRP–P-selectin chimeras is inhibited by BFA , which reduces the SLMV-TI to the level of ssHRP P-selectinL768A in untreated cells . In contrast, SLMV targeting of an endogenous membrane marker protein, synaptophysin/p38, is not affected by BFA , indicating that this protein may be delivered to SLMV directly from the plasma membrane in agreement with Schmidt et al. . Extending the finding of two routes taken by one protein to SLMV , our results strongly suggest that different SLMV proteins may preferentially use one of the two alternative pathways. Whether this choice is dependent on the presence of any particular SLMV targeting signal remains to be investigated. Despite the established phenomenon of dual localization of membrane proteins to both RSO , the targeting requirements needed to achieve this biorganellar distribution have not been analyzed in detail. We have found that YGVF and TNAAF are necessary for the targeting of P-selectin both to SLMV and to DCG. However, in both cases mutation of TNAAF caused a smaller reduction of targeting to either RSO compared with that of YGVF with its critical Tyr777. We would therefore argue that YGVF engages with the sorting machinery, while TNAAF operates as a contextual requirement within which YGVF functions. Nevertheless, these data show that the same determinants are needed for delivery to both RSO. Consistent with our data, a similar observation was previously documented for VAMPII, in which a point mutation within the amphipathic α-helix abrogated targeting of this protein to both insulin-containing secretory granules and SLMV in pancreatic β cells . These findings of a common targeting requirement for sorting to RSO may not be fortuitous. First, despite their different origin, both RSO are involved in stimulation-induced release of neurotransmitters followed by fusion with the same acceptor membrane, the plasma membrane, and exo-endocytic recycling, i.e., they do share aspects of their itinerary. Second, a common targeting requirement may account for the secretagogue-dependent transfer of membrane proteins from one RSO to the other . Third, the two major classes of sorting signals, tyrosine-based and dileucine motifs, have both been documented to operate at more than one location in the cell . We have also shown that the substitution of a sequence centered on Leu768, as well as of DPSP, abolished targeting to SLMV but did not affect targeting to DCG . The existence of these additional sequences needed to target proteins to SLMV presumably reflects the differences in biogenesis of the two RSO: while DCG are formed from the TGN, SLMV biogenesis involves the constitutive delivery of SLMV proteins to the cell surface before they are sorted away to the final destination . Altogether, these data suggest that whereas targeting to both RSO is mediated by a common signal, additional determinants are required for progression of P-selectin through endosomal intermediates en route to SLMV (see above). In this paper, we describe for the first time the sequence-dependent triorganellar targeting within one cell line for a single protein. We have summarized our data in a diagram . Our data are consistent with a view of traffic as flowing along the secretory pathway to the TGN, at which point a proportion of HRP–P-selectin chimera is selected for incorporation into iDCG; a process mediated by a Tyr-based sorting signal. This P-selectin then remains within granules as they are modified into mDCG. The remainder passes to the cell surface and internalizes into the early Trn-positive endosomes before passing along the endocytic pathway towards the lysosomes or to the SLMV. Exit from the Trn-positive endosome requires Leu768. En route to the lysosome some proportion of P-selectin is diverted into SLMV in a DPSP and/or Tyr777-dependent fashion. The effects of mutagenesis as described above are predictable within such a scheme. Thus, impairment of SLMV or DCG targeting, as revealed by ssHRP P-selectinDPSP or ssHRP P-selectinY777A , leads to an increase in lysosomal targeting, whereas knockout of SLMV and lysosomal targeting, as seen for ssHRP P-selectinL768A , results in retaining of the chimera in Trn-positive endosomes. Another important aspect of our findings is of the relative contribution of different pathways to each destination. We have shown that a recycling route operating via the Trn-positive endosome does not make a significant contribution to steady state levels of ssHRP P-selectin in DCG. This conclusion comes from the observation that accumulation in Trn-positive endosomes of ssHRP P-selectinL768A , which would otherwise reach SLMV and lysosomes, does not affect targeting to DCG. These findings suggest that a direct route into granules at the level of the TGN is likely to play the major role in controlling steady state levels of P-selectin in DCG in these cells. By contrast, our finding that Leu768, which mediates exit from Trn-positive endosomes, plus sequences within the C2 domain that modulate lysosomal trafficking are both required for SLMV targeting provide evidence that the endosomal route makes the major contribution in delivery of P-selectin to SLMV. Finally, our data delineating the relationships between the different post-Golgi pathways clearly indicates a hierarchy of trafficking choices operating to direct P-selectin to its multiple destinations. | Other | biomedical | en | 0.999998 |
10385523 | Temperature-sensitive (ts) 1 alleles in VTI1 were introduced into yeast strains BJ3505 and DKY6281 by transformation and loop in-loop out of plasmids containing the vti1 ts alleles and a URA3 marker at the VTI1 locus . Ura + transformants were selected and Ura − clones which were generated in a second selection with 5-fluoroorotic acid were tested for loss of the wild-type VTI1 sequences by their ts growth and CPY-secretion phenotypes . Reagents were as described by Haas , Mayer et al. , and Haas and Wickner . SDS-PAGE, immunoblotting using ECL , and purification of IgGs and his 6 -tagged Sec18p were as described. Rabbit antibodies were generated against Ni-NTA purified His 6 -Ykt6 protein and His 6 -Nyv1p that was overproduced in Escherichia coli. For coimmunoprecipitations, vacuoles were sedimented (10 min, 8,000 g , 4°C) after any priming reaction with ATP, washed with 500 μl PS buffer (10 mM Pipes/KOH, pH 6.8, 200 mM sorbitol), and detergent solubilized in 1 ml of buffer A . The detergent extract was placed onto a nutator for 10 min at 4°C, the insoluble material was removed by centrifugation (10 min, 16,000 g ), and the supernatant was applied to protein A–immobilized IgGs . Incubations, washes, and elution of bound proteins were as described . His 6 -Vam3p was immobilized on Aminolink resin (Pierce) and used as an affinity matrix to purify antibodies to Vam3p. Affinity-purified antibodies (200 ng) and an equal amount of nonimmune rabbit IgGs were covalently linked to 1 ml protein A–Sepharose . Vacuoles were prepared by a batch purification. Cells from 6-liter overnight cultures were lysed with oxalyticase and DEAE dextran as described . After heat shock, cell lysates were chilled on ice, diluted with 15% Ficoll in PS buffer (200 mM sorbitol, 10 mM Pipes/ KOH, pH 6.8) to 4% Ficoll (final concentration), and transferred to 60Ti tubes (Beckman). Lysates were centrifuged (50,000 rpm, 4°C, 60 min, 60Ti rotor) and vacuoles harvested from the top, diluted 20-fold with cold PS buffer, and centrifuged (JA20, 10,000 rpm, 10 min, 4°C). The vacuole pellet was resuspended in PS buffer. For purification of the SNARE complex, 26 mg of vacuoles was lysed in 10 ml of 1.5% Triton X-100, PBS , pH 7.4, 2 mM EDTA, 1× PIC , and 1 mM PMSF (lysis buffer). After 30 min at 4°C on a nutator, the detergent extract was centrifuged for 30 min in a 60Ti rotor at 4°C, 35,000 rpm. The supernatant was collected and incubated on a nutator for 1.5 h at 4°C with 3 ml of a protein A resin bearing nonimmune IgGs. The flow through was collected, reapplied to fresh resin, and incubated as before. Three such sequential preadsorption steps were performed. The sample was then halved. One half was applied to a control resin, the other to the immobilized affinity-purified antibodies to Vam3p. The detergent extracts were incubated with the resins for 18 h on a nutator at 4°C. The flow throughs were collected and the resins were washed with 50 ml of 150 mM, 350 mM, and 500 mM NaCl in lysis buffer. Bound proteins were eluted with 4 ml 0.1 M glycine/HCl, pH 2.6, 0.025% Triton X-100, precipitated by TCA, washed with 1 ml of ice-cold acetone, and dried at 56°C for 5 min. Aliquots were analyzed by SDS-PAGE and Coomassie blue–stained or transferred to nitrocellulose for immunoblotting. Proteins were identified by comparing their tryptic peptide mass maps to the Saccharomyces cerevisiae sequence database . Protein bands were excised from the gel, rinsed, and the protein samples were digested with trypsin in the gel matrix . Extracted peptide mixtures were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (REFLEX; Bruker Daltonics). The peptide mass maps were used to query a comprehensive sequence database for unambiguous protein identification (PeptideSearch software, provided by M. Mann and P. Mortensen, EMBL) . Vacuole fusion is measured by a biochemical complementation assay . Vacuoles from DKY6821 have normal proteases but lack the membrane protein alkaline phosphatase. Vacuoles from BJ3505 accumulate alkaline phosphatase in the unprocessed and catalytically inactive “pro” form due to the deletion of the gene encoding the protease Pep4p. Incubation of a mixture of these vacuoles in reaction buffer at 27°C in the presence of cytosol and ATP leads to fusion, content mixing, and processing of pro-alkaline phosphatase by Pep4p. The active alkaline phosphatase is measured by a colorimetric assay at the end of the fusion reaction. Vacuoles were used immediately after isolation. The standard fusion reaction (30 μl) contained 3 μg of each vacuole type in reaction buffer (10 mM Pipes/KOH, pH 6.8, 200 mM sorbitol, 150 mM KCl, 0.5 mM MgCl 2 , 0.5 mM MnCl 2 ), 0.5 mM ATP, 3 μg/ml cytosol, 3.5 U/ml creatine kinase, 20 mM creatine phosphate, and a protease inhibitor cocktail containing 7.5 μM pefabloc SC, 7.5 ng/ml leupeptin, 3.75 μM o -phenanthroline, and 37.5 ng/ml pepstatin. To reduce proteolysis in the coimmunoprecipitation experiments, only the protease A–deficient BJ3505 vacuoles were analyzed. One unit of fusion activity is defined as 1 μmol p -nitrophenol phosphate hydrolyzed per minute and milligram of BJ3505. To identify proteins that interact with the vacuolar t-SNARE Vam3p, a detergent extract of vacuoles was incubated with immobilized affinity-purified antibodies to Vam3p or with a control IgG resin. Retained proteins were eluted from each column and analyzed by SDS-PAGE and immunoblotting or Coomassie staining . Vam3p was solubilized completely under the experimental conditions and was retained specifically on the anti–Vam3p-affinity column . The Coomassie-stained protein bands were identified by peptide mapping by MALDI mass spectrometry combined with sequence database searching . In addition to Vam3p, Vam7p, and Sec17p, two additional SNARE proteins were specifically eluted from the anti-Vam3p column and identified by MALDI mass spectrometry: Vti1p , an essential v-SNARE implicated in Golgi to vacuole trafficking, and Ykt6p , a v-SNARE previously implicated in trafficking through the Golgi . The vacuole cis-SNARE complex is neither SDS resistant nor as stable as the exocytic complex in neurons . Thus, washing the column in high salt removed a substantial amount of Nyv1p, though its presence in the cis-SNARE complex was confirmed by immunoblotting (data not shown). Both Vti1p and Ykt6p were found in substoichiometric amounts, consistent with their association to Vam3p being salt-sensitive (data not shown). Because of the lability of this cis-SNARE complex during immunoisolation, we cannot be sure that we have identified all its constituents, and the isolation of functional cis-SNARE complex for analysis in a reconstituted fusion assay may reveal additional components. Though Vti1p was found previously in a complex with Vam3p , Ykt6p has so far only been described as a Golgi-specific SNARE . We therefore used coimmunoprecipitation to test whether antibodies to each protein would immunoprecipitate the vacuolar cis-SNARE complex in a manner similar to antibodies to Vam3p. This was indeed the case . Upon ATP/Sec18p/Sec17p-dependent priming, the cis-SNARE complex disassembled (lanes 2, 4, and 6) and cross-immunoprecipitation was lost. While each of the three antibodies immunoprecipitated a disproportionate amount of its cognate protein, reflecting partial SNARE complex disassembly or in vitro lability, each also precipitated the other SNARE complex components (Nyv1p, Vam7p, and Sec17p) in a similar proportion. The composition of the SNARE complex was also examined by an independent approach. All members of the SNARE complex cosediment in a glycerol velocity gradient, suggesting that they are largely in a complex with each other . Vti1p and Ykt6p do not completely copurify with Vam3p, in agreement with their role in other trafficking reactions . They are both present in a complex with Vam3p on the vacuole membrane and behave similarly to previously identified members of the vacuolar SNARE complex. The localization of Vti1p and Ykt6p to the vacuole does not depend on previously characterized members of the SNARE complex . To determine directly whether Vti1p, Nyv1p, and Ykt6p are in the same SNARE complex with each other and with the other SNAREs, successive immunoprecipitations were performed . The same SNAREs were recovered in immunoprecipitates with antibodies to Vam3p , Vti1p (lane 2), Ykt6p (lane 4), or Nyv1p (lane 7). Some Ykt6p remained in the supernatant after immunoprecipitation with antibody to Vti1p, but it was not in complex with Vam3p or Vam7p (lane 3). Similarly, some Vti1p remained in the supernatant after immunoprecipitation with antibody to Ykt6p, but it was not in complex with Vam3p or Vam7p (lane 5). Immunoprecipitation with antibody to Nyv1p (lane 6) removed almost all the Ykt6p, leaving only a small amount that is not in complex with other SNAREs (lane 7). We conclude that the cis-SNARE complex contains Vam3p, Vam7p, Vti1p, Nyv1p, and Ykt6p and is at least pentameric for SNAREs. The above physical data establish that Vtilp and Ykt6p are present in a cis-complex with Vam3p, Nyvlp, and Vam7p. However, we have noted that only a small percentage of the SNAREs enters the trans-complex, and thus functional criteria are needed to establish that each SNARE is part of a functional complex. Such experiments address the possibility that a small percentage of the SNAREs in a tetrameric complex might be the active complex for fusion, while the fifth SNARE was present but irrelevant for function. Three lines of evidence show that Vti1p is directly involved in the fusion reaction. First, antibodies to Vti1p inhibit fusion to a similar extent as Vam3p antibodies when added at different times to an ongoing reaction . Antibodies to Vti1p, Sec17p, or Vam3p were added to aliquots from a fusion reaction at several times and fusion was continued for a total of 90 min at 27°C . All antibodies inhibited the reaction thoroughly when added at the beginning. Whereas Sec17p completes its action during the early priming step , Vam3p acts at a later docking stage and thus antibodies to Vam3p inhibit at later times . Antibodies to Vti1p inhibit the reaction in a kinetic fashion similar to Vam3p antibodies, suggesting that they may act at the same reaction. Second, Vti1p function depends on vacuole mixing. Vacuoles from the two tester strains were incubated in the presence of ATP in separate tubes. At the indicated times, aliquots from each tube were mixed in the absence or presence of antibodies to Vam3p, Vti1p, or Sec17p. Of these three proteins, only Sec17p function can be fulfilled early in the reaction . The reaction remains sensitive to antibodies to Vam3p and Vti1p, showing that the completion of the function of these proteins depends on vacuole contact. Third, previously characterized vti1 ts alleles were introduced into the tester strains and analyzed in the fusion reaction. Vacuoles were purified from all six wild-type and vti1 ts mutant strains and tested in all combinations. To induce the phenotype of the ts allele, vacuoles were mixed and preincubated at the indicated temperatures without ATP for the times shown. The ts alleles are much more thermolabile in the protease-plus DKY background than in the protease-minus BJ vacuoles, perhaps because partially thermally altered mutant Vtilp is more susceptible to proteolysis. When combined with a wild-type partner, only the ts alleles in the DKY background show a ts phenotype which is strongly induced at elevated temperatures. Strikingly, combination of vacuoles with vti1 ts alleles leads to a synthetic fusion phenotype, as even vacuoles that were only preincubated on ice retained only 5–10% fusion activity (DKY vti1-1 /BJ vti1-2 and DKY vti1-2 /BJ vti1-2 ). However, both vacuole partners show up to 60% fusion with the wild-type partner. Though some of the effects observed may be due to enhanced protease sensitivity of the ts alleles of Vti1p, the synthetic fusion phenotype strongly implies that Vti1p is directly involved in the reaction, as priming (as judged by Sec17p release and SNARE complex disassembly) is not altered after induction of the ts phenotype (not shown). Finally, Ykt6p antibodies inhibit with similar kinetics as Vti1p antibodies when added to a fusion reaction and acquisition of resistance to antibodies to Ykt6p requires docking . Thus, three v-SNAREs, Nyv1p, Vti1p, and Ykt6p, are required for the docking stage of vacuole-vacuole fusion. Since they are dissociated from the cis-SNARE complex during priming, these data suggest that inhibition by antibody to each SNARE is not due to steric hindrance of access to another SNARE. Rather, these data indicate that each SNARE has a functional role in the reaction. The complete sensitivity of fusion to antibodies to Vti1p and Ykt6p, or to ts alleles in Vti1p, suggests that each of these proteins is fully involved in the reaction rather than being in redundant tetrameric complexes of Vam3p/Vam7p/ Nyv1p/Vti1p and Vam3p/Vam7p/Nyv1p/Ykt6p. Vti1p and Ykt6p are components of the vacuolar SNARE complex, as both proteins copurify with the SNARE complex and antibodies to both Vti1p and Ykt6p precipitate all the previously identified members of this complex. This does not simply reflect an exchange of SNAREs from other contaminating organelles into association with Vam3p in detergent extracts, as inhibition studies with antibodies to Vti1p and Ykt6p and the synthetic fusion phenotype of ts alleles in Vti1p in our tester vacuoles indicate that both proteins are part of a SNARE complex with a functional role in the fusion reaction. Kinetic inhibition curves with antibodies to Vti1p and Ykt6p are indistinguishable from those reported for antibodies to the previously identified vacuolar SNAREs Vam3p, Vam7p, and Nyv1p . Our data establish that each of these SNAREs— Vam7p, Nyv1p, Vtilp, and Ykt6p—has a role in the reaction, though these roles need not be unique. Before priming, the effects of ts mutants or of deleting SNAREs could be due to allosteric effects on neighboring SNARE complex subunits. Similarly, antibodies which bind to one SNARE could inactivate the function of a pentameric cis-SNARE complex by obstructing access of a crucial protein or ligand to another SNARE. However, these concerns are vitiated by the observation that all SNAREs are disassembled from the complex during ATP-dependent priming and thus are not associated during docking while the reaction remains sensitive to each anti-SNARE antibody during docking . The sensitivities to each of these antibodies is a strong argument that each subunit of the pentameric cis-SNARE complex has some role in the overall reaction. Our data suggest that three v-SNAREs, Nyv1p, Vti1p, and Ykt6p, participate in the fusion reaction. This is not without precedent as Vti1p has been recovered in a complex with Vam3p and Ykt6p has been shown to be a weak multicopy suppressor of Vti1p . What could be the role of three v-SNAREs in the vacuole fusion reaction? The resolution of the crystal structure of the neuronal SNARE complex and the analysis of the exocytic SNARE complex in yeast and neurons have led to the proposal that the core of a SNARE complex consists of four parallel coiled-coil domains provided by three proteins: syntaxin, synaptobrevin, and SNAP-25 and their homologues. The alignment of all SNAREs at their coiled-coil domains identifies a conserved glutamine (Q) in one set of SNAREs (mainly t-SNAREs and some v-SNAREs like Bet1p and Vti1p) and a conserved arginine (R) in another set . Based on these findings, Fasshauer et al. propose that each SNARE complex consists of three Q-SNARE coiled-coils (e.g., one from syntaxin, and two from SNAP25) and one R-SNARE coiled-coil . How does this compare to data for the vacuolar SNARE complex? We already know of five SNAREs in our complex, the t-SNARE Vam3p (or Q-SNARE), the SNAP-25/23 homologue Vam7p (Q), and the v-SNAREs Vti1p (Q), Nyv1p (R), and Ykt6p (R). Vam3p and Vam7p are found in a tight complex on the vacuole . Whereas SNAP-25 provides two coiled-coil domains to the neuronal SNARE complex, Vam7p provides only one . The third Q-SNARE coiled-coil could therefore come from Vti1p, which has been previously considered a v-SNARE. Either Nyv1p or Ykt6p would then be the required R-SNARE. However, both proteins are part of the same cis-SNARE complex and antibodies to either protein inhibit the fusion reaction . Furthermore, vacuoles lacking Nyv1p fuse only poorly, if at all, with each other , suggesting an essential role of Nyv1p in the fusion reaction. In fact, Nyv1p is not required for any of the trafficking reactions to the vacuole , but appears to be exclusively reserved for vacuole fusion. Thus, at least portions of the intracellular pool of all five of these SNAREs are in a complex with each other, which may define a new, five coiled-coil core of a SNARE complex. Not all of the vacuolar SNAREs are recovered in a cis complex. The proportion is highest with salt-washed vacuoles, possibly due to removal of Sec18p (not shown). This might reflect the lability of the complex or, alternatively, that only some of the SNAREs are complexed and a second population may exist in an uncomplexed form or in a complex with unidentified proteins. Vacuoles without Vam3p or Vam7p have no cis-SNARE complex and yet are still capable of fusion at a measurable rate . Furthermore, vacuoles without Vam3p do not need priming by Sec17p/Sec18p/ATP , which suggests that SNAREs that are not in a cis complex can also participate in the homotypic fusion reaction. We have shown by deletion analysis, antibody inhibition, and the generation of ts alleles that each of the subunits has a critical role for the fusion reaction and that a complex of all SNAREs exists on the vacuole . However, we do not know whether the separate SNAREs or the cis-SNARE complex have distinct roles or specific activities. Previous work has shown that a detergent extract which was immunodepleted of SNAREs can be reactivated by addition of a 200-fold purified v-t-SNARE complex . Future work will be necessary to establish the stoichiometry and functional roles of the five SNAREs during this reconstitution reaction. Finding a role for Vti1p and Ykt6p in the vacuole-vacuole fusion reaction adds to a long list of trafficking reactions in which these proteins have been implicated . Ykt6p is unusual as a v-SNARE in that it is prenylated and appears to partition between cytosol and membranes . Subcellular localization of Ykt6p has therefore been difficult. Although Ykt6p was initially identified in a complex with the Golgi t-SNARE Sed5p , and may participate in trafficking between the ER and Golgi membranes , we find a significant portion of Ykt6p on the vacuole, suggesting a vital role for this protein in vacuole function. Vti1p has been recovered in complexes with organellar t-SNAREs along the secretory pathway: with Sed5p, the Golgi t-SNARE, with Pep12p, the endosomal t-SNARE, and with Vam3p . Because of their interactions with multiple t-SNAREs, Vti1p, and Ykt6p cannot be the sole determinants of specificity in vesicular traffic. These proteins are likely to be involved in a retrieval and recycling of trafficking factors from late organelles to, for example, the Golgi apparatus . Two other v-SNAREs have been implicated in retrograde trafficking reactions in yeast: Sft1p in retrograde transport within the Golgi stack , and Sec22p for the trafficking of vesicles from the Golgi apparatus back to the ER . The vacuolar t-SNARE Vam3p has a fundamental role in several trafficking reactions. It has been implicated in trafficking from the endosome to the vacuole , in the trafficking of AP3-dependent Golgi-derived vesicles to the vacuole , in aminopeptidase I transport to the vacuole and autophagocytosis , and in homotypic vacuole fusion as the final step of the inheritance of this organelle . Deletion of Vam3p results in a clear delay of protein trafficking to the vacuole . However, vam3Δ vacuoles can be purified by the same floatation protocol as for wild-type vacuoles, albeit at somewhat lower yield. These vacuoles contain all vacuolar marker proteins at the same steady-state concentration , they fuse with wild-type vacuoles with similar kinetics, and they show the same sensitivities to inhibitors of fusion as wild-type vacuoles . Though vam3Δ vacuoles are fragmented and of much smaller size , their normal protein content and behavior in the vacuole fusion reaction classifies them as vacuoles. This suggests that delivery of proteins to the vacuole, even if slow or of limited efficiency, can occur in a Vam3p-independent fashion and raises the question of how the t-SNARE requirement can be bypassed. The requirement for the vacuole SNARE complex in several reactions implies that other factors are required to add specificity to these trafficking reactions. Defining these factors and their functions may contribute to the understanding of how trafficking to and from this organelle is specified. | Study | biomedical | en | 0.999996 |
10385524 | Polyclonal antibodies were raised against a peptide corresponding to a highly conserved region of cav-3, with a COOH-terminal cysteine for coupling to the carrier (CGFEDVIAEPEGTYSFDE). Antibodies were affinity purified as described previously . Other domain-specific caveolin anti-peptide antibodies have been described previously . Anti-chick caveolin (anti-cavFL) was purchased from Zymed Laboratories . Affinity-purified rabbit antibodies (unconjugated and biotinylated) against p23 were provided by Dr. Manuel Rojo (University of Geneva, Switzerland). Antibodies against the mammalian KDEL-receptor ERD2 and giantin were gifts of Dr. Hans-Peter Hauri (Biozentrum, Basel, Switzerland) and Dr. Hans-Dieter Soeling (Goettingen, Germany). Fluorescein-labeled anti–mouse IgG, Cy3-labeled anti–rabbit IgG and Cy2-labeled streptavidin were from Jackson ImmunoResearch. The hybridoma used to generate monoclonal mouse anti-HA was provided by Professor David James (University of Queensland). The rabbit anti-HA and the cDNA for myc-tagged sialotransferase were both kindly provided by Dr. Tommy Nilsson (EMBL, Heidelberg, Germany). Nocodazole was purchased from Sigma Chemical Co. and was kept at −20°C (stock solution 10 mM in DMSO). BHK cells were grown and maintained as described previously . Primary human fibroblasts were a gift of Professor D. James and were maintained in RPMI supplemented with 10% FCS. C2C12 cells were cultured as described previously . Recombinant Semliki Forest Virus (SFV)-cav-1 and SFV-cav-3 were prepared in BHK cells according to an established protocol . In brief, canine cav-1 and mouse cav-3 were PCR amplified from the original clones with introduced 5′ BamHI and 3′ SmaI restriction sites. After sequencing of both strands, the cDNAs were cloned into the appropriate sites of pSFV1 ( Gibco , Grand Island, NY). RNA was generated by in vitro transcription from pSFV-cav-1, pSFV-cav-3, and pSFV-Helper1 and electroporated into BHK cells. Culture supernatant was harvested after 48 h and frozen at −70°C. The BHK cells used to produce recombinant SFV were harvested and prepared for Western blotting. The expression level of cav-1 was >10-fold higher than endogenous levels. For immunofluorescence experiments cells were infected with undiluted recombinant SFV containing culture supernatant for 1 h at 37°C after which the infection medium was diluted 10-fold with normal culture medium and incubated for a further 12 h. The cav-2-SFV construct was kindly provided by Dr. Elina Ikonen (National Public Health Institute, Helsinki, Finland). BHK cell fractions were kindly provided by Professor Jean Gruenberg. BHK cells were homogenized in isotonic sucrose solution and the membranes of the post-nuclear supernatant were fractionated using a step sucrose gradient to produce a fraction enriched in p23 and ERD2 exactly as described . A series of NH 2 -terminal truncation mutants of cav-3 were generated from the original cDNA clone by PCR. Cav3 DIH (residues 16-151), Cav3 NED (residues 33–151), Cav3 DGV (residues 54–151), and Cav3 KSY (residues 108–151) were generated using the forward primers DIHfor 5′ CGGGGTACCACCATGGACATTCACTGCAAGGAG 3′, NEDfor 5′ CGGGGTACCATGAATGAGGACATTGTGAAG 3′, DGVfor 5′ CGGGGTACCACCATGGACGGTGTATGGAAGGTG 3′ and KSYfor 5′ CGGGGTACCACCATGAAGAGCTACCTGATCGAG 3′, respectively, and the reverse primer RORrev 5′ CCGGAATTCTTAGCCTTCCCTTCGCAGCACCACCTT 3′. Similarly, truncations of cav-1 (residues 135–183) were generated with and without three putative COOH-terminal palmitoylation site cysteines mutated to alanine. Wild-type (WT) cav-1 cDNA template was used to generate Cav1 KSF and cav-1 (Cys-Ala) cDNA template was used to generate Cav1 KSF Δp both using the forward primer CAV1(KSF)for 5′ CGGGGTACCACCATGAAGAGTTTCCTGATTGAGATTCAGTGC 3′ and the reverse primer CAV1HArev 5′ ATAAGAATGCGGCCGCCTGTTTCTTTCTGCATGTTGATGCGG 3′. The resulting mutants were cloned into pCB6KXHA , a derivative of the eukaryotic expression vector pCB6, containing the HA epitope tag (YPYDVPDYA), downstream of an in frame NotI site. The Cav3 KSY region was also amplified with the forward primer, KSYGFPfor 5′ CCGGAATTCGAAGAGCTACCTGATCGAG 3′, and the reverse primer, CAV3rev 5′ TCCCCCCGGGTTAGCCTTCCCTTCGCAGCACC 3′, for in frame insertion into the COOH-terminal green fluorescent protein (GFP) fusion vector, pEGFP-C1 ( CLONTECH Laboratories). Further truncations of Cav3 KSY were similarly prepared using the following combinations of primers; Cav3 IYS (residues 120–151) and Cav3 IRT (residues 125–151) used the forward primers, IYSfor 5′ GGAATTCCATCTACTCACTGTGTATCCGC 3′ and IRTfor 5′ GGAATTCCATCCGCACCTTCTGC 3′ respectively, with the reverse primer CAV3rev. Cav-3ΔC, a COOH-terminal truncation mutant of cav-3 (residues 1–107) was generated using the forward primer, CAV3GFPfor 5′ CCGGAATTCAATGATGACCGAAGAGCACACGG 3′, and the reverse primer, CAVΔCrev 5′ TCCCCCGGGTTAAATGCAGGGCACCACGGC 3′. Full-length cav-3 was similarly produced using the forward primer, CAV3GFPfor, and the reverse primer, CAV3rev. The products were then cloned into pBluescript (Stratagene) and subcloned into pEGFP-C1. The sequences of all constructs were confirmed by sequencing of both strands in pBluescript using the T3 and T7 primers. BHK, FRT, C2C12, and CV-1 cell lines were transiently transfected using Lipofectamine ( GIBCO BRL ) according to the manufacturer's instructions. In brief, cells were grown to ∼50% confluence on coverslips and 10-cm dishes for immunofluorescence and electron microscopy, respectively, or 90% confluence on 10-cm dishes for biochemical analysis. The cells were washed twice with serum-free media before being transfected with a ratio of 1 μg DNA to 5 μl Lipofectamine per 1 ml of Opti-MEM ( GIBCO BRL ). The transfection mixture was left on the cells for 6 h before being washed with, and changed to, normal growth media lacking antibiotics and incubated for a further 18 h before fixation or harvesting. Nocodazole treatment was at a concentration of 10 μM for 2 h at 37°C before fixation. Confluent dishes of cells were washed twice with cold PBS before being scraped into ice-cold HES homogenization buffer (0.25 M sucrose, 1 mM EDTA, and 20 mM Hepes, pH 7.4) containing protease inhibitors. Cells were homogenized by passaging through a 27-G syringe and nuclei and unbroken cells were removed by centrifugation at 1,000 g for 5 min at 4°C. The resulting supernatant was then centrifuged at 100,000 g for 30 min at 4°C to separate cytosol (supernatant) from cellular membranes (pellet). For Western analysis the pellet was extracted with a volume of 1% SDS HES or 1% Triton X-100 HES equal to the volume of supernatant for 10 min at ambient temperature followed by centrifugation at 10,000 g for 5 min at to remove insoluble material. Similarly, salt extractions were performed on the pellet with volumes of 1 M KCl in 50 mM Tris, pH 8.0, or 0.1 M Na 2 CO 3 , pH 11.5, equal to the supernatant. Triton X-114 phase separation was achieved using the method of Bordier , with the exception that membranes (100,000- g pellet) were resuspended in the initial solution. Equal volumes of cytosol (100,000- g supernatant) and the detergent extracted microsomal membranes (100,000- g pellet) were boiled in SDS PAGE sample buffer. After electrophoresis proteins were transferred to Immobilon membrane ( Millipore Corp. ) using a Bio Rad trans blot semidry transfer cell. Membranes were blocked with 5% nonfat dry milk TBS-T (0.15 M NaCl, 0.1% Tween 20, and 20 mM Tris, pH 7.4) followed by incubation in specific antibody diluted in 1% non fat dry milk TBS-T. After washing with TBS-T membranes were incubated with a second antibody diluted in 0.2% BSA TBS-T. Bound antibody was detected using the enhanced chemiluminescence detection system ( Amersham Corp. ). Cells were grown on glass coverslips and fixed with either methanol (−20°C, ≥5 min) or 3% paraformaldehyde (PFA, 20°C, ≥20 min). PFA-fixed cells were permeabilized for 5 min with 0.1% (wt/vol) saponin in PBS and labeled as described previously . For double labeling with mouse and rabbit antibodies, cells were incubated with the mixture of primary antibodies for 30 min, washed three times with PBS, and incubated with a mixture of fluorescein- or Cy3-labeled anti–mouse IgG and Cy3- or fluorescein-labeled anti–rabbit IgG for 20 min. In some experiments, cells were double labeled with two rabbit antibodies (p23 and anti-concav). This was perfomed using the following sequence: anti-concav, Cy3 goat anti–rabbit, a blocking irrelevant rabbit antibody (anti-cholera toxin), then biotinylated rabbit anti-p23 followed by streptavidin-Cy2 (Monarch Medical). Control experiments omitting the primary antibodies showed the specificity of the labeling. Samples were analyzed with a confocal laser scanning microscope (Bio Rad Laboratories) equipped with an argon and a helium/neon laser for double fluorescence at 488 and 543 nm. Fluorescein/Cy2 and Cy3 signals were recorded sequentially (emission filters: BP510-525 and LP590) using 63 or 100× plan-APOCHROMAT oil immersion objectives. For overlay, fluorescein and Cy3 images were adjusted to similar output intensities and merged with Adobe Photoshop 3.0.5 into a composite RGB image using a Power Macintosh 7500/100 computer. Figures were arranged with Microsoft PowerPoint. Colocalization was quantitated by analysis of confocal images of double-labeled nocodazole-treated cells in which individual puncta were clearly evident . Images were digitally captured and overlaid using the RGB function of Adobe Photoshop. Separate images were adjusted to equivalent intensities. IP Lab Spectrum software was then used to analyze the images for the area occupied by the colocalizing elements as a percentage of the total labeled area. Results are expressed as the mean of five fields ± SEM. Note that this technique allows a comparison of the degree of overlap of different markers but underestimates the actual puncta which are positive for both markers . Cells were fixed with 8% paraformaldehyde in 100 mM phosphate buffer, pH 7.35, or with the same fixative containing 0.1% glutaraldehyde for 30 min at RT and then processed for frozen sectioning as described previously . BSA-gold was internalized as a fluid phase marker for 10 min or 30 min at 37°C as described previously . We have previously shown that cav-1 is localized to the Golgi complex and to surface caveolae when expressed in BHK cells . We investigated the use of a transfection approach to express caveolin mutants to study caveolin targeting. As caveolin can form homo-oligomers which could potentially influence the distribution of introduced caveolins, we investigated the use of cav-3 to study caveolin targeting as BHK cells lack endogenous cav-3. Previous work using GST-fusion proteins of caveolin has shown that cav-1FL or COOH- or NH 2 -terminal truncation mutants can oligomerize with full-length cav-1 but not cav-3 . Also in vivo cav-1 is sorted away from cav-3 when expressed in differentiating muscle cells . Epitope-tagged cav-1 and cav-3 were expressed in BHK cells. As shown in Fig. 1 , A and B, cav-1 and cav-3 showed a similar distribution as judged using antibodies against a COOH-terminal epitope tag (VSV-G and HA, respectively). The proteins were localized to the cell surface and to a perinuclear compartment, assumed to represent the Golgi complex . A similar distribution was observed upon expression of a fusion protein comprising GFP fused to the NH 2 terminus of cav-3 . These results suggest that heterologously expressed cav-1 and cav-3 localize to the same compartments in BHK cells and can be used to study caveolin targeting. To further investigate the subcellular localization of caveolin, we examined the distribution of endogenous caveolin using a number of different antibodies and cell lines. Previous studies differ in their analysis of caveolin distribution; some studies have shown that caveolin is only present at the cell surface unless cells are subjected to experimental manipulations , whereas others have concluded that caveolins exist in Golgi complex–associated and surface pools at steady state . BHK, Vero, and MDCK cell lines as well as primary human fibroblasts, which have been extensively studied by others , were labeled with antibodies to the NH 2 terminus of cav-1 (anti-cav1N) and antibodies to the COOH terminus (anti-cav1C). Similar labeling patterns were observed in all the cell types studied; surface staining by anti-cav1N and a striking Golgi-type staining with anti-cav1C antibodies . This suggests that different epitopes are exposed at these two cellular locations. To investigate this possibility further we studied the exposure of a domain of the molecule which has already been extensively studied in terms of interacting proteins, by raising antibodies against the scaffolding domain of the caveolin family. This domain is conserved between caveolins, thereby acting as a signature motif, and is also conserved in evolution . Antibodies were raised in rabbits against a scaffolding domain peptide corresponding to the sequence of cav-3 (see Materials and Methods) and affinity purified on the corresponding peptide column. By Western blotting the affinity-purified antibody (anti-concav) recognized a doublet of ∼21 kD in BHK membranes , a single band in undifferentiated C2C12 myoblast membranes, and a single <20-kD band in differentiating C2C12 myotubes . The signal was completely competed by the specific peptide to which it was raised (not shown). This suggested that the antibody (named anti-concav for consensus caveolin) recognized both cav-1 and cav-3, the predominant isoforms expressed in BHK cells and C2C12 myoblasts, and differentiating C2C12 myotubes, respectively. This was confirmed by blotting of BHK cells overexpressing cav-1 or cav-3 . By immunofluorescence the antibody was shown to recognize overexpressed cav-1, cav-2, and cav-3 consistent with the predicted specificity. The anti-concav gave a characteristic perinuclear staining on cell lines including BHK, CV-1, and MDCK . Primary human fibroblasts revealed the same pattern of labeling which colocalized with the Golgi marker, p23 . This staining pattern was distinct from that obtained with antibodies to the NH 2 terminus of cav-1, which as expected gave a surface staining pattern characteristic of caveolae . This result suggested that the anti-concav antibody was unable to recognize surface caveolin. To test this possibility, we examined the staining pattern for anti-concav in differentiating C2C12 cells. In these cells cav-3 gives a characteristic staining pattern representing the surface-connected T-tubule system . The cav-3(N) antibody showed the characteristic reticular staining pattern as described previously but the anti-concav showed no labeling of the T-tubules (results not shown). We then examined whether the strict specificity for intracellular caveolin was retained after overexpression, when interaction with surface molecules might be expected to be saturated. Even after high overexpression of cav-3 using the recombinant SFV expression system, the strict specificity of the concav antibody for the intracellular caveolin pool was still maintained with no sign of surface staining (compare Fig. 3 J, anti-cav-3(N) with Fig. 3 K, anti-concav). These experiments, in which antibody concentrations were optimized to allow only detection of overexpressed caveolin, also emphasize that the N- and concav antibodies were both recognizing the same heterologously expressed caveolin. In summary, the results suggest that the anti-concav and anti-cavC antibodies specifically recognize Golgi-associated caveolin and that changes in the accessibility or conformation of the epitope on transport of caveolin to the cell surface may inhibit antibody binding. In view of the different models for caveolin cycling, we sought to pinpoint the domain of the Golgi complex with which caveolin was associated. For this analysis we used a monoclonal commercial antibody against purified cav-1 together with either a cis-Golgi marker p23 or a TGN marker . We found that cav-1 immunolabeling colocalized with the TGN marker tST in control cells as judged by confocal microscopy, consistent with previous studies; , but the same high degree of colocalization was found with p23 (not shown). Therefore, we employed the microtubule-depolymerizing agent, nocodazole, which has been shown to disrupt the Golgi complex and has been used to localize proteins to distinct Golgi sub-compartments . Nocodazole treatment caused dispersion of Golgi markers into discrete puncta . Double labeling of nocodazole-treated cells with cis- (p23) and trans-Golgi (tST) markers showed clear, although incomplete, segregation of the two markers . After nocodazole treatment anti-cav1FL showed a relatively low degree of colocalization with tST but almost complete colocalization with p23 . The level of colocalization was also quantitated as the area occupied by the colocalizing elements in the image as a percentage of the total labeled elements (see Materials and Methods for details). Consistent with the qualitative data, these results showed a much higher degree of colocalization for caveolin/p23 (63 ± 4%) than for caveolin/tST (46 ± 6%) or p23/tST (41 ± 4%). This suggests caveolin is present in the early Golgi and is not exclusively present in the TGN. The subcellular location was further analyzed by immunoelectron microscopy. All the available antibodies gave low labeling of the Golgi complex on ultrathin frozen sections of intact cells. However, in frozen sections of BHK cell fractions enriched for p23 and ERD2 , anti-cavC labeling colocalized with p23 on Golgi cisternae . The higher labeling in this preparation presumably reflects greater accessibility of cytosolic epitopes as described for other Golgi proteins . Taken together the results show that under steady state conditions caveolin is detectable within the cis-Golgi. We examined whether a specific region of the caveolin molecule is responsible for the localization of caveolin to the Golgi complex. To avoid potential problems of association with endogenous caveolins we prepared mutants of cav-3 which are not endogenously expressed in BHK cells. A series of cav-3 deletion mutants with a COOH-terminal HA tag were prepared and expressed transiently in BHK cells. As shown in Fig. 6 , C–F, the NH 2 -terminal deletion mutants showed overlapping but distinct localization patterns with varying degrees of surface and intracellular labeling. Most strikingly, Cav3 DGV , which lacks the NH 2 -terminal region up to the scaffolding domain (residues 54–151), showed no hint of surface labeling and this mutant was chosen for more detailed analysis. Cav3 DGV localized to the Golgi apparatus and to punctate structures throughout the cell. This was examined in more detail by immunoelectron microscopy using antibodies to the HA tag . Consistent with the confocal microscopic analysis, Cav3 DGV was shown to associate with the Golgi complex as well as intracellular vesicular elements . The Golgi labeling colocalized with ERD2 (KDEL-receptor) showing that the protein was concentrated in the cis-Golgi complex . In a small proportion of highly expressing cells labeling was also observed within the endoplasmic reticulum (not shown). These studies suggest that Golgi targeting/retention information resides in the COOH-terminal portion of the molecule. To identify the relevant domain we expressed a construct comprising just the putative COOH-terminal cytoplasmic domain of cav-3 (residues 108–151). This domain has previously been assigned as cytoplasmically orientated based on amino acid sequence predictions, on antibody recognition of the COOH-terminal domain in permeabilized cells , and in vitro import experiments . The construct termed Cav3 KSY was expressed in BHK cells with an HA epitope tag. Cav3 KSY was targeted specifically to the perinuclear area of the cell . No surface staining was apparent in contrast to the full-length protein expressed under the same conditions. In very highly expressing cells a cytoplasmic staining in addition to the Golgi complex was apparent (not shown). The specific localization of this mutant was confirmed by immunoelectron microscopy . Expressing cells showed Golgi labeling for the expressed protein. No labeling was associated with the plasma membrane or other organelles. Very high expressing cells showed dispersed cytosolic staining throughout the cell in addition to the Golgi staining but unlike Cav3 DGV -expressing cells, Cav3 KSY expressing cells never showed ER staining. Together with the immunofluorescence results this suggests that the association of this caveolin fragment with the Golgi complex is a saturable process. We then localized the Cav3 KSY mutant with respect to the defined Golgi markers p23, tST, and giantin at the light and electron microscopic levels. The Cav3 KSY colocalized with all three markers in untreated cells . However, after nocodazole treatment, colocalization with cis markers was more complete than with the medial or trans Golgi markers . This confirms that the localization of Cav3 KSY closely follows that of endogenous cav-1 and suggests that the Cav3 KSY is not mislocalized to an irrelevant domain. We also examined the distribution of Cav3 KSY at the electron microscopic level with respect to defined markers. Cav3 KSY showed a clear colocalization with the cis-Golgi markers p23 and with ERD2 (not shown). In extracted cells labeling was clearly shown to be associated with the cytoplasmic face of the membrane. The distribution of Cav3 KSY with respect to endogenous caveolin was also examined. Cav3 KSY showed colocalization, as judged by confocal immunofluorescence microscopy, with antibodies to the COOH terminus of caveolin . In contrast, as expected antibodies to the NH 2 terminus of caveolin which only label the surface caveolin, showed no colocalization with Cav3 KSY (not shown). A consistent observation in the Cav3 KSY -expressing cells was a dramatic decrease in the endogenous Golgi caveolin labeling in a subpopulation (30–40%) of expressing cells . This decrease appeared unrelated to expression levels and was observed with different labeling sequences, in combination with nocodazole treatment and with the three different antibodies against caveolin which recognize different domains of the molecule. This suggests that the association of Cav3 KSY with the Golgi complex either decreases accessibility of caveolin antibodies to the endogenous caveolin or, more likely, decreases the Golgi-associated pool of caveolin. Although the oligomerization of caveolin has been shown to be isotype-specific in vitro , the possibility remained that the Golgi-association of Cav3 KSY could represent association with endogenous Golgi caveolin. To test this in vivo we made use of the epithelial cell line, FRT, which lacks caveolin and caveolae . Cav3 KSY expressed in these cells showed the characteristic staining of the Golgi complex which colocalized with p23 (not shown), strongly suggesting that the association of Cav3 KSY is independent of endogenous caveolin. This was further tested by double transfection of Cav3 KSY and wild-type cav-1 or GFP-tagged cav-3. Cav3 KSY colocalized with the expressed proteins in the Golgi complex but even after high overexpression of full-length cav-3, Cav3 KSY was not recruited to the cell surface (not shown). To examine whether removal of the COOH terminus of Cav-3 caused a loss of Golgi localization, Cav-3ΔC with an NH 2 terminal GFP tag was expressed in BHK cells. The protein showed only a reticular staining pattern consistent with an ER localization (not shown). Although these results are consistent with a role for the COOH terminus in association of caveolin with the Golgi complex they could indicate a general perturbation of folding/oligomerization leading to lack of transport from the ER. Nevertheless our results show that the COOH-terminal cytoplasmic domain of caveolin associates in a saturable fashion with the cis-Golgi complex. To investigate whether the COOH terminus of cav-3 could target a heterologous protein to the Golgi complex, a fusion protein comprising Cav3 KSY fused to the COOH terminus of GFP was prepared. The GFP-Cav3 KSY fusion protein was expressed in BHK cells under a CMV promoter. The fusion protein was specifically targeted to the Golgi complex where it colocalized with p23 (not shown). In the majority of cells cytoplasmic staining was also apparent consistent with saturation of the targeting machinery . In addition, in higher expressing cells GFP-Cav3 KSY labeled the periphery of large vesicular structures in the perinuclear area of the cell . These experiments show that the COOH-terminal domain of cav-3 is sufficient to localize a heterologous cytosolic protein to the Golgi complex. We carried out a preliminary characterization of the regions of the COOH-terminal cytoplasmic domain which were required for Golgi location by expressing COOH-terminal fragments as fusion proteins with GFP. A 32– amino acid segment fused to GFP showed the characteristic staining pattern of the Golgi complex . In contrast, all cells expressing the Cav3 IRT mutant, which lacks the first five amino acids of Cav3 IYS , consistently showed dispersed labeling throughout the cell . The observed differences between the two mutants were independent of expression level. In conclusion we have identified a unique domain of the caveolin molecule which can target a heterologous protein to a specific domain of the Golgi complex. Finally, we examined the nature of the association of Cav3 KSY with the Golgi complex. BHK cells expressing Cav3 KSY were harvested, homogenized, and separated by centrifugation into crude cytosol and membrane fractions. Membranes were initially extracted with buffer containing 1% SDS or 1% Triton X-100. Fig. 12 A shows that both these detergents extracted a peptide of ∼5 kD recognized by the anti-HA antibody which was not present in mock transfected control cells. Treatment with either 1 M KCl or alkaline 0.1 M Na 2 CO 3 both failed to extract Cav3 KSY from the pellet . To confirm the apparent hydrophobicity of Cav3 KSY the membranes were treated with Triton X-114 which enables phase separation of amphiphilic from hydrophilic proteins. Fig. 12 C shows that Cav3 KSY was detected only in the amphiphilic Triton X-114 phase. We then examined the membrane association of the GFP-Cav3 KSY chimera. While expressed WT-GFP was predominantly in the soluble fraction, as predicted for a cytosolic protein, the majority of GFP-Cav3 KSY was targeted to the membrane fraction . Cav-1 has been shown to be palmitoylated and so we examined whether this lipid modification might be required for the Golgi localization of the COOH-terminal caveolin fragment. A full-length cav-1 construct in which the three COOH-terminal palmitoylated cysteines have been mutated to alanine (Cav-1Cys-Ala) has already been described and characterized . We used a WT cav-1 construct to generate the cav-1 equivalent of the Cav3 KSY (Cav-1 KSF ) and the Cav-1Cys-Ala cDNA to generate the corresponding Cys-Ala mutant (Cav-1 KSF Δp). Each construct incorporated a COOH-terminal HA-tag. The constructs were expressed in BHK cells and their distribution examined by immunofluorescence. As expected the Cav-1 KSF was specifically localized to the Golgi complex consistent with the results with the cav-3 constructs . In contrast, cells expressing Cav-1 KSF Δp did not show a characteristic Golgi staining although both Cav-1 KSF and Cav-1 KSF Δp were membrane associated as judged biochemically (not shown). These results suggest a role for palmitoylation in the specific association of the COOH terminus of caveolin with the Golgi complex. Crucial to understanding the role of caveolins is the determination of the exact cellular distribution and intracellular itinerary of this family of proteins. In the present study we have localized caveolin using different specific antibodies and analyzed the targeting information in the caveolin molecule. In particular, we have shown that in addition to surface caveolae, caveolin is also associated with the cis-Golgi in all cell types studied. However, different epitopes are exposed in these locations. We have then used a mutational approach to examine caveolin cycling and targeting. The NH 2 terminus of the protein was shown to be required for caveolae targeting while the COOH terminus contains a Golgi targeting motif which is sufficient to target a heterologous protein to the Golgi. Caveolin has been shown to play an important role in signal transduction at the cell surface and many signaling molecules have been postulated to interact with caveolin directly. Upon cell transformation caveolin levels decrease and it has also been shown that caveolin is phosphorylated upon Rous sarcoma virus transformation or upon stimulation of adipocytes with insulin . In view of these functions, primarily assigned to the plasma membrane, it initially appears somewhat surprising that caveolin is cycling between the cell surface and intracellular compartments. In fact early studies suggested that caveolin was exclusively a surface protein based on immunofluorescence and pre-embedding immunoelectron microscopy . Other studies showed that caveolin is associated with the Golgi complex but this location was only visualized with certain antibodies or with overexpressed protein . This labeling was assigned to the TGN and exocytic vesicles consistent with a conventional cycling pathway between the Golgi and the cell surface. Later work outlined a quite distinct cycling pathway for the caveolin molecule involving transport of caveolin to the ER and cis-Golgi. In these studies caveolin was associated with the plasma membrane in control cells (human fibroblasts) but redistributed to intracellular compartments only upon treatment with cholesterol oxidase or nocodazole . Subsequent studies have raised the possibility that the primary function of caveolin in mammalian cells is to regulate cholesterol transport . To understand these processes it is essential that the caveolin distribution and routing is determined and the molecular machinery defined. This study has started to address these issues and to provide tools for further analysis. First, we have shown that in all cell types studied a pool of caveolin is present in the Golgi complex. However, our studies demonstrate some of the difficulties associated with localizing caveolin. Different antibodies clearly recognize different pools of the protein, at least as seen by immunofluorescence. Of particular interest is a new antibody, characterized here, which was raised against the scaffolding domain of the caveolin molecule. This domain is highly conserved both in evolution and between different mammalian caveolins and consistent with this we have shown that it recognizes all three mammalian caveolins. This domain interacts with a number of signaling molecules and is also involved in self-association to form oligomers . Remarkably, despite these protein-protein interactions, the antibody gave a specific signal by immunofluorescence but only recognized the Golgi form of the protein by this technique. A similar specificity was seen with antibodies against the COOH terminus. The molecular basis for the selectivity is unclear. Oligomerization has been shown to occur early in the biosynthetic pathway immediately after cotranslational insertion into the endoplasmic reticulum. We suspect that higher order complexes of proteins and lipids might restrict antibody accessibility. In fact, in ultrathin frozen sections, antibodies against the COOH terminus or against the conserved domain do recognize surface protein suggesting that epitopes are exposed upon sectioning (results not shown). This suggests that protein–protein interactions might not be responsible for blocking accessibility. Whatever the mechanism, it is apparent that care should be taken in interpreting caveolin localization based on single antibodies. We were also able to define the domain of the Golgi with which the caveolin is detectable as the cis-Golgi complex based on immunoelectron microscopic colocalization with defined markers. This presumably represents a pool of caveolin which cycles through the entire Golgi and TGN to the surface as cav-1 has been detected within Golgi derived exocytic vesicles and is directly implicated in exocytic transport to the apical cell surface of epithelial cells . This implies that caveolin follows an unusual cycling pathway to reach early compartments of the biosynthetic pathway, distinct from that followed by molecules such as furin and TGN38 . In this way the results are consistent with those showing redistribution of caveolin in response to cholesterol oxidase or nocodazole . However, our results differ significantly in other ways. Most notably, in all cell types studied including human fibroblasts as used in the above studies, caveolin is not only detectable on the surface but also in the Golgi complex under all conditions tested. The distribution of surface caveolin was unchanged by nocodazole treatment (not shown). Our subsequent experiments were designed to analyze the Golgi association of caveolin further and to determine the molecular basis of this targeting through mutational analysis of the caveolin molecule. We chose to analyze caveolin targeting using the muscle-specific caveolin isoform, cav-3, expressed in BHK cells. The first striking observation was that truncation mutants lacking the first 54 amino acids no longer associated with surface caveolae as determined by immunofluorescence and immunoelectron microscopy with (COOH-terminal) epitope-tagged constructs and with the (NH 2 -terminally) GFP-tagged fusion protein. In contrast, the removal of the first 33 amino acids had no effect on surface localization. This may indicate that residues 33–54 are required for caveolae targeting or retention or at least implies that removal of the first 54 amino acids disrupts this process. Expression of the entire NH 2 terminus produced a soluble protein with no detectable association with membranes (results not shown). We then went on to analyze the domains of the caveolin molecule required for Golgi association. Removal of the NH 2 terminus had no effect on Golgi localization of the protein. Therefore, we investigated whether the COOH terminus contains Golgi targeting information. Surprisingly, the putative COOH-terminal domain associated with the cis-Golgi even when expressed alone, presumably as a soluble protein. Moreover, this domain was able to target a heterologous soluble protein, GFP, to the Golgi. It is important to note that GFP was fused to the NH 2 terminus of the construct, in place of the putative intramembrane domain. The domain required for targeting GFP to the Golgi was narrowed down to a relatively hydrophobic region of 32 amino acids of which the first five amino acids were essential. This region showed no significant homology with other Golgi associated proteins in amino acid sequence. This small domain is sufficient for Golgi targeting and although normally part of a membrane protein can apparently function in the context of a soluble protein. This property may not be so surprising in view of recent reports that caveolin is not always an integral membrane protein but can exist in a cytosolic complex with cholesterol and chaperones . Our morphological studies suggest that association with the Golgi complex is a saturable process as higher expressing cells showed labeling throughout the cell by both immunofluorescence (particularly with the GFP-tagged construct) and by immunoelectron microscopy. What is the molecular basis of the association with the Golgi complex? Our studies show that the protein does not associate with endogenous caveolins consistent with in vitro studies of caveolin oligomer formation . The caveolin COOH terminus may therefore interact with a specific Golgi component. Our biochemical studies showed a surprisingly tight association with the membrane. Detergent treatment, but not high pH sodium carbonate, released the GFP-Cav3 KSY fusion protein from membranes. One possibility is that like cav-1 , cav-3 is palmitoylated and this modification is involved in Golgi membrane association. Indeed, we showed that the corresponding domain of cav-1 also associates preferentially with the Golgi complex and that this specific localization is lost upon modification of the three COOH-terminal cysteines to alanines. These three cysteine residues have been shown to be palmitoylated in vivo and to play a role in stabilization of higher order oligomers but not to play a role in targeting to caveolae . Cav-1 can be palmitoylated on all three cysteine residues within the COOH terminus . While palmitoylation alone is unable to mediate specific association with the Golgi, the lipid modification may contribute to the tight association with the Golgi membrane in combination with protein interactions. It is interesting to note that a number of other Golgi proteins are palmitoylated such as the glutamic acid decarboxylase isoform, GAD65 , and eNOS but palmitoylation is not required for the Golgi association. Interestingly, eNOS cycles between surface caveolae and the Golgi complex and shows a functional interaction with caveolin . Palmitoylation has been implicated in the lateral segregation of membrane associated proteins into DIGs (see Introduction) but, unlike the full-length caveolins, Cav3 KSY is detergent soluble (results not shown). As detergent insolubility is acquired in the late Golgi/TGN this is consistent with the proposed cis-Golgi location. As well as protein interactions it is possible that the Golgi association of Cav3 KSY relies on specific lipid interactions. Recently it was demonstrated that the cytoplasmic oxysterol-binding protein, which associates with the Golgi complex in response to oxysterols, is targeted specifically to this compartment through interactions with a phosphatidylinositol polyphosphate plus some other Golgi determinant . The COOH-terminal Golgi targeted fragment of caveolin is relatively hydrophobic and secondary structure predictions show a possible helical conformation but it appears unlikely that this domain can insert into the Golgi membrane. Previous studies have shown that a fusion protein containing the COOH terminus of cav-1 does not associate with membranes after synthesis in vitro . This domain of cav-1 is also accessible in vivo as COOH-terminal antibodies specifically recognize the Golgi caveolin in non-detergent permeabilized cells . It has also been shown to interact in vitro with signaling molecules such as n-NOS and c-src . Whatever the molecular mechanism, our experiments show a striking specificity of this domain for the Golgi complex and should provide powerful tools for further analysis of the molecular basis of Golgi localization. In addition, the KSY and DGV mutants described here have functional effects on caveolae-mediated events including infection by Simian Virus 40 and Ras signaling . Interestingly, the present study showed that expression of the COOH-terminal mutant caused a striking loss of detectable caveolin in the Golgi region but only in some cells in the population. This effect was not correlated with expression levels and so the reason for the apparent cell-cell variation is unclear. One possibility is a difference in caveolin cycling in cells at different stages of the cell cycle. The data presented here provide important new insights into the localization and targeting of this important class of proteins which should be taken into account in future models of caveolin function. Mutational dissection of the caveolin molecule appears to be a particularly powerful approach to gain insights into the function and dynamics of caveolin proteins and more detailed mutational studies should provide further clues into the complex and dynamic machinery comprising the caveolae membrane system. | Study | biomedical | en | 0.999996 |
10385525 | The mAb M2 to FLAG tag was purchased from Eastman-Kodak and the anti-CD2 mAb RPA-2.10 from PharMingen . The anti-H–Ras mAb R02120 (clone 18) and anti-p130 CAS mAb P27820 (clone 21) were from Transduction Laboratories. The origin and specificity of the affinity-purified rabbit antibodies to ERK2, phospho-ERK, Src, and GST and of the anti–5-bromodeoxyuridine (anti-BrdU) mAb were described previously . The mAb 3C2 reacting with the gag portion of v-Crk was also described previously . Human fibronectin was from GIBCO BRL and poly- l -lysine from Sigma Chemical Co. 293 human embryonic kidney cells were cultured in DME 10% FCS on gelatin-coated plates. NIH-3T3 mouse fibroblasts were cultured in DME 10% calf serum (CS). Fibroblasts from Src −/− and Fyn −/− embryos were obtained from Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA) and cultured in DME 10% CS. Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics and cultured on gelatin-coated dishes in human endothelial serum-free medium (SFM; GIBCO BRL ) supplemented with 20% FCS ( GIBCO BRL ), 10 ng/ml EGF, 20 ng/ml bFGF, and 1 μg/ml heparin (all from Intergen). The reporter plasmid pcoll TRE-tk-Luc, in which the expression of luciferase is driven by a single copy of the collagen gene 12-O-tetradecanoylphorbol-13-aretate (TPA)-responsive element (TRE) linked to the Hepes simplex virus thymidine kinase minimal promoter, was described previously . Vectors encoding the FLAG-tagged version of JNK 1, glutatione S-transferase (GST)-Jun, dominant-negative Ras (N17), and HA-tagged β-galactosidase were described previously . The CMV promotor-based pCDM8 vectors encoding CD2-FAK (wild-type), CD2-FAK K454R (kinase dead), and CD2-FAK Y397F were described previously . A kinase dead version of chicken c-Src was obtained from Sara Courtneidge (EMBL, Heidelberg, Germany) and subcloned in the cytomegalovirus (CMV) promotor-based vector pRK5. The pEBG vectors expressing GST-tagged MKK4 (wild-type) and MKK4 K129R (kinase dead) from the human elongation factor 1-α promoter were described previously . The Moloney Leukemia Virus (MLV)-LTR based pMEXneo vectors encoding v-Crk (wild-type), v-Crk R273N (SH2 mutant), and v-Crk D386DRHAD (SH3 insertional mutant) were described previously . The pEBG vectors encoding GST-tagged rat p130CAS (short form) and its substrate region deleted form (SD, Δ213– 514) were also described . The TAM-67 transactivation domain mutant form of c-Jun (Jun Δ3–122) was expressed from pCMV and previously characterized . The dominant-negative version of paxillin used in this study carries three phenylalanine permutations at tyrosine 31, 118, and 187 and is unable to bind to Crk. The MLV–LTR-based expression vector pΔraf-22w encodes an activated version of c-Raf 1 lacking an NH 2 -terminal segment of 305 amino acids . The vector encoding activated Ras, pDCR-Ha-ras (G12V), was kindly provided by John Westwick (Signal Pharmaceuticals). NIH-3T3 cells were transiently transfected with Lipofectamine according to the manufacturer's instructions ( GIBCO BRL ). 293 cells were plated at 6 × 10 6 per 15-cm diam dish for 8 h and then transfected overnight with various amounts of plasmid by the calcium phosphate method. All transfections were normalized to the same total amount of DNA with empty vector. Cells were allowed to recover for 12 h before growth factor starvation. To monitor the activation of JNK and ERK during G1, HUVECs were synchronized in G0 by a 24 h incubation in human endothelial SFM containing 0.2% FCS. They were then detached with 0.02% EDTA, collected in SFM containing 0.2% heat-inactivated BSA, washed in the same medium, and kept in suspension at a density of 10 6 /ml for 15 min at room temperature to recover. Aliquots consisting of 1.5 × 10 7 cells were plated on 15-cm diam dishes coated with 20 μg/ml fibronectin and postcoated with 0.2% heat-inactivated BSA in SFM supplemented with ITS+1 ( Sigma Chemical Co. ), EGF (10 ng/ml), bFGF (20 ng/ml), and heparin (1 μg/ml) for the indicated times. Cells from an identical aliquot were pelleted and lysed in suspension as a control. Before biochemical analysis, NIH-3T3 cells were serum starved for 18 h and 293 cells for 24 h in DME containing 0.2% CS or FCS, respectively. After detachment with 0.02% EDTA, cells were collected in DME containing 0.2% heat-inactivated BSA, washed in the same medium, and kept in suspension at a density of 10 6 /ml for 15 min at room temperature to recover. Aliquots consisting of 1.5 × 10 7 cells were plated on 15-cm diam dishes, coated with 20 μg/ml fibronectin and postcoated with 0.2% heat-inactivated BSA for the indicated times. Cells from an identical aliquot were pelleted and lysed in suspension as a control. NIH-3T3 cells were treated with growth factors as indicated. To analyze the activation of JNK, cells were extracted for 30 min on ice with 0.5 ml/dish of modified Triton lysis buffer (25 mM Hepes, pH 7.5, 300 mM NaCl, 0.1% Triton X-100, 0.2 mM EDTA, 20 mM β-glycerophosphate, 1.5 mM MgCl 2 , and 0.5 mM DTT) containing phosphatase and protease inhibitors. Aliquots containing 0.5 mg of total proteins were brought to 0.8 ml with modified Triton lysis buffer and diluted to 1.2 ml with HBB buffer (20 mM Hepes, pH 7.7, 50 mM NaCl, 0.05% Triton X-100, 0.1 mM EDTA, 20 mM β-glycerophosphate, 2.5 mM MgCl 2 , and 10 mM DTT) supplemented with phosphatase and protease inhibitors. Endogenous JNK was precipitated with 5 μg of GST-Jun fusion protein coupled to glutathione agarose beads . The beads were washed four times in HBB buffer, twice in kinase buffer (20 mM Hepes pH 7.5, 20 mM β-glycerophosphate, 10 mM MgCl 2 , and 10 mM DTT), and incubated with 35 μl of kinase buffer containing 10 μCi of γ[ 32 P]ATP (ICN) and 20 μM cold ATP. Recombinant FLAG-tagged JNK 1 was immunoprecipitated with the anti-Flag mAb M2. The beads were washed as above and incubated with 35 μl of kinase buffer containing 5 μg of GST-Jun, 10 μCi of γ[ 32 P]ATP, and 20 μM cold ATP. After 30 min of incubation at 30°C, the samples were boiled in sample buffer and separated by SDS-PAGE. Immunoprecipitation and immunoblotting were performed essentially as described previously . Secondary reagents for immunoblotting included peroxidase-conjugated protein A and affinity-purified rabbit anti–goat IgGs. To measure transcription from the collagen promotor TRE, NIH-3T3 cells were transiently transfected with the reporter plasmid pcoll TRE-tk-Luc. After 24 h of growth factor starvation, the cells were detached, kept in suspension for 30 min, and then solubilized or plated on dishes coated with 20 μg/ml fibronectin for the indicated times in the absence or presence of 20 ng/ml PDGF. Luciferase activity in cell lysates was estimated as described previously . NIH-3T3 cells were transiently transfected with vector encoding β-galactosidase in combination with various doses of the indicated constructs. The cells were allowed to recover in complete medium, synchronized in G0 by growth factor deprivation, and plated at low density on microtiter wells coated with 10 μg/ml poly- l -lysine or 20 μg/ml fibronectin in defined medium (DME supplemented with 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 0.625 ng/ml selenous acid, 1.25 mg/ml BSA, and 5.35 μg/ml linoleic acid) supplemented with 20 ng/ml PDGF and 10 μM BrdU. After 16 h, the cells were fixed and stained with X-gal followed by anti-BrdU mAb and AP-conjugated anti-mouse IgGs. The percentage of X-gal positive cells that had incorporated BrdU was evaluated microscopically after light counterstaining with hematoxylin. Preliminary experiments were conducted to examine if JNK was physiologically activated in primary cells progressing through the G1 phase of the cell cycle. HUVECs were synchronized in G0 by growth factor deprivation and detached to simulate the cell rounding physiologically occurring at mitosis. They were then plated on fibronectin in the presence of growth factors to allow entry into and progression through G1. In vitro kinase assays with the NH 2 -terminal fragment of c-Jun as a substrate showed that JNK is activated to a significant level during mid-G1, before the activation of the D-type cyclin-dependent kinase CDK4 . Peak JNK activity was observed 4 h after entry into G1. In contrast, the activation of ERK was biphasic with a first peak 10 to 20 min after entry into G1 and a second minor peak 8 h later . Since unstimulated cells are known to contain detectable levels of c-Jun, but not c-Fos , the rapid activation of ERK at the onset of G1 may serve to induce serum response element (SRE)-dependent transcription of the c-Fos gene before JNK-mediated transcriptional activation of c-Jun. The AP-1 transcription factor Fos/Jun may then promote the expression of genes necessary for G1 progression. The extent and timing of JNK activation during the cell cycle are thus consistent with a potential role in the control of G1 progression. We next compared the ability of integrins and growth factor receptors to activate JNK in NIH-3T3 fibroblasts. Preliminary experiments showed that the activity of JNK was significantly higher in growth factor deprived, stably adherent cells than in cells that had been detached and immediately lysed . In accordance with the previous observation that several growth factors cause a relatively modest activation of JNK , exposure to mitogenic concentrations of PDGF, bFGF, and insulin increased the activity of JNK only to a limited extent in growth factor starved, stably adherent NIH-3T3 cells. In contrast, PDGF, bFGF, and to a minor extent, insulin, caused a significant activation of ERK under the same conditions . To further examine the relative contribution of integrins and growth factor receptors to the activation of JNK, NIH-3T3 cells were either plated on fibronectin in the absence of growth factors or treated with various doses of PDGF while in suspension. As shown in Fig. 2 B, adhesion to fibronectin induced a rapid, strong, and protracted activation of JNK. By contrast, JNK activity increased only slowly and modestly over time in suspension, perhaps in response to the activation of a stress pathway, as observed by others . The activation of JNK caused by integrin ligation was comparable in intensity to that observed in cells treated with 5 to 10 ng/ml TNF-α , a known activator of JNK . Exposure to a wide range of PDGF concentrations caused little or no activation of JNK in suspended cells . Whereas exposure to 1 μg/ml lysophosphatidic acid (LPA), which is known to activate FAK , amplified the activation of JNK caused by integrin ligation by ∼70% (data not shown), treatment with PDGF did not exert this effect. However, PDGF changed the time course of JNK activation in cells adhering to fibronectin. Specifically, while adhesion to fibronectin caused maximal stimulation of JNK in ∼10 min, simultaneous exposure to PDGF significantly delayed the peak of activation of the kinase . This effect of PDGF may be related to its ability, when used at mitogenic concentrations as were used here, to transiently disrupt the cytoskeleton and thus delay integrin-mediated activation of FAK . Taken together, these observations indicate that JNK is activated by integrins, but only to a limited extent by PDGF, bFGF, and insulin. Exposure to growth factors may, however, contribute to sustain the activation of JNK caused by integrin ligation. Phosphorylation of c-Jun by JNK is required for transcriptional activation of the dimeric transcription factor AP-1 and for the oncogenic cooperation between c-Jun and Ha-Ras . To examine if the activation of JNK caused by integrin ligation could contribute to immediate early gene expression by promoting AP-1 dependent transcription from TRE, NIH-3T3 cells were transiently transfected with a vector encoding the luciferase gene under the transcriptional control of a TRE and plated on fibronectin in the presence or absence of PDGF. As shown in Fig. 2 , D and E, adhesion to fibronectin promoted TRE-dependent transcription with kinetics that closely followed that of JNK activation. Simultaneous exposure to PDGF caused a delay in the transcriptional response to fibronectin, as observed for the activation of JNK. The induction of TRE-dependent transcription by integrins required the transcriptional activity of c-Jun because it was suppressed by the TAM-67 dominant-negative form of the transcription factor (93.3% inhibition). Integrin-mediated activation of ERK and SRE-dependent transcription of Fos can increase the levels of AP-1 available for phosphorylation by JNK. However, TRE-dependent transcription could not have occurred in the absence of JNK-mediated phosphorylation of c-Jun. The mechanism by which integrins activate JNK was examined by introducing dominant-negative versions of various signaling components into human embryonic kidney 293 cells. Since preliminary experiments suggested that the ability to activate JNK is shared by all integrins, irrespective of whether they are able or not to recruit Shc (data not shown), we decided to examine the role of FAK in this process. Inactivating mutations were introduced at either the Src SH2-binding site or the ATP-binding site of CD2-FAK, a chimeric, membrane-anchored form of FAK that localizes efficiently to focal adhesions . We reasoned that membrane attachment would promote the interaction of these FAK mutants with focal adhesion components and thereby facilitate a dominant-negative effect. As shown in Fig. 3 , both CD2-FAK mutants exerted a dominant-negative effect on fibronectin-mediated activation of JNK, indicating that this process requires the kinase activity of FAK and its association with an Src family kinase. Accordingly, integrin-mediated activation of JNK was also suppressed by a kinase dead version of Src. In addition, we observed that the activation of JNK by integrins is partially defective in Src −/− and in Fyn −/− fibroblasts, in accordance with the notion that FAK can combine with both kinases (data not shown). Integrin-mediated activation of JNK was also blocked by a kinase inactive version of MKK4, one of the major enzymes that binds to and phosphorylates JNK . In contrast, it was not inhibited by expression of dominant-negative Ras or by exposure to the PI-3K inhibitors Wortmannin (100 nM) and LY294002 (50 μM) (data not shown). These results indicate that the FAK/Src complex links integrins to MKK4 or a related enzyme, and thereby JNK. In addition, they suggest that Ras and its substrate, PI-3K, which can activate Rac and thus JNK , do not contribute to this process. To examine the mechanism by which the FAK/Src complex activates JNK, we focused on the role of the docking/ adaptor proteins p130 CAS and paxillin, which bind to the FAK/Src complex and become heavily phosphorylated on tyrosine residues upon integrin engagement . The activation of JNK by integrins was inhibited to a significant extent by a mutant form of p130 CAS carrying a deletion of the entire substrate region . In contrast, a mutant form of paxillin carrying phenylalanine substitutions at three tyrosine phosphorylation sites, including all the Crk binding sites, inhibited this event modestly and only when expressed at relatively high levels (data not shown). These results indicate that p130 CAS plays a major role, and paxillin perhaps a minor one, in integrin-mediated JNK activation. 9 out of 15 tyrosine phosphorylation sites within the substrate region of p130 CAS conform to the consensus motif for binding to the SH2 domain of Crk . In addition to the SH2 domain, Crk contains either one or two SH3 domains able to interact with downstream target-effectors . Previous studies have indicated that the adaptor function of Crk is regulated positively by recruitment to the plasma membrane and negatively when tyrosine 222 becomes phosphorylated and associates intramolecularly with the SH2 domain . We reasoned that an SH3 mutant form of the viral version of Crk, which is anchored to the membrane via its gag sequences and truncated before tyrosine 222, would have interacted efficiently, via its intact SH2 domain, with p130 CAS , but not with downstream target effectors. As shown in Fig. 4 , expression of this mutant form of Crk effectively suppressed JNK activation in cells plated on fibronectin. Conversely, the introduction of a control construct with a mutated SH2 domain stimulated the activation of JNK, suggesting that the recruitment of Crk to the plasma membrane and its interaction with downstream target(s) via the SH3 domain are sufficient to activate JNK . These observations indicate that the FAK/Src/ p130 CAS complex activates JNK by recruiting Crk to the plasma membrane. They are also in agreement with previous studies implicating Crk in the activation of JNK . To examine whether FAK signaling to JNK was required for cell proliferation, NIH-3T3 fibroblasts were transiently transfected with various amounts of vectors encoding wild-type and mutant versions of the signaling components of this pathway, in combination with the marker β-galactosidase. The cells were synchronized in G0 and plated on fibronectin in the presence of PDGF. Entry of the transfected cells into S phase was evaluated by double staining with X-gal and anti-BrdU antibodies. While CD2-FAK, which is constitutively active , promoted entry into the S phase to a limited extent, its kinase dead version suppressed it . In both cases, the effects observed were dose dependent. In addition, whereas wild-type p130 CAS did not affect progression through G1, a mutant version carrying a deletion of the substrate region, which includes all the Crk binding sites, partially inhibited transit through G1 . The incomplete effect of this mutant may be due to residual, integrin-induced binding of Crk to paxillin . In accordance with this hypothesis, dominant-negative Crk suppressed entry into S phase as effectively as the kinase dead version of CD2-FAK. Finally, cell cycle progression was also suppressed by dominant-negative versions of MKK4 and Jun . These findings suggest that integrin-mediated activation of the FAK– JNK pathway is necessary for progression through the G1 phase of the cell cycle. Although the details of FAK's interaction with a number of cytoskeletal and signaling components are known, the biological function of this kinase is incompletely understood. Our results suggest that FAK mediates activation of JNK and c-Jun in response to integrin ligation, and by doing so, regulates progression through the G1 phase of the cell cycle. What is the mechanism by which FAK activates JNK? Upon activation, FAK undergoes autophosphorylation at tyrosine 397 and combines with the SH2 domain of Src or Fyn . The most prominent substrates of the FAK/Src complex are the docking adaptor proteins p130 CAS and paxillin . Both contain tyrosine phosphorylation sites conforming to the consensus for binding to the adaptor protein Crk. However, while paxillin has only two such sites and does not appear to associate efficiently with Crk in response to integrin ligation , p130 CAS contains nine Crk-binding motifs and associates well with Crk in cells adhering to fibronectin . Our results indicate that the expression of dominant-negative versions of FAK, Src, p130 CAS , and Crk suppress the activation of JNK by integrins. Together with complementary results of a recent study , these findings provide evidence that integrin-mediated activation of JNK requires the association of FAK with Src (or Fyn) and p130 CAS , and the recruitment of Crk. It is unlikely that the coupling of Ras to Rac mediated by PI-3K contributes in a significant manner to the activation of JNK by integrins because a dominant-negative form of Ras and specific inhibitors of PI-3K did not interfere with activation of this pathway. Thus, it appears that the β1 and αv integrins activate JNK and ERK via two separate pathways . By contrast, the α6β4 integrin, which is presumably unable to activate FAK because it does not contain the sequences required for its recruitment, is coupled to JNK signaling via the Ras–PI-3K–Rac pathway . The mechanism by which Crk activates JNK in response to integrin ligation remains to be examined. Crk is known to interact via one of its SH3 domains with the exchange factor C3G and previous studies have indicated that C3G is required for activation of JNK by the viral oncoprotein v-Crk . Interestingly, the activation of JNK by v-Crk and C3G appears to require the sequential action of the mixed lineage kinases MLK3 and DLK, but not the activity of Rho family GTPases or PAK proteins . In addition, or instead, Crk may activate Rac, and thereby JNK, by promoting the association of C3G with DOCK 180 or mSOS . The identity of genes regulated by JNK is largely unknown, but they must include genes important for cell proliferation. The evidence for this is several fold: first, deregulated expression of c-Jun or its mutated viral version v-Jun is sufficient to cause neoplastic transformation of primary avian and mammalian fibroblasts ; second, primary fibroblasts derived from c-Jun −/− mice display a severe proliferation defect ; and third, several oncoproteins, including v-Src, activated Ras, v-Crk, Bcr-Abl, and Met, potently activate JNK and there is evidence to suggest that this activation is required to cause neoplastic transformation . Despite the clear requirement for c-Jun transcriptional activity in cell proliferation, it has been difficult to identify a physiological, nonstress stimulus for JNK consistent with its role in the regulation of AP-1 transcription. With the notable exception of EGF, mitogenic neuropeptides, and muscarinic receptor ligands, which indeed activate FAK or the related kinase PYK-2 , most growth factors cause a relatively modest activation of JNK . Our results indicating that integrin ligation causes a significant activation of JNK and TRE-dependent transcription provide a physiological stimulus for JNK signaling that is consistent with its role in the control of cell proliferation. Our present results imply that FAK, which appears to be activated by all β1 and αv integrins, is required during G1 progression because of its ability to activate JNK. Recently, it has been shown that a mutant form of FAK, which is truncated at the COOH terminus and thus unable to localize to focal adhesions, interferes with both fibronectin-induced activation of ERK and progression through the cell cycle . On the basis of these results, it has been argued that FAK regulates cell proliferation by stimulating ERK. However, an alternative explanation is that this truncated form of FAK acts as a cytoplasmic sink for all Src-family kinases and thus disrupts not only FAK, but also Shc signaling. In accordance with this hypothesis, two more specific dominant-negative forms of FAK, FRNK and FAK-Y397F, inhibit ERK activation to a much lesser degree, but interfere with cell proliferation nonetheless . Although additional mechanisms cannot be excluded, our observation that dominant-negative forms of FAK, p130 CAS , Crk, MKK4, and Jun all inhibit entry into the S phase provides evidence that FAK regulates cell proliferation by activating JNK. If FAK is required for cell cycle progression, why then do the FAK −/− cells not display an obvious proliferation defect ? There are two potential explanations. First, it is now apparent that the FAK −/− cells originally examined by Ilic and colleagues also lack a functional form of the cell cycle regulator p53 . Some of the cell lines generated more recently do have wild-type p53, but are transformed by the polyoma middle T antigen . It is possible that the lack of p53 or presence of middle T antigen bypasses the requirement for FAK during cell proliferation. In addition, it recently has been shown that FAK −/− cells have elevated levels of PYK-2, which may compensate, at least in part, for the lack of FAK . The mitogenic signaling pathway linking integrins to JNK is likely to be deregulated in, and to contribute to, the transformation of at least some neoplastic cells. Previous studies have provided evidence that FAK is overexpressed in invasive carcinomas and that the constitutively active CD2-FAK induces anchorage-independent growth in MDCK cells . In addition, the viral version of Src is a potent oncogene capable of transforming a variety of cells types, and there is strong genetic evidence that p130 CAS is a necessary substrate of v-Src-induced transformation . In accordance with these findings, we have observed that CD2-FAK and p130 CAS cooperate with activated Raf to induce anchorage-independent growth in NIH-3T3 cells (F. Liu and F.G. Giancotti, unpublished results). Finally, v-Crk and v-Jun are potent oncogenes . Taken together, these observations suggest that the FAK–JNK pathway can contribute to neoplastic transformation. Previous studies have provided evidence that α6β4 and a subset of β1 and αv integrins activate the Ras-ERK signaling cascade by recruiting Shc . Mitogens and Shc-linked integrins synergize to promote transcription from the Fos SRE. Accordingly, ligation of integrins linked to Shc enables these cells to progress through the G1 phase of the cell cycle in response to mitogens, whereas adhesion mediated by other integrins results in growth arrest, despite the presence of mitogens. These mechanisms also appear to operate in vivo, as mice lacking the integrin α1 subunit or the cytoplasmic domain of β4 display cell cycle defects consistent with the lack of Shc signaling . These data suggest that integrin-mediated Shc signaling is necessary for cell cycle progression. The results of this and previous studies support the model that integrins control cell cycle progression primarily by regulating immediate early gene expression . While Shc-linked integrins and growth factor receptors cooperate to activate the Ras-ERK cascade and promote SRE-dependent transcription of c-Fos, all β1 and αv integrins appear to be able to stimulate the FAK–JNK pathway in the absence of a significant contribution from growth factor receptors. It is likely that, in response to integrin ligation, JNK not only acts on preexisting Jun/ATF2 and ATF2/ATF2 dimers, thereby promoting the CRE-dependent transcription of c-Jun, but also activates the Fos/Jun dimers formed in response to the coordinated action of both integrins and growth factor receptors. The existence of a signaling pathway activated by all integrins within a signaling network coordinately regulated by a subset of integrins and growth factor receptors ensures that the control of cell proliferation exerted by the extracellular matrix is both stringent and integrin-specific. | Other | biomedical | en | 0.999997 |
10385526 | L929r2 murine fibrosarcoma cells were treated for 4 h with 1,000 IU/ml mTNF and 10 μg/ml cycloheximide. Poly(A) + -mRNA was extracted and the first strand cDNA was synthesized using random primers . Transformed MC1061 bacteria, containing the L929r2 cDNA library, were grown or lifted on C/P lift membranes (Bio-Rad Laboratories). The membranes were soaked for 7 min in denaturing solution (1.5 M NaCl, 0.5 M NaOH), twice for 3 min in neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2, 1 mM EDTA) and washed in 2× SSC. DNA was fixed on the membranes by UV illumination and baking for 2 h at 80°C. The filters were incubated in prehybridization solution (1 mM EDTA, 0.5 M NaHPO 4 , pH 7.2, 7% SDS) at 60°C. Hybridization was done with the [ 32 P]dCTP-labeled StuI-XhoI fragment of hA20 cDNA. Filters were washed at room temperature with a buffer containing 1 mM EDTA, 40 mM NaHPO 4 , pH 7.2, and 5% SDS, and subjected to autoradiography. Nine individual clones giving a positive signal were obtained. The plasmid of one of these clones contained a cDNA insert of ∼3.7 kb, including besides the complete coding sequence of mA20 also part of the 5′ and 3′ untranslated region. Recombinant mTNF and hTNF were produced by Escherichia coli and purified to at least 99% homogeneity. The preparations used had a specific biological activity of 1.4 × 10 8 IU/mg and 8.8 × 10 6 IU/mg purified protein, respectively, as determined with the international standard (code 88/532 and code 87/650; National Institute for Biological Standards and Control). Recombinant murine IL-1β was produced by E. coli and purified to at least 99% homogeneity. The preparations used had a specific biological activity of 3.65 × 10 8 IU/mg purified protein, as determined with the international standard (code 93/668). TPA was purchased from Sigma Chemical Co. Recombinant green fluorescent protein (rGFP) and polyclonal rabbit antiserum directed against rGFP originated from CLONTECH Laboratories and polyclonal phosphospecific p38 MAP kinase antibody and polyclonal p38 MAP kinase antibody were purchased from New England Biolabs . The cDNAs encoding mutant GFP (S65T) and a fusion protein of GFP (S65T) followed by murine A20 were cloned in the eukaryotic expression plasmid pCAGGS . A20 was cloned as a blunted NcoI-EcoRI fragment in the SmaI site of the multiple cloning site inserted before the stop codon of GFP (pCAGGS-GFP/A20). The eukaryotic expression plasmids for ABIN were made by inserting the corresponding PCR fragment in frame with an NH 2 -terminal E-tag into pCAGGS. PCR fragments encoding TRADD and RIP were cloned in the pCDNA1 vector in frame with a COOH-terminal E-tag. The PCR fragment encoding CD40 was cloned in the plasmid pCDNA3 (pCDNA3-CD40) and was a gift of Dr. S. Pype (Catholic University of Leuven, Leuven, Belgium). Expression plasmids encoding TRAF2 and NIK were gifts of Dr. D. Goeddel (Tularik, San Francisco, CA) and Dr. D. Wallach (Weizmann Institute of Science, Rehovot, Israel), respectively. The plasmids phIL6LUC and p(κB) 3 LUC have been described previously . The plasmid pNFconluc, encoding the luciferase reporter gene driven by a minimal NF-κB responsive promoter was a gift of Dr. A. Israel (Institut Pasteur, Paris, France). The plasmids pAP1-luc and pSRE-luc were obtained from Stratagene. The plasmid pUT651, encoding β-galactosidase, was obtained from Eurogentec and the plasmid pIEX encoding the HTLV-1 Tax under control of the CMV promoter was obtained from Dr. K.-T. Jeang (National Institutes of Health, Bethesda, MD) . An expression plasmid for p300 (pCMV-p300) was provided by Dr. R. Eckner (Institute for Molecular Biology, Zurich, Switzerland) . The yeast plasmid pAS2-A20 was described earlier . L929sA and L929r2 murine fibrosarcoma cells and human embryonic kidney cells 293T were grown as described . Stable transfection of L929sA cells with the expression vectors was carried out by the calcium phosphate–DNA precipitation method. The pSV2neo-plasmid containing the neomycin resistance gene was coprecipitated as a selection marker . Selection of transfected clones was obtained by adding G418 (600 μg/ml) to the growth medium of the cells. Cells were mycoplasma-free, as judged by a DNA fluorochrome assay. L929sA transfectants were seeded in a 6-well plate at 6 × 10 5 cells/ 500 μl medium/well. After 24 h, cells were either stimulated for 6 h with 1,000 IU/ml mTNF or left untreated. Supernatants were harvested and assayed for IL-6 or GM-CSF in a 7TD1 or a FDCp1 cell proliferation assay, respectively . Cells in a 6-well plate were stimulated with 1,000 IU/ml mTNF for 5, 15, or 30 min, and nuclear fractions were prepared as described by Dignam et al. . NF-κB DNA binding was analyzed by incubating 10 μg nuclear proteins for 30 min with the NF-κB specific 32 P-labeled oligonucleotide probe (5′-agctATGTGGGATTTTCCCATGAGCagct-3′) containing the NF-κB site from the promoter of the mIL-6 gene. Protein–DNA complexes were separated on a 4% native polyacrylamide gel as described previously . L929sA cells were seeded at a density of 2 × 10 6 cells/75-cm 2 flask. 24 h later, cells were transfected by the DNA–calcium phosphate cotransfection method with the plasmids pUT651 and phIL6LUC or p(κB) 3 LUC. The plasmids phIL6LUC and p(κB) 3 LUC contain the luciferase gene after the complete hIL-6 promoter or the minimal hIL-6 promoter preceded by three copies of the NF-κB recognition site, respectively. 48 h after transfection, cells were trypsinized and seeded at 1.2 × 10 5 cells/24-well. At 66 h after transfection, cells were either noninduced or induced for 6 h with 1,000 IU/ml hTNF. Inducible promoter activity was determined by measuring the luciferase and β-galactosidase activity present in cell extracts as described previously . Transfection of 293T cells proceeded similarly, except that 293T cells were plated in 6-well plates at 4 × 10 5 cells per well and transiently transfected with 800 ng expression plasmids as well as 100 ng of pNFconluc and 100 ng pUT651. For measurements of AP1- and serum responsive element (SRE)–mediated transcription, cells were transfected with 100 ng of the plasmids pAP1-luc or pSRE-luc instead of 100 ng pNFconluc. 10 6 L929sA cells were seeded in 6-well plates. The next day, cells were either untreated or treated with 1,000 IU/ml mTNF for 5, 15, or 30 min. Cell lysates were prepared by directly adding SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue) to the cells. Proteins were separated by 15% SDS-PAGE and blotted on a nitrocellulose filter. Phosphorylated p38 MAP kinase was detected with a polyclonal phosphospecific p38 MAP kinase antibody and enhanced chemiluminescence ( Amersham International ). The total amount of p38 MAP kinase present in the same cell extracts was revealed with a polyclonal p38 MAP kinase specific antibody. The yeast two-hybrid system was purchased from CLONTECH Laboratories. The screening of a L929r2 cDNA library with pAS2-A20 was described previously . Yeast colonies expressing interacting proteins were selected by growth on minimal media lacking Trp, Leu, and His, in the presence of 5 mM 3-amino-1,2,4-triazole and by screening for β-galactosidase activity. Plasmid DNA was extracted from the positive colonies and the pGAD424 vectors encoding candidate A20-interacting proteins were recovered by electroporation in the E. coli strain HB101 and growth on media lacking Leu. 2 × 10 6 human embryonic kidney 293T cells were plated on 10-cm petri dishes and transiently transfected by calcium phosphate–DNA coprecipitation. 24 h after transfection, cells were lysed in 500 μl of lysis buffer (50 mM Hepes, pH 7.6, 250 mM NaCl, 0.1% NP-40, 5 mM EDTA). Lysates were incubated with 5 μl of rabbit anti-GFP antibody ( CLONTECH Laboratories) and immunocomplexes were immobilized on protein A–Trisacryl ( Pierce Chemical Co. ). The latter were washed twice with lysis buffer and twice with lysis buffer containing 1 M NaCl. Coprecipitating proteins were separated by SDS-PAGE and analyzed by Western blotting with mouse anti–E-tag antibody ( Pharmacia Biotech ). Localization of GFP/A20 was analyzed by means of GFP fluorescence. Therefore, L929sA cells stably expressing GFP or GFP/A20 were seeded on coverslips in 6-well plates and microscopically analyzed for GFP fluorescence (emission at 510 nm) at an excitation wavelength of 485 nm. To identify the subcellular localization of ABIN, 4 × 10 5 293T cells were seeded on coverslips in 6-well plates and transfected with 1 μg plasmid DNA. 24 h after transfection, cells were fixed on the coverslips with 3% paraformaldehyde. Upon permeabilization with 1% Triton X-100, the cells were incubated for 2 h with mouse anti–E-tag antibody (1/1,000) followed by a second incubation with anti-mouse Ig antibody coupled to biotin (1/1,000; Amersham ). After a subsequent incubation with streptavidin coupled to Texas red ( Amersham ), fluorescence can be analyzed via fluorescence microscopy (Axiophot; Zeiss ), using a filter set with excitation at 543 nm and emission at 600 nm. In the same cells, fluorescence of GFP can be detected at a different wavelength, namely excitation at 485 nm and emission at 510 nm. A20 has been described as a TNF inducible inhibitor of NF-κB activation, mainly on the basis of its effect on NF-κB–dependent expression of a reporter gene . Therefore, we first analyzed its effect on the TNF-induced expression of IL-6 and GM-CSF in control (L929SA-neo) and in L929sA cells overexpressing either a fusion protein of A20 with GFP (L929SA-GFP/A20) or GFP as such (L929SA-GFP). Expression of these cytokines has been shown to be NF-κB– dependent . Compared with the amount of IL-6 and GM-CSF found in the supernatant of TNF-treated L929SA-neo and L929SA-GFP cells, their levels were considerably decreased in L929SA-GFP/A20 cells (Table I ). A similar inhibitory effect of A20 on TNF-induced gene activation could be seen when GFP-A20 expressing L929sA cells were transiently transfected with an expression plasmid containing the luciferase reporter gene under control of either the hIL-6 promoter (phIL6LUC) or a minimal promoter with three NF-κB–binding sites [p(κB) 3 LUC], and analyzed for luciferase expression upon stimulation with 1,000 IU/ml mTNF for 6 h . These results demonstrate that A20 interferes with the NF-kB–dependent expression of a reporter gene as well as with the expression of endogenous genes that are NF-κB regulated. NF-κB activation involves the release of the inhibitory protein IκB from NF-κB in the cytosol, leading to the nuclear translocation and binding of NF-κB to its specific recognition sequence in DNA . The latter can be analyzed in a gel shift assay in which binding of active NF-κB to an NF-κB specific DNA probe leads to a slower migration of this probe in a nondenaturing polyacrylamide gel. Nuclear fractions prepared from L929SA-neo, L929SA-GFP, and L929SA-GFP/A20 cells that were either unstimulated or stimulated for 30 min with mTNF were analyzed in such an assay. Remarkably, although A20 completely prevented NF-κB–dependent gene expression as described above, no clear differences in TNF-induced DNA binding of NF-κB were observed . Also, stimulation of these cells for shorter periods, 5 or 15 min, did not reveal an effect of A20 (data not shown). Nevertheless, TNF-induced nuclear translocation and DNA binding of NF-κB could be completely abolished by pretreatment with the proteasome inhibitor MG132 that inhibits NF-κB activation by preventing IκB degradation . These results indicate that A20 inhibits NF-κB–dependent gene induction by specifically interfering with an NF-κB transactivation signal, without affecting the nuclear translocation and DNA binding of NF-κB. By the presence of seven zinc finger structures in A20, it has been originally suggested that A20 may bind to DNA and directly interfere with the transcriptional machinery . However, more recently, it has been demonstrated that A20 transiently expressed in 293T cells exclusively localized in the cytoplasm . In our L929sA cells stably expressing GFP/A20 fusion proteins, a similar speckled cytoplasmic staining was observed via GFP fluorescence and confocal microscopy . In contrast, GFP as such was detected both in the cytoplasm and the nucleus. Furthermore, treatment of the cells with TNF did not induce a detectable relocalization of A20 (data not shown). These results suggest that A20 cannot directly interfere with NF-κB transcriptional activity, but indicate that A20-mediated inhibition of NF-κB transactivation is a cytosolic event. TNF-induced activation of NF-κB involves the recruitment of several cytoplasmic signaling proteins to the TNF-R55 , which is the main signaling receptor for TNF. Moreover, overexpression of some of these proteins is sufficient to induce NF-κB activation. To investigate at which step A20 interferes with NF-κB activation, we tested the effect of A20 on NF-κB–dependent gene expression induced by TNF treatment or by overexpression of the TNF receptor associating proteins TRADD, RIP, TRAF2, and NIK in 293T cells. As expected, A20 completely prevented NF-κB– dependent luciferase expression induced by TNF . A similar protective effect of A20 could be observed when NF-κB was activated by overexpression of TRADD, RIP, or TRAF2. However, NF-κB activation by overexpression of downstream acting NIK was not sensitive to A20. These results clearly demonstrate that A20 interferes with an RIP- or TRAF2-initiated NF-κB transactivation signal, which is different from the NIK-mediated pathway leading to nuclear translocation of NF-κB. Recently, it was shown that activation of NF-κB induced by the HTLV transactivator Tax protein is mediated by NIK and both IKKα and IKKβ . In contrast, overexpression of a TRAF2 dominant negative mutant was not able to block Tax-induced NF-κB activation, excluding a role for TRAF2 in this pathway . Therefore, we tested the effect of GFP/A20 overexpression on Tax-induced expression of an NF-κB–dependent reporter gene in 293T cells. Whereas TNF-induced NF-κB activation was completely prevented by A20, the latter could not block the gene-inducing effects mediated by Tax . These results further indicate that A20 interferes with TNF-induced NF-κB–dependent gene expression at the level of an RIP- or TRAF2-initiated NF-κB transactivation pathway that is independent of NIK. To further substantiate this hypothesis, we also analyzed the effect of A20 on the activation of NF-κB by a number of other stimuli for which NF-κB activation has been shown to be mediated by TRAF2 or TRAF family members. IL-1 engages TRAF6 in its signaling pathway , whereas CD40 initiates NF-κB activation via TRAF2, TRAF5, and TRAF6 . Overexpression of A20 completely prevented IL-1–induced NF-κB activation in 293T cells , which confirms previously published data . Similarly, NF-κB activation induced by overexpression of CD40 was also abolished when A20 was coexpressed . These results are in agreement with the above suggested role for a TRAF-initiated signaling pathway as target for A20. Surprisingly, A20 also inhibited NF-κB activation by the protein kinase C activator TPA . The latter also activates AP1- and SRE-dependent gene expression in 293T cells. Therefore, we analyzed whether A20 could also prevent the TPA-induced expression of a luciferase reporter gene whose expression is controlled by AP1- or SRE-binding proteins. However, in contrast to NF-κB–dependent gene expression, the TPA-induced activation of AP1- or SRE-dependent transcription was not sensitive to A20. These results demonstrate that A20 can also act as a specific inhibitor of NF-κB–dependent gene expression induced by TPA. It remains to be seen whether TRAFs or downstream signaling proteins are also involved in NF-κB activation by this stimulus. Recently, we and others demonstrated an important role for TNF-induced activation of p38 MAP kinase in the transactivation of NF-κB . Moreover, TNF-induced p38 MAP kinase activation was shown to be mediated by TRAF2 . To investigate whether A20 inhibited the TNF-induced transactivation of NF-κB by preventing p38 MAP kinase activation, we analyzed the TNF-induced activation of p38 MAP kinase in GFP/A20–transfected versus GFP-transfected L929SA cells. p38 MAP kinase activation was revealed by immunodetection of phosphorylated p38 MAP kinase with p38 MAP kinase phosphospecific antibodies. TNF induced a transient phosphorylation of p38 MAP kinase in L929SA-GFP as well as in L929SA-GFP/A20 cells . Time kinetics of TNF-induced p38 MAP kinase phosphorylation/ activation were similar in both cell lines, although in some experiments dephosphorylation occurred slightly faster in A20-expressing cells. These results make it unlikely that A20 prevents NF-κB transactivation by inhibiting the TNF-induced activation of p38 MAP kinase. CREB-binding protein (CBP) and p300 are coactivators that link transcriptional activators to the basal transcriptional apparatus. Both CBP and p300 were shown to act as coactivators of NF-κB–dependent gene expression by a direct interaction with the p65 subunit . The latter interaction was recently shown to be enhanced by phosphorylation of p65 by protein kinase A . To analyze whether A20 would prevent NF-κB transactivation by affecting the interaction between p65 and CBP/p300, we analyzed if TNF-induced NF-κB–dependent reporter gene expression could be rescued from A20 inhibition by overexpression of p300 in 293T cells. Basal and TNF-induced NF-κB activity was increased approximately threefold when p300 was coexpressed , which is in agreement with a coactivator function for p300. However, p300 could not rescue TNF-induced NF-κB activation from inhibition by A20. These results make it unlikely that A20 prevents TNF-induced NF-κB–dependent gene expression by abolishing the transactivation of NF-κB by CBP/p300. Although it is known that the NF-κB inhibiting potential of A20 resides in its COOH-terminal zinc finger–containing domain , the underlying mechanism is still unclear. Therefore, we used the yeast two-hybrid system to screen an L929r2 cDNA library for A20 interacting proteins that might be involved in the negative regulation of NF-κB. From 1.3 × 10 6 transformants, 10 clones expressed A20-interacting proteins, including A20 itself and 14-3-3 proteins . Three clones contained COOH-terminal fragments of the same cDNA encoding a protein that we named ABIN. Full-length ABIN cDNA was subsequently isolated from the L929r2 cDNA library by colony hybridization with a fragment cloned by two-hybrid analysis as a probe. Several cDNAs were isolated and in the longest cDNA stop codons were identified in all three frames 5′ of a potential initiator methionine. Two different splice variants were found of ∼2,800 and 2,600-nt long. Northern blot analysis revealed that ABIN is expressed in all murine tissues tested as an mRNA of ∼2,800 bp that is in accordance with the length of the cloned full-length cDNA (data not shown). In contrast to A20, ABIN mRNA is constitutively expressed in both TNF-sensitive and TNF-resistant subclones derived from the parental cell line L929s , irrespective of TNF stimulation. The two splice variants of the ABIN cDNA contained an open reading frame of 1,941 and 1,782 nucleotides respectively, initiating at two different methionines [ABIN (1-647) and ABIN (54-647)] . These cDNAs encode proteins of 72 and 68 kD, containing an amphiphatic helix with four consecutive repeats of a leucine followed by six random amino acid residues, which is characteristic of a leucine zipper structure. Also, full-length ABIN(1-647) and ABIN(54-647) bound A20 in a yeast two-hybrid assay, confirming the original interaction found with the 3′ COOH-terminal fragments ABIN(390-599), ABIN(249-647), and ABIN(312-647). The latter all contain the putative leucine zipper protein interaction motif (397-420). In addition, ABIN also interacted with A20 in mammalian cells since ABIN was able to coimmunoprecipitate with A20 in 293T cells that were transiently transfected with an expression plasmid for chimeric GFP-A20 protein and ABIN with an NH 2 -terminal E-tag . Interestingly, interaction of ABIN with A20 required the COOH-terminal, zinc finger–containing part of A20 [A20(369-775)]. This domain was shown previously to be required for dimerization of A20 and for the interaction of A20 with 14-3-3 proteins . Furthermore, overexpression of this domain is sufficient for the NF-κB inhibiting effects . In contrast, the NH 2 -terminal part of A20 has been shown to interact with TRAF2 , suggesting that A20 acts as an adapter protein between TRAF2 and ABIN. The interaction between A20 and ABIN was not influenced by stimulation with TNF (data not shown). To characterize the subcellular distribution of ABIN, we transiently transfected GFP-A20 and E-tagged ABIN cDNA in 293T cells and analyzed their expression by means of GFP fluorescence and immunofluorescence via the anti–E-tag antibody, respectively. ABIN colocalized with A20 throughout the cytoplasm, both in unstimulated and in TNF-stimulated cells (data not shown). Database similarity searches (BLAST) showed that ABIN is the murine homologue of the human cDNA encoding a human immunodeficiency virus (HIV) Nef–associating factor, NAF1 . HIV–Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T cell function. Interestingly, the effect of Nef on host cell activation has been explained in part by its interaction with specific cellular proteins involved in signal transduction , of which ABIN might be an example. Since both A20 and Nef were shown previously to block the signal transduction pathway leading to NF-κB activation upon stimulation with TNF or IL-1 and T cell receptor stimulation, respectively , we investigated the effect of ABIN on NF-κB–dependent reporter gene expression in transiently transfected 293T cells. GFP and GFP-A20 served as negative and positive controls, respectively. Similar to A20, both splice variants of ABIN were able to block TNF-induced NF-κB activation in these cells, with the shorter NH 2 -terminal truncated isoform being slightly more effective . Overexpression of a combination of suboptimal doses of A20 and ABIN, on their own not sufficient to inhibit NF-κB activation, diminished NF-κB activation upon stimulation with TNF considerably . This suggests that ABIN might mediate the NF-κB inhibiting effect of A20. Furthermore, cotransfection of expression plasmids encoding the TNF receptor–associated signaling proteins TRADD, RIP, TRAF2, and NIK together with the expression plasmid encoding ABIN, showed that the latter completely inhibited NF-κB activation induced by TRADD or RIP, and partially TRAF2-induced NF-κB activation. In contrast, no effect of ABIN was observed when NF-κB–dependent reporter gene expression was induced by NIK or more directly by overexpression of the p65 subunit of NF-κB . Similarly, Tax-induced NF-κB activation that is TRAF-independent and is insensitive to A20, was also not affected by coexpression of ABIN . These results suggest that ABIN, like A20, inhibits TNF-induced NF-κB activation at the level of RIP or TRAF2 proteins, preceding the activation of the NIK–IκB kinase steps. The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of NF-κB– dependent gene expression induced by different stimuli including TNF and IL-1 . Here we show that the TNF-induced expression of GM-CSF and IL-6, as well as the TNF-induced expression of a luciferase reporter gene that is expressed under control of the complete hIL-6 promoter or the minimal hIL-6 promoter preceded by 3 NF-κB recognition sequences, are clearly inhibited in L929sA cells stably transfected with A20. These results are consistent with the fact that NF-κB is required for IL-6 and GM-CSF gene transcription . Surprisingly, gel retardation assays revealed that overexpression of A20 had no effect on the TNF-induced nuclear translocation and DNA binding of NF-κB. Also the constitution of the NF-κB complex was not altered in cells overexpressing A20, and consisted in both cases of a p65 and a p50 subunit as revealed by gel supershift assays (data not shown). Therefore, the inhibition of NF-κB– dependent gene expression by A20 cannot be explained by an A20-induced alteration in the subunits of NF-κB. Ferran et al. showed recently that A20 acts upstream of IκB degradation and prevented the nuclear translocation of NF-κB. The reason for the discrepancy with our results is still unclear. Because activation of NF-κB is an early response after stimulation with TNF, we analyzed NF-κB translocation at 5, 15, and 30 min after TNF stimulation, whereas results of Ferran et al. were obtained 2 h after TNF stimulation. The latter is quite late and might already be regulated by secondary factors that are A20-sensitive. Moreover, NF-κB activation at later times is also regulated by TNF-induced negative regulatory proteins such as IκBα and A20 whose expression is itself under the control of NF-κB, further raising the complexity of NF-κB activation at later time points . Alternatively, we cannot exclude cell type–dependent differences. Until now, the mechanism by which A20 blocks the activation of NF-κB–dependent gene regulation was not known. Up to now, the NIK–IκB kinase pathway has been assumed to be responsible for NF-κB activation upon TNF treatment. However, our finding that A20 has no effect on the translocation of NF-κB to the nucleus argues against this pathway as the target for A20. Moreover we demonstrated that A20 could inhibit RIP- and TRAF2- but not NIK-induced NF-κB–dependent reporter gene activation, suggesting that A20 interferes with NF-κB activation upstream of NIK. These conclusions were confirmed by the inability of A20 to block Tax-induced NF-κB activation since the latter was shown to activate NF-κB by directly interfering with the downstream kinases NIK, IKKα, and IKKβ, independent of TRAF2 . The above results suggest that TNF-induced NF-κB– dependent gene activation requires at least two different pathways: an NIK-mediated pathway leading to translocation of NF-κB to the nucleus, and an NIK-independent pathway leading to transactivation of NF-κB. Our results demonstrate that A20 specifically interferes with the NF-κB transactivation pathway. A20 has also been shown to interact with the TNF receptor–associated protein TRAF2 . Interestingly, TRAF2 as well as some other members of the TRAF protein family, including TRAF5 and TRAF6, were shown to play a positive role in NF-κB activation induced by different cytokines, such as TNF, IL-1, and CD40 ligand, via their interaction with the NF-κB inducing kinase NIK . The fact that A20 directly associates with TRAF2, as well as our observation that A20 not only prevents NF-κB activation by TNF but also by IL-1 and CD40 overexpression, points to a role of a TRAF-mediated signaling pathway as a target for A20. However, gene knockout studies have recently shown that TRAF2 is not absolutely required for NF-κB activation by TNF, although this probably is a consequence of redundancy within the TRAF protein family . Alternatively, RIP may be more important than TRAF2 in mediating activation of NF-κB upon TNF stimulation . The nature of the RIP/TRAF–initiated NF-κB transactivation signal is still unclear. Recently, an important role in the transactivating potential of NF-κB upon TNF stimulation was demonstrated for p38 MAP kinase . This kinase becomes activated by stimulation of cells with TNF as well as by overexpression of TRAF2. In contrast, overexpression of NIK did not induce the phosphorylation of p38 MAP kinase, indicating that a separate pathway initiating at TRAF2 leads to the activation of p38 MAP kinase . Similar to the effect of A20 overexpression, inhibition of p38 MAP kinase with the specific inhibitor SB203580 also prevented NF-κB– dependent gene expression without altering the translocation of NF-κB to the nucleus . As these results suggested that A20 might interfere with the TRAF2-p38 MAP kinase pathway, we investigated if A20 was able to prevent the TNF-induced activation of p38 MAP kinase. However, no significant effect of A20 on p38 MAP kinase phosphorylation, which is a marker for its activation, could be observed. Although these results indicate that A20 does not act upstream of p38 MAP kinase activation, it is still possible that A20 interferes with the TRAF2-p38 MAP kinase/ NF-κB transactivation pathway downstream of p38 MAP kinase. The validation of this possibility awaits the identification of the p38 MAP kinase substrate that is involved in NF-κB transactivation. Alternatively, our results might also fit with the existence of another RIP- or TRAF2-initiated pathway that contributes to NF-κB–dependent transcription, and which is blocked by A20. Our observation that A20 also prevents NF-κB activation by TPA indicates that the A20-sensitive pathway might also be activated by protein kinase C, at least in some cell lines. Similar results were obtained by Cooper et al. . In contrast, stable expression of A20 has been reported to be unable to prevent TPA-induced NF-κB activation in breast carcinoma MCF cells . These controversial results might reflect a cell type specific effect or differences in A20 expression levels upon stable and transient transfection. It should be mentioned that a role for protein kinase C in TNF-induced NF-κB transactivation has been suggested recently based on the inhibition with a protein kinase C inhibitor . Whether protein kinase C functions downstream of TRAF2 or in a totally separate pathway remains to be established. In any case, the protein kinase C–mediated pathway sensitive to A20 seems to regulate specifically NF-κB–dependent gene expression, as TPA-induced transcription that is controlled by an AP1-responsive element or a SRE was not sensitive to A20. The latter result, as well as our finding that Tax-mediated NF-κB activation is not affected by A20, also excludes an aspecific effect of A20 on the general transcription machinery. The transcription activating potential of NF-κB has been primarily attributed to the p65 subunit, whose transactivating potential resides in its COOH-terminal portion . Furthermore, the p65 subunit becomes phosphorylated during the activation of NF-κB upon TNF stimulation . Indeed, a p65 phosphorylating activity was found in the IκB kinase complex . Moreover, it was also shown that IκB is associated with the protein kinase A catalytic subunit that can phosphorylate the p65 in its rel homology domain resulting in enhanced activity of NF-κB . Recently, p65 phosphorylation was shown to promote an interaction between p65 and the coactivators CBP/p300 . The latter were previously shown to synergistically enhance the transcription activating potential of NF-κB . However, it is unlikely that A20 interferes with protein kinase A or another signaling pathway leading to the engagement of the coactivators CBP/p300 in the transactivation of NF-κB because we were unable to rescue NF-κB activation from A20 inhibition by overexpression of CBP/p300. Moreover, activation of the protein kinase A catalytic subunit requires degradation of IκB and the activation of the NIK–IκB kinase pathway which is, however, not modulated by A20. Also, A20 did not interfere with NF-κB–dependent gene expression obtained by overexpression of the p65 subunit as such . In endothelial cells, TRAF2 has been recently shown to translocate to the nucleus, where it might directly regulate transcription . Because A20 can bind to TRAF2 , and exclusively resides in the cytosol, A20 might prevent nuclear localization of TRAF2. Screening of a cDNA library for A20 interacting proteins by the yeast two-hybrid system has revealed some isoforms of the 14-3-3 proteins that interact with the COOH-terminal zinc finger domain of A20 . 14-3-3 proteins were shown to function as adapter proteins between A20 and c-Raf. Moreover, 14-3-3 also functioned as a chaperone in these studies . However, by mutation analysis we previously demonstrated that the interaction of 14-3-3 proteins with A20 is not involved in the effect of A20 on NF-κB activation . By the yeast two-hybrid screening system, we also identified ABIN as a novel A20-interacting leucine zipper protein. The interaction of ABIN with A20 was confirmed in human cells and shown to map to the functional COOH-terminal zinc finger–containing domain of A20. Upon overexpression, ABIN potently inhibits NF-κB activation induced by TNF. Furthermore, also ABIN interferes with TNF-induced NF-κB activation at the level of RIP/ TRAF2. Therefore, the ability of A20 to block TNF-mediated NF-κB activation is likely to involve the binding of the NF-κB inhibitory protein ABIN to the COOH-terminal zinc finger domain of A20. Moreover, the fact that A20 can also interact via its NH 2 -terminal domain with TRAF1 and TRAF2 suggests that A20 can recruit ABIN to the TRAF2 complex in the TNF signaling pathway. In conclusion, A20 appears to prevent NF-κB–dependent gene expression by specifically interfering in the cytosol with a novel RIP/TRAF2–initiated transactivation pathway, thus inhibiting the TNF-induced expression of several cytokines and proinflammatory proteins. Since A20 also inhibits NF-κB activation by IL-1 and CD40, which all signal to NF-κB activation via members of the TRAF family, further identification of TRAF-mediated NF-κB transactivation signals may provide means of achieving more specific antiinflammatory treatments. | Study | biomedical | en | 0.999998 |
10385527 | The plasmid vector containing the full-length human fast skeletal αTm was a gracious gift from Dr. Clare Gooding . An EcoRI fragment containing the full-length αTm cDNA was subcloned into pCA4 plasmid to add additional restriction enzyme sites for future subcloning . A XbaI, HindIII fragment containing the full-length αTm cDNA was subcloned into pSP72 ( Promega ) for mutagenesis. The COOH-terminal FLAG epitope (DYKDDDDK) ( Sigma ) was engineered by PCR mutagenesis using the following primer set to amplify a 204-bp fragment: forward primer 5′ AGAGATCAAGGTCCTTTCCG 3′ and reverse primer 5′ GAAGTGAAGCT * T * AGAAACTTA CTTGTCGTCATCGTCTTTGTAGTC TATGGAAGTCA-TATCGTTGAGAG 3′ (underline indicates FLAG epitope sequence). A HindIII site (asterisks) was engineered into the reverse primer to facilitate subcloning. A Ppu M1, HindIII fragment of the PCR product was ligated to the Ppu M1 site in the αTm cDNA and the αTmFLAG cDNA was verified by DNA sequencing. This subcloning step removed 197 bp of 3′ untranslated sequence. The XbaI, HindIII fragment containing the αTmFLAG cDNA was subcloned into pCA4 (pCA4αTmFLAG). The plasmid (pGEM3ZcTnI) containing the full-length cardiac troponin I cDNA was a gracious gift from Dr. Anne Murphy . A COOH-terminal FLAG epitope was added by PCR mutagenesis using the following primer sets: forward primer 5′ GCCAAGGAATCCTTGGACCTGAGGG 3′ and reverse primer 5′ CAGTGTGAGAGCCATGGCTCA CTTGTCGTCATCGTCTTTGTAGTC GCCCTCG * AACTTTTT- CTTTCGGCC 3′ (underline indicates FLAG epitope sequence). An XmnI site (asterisk) was engineered in the reverse primer to identify the mutagenized clones. An ApaI, NcoI fragment of the PCR product was subcloned into pGEM3ZcTNI and the resulting cTNIFLAG cDNA was verified by DNA sequencing. An EcoRI fragment containing the cTNIFLAG cDNA was subcloned into pCA4 (pCA4cTNIFLAG). Replication-deficient recombinant adenovirus (Ad5ΔE1) vectors were constructed from the cotransfection shuttle vectors pCA4αTmFLAG or pCA4cTnIFLAG with a vector containing the full-length adenoviral genome, pJM17 followed by homologous recombination in HEK-293 cells as previously described . Positive viral lysates were plaque purified and identified by restriction enzyme Southern blot analysis. The viruses were grown to high titer in HEK-293 cells, purified by CsCl centrifugation and the viral stocks (∼10 10 pfu/ml) were stored in single use aliquots at −80°C. Adult rat ventricular myocytes were isolated and cultured as previously described . In brief, female adult rats (∼200 g) were anesthetized with sodium pentobarbitol and the hearts were removed in Krebs-Henseleit buffer containing 1 mM Ca 2+ (KHB+Ca 2+ ). The hearts were perfused for 5 min with KHB+Ca 2+ on a Langendorff perfusion apparatus followed by a 5-min perfusion with KHB, Ca 2+ -free (7–10 ml/ min). Collagenase (162 U/ml; Worthington, Type II) and hyaluronidase (0.125 mg/ml; Sigma ) were then added to Ca 2+ -free KHB and perfusion continued for 15 min. CaCl 2 was then added to a final concentration of 1 mM and perfusion continued for 10–15 min. The ventricles were then minced and digested in the enzyme solution for 2× 10 min. The tissue was then digested for 2× 15 min with gentle trituration and isolated myocytes were collected by centrifugation. The myocytes were resuspended in KHB+1 mM Ca 2+ and 2% BSA and the solution was titrated to 1.75 mM Ca 2+ with three additions of CaCl 2 over 15 min. The resulting myocytes were collected by centrifugation and resuspended in DME, 5% FBS, 1% penicillin/streptomycin (P/S) ( GIBCO BRL ), and plated on laminin coated glass coverslips (1 × 10 5 cells/ml, 200 μl/coverslip) for 2 h. The myocytes were then infected with recombinant adenovirus diluted in 200 μl serum-free DME with P/S at 2.5–5 × 10 7 pfu/ml or a multiplicity of infection (MOI) of 250–500, for 1 h. After gene transfer, 2 ml serum-free DME with P/S was then added, and for the standard culture conditions, myocytes were maintained (media changed every 48 h) in serum-free DME with P/S unless otherwise indicated. In a subset of experiments , in order to generate cultures of redifferentiating adult cardiac myocytes, myocytes were maintained in DME, 20% FBS, P/S from 12 h post infection to the end of time in culture. Intact myocyte samples were prepared by scraping myocytes from coverslips into SDS sample buffer. Permeabilized myocyte samples were prepared by exposing the myocytes on coverslips to relaxing buffer (see below) containing 0.1% Triton X-100 for 3–5 s followed by two washes in 3–4 ml of relaxing buffer. The remaining permeabilized myocytes were scraped into SDS sample buffer. The samples were immediately boiled for 5 min, an equal volume of SDS sample buffer was added, and the samples were stored at −20°C. The protein samples were analyzed on 12% SDS-PAGE followed by transfer to Immobilon-P PVDF membrane ( Millipore ) for 2,000 V⋅ hr. The membranes were blocked overnight in TBS containing 5% nonfat dry milk. The primary antibodies used for detection of myofilament proteins were as follows: Tm, Tm311, 1:100,000 ( Sigma ); TnI, MAB1691, 1:1,000 (Chemicon); troponin T, JLT-12, 1:1,000 ( Sigma ); anti-FLAG M2, 1:2,000 ( Sigma ); sarcomeric actin, clone 5C5, 1:5,000 ( Sigma ). Primary antibody binding was detected with a goat anti–mouse IgG– horseradish peroxidase conjugate followed by ECL detection ( Amersham ). The films were digitized using a transparency scanner and quantitated with Multi-Analyst software (Bio Rad Laboratories). To calculate Tm stoichiometry and to compare the ratios of different myofilament proteins using multiple blots with different exposure times, the ratio of Tm/ actin data was normalized to the mean ratio in the control myocyte samples on each blot ((Tm:actin) sample / (Tm:actin) mean control ). Cardiac myocytes were prepared for confocal imaging as previously described In brief, cardiac myocytes on coverslips were fixed in 3% paraformaldehyde/PBS for 30 min. Myocytes were washed 3× 5 min in PBS and incubated in PBS with 50 mM NH 4 Cl for 30 min followed by washing 3× 5 min in PBS. Myocytes were blocked with 20% NGS in PBS + 0.5% Triton X-100 for 30 min followed by incubation with primary antibody (Ab) for the FLAG epitope (M2, 1:500), sarcomeric Tm (CH-1, 1:200; Sigma ), or TnI diluted in 2% NGS, PBS + Triton X-100) for 1.5 h. Myocytes were washed 3× 5 min in PBS + Triton X-100 and blocked again for 30 min. Myocytes were incubated with secondary Ab (goat anti–mouse IgG, Texas Red, 1:100; Molecular Probes) for 1 h followed by washing 3× 5 min in PBS + Triton X-100. The IgG Ab sites were neutralized overnight with excess whole goat anti– mouse IgG (1:20; Sigma ) and followed by neutralization with goat anti– mouse IgG Fab (1:20; Jackson) for 2 h. The second set of Ab incubations were performed as indicated above with anti–α-actinin (EA53, 1:500; Sigma ) followed by a goat anti–mouse IgG FITC conjugate (1:200; Sigma ). Coverslips were mounted and stored at −80°C. Immunofluorescence was visualized in dual channel mode on a Nikon Diaphot 200 microscope equipped with a Noran confocal laser scanning imaging system and Silicon Graphics Indy workstation and colorized with Adobe Photoshop software. A Leitz Aristoplan fluorescence microscope was used for data presented in Fig. 4 . Cardiac myocyte contractile function was performed on adult cardiac myocytes maintained in serum free media as previously described . Relaxing and activating solutions contained in mmol/liter: 7 EGTA, 1 free Mg 2+ , 4 MgATP, 14.5 creatine phosphate, 20 imidazole, and KCl to yield a total ionic strength of 180 mmol/liter. Solution pH was adjusted to 7.00 with KOH. The pCa (i.e., −log [Ca 2+ ]) of the relaxing solution was set at 9.0 and the pCa of the maximal activating solution was 4.0. Intermediate pCa solutions were generated by mixing the pCa 9.0 and pCa 4.0 solutions as previously described . Coverslips were removed and washed several times with relaxing solution which results in permeabilization of the myocyte membrane. Single rod-shaped cardiac myocytes were attached to micropipettes coated with an adhesive between a force transducer (model 403A; Cambridge Tech) and moving coil galvanometer mounted on three-way positioners . Sarcomere length was set at 2.2 μm by light microscopy. At each pCa, steady state isometric tension was allowed to develop, followed by rapid slackening to obtain the baseline tension. The myocyte was then relaxed. Total tension was measured as the difference in tension just before and after the slack step. Active tension was calculated by subtracting the resting tension (measured at pCa 9.0) Data were acquired on a Nicolet 310 oscilloscope. Tension-pCa curves were fit using the Marquardt-Levenberg nonlinear least squares fitting algorithm and the Hill equation in the form: P = [Ca 2+ ] n / (K n + [Ca 2+ ] n ) where P is the fraction of maximum tension (P o ), K is the [Ca 2+ ] that yields one-half maximum tension, and n is the Hill coefficient ( n H ). Analysis of variance (ANOVA) with a Student-Neuman Keuls post hoc test were used to examine significant differences with P < 0.05 indicating significance. Recombinant replication-deficient adenovirus was generated by homologous recombination in HEK-293 cells. Southern blots of restriction enzyme digests of viral DNA using full-length cDNA digoxigenin-labeled ( Boehringer Mannheim ) probes show the correct insertion of the expression cassettes into the left end of the viral genome . To quantitate the rate of expression and incorporation of newly synthesized αTm in adult cardiac myocytes, protein expression was determined by Western blotting of total cellular protein from AdαTmFLAG infected myocytes at several time points in primary culture . The addition of the epitope resulted in a slower migration pattern of αTmFLAG than the endogenous αTm on SDS-PAGE allowing direct quantitation of expression using an αTm antibody. Note that the expressed αTmFLAG in cardiac myocytes comigrates with the protein expressed in HEK-293 cells infected with the same adenovirus indicating the correct processing size of αTmFLAG in two different cell types. The αTmFLAG protein was first detected in adult cardiac myocytes at day 2 in culture and the ratio of αTmFLAG to total Tm (αTmFLAG + endogenous αTm) increased over time in culture. A summary of densitometric analysis of these Western blots is shown in Fig. 2 B. If we assume that by using a strong viral promoter we can outcompete the endogenous Tm gene expression for sites available on the thin filament, then hypothetically the expression of αTmFLAG would be limited by the rate at which Tm is replaced in the thin filament. In that regard, it is interesting that the proportion of αTmFLAG to total Tm correlates well with the half-life of Tm measured in vivo (5.5 d) . Permeabilization of adult cardiac myocytes in relaxing buffer containing 0.1% TX-100 before collection for Western blot analysis resulted in no apparent change in the proportion of αTmFLAG to total Tm indicating indirectly that the expressed Tm was bound to the myofilaments . At day 5–7 in primary culture, intact cardiac myocytes contained 39.8 ± 3.3% αTmFLAG ( n = 4) and permeabilized cardiac myocytes contained 40.0 ± 2.5% αTmFLAG ( n = 7, P > 0.05). To determine if the newly synthesized Tm was replacing the endogenous Tm, Tm stoichiometry was analyzed by reprobing Western blots with antibodies recognizing sarcomeric actin and normalizing the total amount of Tm (Tm + αTmFLAG) to the amount of actin in each lane . To compare several different Western blots from different experiments the ratios of Tm/actin were normalized to the average of the Tm/actin ratio in control myocytes on each blot (see Materials and Methods). There was no significant change in the ratio of total Tm to actin in uninfected cells (1.00 ± 0.03, mean ± SEM, n = 4) compared with the total Tm in cells at days 5–7 expressing αTmFLAG (1.21 ± 0.12, n = 8, P > 0.05). In addition, there were no detected changes in isoform expression of troponin I or troponin T (data not shown) and no induction of βTm in controls and AdαTmFLAG infected cells after 7 d in culture indicating that the adult cardiac myocytes were maintained in fully differentiated state throughout the experiments. In support of this result, it has been shown previously that similar culture conditions and adenoviral mediated gene transfer of reporter proteins has no effect of the stability and differentiated state of adult rat cardiac myocytes over 7 d in culture . Indirect immunofluorescence using an anti-FLAG mAb and confocal microscope imaging was used to follow the incorporation of the expressed αTmFLAG into the myofilaments of adult cardiac myocytes over time in culture. No αTmFLAG protein was detected by indirect immunofluorescence in AdαTmFLAG infected myocytes at day 1 after infection (data not shown). In Fig. 3 the immunofluorescence three-dimensional reconstructions of representative myocytes at days 2 and 4 after treatment with AdαTmFLAG are shown. Several interesting points can be noted from these experiments. First, the αTmFLAG incorporation is uniform throughout the entire width, length, and depth of the cardiac myocytes. Second, the αTmFLAG decorates the thin filament between, but not including, the Z-line structures (as noted by the α-actinin staining). Finally, αTmFLAG immunofluorescence always appears first at the center of the sarcomere , with absence of αTmFLAG immunofluorescence between the center of the sarcomere and the Z-line. This is quite different from the immunofluorescence pattern of endogenous Tm in uninfected cells (data not shown) and the immunofluorescence pattern of cardiac TnI which shows labeling of the entire thin filament from Z-line to Z-line . It should be noted that the resting sarcomere length in cultured fully differentiated adult cardiac myocytes is 1.8–1.9 μm. Because at this sarcomere length the thin filaments are partially overlapped, one would not expect to see a gap at the center of the sarcomere as seen in immunofluorescence of thin filament proteins in skeletal muscle fibers or neonatal cardiac myocytes where clearly segregated I-bands are evident. The region that is void of αTmFLAG immunofluorescence, as shown in Fig. 3 , C and D, appeared to decrease slightly over time in culture which is associated with increased αTmFLAG protein expression by Western analysis. These results suggest the αTmFLAG incorporates most readily into the pointed end of the thin filament and incorporates in a direction from the pointed end to the barbed end of the thin filament. To determine if the addition of the eight–amino acid (DYKDDDDK) epitope somehow alters or limits the incorporation of Tm into myofilaments, a second experimental protocol was used. In this protocol, cardiac myocytes infected with AdαTmFLAG were treated with 20% serum added to the media. Treatment of cardiac myocytes with 20% serum increases myofilament protein turnover and results in redifferentiation, or the breakdown of existing myofibrils and myofibrillogenesis with reinduction of embryonic myofilament protein isoform expression . Western blot analysis shown in Fig. 4 C indicates that treatment of AdαTmFLAG infected myocytes with 20% serum resulted in nearly complete replacement of the endogenous Tm with αTmFLAG after 6 d in culture. Small amounts of expression of βTm, the fetal Tm isoform, was induced by the treatment of cells with 20% serum, but was not different between control serum–treated cells and AdαTmFLAG serum–treated cardiac myocytes. Indirect immunofluorescence on AdαTmFLAG-infected myocytes showed αTmFLAG immunofluorescence patterns that now resemble the pattern of Tm immunofluorescence in uninfected serum–treated myocytes with striated wide bands of staining from Z-line to Z-line around the perinuclear mature myofibrillar region of the redifferentiating cardiac myocytes and αTmFLAG staining premyofibrils in the periphery . These results together suggest that the FLAG epitope does not limit the structural replacement of the endogenous Tm with the adenoviral delivered αTmFLAG protein. In addition, if the epitope was altering the structural integrity of Tm, it might be expected that myocytes expressing and incorporating TmFLAG might show altered mechanical function. To determine if the FLAG epitope alters Tm regulation of mechanical function, single cardiac myocyte isometric force measurements were used to determine if expression of αTmFLAG in fully differentiated, serum-free cardiac myocytes, resulted in alterations in contractile functions. As shown in Fig. 5 , no significant changes in contractile function (maximum force, pCa 50 , and Hill coefficient, P > 0.05) were detected in AdαTmFLAG infected myocytes compared with control uninfected cardiac myocytes. This result was likely not due to selection of a large population of uninfected cardiac myocytes, because of the high efficiency of adenoviral-mediated gene transfer to adult cardiac myocytes in vitro using similar preparations of cardiac myocytes and infection protocols . In support of this point, immunofluorescence staining of time-paired myocytes indicated >85% of the AdαTmFLAG infected rod-shaped myocytes were expressing αTmFLAG (data not shown). To assess if the mechanism of incorporation of Tm into myofilaments was unique to Tm or if a similar mechanism exists for all thin filament regulatory proteins, myocytes were treated with the AdcTnIFLAG vector and analyzed for protein expression and myofilament protein incorporation. Fig. 6 shows the expression of cTnIFLAG in adult cardiac myocytes over time in primary culture. The ratio of cTnIFLAG to total TnI increases over time in culture indicating the cTnIFLAG protein is being expressed. Note that the ratio of cTnIFLAG to the endogenous TnI over time in culture is much greater than the expression of αTmFLAG which is consistent with the greater turnover (shorter half-life) of this protein in cardiac myocytes . Permeabilization of the myocytes before sampling does not appear to affect the ratio of cTnIFLAG to the endogenous cTnI indicating that the cTnIFLAG protein was bound to myofilaments . In addition, there was no significant change in the ratio of total TnI to actin in untreated cells (1.00 ± 0.08, mean ± SEM, n = 8) compared with the total TnI in cells at days 5–7 expressing cTnIFLAG (0.95 ± 0.16, n = 10, P > 0.05). Confocal three-dimensional reconstructions of a representative AdcTnIFLAG-treated cardiac myocyte is shown in Fig. 7 . Most notably, the first detectable immunofluorescence from cTnIFLAG at day 2 after treatment with viral vector extends the entire length of the thin filament from Z-line to Z-line . This pattern of immunofluorescence does not change over time in culture and appears to be identical to the immunofluorescence pattern of cTnI labeling in control cells (data not shown). This indicates that TnI turnover and incorporation occurs randomly along the entire length of the adult cardiac thin filament. To more clearly highlight the protein-specific mechanisms of replacement of endogenous myofilament proteins with newly synthesized myofilament proteins, Fig. 8 compares the immunofluorescence pattern of αTmFLAG and cTnIFLAG at day 2 after gene transfer. It should be noted that day 2 is the first day at which TmFLAG or cTnIFLAG can be detected in adult cardiac myocytes by Western blotting or immunofluorescence analysis. This comparison clearly shows that while the initial replacement of the endogenous TnI with cTnIFLAG occurs along the entire length of the thin filament from Z-line to Z-line (Z-lines marked by arrows), the initial replacement of αTm with αTmFLAG occurs at distinct regions of the thin filament near the pointed end . In this study, the mechanism of sarcomere maintenance, collectively defined as the processes of myofilament protein synthesis, incorporation, and degradation, in a fully differentiated muscle cell, the adult cardiac myocyte, was examined. Using adenoviral-mediated gene transfer into adult cardiac myocytes in vitro, the expression and incorporation of newly synthesized epitope-tagged contractile proteins into myofilaments was visualized in order to understand the process of sarcomere maintenance in fully differentiated contractile cells. The results from the expression of epitope-tagged αTm and cTnI in adult cardiac myocytes suggest common mechanisms and protein specific mechanisms of sarcomeric maintenance that shed new light on how sarcomeric maintenance occurs while the cell is still able to maintain force production. The results presented here suggest several common mechanisms of sarcomeric maintenance. Given the high efficiency of adenoviral mediated myofilament gene transfer into adult cardiac myocytes in vitro , it is possible to estimate the average protein replacement in single cardiac myocytes by measuring the protein expression in a large sample of cardiac myocytes. From the results shown in Figs. 2 and 6 , it is apparent that the expression of both epitope-tagged αTm and cTnI proteins results in expression and incorporation rates that are similar to their measured half-life in vivo . Interestingly, in both cases the expression of exogenous cTnI or Tm does not change the total amount of either TnI or Tm indicating that the expression of the endogenous protein is downregulated. A similar result was obtained previously using adenoviral-mediated gene transfer of slow skeletal TnI into adult cardiac myocytes in vitro . This maintenance of myofilament protein stoichiometry during changing levels of gene expression has been seen in several mouse models where ablation of one allele of the cardiac expressed αTm1 gene or overexpression the fetal isoform βTm of in the heart results in no change in the total amount of myofilament bound Tm protein . Interestingly, while ablation of one allele of the αTm gene has no effect on total Tm in the heart and doesn't result in any marked changes in cardiac function , ablation of a single copy of αTm gene in yeast disrupts cellular function , and in Drosophila a similar disruption of one Tm allele results in impairment of indirect flight muscle function . This suggests that the ability of myofilament proteins and the contractile apparatus to adapt to altered levels of myofilament gene expression may not be evolutionarily conserved. Taken together, these results also suggest that the rate of myofilament incorporation of newly synthesized contractile proteins is limited by the turnover rate of the endogenous myofilament protein already residing on sites on the myofilament lattice. If sites on myofilaments are unavailable, the newly synthesized contractile protein is likely not made or is rapidly degraded. In support of these results, a previous study by Dome et al. showed that binding of fluorescently labeled brain Tm to permeabilized muscle fibers could only occur if the endogenous Tm was extracted by high salt treatment. The mechanism of regulation of myofilament protein stoichiometry in adult muscle cells is not well understood, although mouse models of overexpression and ablation of myofilament genes in the heart suggest that multiple regulatory mechanisms, including transcriptional and translation regulation, may be involved . Another common feature of sarcomere maintenance shown in this study is that replacement of endogenous myofilament proteins is uniform throughout the entire cell. This suggests that in mature adult cardiac myocytes where the myofilament lattice is already formed, the myofilaments throughout the muscle cell are being replaced simultaneously. A previous report using 3 H-leucine pulse–chase techniques suggested that unidentified newly synthesized protein in cultured, growing skeletal muscle myotubes appears throughout the muscle fiber but also showed that newly synthesized proteins appear somewhat more readily around the periphery of myofibrils . Although we did not find any direct evidence in support of this latter result in fully differentiated adult cardiac myocytes, the resolution needed to address this question is probably beyond the limits of confocal microscopy especially in the axial direction. In this study, the visualization of the incorporation of newly synthesized αTmFLAG and cTnIFLAG with confocal microscopy yielded some interesting and surprising results. As shown in Fig. 3 , the incorporation of newly synthesized Tm appears at the free end or pointed end of the thin filament. Two important conclusions can be drawn from this result. First, the newly synthesized αTmFLAG protein is capable of binding to the appropriate location of Tm in the sarcomere, namely the thin filament regions with no binding to the Z-line . Second, the replacement of the endogenous Tm with newly synthesized Tm occurs more readily at the pointed end of the thin filament and continues toward the Z-line. Several explanations could explain this result of preferred pointed end replacement of the endogenous Tm with newly synthesized Tm. The first and most likely explanation is that Tm turnover occurs more readily at the pointed end of the thin filament. We speculate that this is due to the structural properties of Tm, in that it may be more favorable to remove Tm from one end of the head-to-tail polymer than in the center of the polymer, especially if one end of the polymer is anchored in the Z-line by binding to α-actinin or other Z-line proteins. Interestingly, Trombitas et al. reported that when localizing Tm in frog skeletal muscle with immunoelectron microscopy, there were differences in the ability of certain Tm antibodies to recognize the Tm proteins nearest the Z-line. In particular, the 24th Tm (nearest the Z-line) was only recognized by an antibody that preferentially binds to phosphorylated Tm. These results suggest there may be structural differences in Tm that may affect how rapidly Tm can exchange with newly synthesized Tm along the length of the thin filament. A second possible explanation is that the result is an artifact due to the addition of the COOH-terminal FLAG epitope. Previous biochemical work has shown that modification of the ends of Tm by acetylation or deletion can alter actin binding affinity . If the addition of the COOH-terminal FLAG epitope merely disrupted end-to-end interactions of Tm preventing polymerization, we would expect to be able to replace Tm equally well on both ends of the thin filament, a result which was not seen. To confirm further that the epitope does not limit or alter the incorporation of Tm into myofilaments two additional control experiments were performed. First, under serum-treated conditions, the αTmFLAG protein nearly completely replaced the endogenous Tm protein from Z-line to Z-line in the mature myofibrillar regions indicating that the epitope itself is not limiting the incorporation of the αTmFLAG protein into the myofilaments. Second, if the epitope itself was significantly altering the Tm structure, we would expect contractile function would be altered in myocytes expressing the epitope-tagged Tm. This result was not seen as there was no significant difference in Ca 2+ -activated contractile function (e.g., pCa 50 , P 0 , n H ) between control cardiac myocytes and cardiac myocytes expressing αTmFLAG. In support of this result, it was also observed that serum-treated adult cardiac myocytes expressing the TmFLAG protein spontaneously beat in culture similar to control serum-treated cardiac myocytes while in these cells nearly all the Tm in these cardiac myocytes has been replaced with αTmFLAG. A third possible explanation for the preferred pointed end replacement of the endogenous Tm with newly synthesized Tm is that this process of Tm replacement is not characteristic of the turnover of Tm protein alone but a visualization of what is happening globally to the thin filaments. In other words, is what is visualized with Tm incorporation specific to Tm or a manifestation of thin filaments completely breaking down and reforming from their pointed ends? If the latter were the case, it would be hypothesized that other newly synthesized thin filament proteins would show similar patterns of incorporation into myofilaments. The results presented here show that incorporation of the newly synthesized cTnIFLAG protein does not show preferred pointed end incorporation. Rather, cTnIFLAG is incorporated in all sarcomeres throughout the cell in a stochastic fashion across the length of the thin filament. Interestingly, the half-lives of the subunits of the troponin complex in the rat heart in vivo are varied with TnI and TnT at ∼3 d and TnC at ∼5 d . This would suggest, along with our results, that the replacement of Tn subunits may either occur rapidly while the complex remains attached to the thin filament, or that dissociated TnC subunits are capable of reassociating with newly synthesized TnI and TnT subunits followed by rebinding stochastically to the thin filament. The lack of kinetic evidence from isotope studies for precursor pools of TnT and TnC in adult rat heart favors that subunit exchange might take place while the troponin complex remains attached to actin . The pointed end incorporation of newly synthesized αTm in fully differentiated adult cardiac myocytes presented here differs significantly from recent studies in serum-treated neonatal cardiac myocytes. The expression of transfected green fluorescent protein-tagged Tm (Tm-GFP) in neonatal myocytes showed localization of Tm after 48 h along the entire length of the thin filament. There are several possible explanations for these different observations. First, since complete replacement of the endogenous Tm by newly synthesized Tm does not occur in our 7-d culture period in fully differentiated adult cardiac myocytes, we would hypothesize that if culture conditions could be extended further, complete replacement of Tm from Z-line to Z-line would eventually occur (see model below). If the turnover of myofilaments is much faster in neonatal cardiac myocytes (which is likely since neonatal myocytes undergo shape changes and cell division) full replacement of Tm from Z-line to Z-line could be complete in 48 h. However, the average percentage of Tm replaced by Western blotting cannot be estimated in the Tm-GFP experiments due to the low transfection efficiency of neonatal cardiac myocytes . Second, Morkin found in growing muscle myotubes (neonatal skeletal muscle) that new myofibrillar proteins were added preferentially to the periphery of the myofibrils. If myofibrils are adding more filaments in parallel as they grow wider in differentiating neonatal muscle cells, the addition of new filaments from the Z-line could explain the end to end incorporation. Indeed, our results of αTmFLAG incorporation into myofilaments of serum-treated redifferentiating adult cardiac myocytes show very similar incorporation patterns to GFP-Tm incorporation into neonatal adult cardiac myocytes . By switching adult cardiac myocytes from a state of sarcomere maintenance (i.e., serum-free conditions) to increased turnover and myofibrillogenesis (i.e., serum conditions) incorporation of αTmFLAG from Z-line to Z-line and complete replacement of the endogenous Tm can and does occur . The differential incorporation of newly synthesized Tm and TnI proteins into sarcomeres of adult cardiac myocytes not only suggests that there are contractile protein specific mechanisms for sarcomere maintenance but also suggests some important basic mechanisms for sarcomere maintenance in fully differentiated contractile cells. Fig. 9 shows several possible models of how replacement of thin filament proteins during sarcomere maintenance could occur. The results from this study favor the model shown in Fig. 9 A in which sarcomere maintenance occurs while maintaining a nearly intact thin filament. More specifically, endogenous contractile proteins of the thin filament are capable of rapidly exchanging with newly synthesized contractile proteins, so rapidly that the thin filament structure and function is not dramatically altered. For instance, it has been shown previously that extraction of myofilament proteins such as TnC from skeletal muscle fibers results in a dramatic alterations in the ability to produce force in response to a change in [Ca 2+ ] . Consequently, if sites remained vacant for a substantial period of time, this would lead to a dramatic destabilization of sarcomeric structure and alterations in force production of the cell, processes which did not occur as shown in Fig. 5 . In addition, this maintenance of intact thin filaments could explain how myofilament protein turnover can occur while maintaining continuous and near maximal force production of the adult cardiac myocyte in vivo. The mechanisms of exchange of newly synthesized regulatory proteins with endogenous proteins of the thin filament in the first model are protein isoform specific. Cardiac TnI, as a subunit of the ternary troponin complex exchanges stochastically along the length of the thin filament, possibly while the complex remains attached to the thin filament through interactions of TnT and tropomyosin. Tm, which forms head to tail polymers, exchanges with the endogenous Tm in a more ordered fashion. Assuming the Tm polymer is anchored into the Z-line by binding α-actinin, or the Tm nearest the Z-line is structurally different, it is more favorable to remove and replace Tm proteins from the pointed end. Whereas the results presented here support the model detailed in Fig. 9 A, there are at least two other possible models of sarcomere maintenance that could be considered. In the second model shown in Fig. 9 B, older thin filaments are replaced by formation of entirely new thin filaments. This process could occur by nucleation of new actin polymers from the Z-line, polymerization of new thin filaments with coordinate addition of regulatory proteins followed by removal and breakdown of older thin filaments. This model was proposed by LoRusso et al. to explain the rapid incorporation (30 s) of microinjected fluorescently labeled actin into myofilaments of freshly isolated adult cardiac myocytes. If sarcomere maintenance occurred by formation of new thin filaments, we would have expected to see the newly synthesized Tm and TnI first binding near either side of the Z-line and extending toward the pointed end over time in culture as these new thin filaments polymerized, a result which was not obtained. It could be argued that actin cables are fully formed from the Z-line followed by addition of newly synthesized Tm from the pointed end. However, this would require a large portion of actin polymers to remain stable in the absence of any Tm binding for several days, and it has been shown previously in yeast and in the hearts of mutant axolotls that in the absence of Tm, actin filaments are not stable and/or do not readily form . In the hypothetical third model of sarcomere maintenance shown in Fig 9 C, the pointed ends of the thin filament are most readily turned over by a process of breaking down thin filaments from the free end, followed by repolymerization of the actin filament and association of regulatory proteins. If the extent or length of myofilament breakdown from the pointed end was stochastic, gradually over time, more and more of the thin filaments on average would be replaced. This model could explain the results seen for the incorporation of newly synthesized Tm into myofilaments. However, if this model was accurate in describing sarcomere maintenance, it would be expected to see similar pointed end incorporation of newly synthesized TnI, a result which was not obtained. In conclusion, the results presented here support a model for sarcomere maintenance in which the replacement of thin filament proteins by newly synthesized proteins (a) is a ordered process for Tm and a stochastic process for TnI, and (b) exchange with newly synthesized proteins occurs rapidly enough for thin filament structure to be maintained which allows the adult cardiac myocyte to undergo sarcomere maintenance while maintaining the continual ability to produce force and motion. These results and this model suggest that Tm proteins nearest the Z-line are older than the Tm on the pointed ends of the thin filament. The functional consequences of this property of Tm replacement are unknown. However, it is well known that in cardiac muscle there is a sarcomere length dependence of Ca 2+ activation where at longer sarcomere length the myofilaments are more sensitive to activating Ca 2+ . Whether or not structural, functional, or age differences in Tm along the length of the thin filament contribute to or reflect properties of this phenomenon remains to be tested. In addition, it is unknown how other contractile proteins of the contractile apparatus are replaced in fully differentiated cardiac myocytes. For example, it will be interesting in future studies to determine how the subunits of the troponin complex are replaced. The components of this complex have been shown to have different turnover rates in vivo . This suggests that a single troponin subunit may either turnover while the other subunits remain attached to the thin filament (allowing different rates of total protein turnover), or that troponin subunits with longer half-lives (TnC ∼5 d) can reassociate with newly synthesized troponin subunits and rebind to the thin filament. Finally, another interesting outcome of this work will be to determine if sarcomere maintenance of contractile proteins is altered under pathophysiological conditions such as heart failure and aging. | Study | biomedical | en | 0.999998 |
10385528 | CV-1 monkey kidney cells and A431 human epidermoid cells were obtained from Health Science Research Resources Bank. Mouse fibroblastic L cells and MTD-1A epithelial cells were provided by M. Takeichi (Kyoto University, Kyoto, Japan). These cells were cultured in DME supplemented with 10% FCS. Mouse anti-ERM mAb (CR22) with higher affinity to moesin than to ezrin/radixin , rat anti-ezrin mAb (M11), rat anti-radixin mAb (R2-1) and rat anti-moesin mAb (M22) , rat anti–COOH-terminal phosphorylated ERM (CPERM) mAb (297S) specific for phosphorylated ERM proteins at T567, T564, or T558 for ezrin, radixin, or moesin, respectively , and rabbit anti-ERM polyclonal antibodies (pAbs) (TK88 and TK89) were used. TK88 was raised in a rabbit against synthesized peptides corresponding to the mouse ERM proteins' common sequence (293–302) and detected all ERM proteins. Rabbit anti–E-cadherin pAb and rat anti–E-cadherin mAb were provided by M. Takeichi. IM7.8.1 is a rat anti–mouse CD44 mAb . Mouse anti–phosphotyrosine mAb (clone 4G10) and mouse anti-vesicular stomatitis virus glycoprotein G (VSVG) mAb (clone P5D4) were purchased from Upstate Biotechnology Inc. and Sigma Chemical Co. , respectively. Some vectors used in this study were described previously . E-43, E-44, or E-ICAM-2 represents a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and transmembrane/cytoplasmic domain of rat CD43, mouse CD44, or mouse ICAM-2, respectively. The extracellular domain of E-cadherin was used as an epitope tag to detect the cell surface expression of each chimeric molecule in L fibroblasts expressing no detectable E-cadherin. Several mutant proteins, E-43/1-31, E-44/1-19, E-44/20-70, and E-43/KRR:NGG, were described in detail previously . To obtain cDNA encoding VSVG-tagged ezrin (pSK/mEz-VSVG), an oligonucleotide encoding 11 COOH-terminal amino acids of VSVG was ligated into the 3′ terminal end of mouse ezrin cDNA according to Algrain et al. . A cDNA fragment of pSK/mEz-VSVG was inserted into the HindIII-HpaI fragment of the pβact-CAT9 to construct its expression vector (pA/mEz-VSVG). Plasmids encoding ezrin mutants on the COOH-terminal threonine (T567) were generated by PCR with appropriate primers from pSK/mEz-VSVG. Alanine or aspartic acid was substituted for T567 (pA/mEz-T/A-VSVG or pA/mEz-T/D-VSVG, respectively). Cells were transfected with DNA using Lipofectin or Lipofectamine ( GIBCO BRL ). Cultured cells on coverslips were washed twice with Opti-MEM ( GIBCO BRL ), and incubated for 3–5 h with 1 ml Opti-MEM containing 1 μg of plasmid DNAs and 10 μl of the reagents, followed by addition of 3 ml of normal medium containing FCS. Cells were then cultured for 3–5 d. Since transfection with Lipofectin or Lipofectamine occasionally caused damage to the cell surface structures of transfected cells, cells were also transfected by microinjection using a set of manipulators (MN-188 and MO-189; Narishige) connected to an Eppendorf microinjector 5242 (Eppendorf, Inc.). Expression vectors in injection buffer (100 mM KCl, 10 mM Hepes buffer, pH 7.5) were injected into the nuclei of cells cultured on coverslips. Cells were examined 12–24 h after injection. Subconfluent A431 cells on coverslips in DME with 10% FCS were transferred to DMEM without FCS and cultured for 6–12 h. Recombinant human EGF ( GIBCO BRL ) was added at a final concentration of 100 ng/ml. Cells were incubated for 30 s, and quickly fixed and processed for immunofluorescence microscopy or SDS-PAGE followed by immunoblotting. In some experiments, cells were microinjected with expression vectors, allowed to express proteins for 6–18 h, serum-starved, treated with EGF, and then processed for immunofluorescence microscopy. Cells were fixed with 1–4% fresh formaldehyde in 0.1 M Hepes buffer (pH 7.5) for 15 min. After three washes with PBS containing 30 mM glycine (G-PBS), cells were soaked in blocking solution (G-PBS containing 2% normal goat serum) for 5 min and incubated with anti– E-cadherin mAb (ECCD-2) diluted with the blocking solution for 10 min. Cells were then washed three times with G-PBS, treated with 0.2% Triton X-100 in G-PBS for 10 min, and washed with G-PBS. Cells were then soaked in blocking solution for 10 min, incubated with CR22 for 30 min, washed three times with G-PBS, and incubated with secondary antibodies for 30 min. DTAF-conjugated goat anti–rat IgG antibody (Chemicon) and Cy3-conjugated goat anti–mouse IgG antibody ( Amersham International ) were used as secondary antibodies. For triple labeling, FITC-phalloidin (Molecular Probes, Inc.), Cy3-conjugated goat anti–rat IgG antibody ( Amersham International ), and Cy5-conjugated goat anti–mouse IgG antibody ( Amersham International ) were used. Cells were washed three times, then mounted in 90% glycerol-PBS containing 0.1% para-phenylenediamine and 1% n -propylgalate. Specimens were observed using a Zeiss Axiophot photomicroscope ( Carl Zeiss ) with appropriate combinations of filters and mirrors. Photographs were taken on T-MAX 400 film ( Eastman Kodak Co. ), or recorded with a cooled CCD camera controlled by a Power Macintosh 7600/132 and the software package IPLab Spectrum V3.1 (Signal Analytics Corp.). Cells were fixed and doubly stained with anti–E-cadherin mAb (ECCD-2) and anti-ERM mAb (CR22) as described above. Alternatively, cells were microinjected with expression vectors, washed with cold culture medium the next day, and incubated with cold hybridoma culture medium containing anti-CD44 mAb (IM7.8.1) and anti– E-cadherin pAb for 15 min at 4°C. After washing twice with cold culture medium, cells were fixed with 1% formaldehyde in 0.1 M Hepes buffer (pH 7.5) for 15 min at room temperature. Cells were washed, permeabilized, and soaked in blocking solution. Cy2-conjugated goat anti–rabbit IgG antibody ( Amersham International ) and Cy3-conjugated goat anti– rat IgG antibody were used as secondary antibodies. For double labeling of cells with 297S and TK88 to detect CPERMs and total ERM proteins, respectively, cells were fixed with 10% trichloroacetic acid for 15 min on ice . In this case, TK89 which also detected all ERM proteins was not used, since the epitope for 297S is overlapped with that for TK89. To label cells with ECCD-2, TK89, P5D4, and/or FITC-phalloidin, they were fixed with 4% formaldehyde in 0.1 M Hepes buffer (pH 7.5) for 15 min. Cells were washed, permeabilized, and soaked in blocking solution. Cy2-conjugated goat anti–rabbit IgG antibody, Cy2-conjugated donkey anti–mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc.), Cy3-conjugated goat anti–mouse IgG ( Amersham ), Cy3-conjugated goat anti–rat IgG antibody, and/or Cy5-conjugated goat anti-mouse IgG antibody were used as secondary antibodies. In A431 cells, cell surface expression of E-cadherin-chimeric molecules was hard to detect because these cells express considerable amounts of endogenous E-cadherin. However, E-cadherin-chimeric molecules could be detected by staining cells with ECCD-2 after permeabilization with Triton X-100 because these molecules, but not endogenous E-cadherin, were accumulated in the cytoplasm in large amounts. Polystyrene latex beads with a sulfate group on their surface (0.1 μm FluoSpheres; Molecular Probes) were coated with avidin according to the method of Molday et al. . 1 d after microinjection with the expression vector encoding E-43, CV-1 cells on coverslips were fixed with 4% formaldehyde in 0.1 M Hepes buffer (pH 7.5) for 20 min, washed with G-PBS three times, soaked in blocking solution for 10 min, and then incubated in blocking solution containing anti–E-cadherin mAb, ECCD-2 for 20 min. Cells were washed with G-PBS, incubated with biotinylated goat anti–rat IgG antibody ( Amersham International ) in blocking solution, and then washed with G-PBS. Samples were incubated with the avidin-coated beads suspended in blocking solution for 1 h. Cells were washed with G-PBS and fixed with 2.5% glutaraldehyde in 0.1 M Hepes buffer (pH 7.5) for 2 h followed by postfixation with the same buffer containing 1% OsO 4 at 4°C for 1 h. Samples were then washed with distilled water, dehydrated through a graded series of ethanol, transferred into isoamyl acetate, and dried in a critical point drier (Hitachi Co.) after substitution with liquid CO 2 . Dried samples were coated with gold using a gold sputter coater (Eiko Engineering), and were examined under a scanning electron microscope (model S-800; Hitachi Co.). Subconfluent A431 cells cultured on a 35-mm dish were serum-starved for 6 h as described above. With or without EGF treatment (100 ng/ml) for 30 s, cells were washed with ice-cold PBS twice, and then recovered in 200 μl of SDS-PAGE sample buffer by scraping. Proteins in 20 μl of the sample were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were first incubated with anti-phosphotyrosine mAb (4G10), which were localized using the enhanced chemiluminescence Western blotting detection system ( Amersham ). Bound antibodies were then removed from the membranes by incubating with 7 M guanidine-HCl, 50 μM EDTA, 20 mM 2-mercaptoethanol for 10 min at room temperature. These membranes were incubated with anti-CPERM mAb (297S) to detect threonine-phosphorylated ERM proteins, then bound mAb was again removed. Finally, these membranes were incubated with anti-ERM protein pAb (TK89) to total ERM proteins. In our previous study, we transfected L cells with cDNAs encoding several chimeric molecules containing the extracellular domain of E-cadherin and the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2 (E-43, E-44, or E-ICAM-2, respectively), and showed that these chimeric molecules were concentrated in microvilli together with ERM proteins . Then, we attempted to examine the abilities of these chimeric molecules to elongate microvilli using stable transfectants. However, the microvillar elongation was not clearly observed in these cells, probably because the stable transfectants expressed relatively low levels of chimeric molecules. Next, we examined the effects of transient overexpression of these chimeric molecules on L cell surface morphology. The expression and localization of each chimeric molecule was detected with anti–E-cadherin mAb, ECCD-2, which recognized the extracellular domain of E-cadherin, and microvilli were specifically visualized by immunofluorescence staining with anti-ERM protein mAb, CR22. As shown in Fig. 1 , a and b, when the expression level of E-43 was sufficiently high, expressed E-43 was not only colocalized with ERM proteins in microvilli but also caused elongation of microvilli, as compared to nontransfected L cells . The number of microvilli did not appear to increase. Overexpression of E-44 or E-ICAM-2 gave the same results . Overexpression of E-43/1-31 also induced microvillar elongation (data not shown). Immunofluorescence microscopy with ezrin-, radixin-, or moesin-specific mAbs revealed that in all cases ezrin, radixin, and moesin were recruited to the elongated microvilli without any bias (data not shown). Two distinct control experiments were performed. First, wild-type E-cadherin was introduced into L cells. As previously reported , E-cadherin was distributed diffusely over the cell surface , and the length and number of ERM-positive microvilli were not changed . Second, two chimeric molecules lacking the ERM-binding ability were introduced into L cells: E-44/20-70 in which the juxta-membrane ERM-binding region was deleted from the E-44 cytoplasmic domain and E-43/KRR:NGG in which the juxta-membrane ERM-binding region of E-43 was disrupted by site-directed mutation . These chimeric molecules did not cause elongation of microvilli in transient transfectants . Furthermore, the effects of overexpression of E-43 on the cortical actin cytoskeleton organization were examined . In the dorsal cortex of nontransfected L cells, stress fibers and relatively short microvillar core actin bundles were observed . In the dorsal cortex of E-43–expressing cells, stress fibers were disrupted with concomitant formation of relatively long microvillar actin bundles , whereas ventral stress fibers were not affected (data not shown). In E-cadherin–overexpressing cells, dorsal stress fibers were not affected (data not shown). Next, another fibroblastic cell line, CV-1, was used. CV-1 cells bear short microvilli and were used to examine the effects of villin on microvillar elongation . When E-43 was transiently introduced into CV-1 cells, microvillar elongation was more clearly demonstrated than in L cells . The length of microvilli was then quantitatively compared between nontransfected and E-43–expressing CV-1 cells using anti–ERM mAb-stained images . In nontransfected cells, microvilli were mostly shorter than 1 μm (0.53 ± 0.33 μm; n = 320), whereas in E-43–expressing cells most microvilli were 3–4 μm in length (3.5 ± 1.4 μm; n = 300). These results are comparable with those in villin-induced microvilli (2.4 μm) . Finally, the surface morphology of these cells was examined by immunoscanning electron microscopy . To detect cells transiently expressing E-43, the extracellular domain of E-cadherin was detected with anti–E-cadherin mAb labeled with biotinylated secondary antibody followed by avidin-conjugated beads. The surface of nontransfected cells lacking bead labeling was characterized by numerous short microvilli . In contrast, many elongated microvilli were observed on the surface of E-43–expressing cells together with scattered beads . When E-43 was introduced into cultured MTD-1A epithelial cells, E-43 was concentrated at ERM-positive microvilli at their apical membrane surface, but did not induce apparent elongation of microvilli . Instead, in these E-43–overexpressing cells, the endogenous microvillar integral membrane protein, CD44, was significantly excluded from microvilli . Overexpression of E-44 also gave the same results, whereas that of E-cadherin did not affect the microvillar localization of endogenous CD44 . This effect appeared to be dependent on the level of E-43 or E-44 expression. In cultured fibroblastic L cells, E-43, E-44, or E-ICAM-2 did not cause exclusion of endogenous CD44 from microvilli (data not shown). Another epithelial cell line, A431, was examined. Similar to MTD-1A cells, overexpression of E-43 , E-44, or E-ICAM-2 (data not shown) did not induce elongation of microvilli. In A431 cells, EGF treatment was reported to induce morphological changes in the cell surface such as microvillar elongation and ruffling with concomitant activation of ezrin . Interestingly, as shown in Fig. 8 , c and d, when E-43–overexpressing cells were treated with EGF, within 30 s significantly longer microvilli appeared on the cell surface as compared to surrounding nontransfected cells. Ezrin was clearly recruited into and concentrated in these overelongated microvilli. This type of EGF-dependent overelongation of microvilli was not observed in A431 cells expressing E-43/KRR:NGG that has no binding ability to ERM proteins . The difference in the effects of overexpression of ERMBMPs between fibroblastic and epithelial cells suggested that in fibroblasts ERM proteins are more efficiently activated than in epithelial cells . If this is the case, it is likely that in A431 cells EGF effectively activates ERM proteins. ERM proteins phosphorylated at the COOH-terminal threonine residue (T567, T564, and T558 in ezrin, radixin, and moesin, respectively) have been proposed to function as active forms in cross-linking ERMBMPs to actin filaments . We examined the effects of EGF on the threonine phosphorylation of ERM proteins in A431 cells . As reported previously , immunoblotting of EGF-treated A431 cells with anti-phosphotyrosine mAb revealed that ezrin as well as EGF receptor were heavily tyrosine-phosphorylated . In A431 cells with or without EGF treatment, anti-ERM pAb (TK89) identified almost equal amounts of ezrin and moesin, but only a trace amount of radixin . Previously, we generated mAb 297S which specifically recognized CPERMs . Immunoblotting with this mAb clearly revealed that the amount of CPERMs was increased in A431 cells after EGF treatment . Importantly, not only ezrin but also moesin were phosphorylated at their COOH-terminal threonine residues. MTD-1A cells and L cells contained CPERMs in comparable amounts to EGF-treated A431 cells . We then compared the behavior of CPERMs with that of total ERM proteins by immunofluorescence microscopy during the activation of ERM proteins induced by EGF treatment . Before EGF treatment, CPERMs were hardly detected with some exceptional signals from the cell surface . Total ERM proteins were distributed diffusely in the cytoplasm and in a punctate manner on the dorsal surface . When cells were treated with EGF for 30 s, the staining intensity of CPERMs was increased markedly on cell surface structures including elongating microvilli but not in the cytoplasm . In contrast, the total ERM proteins were still detected in large amounts in the cytoplasm in addition to the cell surface . These observations favored the suggestion that CPERMs represent activated ERM proteins. To further evaluate this suggestion experimentally, we performed site-directed mutagenesis on ezrin which was predominant among ERM proteins in A431 cells. The COOH-terminally VSVG-tagged ezrin (Ez-VSVG) was shown previously to behave similarly to endogenous ezrin . Also, in serum-starved A431 cells Ez-VSVG was codistributed with endogenous ERM proteins . When the COOH-terminal threonine residue of ezrin (T567) was mutated to alanine (Ez-T/A-VSVG; nonphosphorylatable ezrin), the expressed mutant ezrin was again codistributed with endogenous ezrin in the cytoplasm as well as on the cell surface of serum-starved A431 cells . In sharp contrast, when T567 was mutated to aspartic acid (Ez-T/D-VSVG), which was expected to mimic the T567-phosphorylated form of ezrin, the expressed mutant was preferably enriched at the cell surface to elongate microvilli in serum-starved A431 cells . These elongated microvilli contained actin filaments along their length , and the expressed Ez-T/D-VSVG often formed large aggregates on the cell surface or in the cytoplasm where actin filaments were recruited . In contrast, when Ez-T/D-VSVG was introduced into L cells, no elongation of microvilli was detected (data not shown). Although microvilli are ubiquitous membrane surface structures, their physiological functions and the molecular mechanism behind their organization have not been fully elucidated. It has been reported that overexpression of some cytoskeletal or lipid-binding proteins results in the elongation of microvilli , but no information regarding the involvement of integral membrane proteins in the microvillar organization is available. In this study, we first showed that overexpression of several ERMBMPs such as E-43, E-44, and E-ICAM-2 induced the elongation of microvilli in fibroblasts such as L and CV-1 cells. This activity of these integral membrane proteins was dependent on their direct binding to ERM proteins, since the removal of the ERM-binding domains from their cytoplasmic domains resulted in loss of activity. Furthermore, as shown in Fig. 3 , this ERMBMP-dependent elongation of microvilli was associated with disruption of the dorsal stress fibers, suggesting that the expressed ERMBMPs recruited not only ERM proteins but also actin filaments. Of course, the possibility cannot be excluded that unidentified cytoplasmic proteins other than ERM proteins are also involved in such integral membrane protein-induced microvillar organization in some types of cells. In human RPM-MC melanoma cells, CD44 mutants lacking the ezrin-binding domain were reported to still localize at microvilli together with ezrin , and in some types of tissues CD44 and ERM proteins are not necessarily colocalized . Interestingly, in epithelial cells such as MTD-1A and A431 cells, these ERMBMPs did not induce the elongation of microvilli. Instead, they caused exclusion of endogenous CD44 from preexisting microvilli in MTD-1A cells. This differential behavior of ERMBMPs in epithelial and fibroblastic cells could be explained if the pool size of activated ERM proteins is larger in fibroblastic cells than in epithelial cells. However, immunoblotting did not identify any significant difference in the amount of CPERMs (which may correspond to activated ERM proteins as discussed below) between fibroblastic L cells and epithelial MTD-1A cells . Thus, as shown schematically in Fig. 11 , A and B, we postulated that in epithelial cells used in this study the ability to convert ERM proteins from inactive form to active form was suppressed as compared to fibroblastic cells. In response to increases in the amount of ERMBMPs, ERM proteins in the cytoplasm of fibroblasts would be efficiently activated with their concomitant recruitment to organize microvilli. In contrast, since in epithelial cells the activation of ERM proteins in response to increases in the amount of ERMBMPs was suppressed, the introduced ERMBMPs would compete out endogenous CD44 from the membrane-bound activated ERM proteins in preexisting microvilli. This interpretation was supported by the observations in EGF-treated A431 cells . Bretscher and colleagues reported that EGF treatment increased the levels of tyrosine phosphorylation and oligomerization of ezrin in A431 cells, resulting in the elongation of microvilli and membrane ruffles by recruiting ezrin . These findings suggest that in these cells, EGF directly or indirectly released the suppression of the ezrin (and probably ERM protein) activation process . The present results indicated that in these EGF-treated A431 cells, in which ERM proteins could be efficiently activated in response to increases in the amount of ERMBMPs, the overexpression of E-43 caused marked elongation of microvilli . Furthermore, introduction of Ez-T/D-VSVG, which mimicked activated ERM proteins, into serum-starved A431 cells but not L cells induced microvillar elongation. These findings suggest that in L cells the elongation of microvilli is regulated by the amount of ERMBMPs, whereas in serum-starved A431 cells it is regulated by the activation of ERM proteins as shown in Fig. 11 . Based on these observations, we concluded that ERMBMPs such as CD43, CD44, and ICAM-2 can serve as organizing centers for microvilli in collaboration with activated ERM proteins. Of course, the role of ERMBMPs remains undetermined in the normal organization of microvilli in a variety of cells because our conclusion resulted from overexpression experiments. In support of our model, lymphocytes from patients with Wiskott-Aldrich syndrome, where CD43 is defective, were relatively devoid of microvilli . The question has naturally arisen as to the nature of the activated state of ERM proteins and how they are activated. Ezrin has been reported to be tyrosine-, serine-, and also threonine-phosphorylated in vivo. Ezrin is heavily tyrosine-phosphorylated in response to EGF in A431 and to hepatocyte growth factor in LLC-PK1 cells . However, tyrosine phosphorylation of ezrin did not alter the association of ezrin with the cytoskeleton . Furthermore, even when two tyrosine residues in ezrin that are known to be major phosphorylation sites by EGF receptor were mutated to phenylalanine, transfected LLC-PK1 cells became unresponsive to hepatocyte growth factor in terms of cell motility and tubulogenesis, but the localization of ezrin at microvilli was not affected . These findings suggest that tyrosine phosphorylation of ezrin (and probably ERM proteins) is not required for ezrin activation as a membrane-cytoskeleton linker. Several lines of evidence suggest that serine/threonine phosphorylation of ERM proteins is associated with their activation . Moesin was reported to be threonine-phosphorylated at its COOH terminus during thrombin activation in platelets . Phosphorylation at the COOH-terminal threonine residue was shown to affect the direct interaction between the NH 2 - and COOH-terminal halves of radixin in vitro , suggesting that this phosphorylation keeps ERM proteins in an opened (activated) form. The results of our previous investigations and of the present study indicated that CPERMs represent the activated ERM proteins shown in Fig. 11 . Immunofluorescence microscopy with a mAb specific for CPERMs showed that they were highly concentrated at microvilli of cultured fibroblasts and also at elongated microvilli of L and CV-1 cells overexpressing ERMBMPs (data not shown). When A431 cells were treated with EGF, levels of CPERMs were shown to be increased by immunoblotting, which were concentrated in elongating microvilli on the cell surface , although it remains unclear how the activation of EGF receptor tyrosine kinase resulted in the phosphorylation of COOH-terminal threonine of ERM proteins. As expected, CPERMs were also highly concentrated at overelongated microvilli in EGF-treated A431 cells overexpressing ERMBMPs (data not shown). Furthermore, transfection experiments with site-directed mutants of ezrin revealed that Ez-T/D-VSVG (which mimicked CPERMs) was preferably recruited to the plasma membranes of serum-starved A431 cells to elongate microvilli. Similar results with mutated moesin were recently reported in COS7 cells . In contrast, Ez-T/A-VSVG, a nonphosphorylatable form at the COOH- terminal threonine, was codistributed with endogenous ERM proteins at the cytoplasm as well as plasma membranes . Since ERM proteins form dimers or oligomers , the localization of Ez-T/A-VSVG, i.e., inactivated form of ERM protein, on plasma membranes can be explained if these mutant molecules are incorporated into oligomers of endogenous ERM proteins. Current knowledge regarding how ERM proteins are activated, i.e., how the COOH-terminal threonine residues of ERM proteins are phosphorylated in vivo, is still limited. Rho-associated kinase and/or protein kinase C-θ are good candidates for the kinase responsible for production of CPERMs in vivo . Although protein kinase C-θ but not Rho-associated kinase was shown in vitro to phosphorylate full-length ezrin and moesin to directly open the closed form of ERM proteins , further analyses would be required to determine conclusively which serine/threonine kinase by itself functions as an activator for ERM proteins in vivo. Phosphoinositides such as phosphatidylinositol 4,5-bisphosphate were also thought to be key factors in opening of the closed ERM proteins . Further detailed analyses of how ERM proteins are activated in response to increases in amounts of ERMBMPs in fibroblasts and how this activation process is suppressed in epithelial cells will lead to a better understanding of the molecular mechanism of cell surface morphogenesis including the formation and destruction of microvilli in general. | Study | biomedical | en | 0.999995 |
10385529 | The BALB/c mouse B lymphoma line, A20, was cultured in RPMI 1640 supplemented with 10% fetal calf serum and 50 μM β-mercaptoethanol. Rabbit anti–mouse whole Ig antibody and F(ab′) 2 fragment were obtained from Zymed. The serine 473 phosphospecific PKB antibody was purchased from New England Biolabs , the phosphospecific GSK3α and the pan anti-PKBα were from Upstate Biotechnology, the pan anti-GSK3α antisera were from Santa Cruz Biotechnology . Rabbit antisera reactive with the COOH-terminal residues 904–918 of PKD/PKCμ were generated by standard protocols. Anti–sheep and anti–goat HRP-conjugated antibodies were obtained from Chemicon, and anti–mouse HRP and anti–rabbit HRP were from Sigma Chemical Co. The rat CD2 monoclonal antibody OX34 and the 12CA5 monoclonal reactive with the Ha epitope tag were purified from hybridoma supernatants by standard protocols. The FcγRIIB blocking antibody (FcBlock™) was purchased from PharMingen . The PI3K inhibitor LY294002 was purchased from BIOMOL Research Laboratories, the histone H2B was from Boehringer Mannheim , the protein kinase inhibitor and ATP were from Sigma Chemical Co. , and the [γ- 32 P]ATP was from Amersham . A chimeric protein of the extracellular and transmembrane domain of the rat CD2 molecule fused with the p110a catalytic subunit of PI3K (rCD2p110) targets PI3K to the membrane and creates a constitutively active enzyme that generates PI(3,4,5)P 3 and PI(3,4)P 2 . The rCD2p110 construct and a catalytically inactive form with a mutated ATP-binding site (rCD2-p110R/P) were described previously . The GFP-tagged PH domain of PKB and its non–lipid-binding R25C mutated form were generated, respectively, from subcloning HindIII/BamH1 from pCMV6 containing HA-tagged murine PKBα PH domain (HA-AH) or HA-R25C into -C1 ( Clontech ), the BsrG1/Sal1 fragment was then replaced by a linker of six glycine residues. Full length PKB was COOH-terminally GFP-tagged using standard protocols. Oligonucleotides introduced in pEGFP ( Clontech ) a sequence corresponding to the COOH-terminal sequence of PKB from the BclI site to the end of the protein at the NH 2 terminus of GFP and a BglII site and a stop codon at the COOH terminus of GFP. This resultant fragment was purified and ligated to pSG5 HA-PKB digested with BclI and BglII. All constructs were verified by DNA sequencing. The GFP-PKB fusion protein undergoes activation in response to BCR ligation (data not shown). A20 cells were washed and resuspended at 2 × 10 7 /ml in RPMI 1640. The cells were incubated at 37°C with either 10 μg/ml of anti–mouse F(ab′) 2 fragment or 15 μg/ml of intact Ig, for the indicated periods of time. Some samples were preincubated with 5 μM of Ly294002 or 2.5 μg/ml anti-FcγRIIb for 30 min before stimulation. Cells were quickly pelleted at 4°C and lysed in 50 mM Hepes, 10 mM NaF, 10 mM iodoacetamide, 75 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM Na 2 VO 3 , and 1 μg/ml of each for leupeptin, pepstatin A, and chymotrypsin. Nuclear debris were eliminated by 20 min centrifugation at 14,000 rpm and the supernatant proteins precipitated for 1 h at −70°C in 1.5 vol acetone. Precipitates were pelleted by 20 min of centrifugation at 14,000 rpm, resuspended in Laemmli buffer, boiled, and fractionated on 10% SDS-PAGE, with the exception of samples for GSK3 blotting, which were analyzed on 7.5% gels. Immunoblotting was performed by standard protocols and revealed by chemiluminescence (ECL; Amersham ). For transfection, 500 μl of cells was aliquoted with DNA, electroporated at 310 V and 960 μF using a Bio-Rad gene pulser. Thereafter cells were cultured for 12–14 h before stimulation, microscopic analysis, or lysis. 5 × 10 6 cells transfected with HA-tagged PKB were stimulated as indicated and lysed in 120 mM NaCl, 50 mM Hepes, 10 mM NaF, 1 mM EDTA, 40 mM β-glycerophosphate, 1% NP-40, 1 mM Na 2 VO 3 , and 1 μg/ ml of each for leupeptin, pepstatin A, and chymotrypsin. After elimination of nuclear debris, the supernatant was cleared by addition of 1/25 vol of a 30% solution of Sepharose-coupled protein G. Supernatants were incubated for 2 h with 10 μg/ml of the anti-HA antibody 12CA5, and another 45 min after addition of 1/10 vol of the Sepharose–protein G solution. Pellets were washed once in lysis buffer, twice in 500 mM LiCl, 100 mM Tris, pH 7.5, 1 mM EDTA, and once in assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl 2 , and 1 mM DTT). Dried samples were assayed by incubation for 30 min at room temperature with 2.5 μg H2B, 5 μM protein kinase inhibitor, 50 μM ATP, and 3 μCi of [γ- 32 P]ATP in a final volume of 15 μl. Boiled samples in Laemmli buffer were fractionated by SDS-PAGE. The level of H2B phosphorylation was analyzed by exposing the lower part of the gel on a sensitive film, and the amount of PKB protein immunoprecipitated was determined by Western blot analysis with a pan PKB antibody. After stimulation, cells were disrupted in 10 mM Hepes, 15 mM KCl, 2 mM MgCl 2 , 0.1 mM EDTA, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 1 mM Na 2 VO 3 , 0.15% NP-40, and the nuclear fraction pelleted at 14,000 rpm for 1 min. Cytosolic extracts were removed and NaCl was added to obtain a final concentration of 120 mM. These cytosolic extracts were then concentrated by acetone precipitation. The nuclear pellets were washed three times with lysis buffer and then resuspended in 20 mM Hepes, 1.5 mM MgCl 2 , 0.2 mM EDTA, 20% glycerol, 0.42 M NaCl, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 1 mM Na 2 VO 3 , 0.15% NP-40, and lysed for 30 min at 4°C. Debris was eliminated by 20 min of centrifugation at 14,000 rpm and supernatants, corresponding to nuclear extracts, were acetone precipitated. After transfection, cells were resuspended at 10 6 /ml in 35-mm glass bottom dishes (MatTek Corp.). Before analysis dishes were gently rinsed with warm Hanks' medium without phenol red. Dishes were mounted in the warm chamber of the confocal microscope ( Zeiss Laser Scanning Microscope 5.10). Samples were excited at 488 nm by an argon laser and detected with a 63 × 1.4 NA oil immersion objective. The first image was recorded just before stimulus addition directly into the dish, and then scans were made automatically every 5 s by using Zeiss LSM software. Images shown are representative of a minimum of five experiments. In Fig. 2 , anti-FcγRIIB and LY294002 were added to the dishes in Hanks' medium 30 min before microscopic analysis. The B lymphoma cell line A20 was either left unstimulated or activated by cross-linking the BCR with F(ab′) 2 fragment of anti–mouse IgG. Active PKB is phosphorylated on two residues; threonine 308 and serine 473 by PI3K-dependent protein kinases . To monitor PKB activity in cells, total cell extracts were prepared and fractionated by SDS-PAGE and processed for Western blot analysis with a specific antisera that recognizes active PKB molecules phosphorylated on serine 473. The phospho-PKB antisera did not react with PKB present in cell lysates from quiescent cells, whereas in cells activated by cross-linking the BCR with F(ab′) 2 fragment of anti–mouse IgG there was a strong reactivity of PKB with the phospho-PKB antisera . The data in Fig. 1 B show the in vitro catalytic activity of immune complexes of PKB isolated from quiescent or BCR-triggered cells, assayed using histone H2B as a substrate. These data confirm that stimulation of B cells via the BCR activates the catalytic activity of PKB. This result was confirmed also by analysis of the effects of BCR ligation on the phosphorylation of GSK3, an endogenous substrate for PKB . Phosphospecific antisera with selectivity for GSK3 molecules phosphorylated on the PKB substrate sequence do not recognize GSK3 proteins isolated from quiescent B cells but are strongly reactive with the GSK3 present in cell lysates prepared from BCR-triggered cells . Cross-linking of the BCR with the FcγRIIB inhibits BCR signal transduction pathways by recruiting the inositol 5′ phosphatase SHIP into the BCR complex . The data in Fig. 1 A show that B cell activation with intact IgG, which cross-links the BCR with the FcγRIIB, fails to stimulate PKB phosphorylation or PKB catalytic activity as judged by in vitro and in vivo assays of PKB function. In the presence of an anti-FcγRIIB antibody which blocks the interaction of IgG with the FcγRIIB receptor, intact IgG is able to trigger the BCR without coligating inhibitory FcγRIIB into the BCR complex. The presence of the blocking antibody to the FcγRIIB then allows intact IgG to ligate the BCR and stimulate PKB activity . Membrane localization of PKB is known to be essential for the activation of the enzyme but there are conflicting reports as to whether activated PKB is present in the nucleus or maintained at the cell membrane . To examine the localization of PKB carefully in intact B cells we expressed a GFP-tagged construct of wild-type PKB in A20 cells and analyzed the cellular localization of this enzyme in quiescent and BCR-activated cells. Membrane targeting of PKB is mediated by interactions of the PH domain with either PI(3,4,5)P 3 or PI(3,4)P 2 . Accordingly, we examined also the cellular localization of a GFP-tagged PKB-PH domain. Fig. 2 shows midsection confocal images of A20 cells transiently transfected with GFP wild-type PKB or the GFP-tagged PKB-PH domain. In unstimulated A20 cells, GFP-PKB was uniformly distributed throughout the cytosol of the cell and was also present in the nucleus. The GFP-tagged PKB-PH domain of PKB was similarly distributed. Western blot analysis of nuclear extracts prepared from A20 cells revealed that endogenous PKB was present in the nuclei of these lymphocytes . A20 cells expressing GFP-PKB constructs were stimulated with F(ab′) 2 fragment of anti–mouse IgG and confocal images of a midsection of the cells were taken at 10-s intervals after BCR ligation. This allows the effects of BCR triggering on the cellular localization of PKB to be monitored in live cells in real time. These images in Fig. 2 show that triggering of the BCR induces a rapid membrane localization of both full length PKB and the PKB-PH domain. The membrane localization of both PKB constructs was detected within 10 s of BCR ligation. Consistently, it was seen that wild-type PKB recycles from the membrane within 40–60 s of BCR triggering. In contrast, the translocation of the PKB-PH domain to the membrane was sustained . The motility of the activated B cells makes longer periods of confocal imaging live cells difficult but there was no indication of recycling of the PKB-PH domain away from the membrane over a period of minutes. Thus, BCR-induced translocation of the PKB-PH domain to the plasma membrane is stable compared with the response of the full length GFP-tagged wild-type PKB. A striking feature of the effect of the BCR on PKB membrane localization is its speed and transience. Kinetic analysis of the effect of the BCR on PKB activity reveals that this is also a rapid response detectable within 30 s of BCR triggering (data not shown). The data in Fig. 2 B show that the effect of BCR ligation is to induce a stable activation of PKB in a response that is maximal at 1 min and sustained for at least 60 min . It is also clear that BCR induced phosphorylation of GSK3; an endogenous substrate for PKB is a response that is maintained for a period of 1–2 h . In summary, analysis of PKB cellular localization and activity shows that quiescent B cells express PKB in the cytosol and the nucleus. BCR triggering induces rapid membrane localization and activation of PKB. PKB targeting to the membrane in response to BCR ligation is very transient and can be detected within 10 s but is finished by 40–50 s. In contrast, the stimulatory effects of the BCR on PKB activity are sustained for at least 1 h. 1 min after BCR triggering there is no discernible plasma membrane localized PKB, rather the active PKB is present in either the cytosol or the nucleus. The question of the localization of active PKB is important because it affords insight as to the potential site of action of this kinase. To determine whether active PKB is present in the nuclear or cytosolic compartment of B cells, A20 cells were triggered via the BCR and cytosolic and nuclear cell fractions were resolved by SDS-PAGE and analyzed by Western blot with the phospho-PKB antibody that recognizes active enzyme or with the pan PKB antisera. These data show that endogenous PKB is found in both the nucleus and cytosol of quiescent or activated B cells. In cells activated by cross-linking the BCR with F(ab′) 2 fragment of anti–mouse IgG there was a strong reactivity of both cytosolic and nuclear PKB with the phospho-PKB antisera. These results show that active PKB is found in both the cytosol and nucleus of activated B lymphocytes. PI3K signals are essential and sufficient for activation of PKB in T lymphocytes . A comparison of the data in Fig. 4 , A and B, shows that the BCR-induced transient membrane localization of GFP-PKB is abolished by the PI3K inhibitor Ly294002. The data in Fig. 4 C show that Ly294002 also prevents BCR activation of PKB. Hence, the ability of the BCR to regulate the cell localization and activity of PKB is dependent on PI3 kinase activity. To determine whether PI3K signals are sufficient for membrane targeting and activation of PKB we examined the effects of expression of constitutively active PI3 kinase on PKB cellular localization and activity. The BCR stimulates the activity of a PI3K complex that comprises a regulatory p85 and a catalytic p110 subunit. A chimeric protein of the extracellular and transmembrane domain of the rat CD2 molecule fused with the p110α catalytic subunit of PI3K (rCD2p110) targets PI3K to the membrane and creates a constitutively active enzyme that generates PI(3,4,5)P 3 and PI(3,4)P 2 when expressed in cells. The data in Fig. 5 show that expression of the constitutively active PI3K, rCD2p110, induces a strong activation of PKB. In these experiments A20 cells were transfected with increasing concentrations of the rCD2p110 expression vector resulting in increasing levels of expression of rCD2p110. The effect of the active rCD2p110 constructs on endogenous PKB activity was monitored using the phospho-PKB antisera. The results show that there was a dose-dependent stimulation of PKB activity by the expressed active PI3K. This stimulatory effect of rCD2p110 was dependent on the kinase activity of the chimera since expression of a kinase inactive mutant, rCD2p110 R/P, did not stimulate PKB activity. Moreover, treatment of A20 cells with the PI3K inhibitor LY294002 abrogated PKB activity in cells expressing rCD2p110 . Confocal microscopy of cells cotransfected with rCD2p110 and GFP-tagged PKB constructs shows the localization of PKB in cells expressing constitutively active PI3K. These data reveal that membrane-localized PKB can be detected in cells expressing active PI3 kinase . However, these cells also contain significant levels of cytosolic and nuclear GFP-PKB. In marked contrast, the total cellular pool of the GFP-tagged PH domain of PKB is constitutively present at the membrane of cells expressing active PI3K . In A20 cells stimulated via the BCR we had noted that the wild-type PKB is only transiently present at the cell membrane, whereas the BCR-induced translocation of the isolated GFP-tagged PKB-PH domain to the plasma membrane was sustained. The results in Fig. 6 show clearly that in cells expressing a constitutively active PI3K there is a stable and sustained plasma membrane localization of the total cellular pool of the PKB-PH domain but not a sustained membrane recruitment of intact PKB. The ability of active PI3K to sustain membrane localization of the PKB-PH domain is dependent on the catalytic activity of the enzyme. The application of the PI3K inhibitor, Ly294002, to cells coexpressing rCD2p110 and the GFP-tagged PKB-PH domain initiates a rapid relocation of the PKB construct from the plasma membrane into the cytosol and the nucleus. This striking response was complete within 30 s of adding the PI3K inhibitor to the B cell population . BCR activation of PKB is subject to a negative feedback control mechanism initiated by the FcγRIIB and mediated by SHIP . The stimulation of PKB catalytic function requires membrane localization of the kinase plus its phosphorylation at two residues: threonine 308 and serine 473 by PI3K-dependent protein kinases . The FcγRIIB/SHIP complex will influence both the kinetics and magnitude of PIP3 levels during B cell activation and could remove a membrane targeting signal for PKB. However, SHIP dephosphorylates PI(3,4,5)P 3 to form PI(3,4)P 2 which is also able to bind to the PH domain of PKB . Thus, it is possible that PKB will still be recruited to the plasma membrane when the BCR is coligated with the FcγRIIB complex and that SHIP terminates PKB-mediated responses by preventing the phosphorylation and activation of membrane-localized PKB. For example, osmotic stress can prevent PKB activation without preventing membrane localization of Akt/PKB . To study the effects of BCR coligation with the FcγRIIB on the cellular localization of PKB, A20 cells expressing the GFP-PKB constructs were stimulated with either F(ab′) 2 fragment of anti–mouse IgG or with intact IgG. Confocal images of a midsection of the cells were then taken at 5-s intervals after addition of the stimulus. The results in Fig. 7 show that B cell activation with intact IgG, which cross-links the BCR with the FcγRIIB, fails to stimulate PKB membrane translocation. Fig. 7 shows confocal images of cells 15 s after BCR triggering but cells were monitored for several minutes and no membrane localization of PKB could be seen in BCR/FcγRIIB coligated cells at any time point. In the presence of an anti-FcγRIIB antibody which blocks the interaction of IgG with the FcγRIIB receptor, intact IgG is able to trigger the BCR without coligating inhibitory FcγRIIB into the BCR complex. The results show that presence of the blocking antibody to the FcγRIIB then allows intact IgG to induce plasma membrane localization of PKB. This study has used GFP-tagged PKB and time lapse confocal microscopy of live cells to follow the cellular localization of PKB during antigen receptor activation of B lymphocytes. The data show that BCR regulation of PKB is a dynamic process; there is an initial rapid and transient recruitment of PKB to the plasma membrane followed by a sustained activation of the enzyme. PKB localization to the plasma membrane can be detected within 10 s of BCR triggering but the response has finished after a further 40– 50 s. Stimulation of PKB catalytic activity is similarly rapid and can be detected within 30 s of BCR triggering but the activation of PKB is maintained for at least 1 h after BCR triggering. PKB is distributed throughout the cytosol and the nucleus of both quiescent and BCR-triggered B cells. More importantly, in BCR-triggered B cells, phosphorylated active PKB is found in both the cytosol and nucleus. The question of the localization of active PKB is important because it affords insight as to the potential site of action of this kinase during B cell activation. The present results show that PKB has three potential sites of action during B cell activation. Transiently after BCR triggering, PKB could function to phosphorylate plasma membrane localized targets but during the sustained B cell response PKB is acting in the nucleus and the cytosol. PKB is always present in the nucleus of the A20 cells but we have no way to test whether the PKB that one sees in the nucleus at time zero is the same PKB found in the nucleus several minutes after BCR triggering. Mutations in the PKB-PH domain that prevent PKB plasma membrane targeting prevent PKB activation and we therefore assume that the active PKB that we see in the nucleus has been activated at the membrane. This would mean that there is a dynamic turnover from the cytosol/nucleus to the membrane and back to the cytosol and nucleus. The present results show that this cycle could be completed within 30–40 s. The results in Fig. 2 show that PKB can move to the plasma membrane within 10 s of activating the BCR. The data in Fig. 6 show that the PKB-PH domain can leave the membrane and enter the nucleus within 20–30 s. So PKB could do a full cycle, cytosol/plasma membrane/nucleus, within 30–40 s. The FcγRIIb mediates vital homeostatic control of B cell function by recruiting an inositol 5 phosphatase, SHIP, into the BCR complex. The importance of the FcγRIIB/ SHIP complex for B cell homeostasis is illustrated by the phenotype of mice lacking expression of either FcγRIIB or SHIP which are prone to inflammatory disease and show a higher sensitivity to anaphylaxis . FcγRIIB ligation with the BCR prevents activation of PKB; this response was revealed by quantitation of PKB activity in vitro but was confirmed also by analysis of the effects of BCR and FcγRIIB coligation on the phosphorylation of GSK3, an endogenous substrate for PKB. SHIP dephosphorylates PI(3,4,5)P 3 to produce PI(3,4)P 2 and is essential for the inhibitory action of the FcγRIIB . The PH domain of PKB, which mediates membrane targeting of the enzyme, is able to bind the product of SHIP, PI(3,4)P 2 , in vitro. Nevertheless, the present data show that coligation of the BCR with the inhibitory FcγRIIB prevents membrane targeting of PKB. This was an intriguing result that reveals a mechanism for the inhibitory action of the FcγRIIB on the BCR/ PKB response. It also suggests that binding of PI(3,4)P 2 to the PH domain of PKB in vivo is insufficient to target PKB to the membrane. However, it should be emphasized that accumulation of PI(3,4)P 2 in BCR/FcγRIIb activated B cells and has not been formally shown. The PKB-PH domain was recruited stably to the membrane in BCR-activated cells and also in cells expressing constitutively active PI3K. The PKB-PH domain binds to PIP3 and its translocation to the plasma membrane in B cells was absolutely dependent on the continued presence of D3 phosphoinositides: inhibition of the catalytic activity of PI3K with LY294002 causes immediate loss of the PKB-PH domain from the plasma membrane. It was particularly striking that within 30–40 s of adding the PI3K inhibitor, the PKB-PH domain moves from the plasma membrane into the cytosol and the nucleus of the B cell. The stability of the membrane localization of the PKB-PH domain was in marked contrast to the transient membrane residence of intact wild-type PKB under conditions of continuous PI3K activation or BCR triggering. The loss of wild-type active PKB from the plasma membrane within 1 min of BCR ligation is intriguing and cannot be explained by limits in levels of cellular PIP3 because PIP3 levels are elevated in A20 cells for at least 15 min after BCR ligation (data not shown). Moreover, PKB is not maintained at the membrane in cells expressing constitutively active PI3K although the stability of the membrane targeting of the PKB-PH domain confirms that there are sufficient levels of D-3 phosphoinositides in activated B cells to tether PKB to the membrane. Previous models have suggested that PKB colocalizes with active PI3K to membrane sites with elevated levels of D-3 phosphoinositides. The present confocal imaging data comparing the localization of full length PKB and the PKB-PH domain in B cells show that the PKB-PH domain follows this model but the full length PKB molecule does not. These studies thus reveal the existence of a molecular mechanism that must cause active PKB to dissociate from the plasma membrane in B cells despite the continued generation of PIP3. One possibility is that phosphorylated and/or active full length PKB undergoes a conformational change that prevents continued lipid binding. Alternatively, PKB interactions with substrate may relocalize the active kinase to the cytosol and nucleus. In summary, this study has used confocal imaging coupled with more classical biochemical analyses to study the dynamic PI3K-regulated processes that occur during B cell activation. The BCR triggers a transient membrane localization of PKB but a sustained activation of the enzyme; active PKB is found in the cytosol and nuclei of BCR-stimulated B cells. This result affords new information as to the potential site of action of PKB during B cell activation. Recent genetic studies have shown that PI3K is important for B cell development and for B cell function in the peripheral lymphoid compartment. The present results show that PI3K signals are both necessary and sufficient for sustained activation of PKB in B lymphocytes which firmly positions Akt/PKB in PI3K-mediated signaling pathways in B lymphocytes. There has been much recent information about the effects of antigen receptors on membrane-localized signaling pathways in lymphocytes; the significance of the present report is that the PI3K/PKB pathway couples signaling events triggered at the lymphocyte membrane to the cell nucleus. | Study | biomedical | en | 0.999997 |
10398688 | The ClC-0 chloride channel from Torpedo electric organ is thought to be a homodimeric protein containing two ion-permeating pores . Two different gating mechanisms, fast and slow gating, have been known to control the opening of this channel . Each pore of the channel can independently open and close on a millisecond time scale, via the fast-gating process. The slow gate, on the other hand, controls the two pores at the same time, with an average transition time on the order of seconds. The opening of the slow gate allows the pore openings to be controlled by the two fast gates, whereas its closure shuts two pores simultaneously and results in a long-lived, zero-conductance inactivated state. At the single-channel level, the inactivated intervals thus interrupt the millisecond opening–closing transitions, leading to a burst-like behavior of channel opening. Several factors, such as membrane potential and temperature, as well as the chloride ion have been shown to affect the slow gating. Early studies on the channel purified from Torpedo electric organ revealed that the slow gate of the channel favors a nonconducting (inactivated) state upon membrane depolarization , much like the inactivation process of voltage-gated cation channels. Moreover, the operation of the slow gate is also affected by the chloride gradient across the channel pore , and reduction of the extracellular chloride concentration favors the inactivated state . Recently, it was found that the slow-gating process of ClC-0 is extremely temperature dependent . The inactivation rate of the channel is speeded up by ∼40-fold as the temperature is increased by 10°C. It was argued based on the high temperature dependence that the gating process may involve a large conformational change in the channel structure . We have recently found that the extracellular zinc ion (Zn 2+ ) inhibits the wild-type ClC-0 channel with a similar degree of temperature dependence, leading to a conclusion that Zn 2+ inhibits the channel by inducing the closure of the slow gate . That the slow gating may be a very complex process and may involve different parts of the channel protein is also suggested by mutational studies. Several single-point mutations scattered around the whole channel sequence as well as deletion mutations of the COOH terminus of the channel have been shown to alter the voltage dependence or the kinetics of the slow gating . Taking advantage of the finding that Zn 2+ inhibits the channel via an effect on the slow gate, we probe the channel's inactivation process with extracellular Zn 2+ . In the present study, we focus on cysteine residues because of numerous examples of the involvement of cysteine residues in forming the Zn 2+ -binding site . With this approach, we have now found that C212 of ClC-0 is important in controlling the Zn 2+ sensitivity of the channel. Mutation of C212 to serine or alanine greatly reduces the Zn 2+ sensitivity. At the same time, the mutations appear to eliminate the slow-gating process. These results further support the assertion that the high apparent affinity of Zn 2+ to inhibit ClC-0 is indeed due to the effect on the slow gating of the channel. Site-specific mutants were generated using recombinant PCR mutagenesis. All 12 cysteine residues of ClC-0 were mutated, one at a time, to serine except C39, C100, and C213, where the substituted amino acids were arginine, threonine, and glycine, respectively. Wild-type ClC-0 and all the mutants were constructed in the expression vector pBlueScript and the regions generated by PCR were sequenced completely to exclude errors introduced by the polymerase. Capped RNAs of ClC-0 and all the mutants were synthesized from ScaI-linearized DNA (New England Biolabs Inc.) with T3 polymerase (Ambion Inc.). Xenopus oocytes were prepared and injected with RNAs as previously described . Whole oocyte ClC-0 current was recorded with a two-electrode voltage clamp amplifier (725C; Warner Instruments) and digitized with Digidata 1200 data acquisition board and pClamp6 software (Axon Instruments). Detailed recording techniques and the confirmation of successful channel expression in Xenopus oocytes have been described . To assay the sensitivity of Zn 2+ inhibition, the bath (external) solution containing Zn 2+ was ND 96 containing (mM): 96 NaCl, 2 KCl, 1 MgCl 2 , 0.3 CaCl 2 , 5 HEPES, pH 7.6. The solution to wash out Zn 2+ had an additional 1 mM CaCl 2 and 1 mM EGTA. The membrane potential of the oocyte was held at −30 mV and the current was monitored with a 100-ms voltage pulse to +40 mV, given every 4–8 s. The steady state P o –V curve of the fast gate and the activation curve of the slow gate were constructed following previously described protocols . In brief, for the P o –V curve of the fast gate, a −120-mV voltage step was first given to activate the slow gate. The fast gate was then examined with a voltage pulse to +50 mV, followed by different test voltages from +50 or +60 mV to −170 or −160 mV in −20-mV steps. The tail current was measured at −100 mV and the extrapolated value to the beginning of the pulse was normalized to that obtained with the +70- or +80-mV test pulse, yielding the relative P o s at the corresponding membrane potential in the test period. To construct the activation curve of the slow gate, a 7-s prepulse from 0 to −130 or −150 mV in −10-mV steps was given to reach a quasi–steady state opening of the slow gate. The oocyte current was then examined by a 0.8-s test pulse at +40 mV. The current measured at the end of the 0.8-s test pulse was normalized to the maximal value obtained from each oocyte. Because of a paradoxical behavior of the slow gate at membrane potentials more negative than −120 mV , only data points in the voltage range from −120 to 0 mV were shown. Because the channel does not close completely even at very negative membrane potential , records were not leak-subtracted. ZnCl 2 (J.T. Baker, Inc.) was dissolved in water as 100 mM stock solution, and then added to the ND96 solution to obtain the indicated concentration. The temperature of the bath solution was monitored and controlled by a heater controller (TC 324A; Warner Instruments). Inside-out patches were obtained with glass electrodes fire-polished to a resistance of 2–7 MΩ. Single-channel currents were recorded with an Axopatch 200B (Axon Instruments) amplifier. The output of the amplifier was filtered at 200 Hz (−3 dB corner frequency, four-pole Bessel; Dagan Corp.) and digitized at 1 kHz by a Microstar DAP 800 acquisition board (Microstar Laboratories, Inc.) installed in a Pentium computer using home-written software . The external (pipette) solution contained (mM): 110 N -methyl- d -glucamine (NMDG) – Cl, 5 MgCl 2 , 1 CaCl 2 , 5 HEPES, pH 7.6. The bath solution contained (mM): 110 NaCl, 5 MgCl 2 , 5 HEPES, 1 EGTA, pH 7.6. To display the slow-gating transition with long single-channel trace, every 500 sampling points (equivalent to 0.5 s) were averaged and shown as one data point . In such a compressed time window, the fast-gating transition is no longer visible, but any inactivation event with duration longer than ∼200 ms can be seen as an upward deflection . The displays of the fast gating are the original recording traces sampled at 1 kHz. Data analysis for macroscopic current was conducted with software programs in pClamp6 (Axon Instruments) and Origin 4.0 (Microcal Software, Inc.). Analysis of the single-channel recording trace was performed with a home-written program . Due to a small single-channel current amplitude at voltages close to the reversal potential, the single-channel traces were further digitally filtered at 200–300 Hz, leading to a final filter frequency at ∼140–170 Hz (−3 dB). Missed events were not corrected. The methods for calculating the open probability and the opening and closing rate constants of the fast gate have been described previously . For the wild-type channel, except where indicated, the inactivation events were first eliminated by eye to isolate the bursts of channel openings. The bursts of channel activity always show three equally spaced conductance states, labeled D, M, and U, corresponding to the opening of 0, 1, and 2 pores, respectively. The probability for the channel to stay at each state, f D , f M , and f U , and the time constants of the dwell-time histograms for those events in the three levels, τ i , were determined. The open probability of the fast gate was then calculated from the observed state probabilities: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{{\mathrm{o}}}={f_{{\mathrm{M}}}}/{2+f_{{\mathrm{U}}}{\mathrm{.}}}\end{equation*}\end{document} The time constants of the distributions, τ i , were used to determine the opening and closing rate constants, α and β, respectively, of each individual pore: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\alpha}}}={1}/{ \left \left(2{\mathrm{{\tau}}}_{{\mathrm{D}}}\right) \right {\mathrm{,}}}\end{equation*}\end{document} and 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\beta}}}={1}/{ \left \left(2{\mathrm{{\tau}}}_{{\mathrm{U}}}\right) \right {\mathrm{.}}}\end{equation*}\end{document} The gating parameters, P o , α, and β, for C212S were calculated in the same way except that no event at level D (or nonconducting level) was eliminated. Single-channel current amplitudes were measured from all-points amplitude histograms. To test the binomial distribution of the three current levels in C212S, theoretical values of the state probabilities were calculated from P o based on binomial distribution: 4a \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{0}= \left \left(1-P_{{\mathrm{o}}}\right) \right ^{2}{\mathrm{,}}\end{equation*}\end{document} 4b \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{1}=2P_{{\mathrm{o}}} \left \left(1-P_{{\mathrm{o}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} and 4c \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{2}=P_{{\mathrm{o}}}^{2}{\mathrm{.}}\end{equation*}\end{document} These calculated values were compared with the measured state probabilities f D , f M , and f U , as shown in Fig. 8 C. All curve fittings were performed with an unweighted least-squares method. Results are presented as mean ± SEM. We started with examining the expressions of various cysteine mutants as well as studying their slow-gating behaviors. Fig. 1 A shows the positions of the 12 cysteine residues of ClC-0 based on the putative membrane topology. Among these mutations, 9 of 12 cysteine mutants generated voltage-dependent chloride current similar to that of the wild-type channel. Fig. 2 A shows the quasi–steady state activation curve of the slow gate of wild-type ClC-0. Consistent with previous studies , the open probability of the slow gate is higher at more hyperpolarized membrane potentials. This voltage dependence of the slow gating is different in C212S, C213G, and C480S. These mutants reveal either no voltage dependence , a reverse voltage dependence with lower open probability at negative potential , or only slight voltage dependence . As shown in Fig. 1 B, C212 and C213 of the Torpedo channel are also conserved in ClC-1 and ClC-2, but this conservation occurs only within this branch of the channel family. The channel in the closest branch, for example, ClC-Ka, already loses this conservation. C480 has less conservation, with a corresponding cysteine present only in ClC-1, but not ClC-2. The significance of the conservation of these cysteine residues remains to be determined. The Zn 2+ sensitivities of wild-type ClC-0 and the three cysteine mutants are compared in Fig. 3 . All of the channels except C212S have a relatively high sensitivity to Zn 2+ inhibition, with half effective concentrations <10 μM. The remaining current after the inhibition by saturating Zn 2+ concentration is usually <10–20% of the total current. In contrast, C212S has a lower sensitivity for zinc inhibition. The dose–response curve of zinc inhibition reveals a K 1/2 of ∼50 μM. The most striking observation, however, is the maximal inhibition by Zn 2+ . With a Zn 2+ concentration as high as 3 mM, the maximal inhibition is <40%. Thus, there seems to be at least two different ways for Zn 2+ to inhibit these channels. The one with higher apparent affinity ( K 1/2 < 10 μM) in wild-type ClC-0, C213G, and C480S is eliminated by a mutation at C212. This Zn 2+ effect has previously been shown to be due to an effect on the entropy of the free energy involved in the slow-gating transition. Because a change of entropy is equivalent to a change in temperature in terms of their contributions to the free energy of the inactivation process , we examine the slow gating with the temperature-jump experiment as shown in Fig. 4 . For the wild-type channel, raising the temperature of the bath solution leads to an initial increase in the current amplitude, followed by a current reduction . The initial increase of the current is likely due to a larger single-channel conductance, whereas the current reduction is due to the closure of the slow gate . In the three mutants shown here , only C212S does not show the current reduction phase, suggesting that the slow gating of C212S may be eliminated by a single conserved mutation, thus rendering the channel insensitive to the Zn 2+ inhibition. The conclusion is further supported by examining the temperature dependence of the current increase upon raising the temperature. The Q 10 of the current increase seen with C212S is ∼1.4 , a value similar to the temperature dependence of the diffusion rate . Thus, the increase of the steady state current in C212S upon temperature jump appears to result from the increase of the single-channel conductance. To examine whether the inactivation of C212 mutants is indeed eliminated, we employed single-channel recordings on inside-out membrane patches excised from Xenopus oocytes. In these recordings, symmetrical chloride concentrations were applied on both sides of the membrane patch and the membrane potential was held at −50 mV. In the wild-type channel, there is a gating transition with average time on the order of seconds, a typical inactivation process that is also present in C213G and C480S . The probability of the inactivated state and the kinetics of the slow-gating process in C213G and C480S, however, are different from those of wild-type ClC-0. Although an accurate determination of the inactivation probability may require a longer recording trace because of the slow behavior of the process, several minutes of the recording can provide a rough estimate. For C213G, the inactivation probability is usually >50%. For C480S, the probability of the inactivation state is rather small (∼0.1–5%), consistent with the small amount of current reduction in the temperature-jump experiment . In the C212 mutants, the slow-gating transitions of both C212S and C212A are absent . In all our recordings with good quality (∼100 min for C212S, ∼20 min for C212A), we did not observe any nonconducting event with duration longer than 500 ms. There are, however, very rare occasions of long closings of an individual pore as described previously in the wild-type channel . The absence of a relatively long nonconducting event in the C212 mutants, though indicative of a change in the slow-gating process, may not truly reflect the absence of the inactivation event. One could argue that the inactivation event may be shortened by the mutation so that it is not possible to differentiate the closed from the inactivated state based on the duration of nonconducting events. This situation was recently found in the ClC-1 channel, in which the average time of the slow-gating transition is only threefold larger than that of the fast-gating process, rendering a clear dissection of these two gating processes rather difficult . However, it was shown in the same study that if all nonconducting events were considered as closed rather than inactivated states, the dwell-time distribution of these events could not be described by a single-exponential function and the probabilities of the three current levels of ClC-1 would depart from a binomial distribution. We therefore analyzed the single-channel recording trace of C212S in more detail. Fig. 7 C shows that, without eliminating any nonconducting event, the dwell-time distribution of zero-current events of C212S is well described by a single-exponential function, whereas that of the wild-type channel is not. In addition, the three current levels of C212S follow a perfect binomial distribution . These results indicate that the nonconducting events of C212S indeed do not include inactivation events. Most (or all) of them appear to result from the simultaneous closure of the two independent fast gates. The effect of C212 mutation appears to be quite specific for the slow-gating process of the channel. The fast-gate open probability , the rate constants of the activation , and the deactivation of the fast gate as well as the single-channel conductance are almost identical between wild-type ClC-0 and C212S. The elimination of the slow gating in C212S, therefore, explains why the channel is not sensitive to Zn 2+ inhibition. Thus, probing the channel's inactivation process with Zn 2+ indicates that the high affinity block of the channel by Zn 2+ is present only when the inactivation process exists. We have also tested this hypothesis with a previously identified slow-gating mutant, S123T . Although the voltage dependence of the slow gating was altered, inactivation events were still present in this mutant as examined at the single-channel level . Fig. 10 shows the voltage and temperature dependence of the slow gating as well as the Zn 2+ sensitivity of S123T. Although the voltage dependence is reversed, the temperature dependence of the slow gating is obviously present, likewise the high apparent affinity for Zn 2+ inhibition. These results are consistent with the hypothesis that the effect of Zn 2+ is on the temperature-dependent process of the slow gating. The present study was initiated with an intention to search for the possible Zn 2+ -binding site responsible for the extracellular Zn 2+ inhibition . We started with mutating all cysteine residues of ClC-0 one at a time, followed by examining their Zn 2+ sensitivity. We have indeed found a cysteine mutant with an altered Zn 2+ sensitivity. As shown in Fig. 3 , a conserved point mutant C212S still retains >60% of the current after applying 3 mM extracellular Zn 2+ . On the other hand, the inhibition of Zn 2+ on the wild-type channel, which has been shown to be due to an effect on the slow gating of the channel, has an apparent affinity of only ∼1 μM . Thus, it seems that there are at least two ways for Zn 2+ to inhibit the channel. Zn 2+ at a low micromolar concentration induces the closure of the slow gate of ClC-0, resulting in the steady state current inhibition . On the other hand, the partial inhibition seen on C212S requires a higher concentration of Zn 2+ . The on and off rates of this partial inhibition have little temperature dependence (data not shown), a phenomenon also different from that found in the wild-type channel . Although we do not understand the exact mechanism for the partial inhibition of C212S, there are several possibilities. First, the inhibition of C212S by high concentrations of Zn 2+ may be due to a mechanism totally unrelated to the slow gating, such as a reduction of the single-channel conductance or a change of an unrelated gating process. On the other hand, it is equally possible that the Zn 2+ inhibition of C212S is on the remaining inactivation process. For example, if the inactivation process of wild-type ClC-0 is composed of multiple sequential steps, it is fully conceivable that a high concentration of Zn 2+ is necessary for the channel to achieve the first step of very short-lived closure, which is followed by a highly temperature-dependent gating step leading to a deep inactivated state. For the C212S mutant, because the latter step is removed, Zn 2+ is unable to drive the channel into the deep inactivated state. Although we have found that mutation of a cysteine residue appears to greatly reduce the sensitivity of the channel to Zn 2+ inhibition, we are unable to conclude that C212 is responsible for the Zn 2+ binding site for an obvious reason. Mutation at the C212 position appears to eliminate the slow-gating transition, it is thus expected that the channel loses its sensitivity to Zn 2+ inhibition if the effect of Zn 2+ is indeed on the slow gate. In the >100-min recordings we made on C212S and C212A with good quality, we did not observe any nonconducting event with duration longer than 500 ms. We did, however, observe long closings of an individual pore, which was also found in the wild-type channel . It was speculated that such rarely occurring channel behavior that deviates from the binomial distribution may be due to a different gating process . The conserved mutation at the 212 position appears not to alter this obscure gating mechanism. Inspection of the single-channel recording trace thus provides a direct way to evaluate the presence of the long-lived inactivated state. Nonetheless, one may argue that the duration of the inactivation events could be shortened by the mutation and it may then be difficult to visually dissect the inactivated from the closed events. However, if the nonconducting level of C212S indeed contains two populations of events that do not have the same average event duration, the dwell time of these nonconducting events would not have revealed a single-exponential distribution . Furthermore, analysis of the single-channel recording trace of C212S without eliminating any nonconducting event reveals that the three current levels follow a perfect binomial distribution . This indicates that if the slow gate of C212S or C212A is still present, its probability at the nonconducting state must be extremely low. We have also applied the same three tests to examine the slow gating of S123T, a previously identified mutant of slow gating . Consistent with the previous report, the voltage dependence of the slow gating for this mutant has been altered, but the channel still retains relatively high sensitivity to Zn 2+ inhibition and shows a current reduction phase in the temperature-jump experiment . This is consistent with previous results that the channel still showed prominent inactivation at the single-channel level . The high sensitivity of Zn 2+ inhibition on the slow-gating mutants correlates well with the temperature dependence of the macroscopic current reduction and the presence of the inactivation process. These results thus provide further support to the conclusion that the inhibition of ClC-0 by low concentration of Zn 2+ is due to the facilitation of the inactivation process via a change in the entropy of the free energy involved in the slow-gating process . The conserved change of cysteine to serine or alanine at position C212 appears to have a rather specific effect on the slow gating of the channel. The steady state P o –V curve and the kinetics of the fast-gating process of C212S does not show significant difference from those of the wild-type channel . The single-channel recording not only confirms this conclusion, but also reveals that the conductance of C212S is the same as that of wild-type ClC-0 . The only difference that we can so far identify between the wild-type channel and C212S is the presence of the inactivated state. That a single conserved mutation of ClC-0 specifically eliminates a gating transition with a very high activation energy raises an interesting question regarding the underlying inactivation mechanism. Clearly, C212 by itself does not account for the structural basis of the slow gating because mutations at other places in the channel alter the properties of the slow gating . Furthermore, the residue is conserved in ClC-1 , but this muscle chloride channel shows a common gate very different from the slow gating of ClC-0 in the gating kinetics and voltage dependence . The slow-gating transition of ClC-0 is thought to be related to the chloride flux across the channel pore . Although C212S does not show difference in the single-channel conductance from the wild-type channel, the possibility that the 212 position is located in the channel pore has not been ruled out, given the nearby regions in ClC-1 channel were implicated as pore segments . It would be interesting to further examine this possibility by studying the permeation properties of C212 mutants with respect to different permeant ions. | Study | biomedical | en | 0.999995 |
10398689 | The highly selective epithelial sodium channel (ENaC) 1 in the apical membrane of epithelial cells represents the predominant pathway in mediating sodium reabsorption in the distal nephron, the colon, and the lung . This electrogenic vectorial transport of Na + is accomplished by a two-step transport system involving the apical ENaC and the basolateral Na + -K + pump. In the distal nephron, ENaC activity is regulated by aldosterone and vasopressin, serving to maintain Na + balance, extracellular volume, and blood pressure. The functional characteristics of ENaC have been studied in isolated renal tubular segments and in recombinant expression systems using patch-clamp techniques. ENaC is a small 4–6-pS conductance channel in isotonic NaCl with high selectivity for Na + and Li + over K + (permeability ratios P Li / P Na > 1 and P Na / P K > 100) and slow gating kinetics. ENaC currents are blocked by submicromolar concentrations of amiloride. ENaC belongs to a new class of channel proteins called the ENaC/DEG superfamily, which includes a variety of proteins involved in mechanotransduction and neurotransmission, and is found in nematodes, flies, snails, and mammals . Subunits of this superfamily coassemble usually within subfamilies into Na + -preferring or -selective multimeric channels that are either constitutively active (e.g., ENaC), activated by mechanical stimuli (as postulated for Caenorhabditis elegans degenerins), activated by a peptide , or activated by protons (ASIC). ENaC is a heterotetramer, and made of two α, one β, and one γ homologous subunits arranged around the channel pore in an αβαγ configuration . Each homologous subunit has two transmembrane spanning segments (M1 and M2) with intracellular NH 2 and COOH termini leaving a large extracellular hydrophilic loop, as illustrated in Fig. 1 A . Based on sequence comparisons, current models predict that the second transmembrane spanning segment of ENaC forms an α helix starting with a conserved Ser residue (αS589) and extending 22 residues further downstream in the defined ENaC sequence . A pre-M2 segment can arbitrarily be defined as a sequence delineated by a conserved Gly (αG579) residue at the 5′ end and αS589 at the 3′ end that initiates the M2 segment. Fig. 1 C shows a model of the narrow pore region of ENaC, based on this and previous work. Previous mutagenesis experiments provided evidence that the pre-M2 forms the outer pore of ENaC . Amino acid residues mutated in those experiments are presented in bold on a gray background in Fig. 1 B. These experiments showed that mutations of Gly residues in β (G525) and γ (G537) subunits decrease the affinity for the pore blocker amiloride and change single-channel conductance. In addition, a Cys substitution at the homologous position of αENaC (αS583C) generated a high affinity Zn 2+ binding site that leads to channel block by Zn 2+ . Recently, we have shown that mutations of the conserved Ser residue αS589 allow larger ions such as K + , Rb + , and Cs + as well as divalent cations to pass through the channel . Ion substitution experiments indicate that αS589 determines the molecular cutoff of the channel pore at its narrowest point, the selectivity filter. Thus, in the pre-M2 segment that lines the outer channel pore, the amiloride binding site precedes the selectivity filter in the sequence. In this study, we have analyzed the role of conserved amino acid residues located between the amiloride binding site (corresponding to αS583) and the selectivity filter (αS589) in channel permeation properties and blocking by amiloride. We have focused our interest on the conserved aromatic residues at the position corresponding to W585 in the αrENaC sequence, and on a cluster of Ser and Gly residues . Mutations were introduced in rat ENaC cDNA as described previously . Complementary RNAs of each α, β, γ subunit were synthesized in vitro. For binding experiments, α, β, and γ subunits that had been tagged as described by Firsov et al. 1996 were used with the FLAG reporter octapeptide in the extracellular loop, directly COOH terminal of the first transmembrane segment of each subunit. Healthy stage V and VI Xenopus oocytes were pressure injected with 100 nl of a solution containing equal amounts of αβγ ENaC subunits at a total concentration of 100 ng/μl. For simplicity, ENaC mutants are named by the mutated subunit only, although always all three subunits (α, β, and γ) were coexpressed. The FLAG reporter octapeptide, which had been introduced in α, β, and γ subunits, is recognized by the anti–FLAG M 2 mouse monoclonal antibody (M 2 Ab) (Eastman Kodak Co.). M 2 Ab was iodinated as described by Firsov et al. 1996 . Iodinated M 2 Ab had a specific activity of 5–20 · 10 17 cpm/mol and were used up to 2 mo after synthesis. On the day after mRNA injection, oocytes were transferred to a 2-ml Eppendorf tube containing modified Barth's saline (mM: 10 NaCl, 90 N -methyl- d -glutamine HCl, 0.8 MgSO 4 , 0.4 CaCl 2 , 5 HEPES, pH 7.2) supplemented with 10% heat-inactivated calf serum, and incubated for 30 min on ice. The binding was started upon addition of 12 nM 125 I-M 2 Ab (final concentration) in a volume of 5–6 μl/oocyte. After 1 h of incubation on ice, the oocytes were washed eight times with 1 ml modified Barth's saline supplemented with 5% heat-inactivated calf serum, and then transferred individually into tubes for γ counting containing 250 μl of the same solution. The samples were counted and the same oocytes were kept for subsequent measurement of the whole-cell current. Nonspecific binding was determined from parallel assays of noninjected oocytes. Theoretically, it might be possible that our mutations affect the accessibility of the FLAG epitope for the M 2 antibody by changing the conformation of the extracellular loop. However, this possibility seems rather unlikely since most of the mutations did not affect 125 I-M 2 Ab binding. Electrophysiological measurements were taken at 16–20 h after injection. Two-electrode voltage-clamp recordings were obtained using a TEV-200 amplifier (Dagan Corp.). The standard bath solution contained 110 mM NaCl, 1.8 mM CaCl 2 , 10 mM HEPES-NaOH, pH 7.35. For selectivity measurements, Na + was replaced by Li + , K + , Rb + , or Cs + at the same concentration. Macroscopic amiloride-sensitive currents (I) are defined as the difference between ionic currents obtained in the presence and absence of 5 μM (or higher concentrations for some mutants, as indicated) of amiloride (Sigma Chemical Co.) in the bath. All macroscopic currents shown are amiloride-sensitive currents as defined above. Pulses for current–voltage curves were applied, and data were acquired using a PC-based data acquisition system (Pulse; HEKA Electronik). The cell-attached or outside-out configuration of the patch-clamp technique was used to obtain macropatch and single-channel data. Before recording, the vitelline layer of the oocyte was removed. For cell-attached patches, the oocytes were kept in a standard bath K + solution to depolarize the membrane potential, and pipette solutions were Na + or Li + , as described above. For outside-out patches, extracellular solutions were as described above. Changes of external solutions of outside-out patches were made using the Rapid Solution Changer RSC-200 (BioLogic International Ltd.). In this system, the perfusion solutions are driven by gravity to the rotating head containing in these experiments up to 11 glass tubes. The solution exchange is performed by a highly precise and fast rotation of the RSC head, which exposes the patch pipette to the flow of one of the tubes. Times of rotation from one tube to the adjacent one were a few milliseconds. The pipet solution contained 75 mM CsF, 17 mM N -methyl- d -glucamine, 10 mM EGTA, and 10 mM HEPES, pH 7.35. Pipettes were pulled from Borosilicate glass (World Precision Instruments, Inc.). In patch-clamp experiments, currents were recorded with a List EPC-9 patch clamp amplifier (HEKA Electronik) and filtered at 100 Hz for analysis. Data are shown as mean ± SEM, or as indicated. To analyze titration curves for inhibition of macroscopic Li + or Na + currents (I), the ratio I/I 0 measured in the presence (I) of a particular blocker B to that in the absence of the blocker (I 0 ) is described by the inhibition equation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{I}}/{\mathrm{I}}_{0}\;=\;K_{{\mathrm{i}}}^{{\mathrm{n}}}^{\prime}/(K_{{\mathrm{i}}}^{{\mathrm{n}}}^{\prime}\;+\;[{\mathrm{B}}]^{n}^{\prime})\end{equation*}\end{document} , where K i is the inhibitory constant of the blocker, [B] is the concentration of the blocker, and n′ is a pseudo–Hill coefficient. For the fit of Li + block of Na + current through γS541A ENaC , n′ was set equal to 1, and a nonblockable fraction of the amiloride-sensitive current (I) was introduced. This nonblockable fraction of I was set equal to the amiloride-sensitive current carried by 140 mM Li + alone, normalized to the amiloride-sensitive current carried by 20 mM Na + \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{e.g}}.,\;0.21\;{\pm}\;0.01\;{\mathrm{at}}\;-150\;{\mathrm{mV}},\;n\;=\;6)\end{equation*}\end{document} . A reaction rate theory treatment of transmembrane ionic diffusion considers ion movement as a series of discrete steps between energy minima (wells) separated by energy maxima (barriers). One may simply account for saturation or ionic block by assuming that only one ion at a time may reside in a particular energy well, which corresponds to a discrete ion-binding site in the channel, and that individual ions cannot pass each other in the channel. To fit our current–voltage (I/V) data collected at various Na + or Li + concentrations to discrete-state barrier models, we have used a version of the AJUSTE program, originally developed by Alvarez et al. 1992 and modified by French et al. 1994 . The theoretical basis of barrier models is described extensively in Alvarez et al. 1992 , French et al. 1994 , and Hille 1992 . We used a discrete-state permeation model based on a kinetic scheme for a three-barrier–two-site (3B2S) channel that includes double-ion occupancy and ion–ion repulsion. The energy diagram in Fig. 9 A (below) summarizes the adjustable parameters of the model. The energies of the unoccupied channel at zero voltage are defined by three peak energies (G1, G2, and G3) and two wells or site energies (U1 and U2) for Na + and Li + . The subscripts of the parameters refer to the position with respect to the inside solution as shown in Fig. 9 A. The program can deal with the simultaneous presence of three types of ions. The distances D1–D6 refer to the fraction of the electric field that separates peak and well positions with the requirement that \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{D}}1\;+\;{\mathrm{D}}2\;+\;{\mathrm{D}}3\;+\;{\mathrm{D}}4\;+\;{\mathrm{D}}5\;+\;{\mathrm{D}}6\;=\;1\end{equation*}\end{document} . In addition to the above energy and distance parameters, there is an interaction energy A(ion x, ion y), which models the effects of ion–ion interactions between the same or different types of ions. The reference energy state used in the model is 1 mol fraction, which corresponds to 55.5 M. Conversion to a 1-M reference state is readily accomplished by addition of 4.0 RT units to the reported peak and well energies. The translocation rate constant K for translocation from well U over peak G is related to the translocation barrier height \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\Delta}}}G\;=\;G_{{\mathrm{G}}}\;-\;G_{{\mathrm{U}}})\end{equation*}\end{document} according to \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K\;=\;(kT/h)\;{\mathrm{exp}}(-{\mathrm{{\Delta}}}G/RT)\end{equation*}\end{document} , where k is Boltzmann's constant, T is the temperature in °K, and h is Planck's constant \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(kT/h\;=\;6.2\;{\times}\;10^{12}{\mathrm{s}}^{-}1\;{\mathrm{at}}\;25{^\circ}{\mathrm{C}})\end{equation*}\end{document} . For the modeling of energy barrier profiles, we used single-channel data from outside-out patches of Xenopus oocytes expressing wild-type (wt) or mutant ENaC. Data sets had been obtained at three to five different negative membrane potentials (less than −50 and more than −180 mV) and four to six different external concentrations of the permeant ion (either Na + or Li + at 15–200 mM) per channel type, and in the absence of any permeant ions in the internal solution. As we have analyzed inward, but not outward, currents for the modeling, the part of the energy barrier profiles on the extracellular side (G2, G3, U2) are well defined, whereas the quality of the fit was relatively insensitive to changes of the energy parameters U1 and G1. The procedure for fitting single channel data consisted first in setting barrier profiles for ENaC wt with either Na + or Li + as the single permeant ion. The quality of fits was evaluated both visually and by SUMSQ, which is the weighted sum of squared differences between experimental and theoretical data minimized by the fitting routine. The parameter values for the electrical distance D were arbitrarily constrained by a requirement of symmetry about the central barrier located at an electrical distance of 0.5. The optimal arrangement found for wt ENaC was \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{D}}1\;=\;{\mathrm{D}}6\;=\;0.1,\;{\mathrm{D}}2\;=\;{\mathrm{D}}5\;=\;0.15,\;{\mathrm{and\;D}}3\;=\;{\mathrm{D}}4\;=\;0.25\end{equation*}\end{document} , and this arrangement was used for fitting of all mutants. The quality of the fit was relatively insensitive to the ion–ion interaction parameter A. This can be readily explained by the fact that the data used for constructing the model was obtained at relatively low ion concentrations (typically <160 mM). In this concentration range, the probability of double occupancy of the channel was <0.01, even with A set to a low value (2.6). This is consistent with flux-ratio experiments indicating that ENaC forms a one-ion pore . The model obtained for Na + permeation of wt channels is shown in Fig. 9 B (below). The model fits the conductance/ion concentration relationship well, as shown by the comparison of model prediction (solid line) and data points (symbols) in Fig. 7 (below). The outer well (U2) is deeper than the inner well (U1). This difference allowed fitting of mutant data, in which the apparent channel affinity for Na + decreased (γS541A, αG587A) by just changing one well energy (one binding site) with regard to wt. This energy profile predicts an almost ohmic behavior of the channel, with outward currents <111% and >90% of inward currents over a voltage range of ±150 mV. Based on the energy profile obtained for the wt Na + permeation, the profile best suited for Li + permeation of wt ENaC was obtained by increasing U2 and G3 . Starting from these two models for Li + and Na + permeation, we attempted to fit the mutant data by changing only energy values of one well and one barrier at the extracellular side of the permeation pathway, either U2 and G2, or U2 and G3, assuming that single point mutations in the external channel pore would not affect binding sites on both sides of the highest barrier of the selectivity filter. Adjustment of at least one barrier was necessary to adjust the model to the experimental conductance values. By changing U2 and G2, we obtained better fits of our experimental data than by changing U2 and G3. For this reason, we report here models obtained by only the first method. Finally, we used the 3B2S model to fit the block of 20 mM Na + currents in the γS541A mutant by increasing Li + concentrations . In this case, the model takes into account the simultaneous presence of Na + and Li + in the external solution. The use of the 3B2S model requires that macroscopic currents are converted to unitary currents. The macroscopic current data of Li + block in macropatches were normalized at each voltage to the condition with 20 mM Na + alone and, for each voltage, these normalized currents were multiplied by the single-channel current for 20 mM Na + predicted by the γS541A-Na + model. These modified data were then used to obtain a model for Li + permeation of the γS541A channel. The sequence alignment of the pre-M2 segment and the NH 2 -terminal part of M2 of ENaC subunits and homologous proteins is illustrated in Fig. 1 B. Residues, which have previously been shown to be important for ion permeation and/or blocking of ENaC currents by amiloride are shown in bold on a gray background and amino acids that have been analyzed in the present study are shown in white on a dark background. αS583 and the corresponding residues βG525 and γG539 line the outer channel pore as shown in Fig. 1 C at a site where amiloride plugs the channel . αS589 at the NH 2 terminus of M2 is part of the narrowest region of the pore at the selectivity filter . Other amino acid residues in this region, αW585 and αV590 and their analogues in β and γ subunits, are conserved and might be important for the ion permeation properties. We have mutated the αW585, βW527, and γW539 residues to various amino acids and did not observe any significant changes in ion selectivity, unitary conductance, or block by amiloride (Tables I–III). The level of cell surface expression of wt and mutant ENaC was determined on intact oocytes by specific binding of an iodinated monoclonal antibody to a FLAG epitope inserted in the extracellular domain of ENaC subunits (see methods ). Some mutations resulted in a low expression of the channel at the cell surface ( Table ). Similar to Trp mutations, Ala substitutions of the conserved αV590, βV532, and γV544 residues were without effects on the biophysical and pharmacological properties of the channel (Tables I–III). This lack of effects of mutations of the conserved Trp and Val residues suggests that these amino acids may not line the channel pore. Next, we looked at the cluster of Ser and Gly residues, αG587 and αS588 and the analogous amino acid residues in β and γ subunits . The αS588I mutation is known to increase single channel conductance for Na + ions and to decrease channel affinity for blocking by amiloride . More conserved mutations, however, such as Ala substitutions of αS588 and the homologous residues βG530 and γG542 did not change ion conductance properties or channel affinity for amiloride (Tables I–III). The effect of αS588I on channel conductance corroborates the previous observation that αS588 is close to the selectivity filter . Conserved Gly residues in the α and β subunits at positions α587 and β529, and the unique Ser residue in the γ subunit (γS541) were substituted with the polar Ser, the nonpolar residues Cys, Gly, or Ala or the negatively charged Asp. Analysis of the concentration-dependent block of macroscopic Na + currents by amiloride ( Table ) showed that specific mutations in α and β subunits decreased channel affinity for amiloride. The βG529A, βG529S, and βG529C mutations resulted in a 40- to 130-fold increase in K i for amiloride, and a significant sevenfold increase in amiloride K i was measured for αG587S. Some substitutions of these residues had no effect on block by amiloride (αG587A, βG529D, βG529R; Table ), suggesting that these amino acids are not part of the amiloride binding site, but rather are indirectly involved in channel block. No I Na was detected for αG587D and γS541N or γS541F mutants, and only a small I Na (<0.3 μA) was detected for γS541R even using high (1 mM) amiloride or benzamil concentrations. In the case of the αG587D mutant, I Na was not detected despite a 5.5-fold higher anti–FLAG binding signal compared with the background signal. The specific anti–FLAG binding to oocytes expressing the αG587D mutant was only slightly lower than binding to oocytes expressing the functional αG587A mutant ( Table ). Similarly, specific anti–FLAG antibody binding was about the same for the nonconducting γS541N and γS541F mutants and for γS541R compared with the functional γS541A mutant ( Table ). These observations indicate that αG587D, γS541N, γS541F, and γS541R mutants are expressed at the cell surface, but are nonconducting channels or channels with minuscule conductance (γS541R), consistent with the notion that mutations of αG587 and γS541 residues affect ion permeation through the channel. Thus it appears that ENaC can tolerate relatively conservative substitutions at these positions and that less conservative replacements can make the channel nonfunctional. We tested whether αG587A, βG529A, or γS541A mutations affect channel selectivity for Li + over Na + ions. Representative current traces in the presence of extracellular Na + solution at different voltages are shown in Fig. 2A and Fig. B , for wt and the γS541A mutant. When Li + replaced Na + in the external medium, the amiloride-sensitive inward current increased in oocytes expressing ENaC wt , but clearly decreased in oocytes expressing γS541A . The macroscopic current–voltage relationships of ENaC wt and the αG587A, βG529A, or γS541A mutants in the presence of external Na + , Li + , or K + ions are plotted in Fig. 2 C. The currents of the I/V curves are normalized to I Na of each channel type measured at −100 mV. In external Na + solution, the I/V behavior of wt and mutant channels was identical. The I Li /I Na ratio was lower for the three mutants compared with wt, and followed the sequence wt > αG587A > βG529A > γS541A. The current ratio I Li /I Na at −100 mV of all the αG587, βG529, and γS541 mutants is shown in Table . In addition to mutants shown in Fig. 2 , βG529C also exhibited a decreased I Li /I Na ratio, whereas other mutants such as βG529D, βG529R, or γS541G maintained I Li /I Na ratios > 1, similar to ENaC wt. Thus, the different effects by different βG529 substitutions cannot be correlated with properties of the amino acid side chain. The fact that substitution of βG529 by the much larger Arg did not change ion permeation indicates that the side chain may point away from the channel pore (see discussion ). The I/V relationship in external K + did not provide evidence for a detectable K + permeability for the αG587A, βG529A, and γS541A mutants even at hyperpolarized membrane potentials . However, βG529S generated a significant amiloride-sensitive inward K + current as illustrated by the current–voltage behavior in Fig. 3 . In the presence of external Na + the I/V relationship of the βG529S mutant was identical to wt. When K + replaced Na + ions in the external medium, βG529S channels exhibited measurable inward currents at negative membrane potentials. At −100 mV, I K represented 22% of I Na \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{I}}_{{\mathrm{K}}}/{\mathrm{I}}_{{\mathrm{Na}}}\;=\;0.22\;{\pm}\;0.2,\;{\mathrm{Table\;II}})\end{equation*}\end{document} . The βG529S mutant allows to a lesser extent Rb + ions to pass through the channel \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{I}}_{{\mathrm{Rb}}}/{\mathrm{I}}_{{\mathrm{Na}}}\;=\;0.04\;{\pm}\;0.01,\;n\;=\;8)\end{equation*}\end{document} , but not larger ions like Cs + \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{I}}_{{\mathrm{Cs}}}/{\mathrm{I}}_{{\mathrm{Na}}}\;<\;0.01,\;n\;=\;8)\end{equation*}\end{document} . The permeability of the βG529S mutant to K + and Rb + suggests that this mutation increased the molecular cutoff of the channel. Similarly, the βG529C mutant was also slightly permeant to larger ions with an I K /I Na ratio of 0.06 ± 0.0 ( Table ) and an I Rb /I Na ratio of 0.03 ± 0.01 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document} . The βG529D mutant, which exhibited a I K /I Na ratio of 0.19 had a very low I Na expression level and was not analyzed further. None of the αG587 or γS541 mutants showed significant K + permeability ( Table ). We conclude from the analysis of these macroscopic current data that (a) the residues αG587 and βG529 in the pre-M2 segment play a role in channel block by amiloride, (b) αG587, βG529, and γS541 are involved in defining channel Li + /Na + permeability ratio, and (c) βG529 codetermines the molecular cutoff of the channel that normally prevents K + ions from going through the channel. Measurements of channel unitary currents are necessary to ultimately demonstrate changes in ion permeation through the channel. Our single-channel analysis was focused mainly on channel mutants that exhibit important changes in macroscopic current properties. Many of the single-channel recordings were performed in the excised outside-out configuration to allow the determination of the channel sensitivity to amiloride to ascertain that unitary currents detected in the patch indeed represented the activity of ENaC mutants. Representative recordings are shown in Fig. 4 . As anticipated from macroscopic currents, αG587A and βG529A showed reduced unitary currents with Li + ions as charge carrier, and no obvious changes in unitary Na + current (i Na ) or channel gating were observed. The corresponding γS541A mutation decreased i Na , and the unitary Li + current (i Li ) was considerably reduced so that transitions between open and closed states could not be precisely determined under our recording conditions . Single-channel conductance values, which are summarized in Table , show that the unitary Li + conductance (g Li ) for the mutants follows the order wt > αG587A > βG529A > γS541A. The g Li /g Na ratios agree with the Li + /Na + permeability ratios measured for macroscopic amiloride-sensitive currents ( Table ). The higher Li + /Na + permeability of the βG529R and the γS541G mutants compared with wt measured by the macroscopic I Li /I Na ratio \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{I}}_{{\mathrm{Li}}}/{\mathrm{I}}_{{\mathrm{Na}}}\;=\;2.37\;{\mathrm{and}}\;3.11,\;{\mathrm{respectively,\;compared\;with}}\;1.4\;{\mathrm{for\;wt}})\end{equation*}\end{document} , could not be confirmed by changes in either unitary currents for Na + or Li + ions, indicating that βG529R and γS541G mutations do not increase channel permeability to Li + relative to Na + ions. Comparison with other members of the ENaC gene family shows that at the position homologous to αG587 all known genes have a Gly residue, except for S541 in the ENaC γ subunit. Substitution of the γS541 by Gly did not change the conductance or the ion selectivity of the channel. We also tested whether Gly and Ser residues are interchangeable at the homologous positions in α and β subunits. The αG587S mutant showed a decreased unitary Na + and Li + conductance, but the g Li /g Na ratio remained unchanged ( Table ). The most dramatic effect was observed with βG529S. As shown before , this mutation makes the channel permeable to K + and preserves a macroscopic I Li /I Na ratio >1. At the single-channel level, βG529S decreased unitary Na + and Li + current amplitudes to such an extent that transitions between open and closed states could not be detected. As shown in Fig. 5 in a representative experiment, channel openings or closures could not be detected under our conditions in excised outside-out patches containing channels with the βG529S mutation and exhibiting amiloride-sensitive Na + and Li + currents. This indicates that g Li and g Na of the βG529S mutant are more than likely <1 pS. Thus the analysis of the single-channel currents of the αG587, βG529, and γS541 mutants indicate that substitution of these residues by Ala, Ser, or Cys change the ion permeation properties by decreasing channel conductances for either Li + ions alone or for both Li + and Na + . The decrease in unitary Li + current in the mutants reflects a slower movement of Li + ions along the pore of ENaC mutants. Strong binding interactions between the permeant ion and the residues lining the channel pore can account for a slow ion permeation through the pore. Alternatively, high energy barriers for ion translocation along the conduction pore are also expected to slow ion permeation. We addressed these possibilities by measuring the changes in unitary current i Na and i Li with increasing concentrations of the permeant ion, as illustrated in typical recordings in Fig. 6 . From i Na and i Li measurements at different holding potentials, values for single-channel conductance (g Na and g Li ) were obtained for ion concentrations ranging from 10 to 200 mM. Unitary conductances are plotted versus the concentration of the permeant ion (Na + or Li + ) in Fig. 7 . ENaC wt shows saturation of g Na around 5 pS for Na + concentrations >100 mM, whereas g Li does not saturate even at concentrations >100 mM. Values of ion concentration for half-maximal unitary conductance ( K M ) were determined from fits of the conductance–ion concentration relationship to the Michaelis-Menten equation and are given in Table . In the case of ENaC wt, K M values were 38 mM for Na + and 118 mM for Li + . The g Na and g Li saturation curves of wt ENaC suggest that the difference in Li + versus Na + permeability is due to differences in channel affinity for the two ions, implying that the I Li /I Na ratio strongly depends on the absolute concentration of Na + and Li + . In the αG587A mutant, which is equally permeant to Na + and Li + , the apparent affinity for Na + is slightly lower and the apparent affinity for Li + ions is increased . The K M of 114 mM for Na + ions is close to that for Li + ions (90 mM) ( Table ). The αG587S mutant is characterized by a dramatic decrease in maximal conductances for Na + and Li + ions and a lower apparent K M for Li + . The unitary Na + conductance of αG587S saturates at concentrations <100 mM. Due to the low g Na , the K M for Na + permeation was not determined more precisely. The βG529A mutant shows a g Li that saturates between 2 and 3 pS with an apparent affinity for Li + ions around 40 mM, thus the apparent affinity is increased threefold compared with wt. It is remarkable that the change in apparent affinity and maximal conductance of the βG529A mutant affects only Li + ions, whereas the K M for Na + , and maximal g Na , remained basically unchanged ( Table and Table ). Interestingly the βG529R mutation has no apparent effects on the permeation properties of Na + and Li + ions . The barely detectable single-channel Li + currents of the γS541A mutant made the determination of g Li impossible. The γS541A mutant showed a slight decrease in g Na , with a slightly lower apparent affinity for Na + ions compared with ENaC wt. Thus mutations of αG587 and βG529 change the apparent affinity for Li + and/or Na + ions, resulting in alterations in the single channel conductance and the microscopic conductance ratio g Li /g Na . What is the affinity of the γS541A mutant for Li + ions? In a few excised outside-out macropatches, we measured macroscopic Li + conductance of the γS541A mutant at five different Li + concentrations, and obtained an apparent K M for Li + as low as 11 ± 1 mM \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;6)\end{equation*}\end{document} . From this high apparent channel affinity for Li + ions and low Li + permeability of the γS541A mutant, we expected that Li + ions should block Na + currents. The experiment illustrated in Fig. 8 shows that this is indeed the case. Macroscopic, amiloride-sensitive inward currents were measured in 20 mM external Na + and increasing Li + concentrations from excised outside-out macropatches from oocytes expressing the γS541A mutant. 80 mM Li + blocked 60% of the Na + current in the presence of 20 mM external Na + , a Na + concentration below the K M for Na + of the γS541A mutant. The inward amiloride-sensitive current is not completely blocked by Li + because Li + still carries current in the γS541A channel. From the fit of the Li + inhibition curve of the I Na to a simple inhibition scheme that takes into account a nonblockable fraction (see methods ), we obtained an apparent Li + affinity for I Na inhibition of 28 mM. In contrast to the situation in the γS541A mutant addition of 80 mM Li + to the extracellular solution increased in the ENaC wt the amiloride-sensitive current by 3.1 ± 0.1-fold \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;5)\end{equation*}\end{document} at −150 mV. We conclude that the low unitary conductance of the γS541A mutant is related to a higher apparent channel affinity for Li + ions. To interpret the changes in the maximal open state conductance and apparent affinity for Na + and Li + ions induced by αG587, βG529, or γS541 mutations, we have used a simple model of ion permeation through ENaC, based on reaction rate theory of ion permeation . According to this theory, the movement of ions through the single filing region of the pore is described as a series of discrete steps between energy minima (wells), separated by energy maxima (barriers). Our model incorporates a 3B2S kinetic scheme, outlined in Fig. 9 and described in methods . Single-channel current amplitudes, determined at various negative voltages and different concentrations of the permeant ion (either Na + or Li + ), were used to construct energy barrier profiles. The best fit of our I/V data and the conductance–ion concentration relationship to a 3B2S model provided energy profiles for wt and mutants shown in Fig. 9 B and listed in Table . The conductance–ion concentration relationship predicted by the model for the different channel types in the presence of Na + or Li + are shown as solid lines in Fig. 7 . The extracellular part of the energy profile is much better defined by our data than the intracellular side (G1, U1) because we have obtained the energy barrier profiles by fitting exclusively inward currents. The best fit of our data predicts energy profiles for Na + and Li + permeation in wt ENaC that are not absolutely symmetrical, but still result in a near ohmic behavior of the I/V relationship over a voltage-range of ±150 mV. For the wt, the higher permeability of Li + versus Na + ions observed at concentrations >50 mM is mainly due to a higher energy of the outer well (U2) for Li + permeation, corresponding to a lower affinity for Li + binding. The use of Eyring reaction rate theory has several limitations, among them the fact that it describes ion movements over well-to-well distances of fractions of Angstroms, whereas in our 3B2S model the ion translocation over the three energy barriers takes place over a distance of several Angstroms. In discrete-state permeation models, the calculation of the barrier energies depends on the use of a prefactor, kT / h according to the Eyring absolute reaction rate theory . In a different approach, an approximation to a continuum model, a prefactor including the ion's diffusion coefficient and information on the geometry of the barrier and well has been used to calculate barrier energies . The use of this prefactor yields barrier and well energies ∼4.6 RT units more negative than the values reported in Fig. 9 B and Table . Because of the uncertainty about absolute energies of barriers and wells, we illustrate in Fig. 9 C the changes in barrier and well energies (G2 and U2) obtained for the αG587A, βG529A, and γS541A mutants relative to wt. These relative changes are much less model dependent. The small changes in the Na + permeation through these channel mutants are simply due to a parallel positive shift in G2 and U2 , corresponding to a slight decrease in channel affinity for Na + ions but a conserved maximal g Na . The gradual decrease in Li + permeability and the changes in the g Li –[Li + ] relationship observed in αG587A, αG587S, and βG529A mutants compared with wt are essentially due to a substantial increase in the peak energy of the middle barrier (G2) relative to wt and only small changes in the energy of the outer well (U2) . This results in a higher energy (Δ G ) required for Li + ions to hop over G2 from an outer binding site U2 to the inner binding site U1, and a consequently slower translocation rate of Li + ions from U2 to U1. We have used the macroscopic current data of Na + current block by external Li + to obtain an energy profile for Li + permeation through γS541A ENaC, as described in methods . The prediction of the Li + block by the model is shown as a dotted line in Fig. 8 , and the energy parameters for the fit are listed in Table and shown in Fig. 9 B. As for the αG587A and βG529A mutants, the corresponding energy profile predicted for Li + permeation through γS541A shows a higher peak energy of the middle energy barrier that increases the energy required for translocation of Li + ions from U2 to U1 . Thus, when Li + occupies the pore of the γS541A in the single filing region, it slows the movement of Na + ions. The question remains as to whether amiloride binds to a unique site in the external pore, and whether the ion selectivity filter is confined to the region we identified in the pre-M2 segment. Recently, Ismailov et al. 1997 identified a WYRFHY sequence rich in aromatic residues in the large extracellular loop of αENaC as a putative amiloride-binding domain. Later, it was reported that conserved Lys residues at positions 550 and 561 in αENaC are critical for channel sensitivity to amiloride and for ion selectivity . Since these studies were done on channels presumably made of αENaC subunits reconstituted in lipid bilayers, we tested the relevance of these mutations for the pharmacological and functional properties of ENaC composed of the three types of subunits, α, β, and γ. Following expression of an α mutant with deletion of the WYRFHY sequence together with β and γ subunits, no I Na could be measured and no ENaC cell surface expression could be detected using anti–FLAG antibodies directed against β and γ subunits. The point mutation within this sequence, αH282D, reported to be responsible for ENaC insensitivity to amiloride did not result in changes in amiloride sensitivity or macroscopic ionic selectivity of channels made of αH282Dβγ ( Table and Table ). The αK550E and the αK561E mutants, when coexpressed with β and γ subunits, exhibited the same sensitivity to block by amiloride as ENaC wt and showed normal macroscopic Na + /Li + selectivity and unitary currents (Tables I–III). From these results, we conclude that the putative amiloride-binding sequence described by Ismailov et al. 1997 and the Lys residues in the large extracellular loop are not relevant for the amiloride sensitivity and ionic selectivity of the native ENaC made of α, β, and γ subunits. Our mutagenesis experiments have identified amino acid residues at homologous positions in α, β, and γ ENaC subunits, located two residues upstream of the predicted NH 2 terminus of the second transmembrane α helix, that play an important role in ion conduction. Mutations of these residues αG587, βG529, and γS541 resulted in the following changes in ion permeation and channel ionic selectivity. First, the mutations αG587D, γS541N, γS541F, and γS541R resulted in channels, which were expressed at the cell surface at normal densities, but did not conduct ionic current or showed only a minuscule ionic current (γS541R). Second, αG587A, βG529A, βG529C, and γS541A mutations decreased Li + permeability with relatively small changes in Na + permeability. Third, introduction of a Ser residue in the α (αG587S) and β subunits (βG529S) resulted in a decrease in both Na + and Li + unitary currents with no apparent changes in the Li + /Na + permeability ratio. Fourth, the βG529S (and similarly βG529C and βG529D) mutant was permeant to K + ions and to a lesser extend to Rb + ions. Finally, the mutants αG587S, βG529A, βG529C, and βG529S with major changes in ion permeation or selectivity also showed resistance to amiloride block. These observations indicate that αG587, βG529, and γS541 interact closely with the permeant Na + and Li + ions, and participate at least in part in ion discrimination at the selectivity filter. The selectivity filter appears in close vicinity of the amiloride binding site. The amiloride-sensitive ENaC, target of aldosterone action in the distal nephron and colon, is made of homologous αβγ subunits. This channel is highly selective for Na + over K + ions and has the unique characteristic among the other members of the ENaC/DEG gene family of being more permeant to Li + than to Na + ions. The higher permeability of ENaC for Li + compared with Na + ions was evident from early macroscopic current measurements in flat epithelia like frog skin or toad urinary bladder. In these preparations, it was generally not possible to detect currents through ENaC carried by K + ions. The only ion other than Na + and Li + allowed to pass through the channel was H + . The selectivity profile H + > Li + > Na + of ENaC suggested that the pore discriminates among cations mainly on the basis of the size of the dehydrated ion allowing small cations to pass through the channel. Larger cations such as K + , Rb + , or Cs + go only part-way along the ion conduction pathway, and block the channel pore at high concentrations, but are not able to pass the most constricted region of the pore, the selectivity filter . In support of this hypothesis for ion selectivity in ENaC is our recent finding that mutation of the highly conserved αS589 residue alters the molecular sieving properties of ENaC . Specific αS589 mutants exhibit different I K /I Na ratios. In particular, the αS589D mutant with the highest K + /Na + permeability ratio showed a permeability profile for alkali metal cations following the order K + > Rb + > Cs + , indicating that αS589 mutations result in an increase in the molecular cutoff of ENaC. It was concluded that αS589 is part of the selectivity filter, which acts as a molecular sieve to discriminate between small Na + or Li + ions and larger K + ions. In the present study, the mutants that show a significant K + permeability are βG529S, βG529C, and βG529D; the permeability relative to Na + of these mutants was higher for K + ions than for Rb + , and no current carried by Cs + could be detected. The increase in the channel molecular cutoff by the βG529 mutations is consistent with the notion that, like the αS589 mutation, they enlarge the pore diameter at the selectivity filter and alter the molecular sieving properties of the channel . Thus the changes in the pore geometry at the selectivity filter that allow K + or Rb + ions to pass through the pore can be achieved by specific mutations of αS589 or βG529 residues. It suggests that these two residues are important for the steric selectivity of the channel by maintaining the proper pore geometry to tightly accommodate the permeating ion and exclude larger ions. Beside the changes in ion selectivity of the βG529S, βG529C, and βG529D mutants, mutations of the αG587, βG529, and γS541 residues resulted in important changes in the permeability properties of the channel for Na + and Li + ions. These effects involve both the changes in the maximal open state conductance and/or in the apparent channel affinity for Na + and Li + ions as determined by K M values for channel conductance. Recordings of individual channels in the native tissue have shown that the open state conductance of ENaC saturates with increasing concentrations of Na + and Li + ions, indicating that Na + and Li + ions bind to specific sites in the ion permeation pathway. This saturation process arises when the binding–unbinding steps of the ion conduction at specific binding sites along the pore become rate limiting. At high ion concentrations, the channel pore is occupied most of the time and the rate of ion translocation is determined by the maximal rate at which the permeant ions dissociate from their binding site and the ion flux approaches saturation. For ENaC wt, the dependence of open state conductance on the concentration of the conducting ion gives an apparent affinity ( K M ) of 38 mM for Na + and 118 mM for Li + . This difference in channel affinity for Na + and Li + ions can account for the higher Li + over Na + permeability usually observed at an ion concentration around 100 mM since the flux of Na + ions already reaches saturation at these concentrations . Specific binding sites for Na + have been postulated on the basis of competitive interactions between Na + and amiloride or K + ions that inhibit Na + current through the channel in a voltage-dependent manner. In addition, the lack of voltage dependence of channel occupancy by Na + ions was consistent with the presence of multiple ion binding sites along the channel pore. Reasonable models for Na + permeation have been proposed that are consistent with these experimental observations. These models involve at least two common binding sites for Na + , Li + , K + , and amiloride in the outer mouth of the channel . How can the decrease in Li + permeability relative to Na + of the αG587A, βG529A, or γS541A mutants be explained in molecular terms? High affinity binding of ions in the channel pore tends to slow ion permeation provided that the channel is occupied by a single ion at a time. A low Li + permeability was observed in αG587A, βG529A, or γS541A mutants characterized by a lower maximal g Li and an apparent higher affinity for Li + compared with wt. Alternatively, the mutations might result in steric changes within the narrow region of the pore, making it more difficult for Li + ions to pass through. For the interpretation of the changes in ion permeation in ENaC mutants, we need to consider ion conduction through ENaC consisting basically of three fundamental processes: (a) diffusion of an ion up to the outer entrance of the channel pore, (b) dehydration and solvation of the ion by polar chemical groups lining the pore, and (c) translocation through the selectivity filter. Models of ion permeation in terms of energy profile and Eyring rate theory can formulate these processes . We have empirically chosen an energy profile for Na + and Li + translocation through ENaC that consists of two energy wells representing two binding sites and three energy barriers for ion translocation. It has previously been shown that this type of model (3B2S) for ion conduction can account for the electrical properties of ENaC . In energy barrier models, the K d of the permeant ion is defined by the value of the deepest energy well; thus, the deeper the well, the lower the concentration needed to reach saturation of the ion flux. By contrast, the higher the energy barriers, the lower the maximal conductance of the channel. While such models cannot provide an accurate representation of the structure of the ion conduction pathway, they can help us to determine whether alterations in ion conduction of αG587A, βG529A, or γS541A mutants can be related to particular permeation mechanisms. In the ENaC wt, the energy barrier profile obtained for Li + permeation compared with Na + is characterized by a higher outer energy well consistent with a lower affinity for Li + at an external binding site , accounting for the differences in Li + versus Na + permeability. In our models for Li + permeation through the αG587A, βG529A, or γS541A mutants, the predominant changes in Li + versus Na + conductance are due to an increase in the middle energy barrier, with little changes in the outer energy well. It suggests that translocation of Li + ions in the single filing region at the central energy barrier requires more energy in mutants compared with wt. Thus, the αG587, βG529, or γS541 residues can be assigned to a region of the pore that represents a significant barrier for ion translocation. It is possible that βG529 and γS541 have preferential interactions with Li + ions since mutations of these residues did not affect Na + permeation much. We observed that the βG529S, βG529C, and βG529D mutations change the channel molecular cutoff allowing K + and Rb + ions to pass through the channel. Thus, βG529 plays a role in defining the molecular cutoff of the channel and is at the same time important for the Li + /Na + selectivity. The dual role of βG529 underlines that the discrimination between Li + and Na + , and between Na + and larger ions, occurs at overlying sites in the selectivity filter. Our data suggest that αG587A, βG529A, and γS541A mutations result in steric changes at the selectivity filter that impair translocation of Li + ions through the narrowest part of the channel pore. In moving from the outer binding site U2 into the narrowest region of the pore, the permeant ion likely has to lose a few more molecules of its hydration shell. This loss of the hydration shell raises the energy of the permeant ion. Our data are consistent with the hypothesis that the energy cost for Li + dehydration at the U2 → G2 translocation is raised in the αG587A, βG529A, and γS541A mutants because it cannot be sufficiently compensated by the interactions between the permeant ion and the polar groups lining the pore. Mutations such as αG587S and βG529S decrease unitary conductance for both Na + and Li + ions, although the low unitary currents made it difficult for αG587S and impossible for βG529S to fit the I/V data to our 3B2S model. The energy profile of αG587S for Li + conduction predicts an increased peak energy of the middle barrier, consistent with what we observed for the other mutants discussed above. The conductance data of βG529S are also compatible with a mutation resulting in a considerable increase in the peak energy of the middle barrier for Na + and Li + permeation, but a decrease in the energy barrier for K + conduction. The binding site U2 cannot be assigned yet to a molecular structure, but is likely to be in the close vicinity of the selectivity filter. It is somewhat surprising that the effects of substitutions of αG587, βG529, and γS541 did not correlate in a predictable way with general physical–chemical properties of the substituting amino acid residues. The fact that ion permeation is not altered by the βG529R mutation indicates that charges do not play a crucial role in ion coordination at the selectivity filter, and basically excludes the possibility that the βG529 side chain faces the lumen of the channel pore. As already suggested for αS589 mutations , the amino acid side chain of βG529 (and probably of its analogues) are likely to point away from the channel pore, in a similar way as has been shown for the selectivity filter of the KcsA channel . If this is the case, the side chains of these amino acid residues are likely to interact with other amino acid residues, to keep the backbone of αG587, βG529, and γS541 in place and maintaining the proper conformation of the pore at the selectivity filter. In ENaC wt, Gly, and Ser, amino acid residues with short side chains make these interactions, and the positive charge in the βG529R mutant (four carbon atoms between the α-carbon atom and NH 3 group) might be too far away to disrupt this interaction. In mutant channels that are permeable to K + (αS589 and some βG529 mutants), the mutated side chain might push parts of the pre-M2/M2 domains of the subunits away from each other or tilt them, resulting in a widening of the pore. In summary, our mutagenesis experiments indicate that αG587, βG529, γS541, and αS589 residues are part of the narrow region of the pore that constitutes the selectivity filter of ENaC . It seems that the important steps of ion conduction that involve ion dehydration, solvation, and translocation occur in a very restricted region of the ion permeation pathway that encompasses the αG587 and analogues and αS589 residues. For comparison, the crystal structure of the KcsA channel reveals that the narrow selectivity filter lined by the backbone of VGYG residues is only 12-Å long, whereas the overall length of the pore is 45 Å . Finally, the mutations αG587S, βG529A, and βG529S that change ion selectivity and ion permeation properties also decrease channel affinity for amiloride, whereas the βG529 mutation without consequences on ion permeation properties did not change channel blocking by amiloride. It is unlikely that αG587 and βG529 are involved in specific binding interactions with amiloride since nonconserved substitutions have no effect on channel affinity for amiloride. The αG587A, βG529A, or γS541A mutations may indirectly impair the binding interaction of amiloride with the channel pore by steric changes that result in alterations in the pore geometry. According to its size, amiloride is not supposed to penetrate deep into the narrowest part of the selectivity filter. We propose that αG587 and βG529 are located at the external entrance of the narrow selectivity filter where steric alterations of the pore have consequences on amiloride binding in the channel outer vestibule. Fig. 1 C, showing the ion pore of ENaC, illustrates some relevant experimental observations regarding the structures involved in ion conduction. The Gly 525 and 537 residues of β and γ subunits and to a lesser extent the corresponding αS583 residue form the amiloride binding site in the pore . Mutation of the two αS583 residues in the ENaC tetramer to Cys creates a high-affinity binding site for Zn 2+ . Because the α subunits are most likely on opposite sides of the channel pore , and the optimal distance of two sulfhydryl groups for coordinated ligation of Zn 2+ is ∼5 Å , we estimate the pore diameter at this site to be around 5 Å. The pore then narrows down to its narrowest part that constitutes the selectivity filter. This narrow region is relatively short and may be lined by only three amino acid residues involving the residues Ser or Gly in the αβγ subunits. We suggest that, in analogy to the KcsA channel, the cytoplasmic part of the pore is lined by residues from the second transmembrane α helix. | Study | biomedical | en | 0.999998 |
10398690 | Excitation–contraction coupling in striated muscle takes place by the release of stored calcium in response to depolarization of the sarcolemma, via ryanodine receptor channels of the sarcoplasmic reticulum. When studied in frog skeletal muscle by confocal microscopic imaging of the fluorescence of fluo-3, this release is seen to occur largely in the form of discrete events, first described in cardiac muscle and termed Ca 2+ sparks. A continuous form of release, not constituted by resolvable discrete events, is of comparatively minor magnitude in the frog, where it may have an important mechanistic role , and it is the sole component seen in adult rat muscle under voltage clamp . It is still not clear whether the sparks of cardiac and skeletal muscle are the result of opening of individual channels or of concerted openings of channel clusters . One of the approaches that could help elucidate this question is to determine the flux or current of Ca 2+ release underlying sparks. Blatter et al. 1997 adapted release calculation methods that had been developed earlier for signals from macroscopic cell segments and applied them to sparks of cardiac myocytes. In the present paper, we apply this method to sparks recorded in frog skeletal muscle under different conditions. Taking a clue from earlier work with whole cell methods , we first tested the technique by simulating sparks (as the result of release from spatially and temporally discrete sources), and then applying the release calculation algorithm to the simulated sparks. By this combination of forward (i.e., simulation of sparks) and backward calculations (the release algorithm), we found that the method recovers adequately the current of release flux. We also uncovered limitations of the method, manifested in errors in the determination of release magnitude, source diameter, or release flux duration, and established circumstances that make these errors greater or reduce them. When the release calculation algorithm was applied to sparks obtained experimentally in skeletal muscle under different conditions, the resulting flux was large by comparison with Ca 2+ flux measured through single channels in bilayers under what are thought to be near-physiological conditions , suggesting a multichannel origin for sparks. The point spread function (PSF), 1 a function P (x,y,z), was determined generally following procedures discussed by Agard et al. 1989 . We used fluorescent beads of subresolution size (0.1 μm diameter; Molecular Probes, Inc.) with the laser scanner and microscope operating under the same conditions as in the experiments, and the same objective (C-apochromat 40×, 1.2 N.A., water immersion lens; Carl Zeiss, Inc.), filters, and glass (#0 coverslip, with the correction collar of the objective set for minimum glass thickness). Before the determination of the PSF, the alignment of the system was optimized following the manufacturer's recommended procedure. Two techniques were used to immobilize the beads. In one, the bead-containing suspension was painted and dried on a glass slide, antifade solution and a coverslip were added, and the beads were then imaged through the coverslip. In the other, the beads were immobilized in an aqueous 4% agar gel. The second method gave a slightly more symmetrical PSF. The PSF was determined by serial optical sections (or xy scans) at different axial (z axis) levels. The z axis level was determined by the stepping motor acting on the vertical nosepiece focusing of the microscope (Axiovert 100-TV; Carl Zeiss, Inc.), which in turn was calibrated by focusing on an object of known dimensions. The three-dimensional array of fluorescence intensities thus obtained was simplified (under the assumption of cylindric symmetry) to a two-dimensional array. For this, the average per pixel was calculated at each value of z, in annular bins in the xy plane, at 0.1425-μm intervals of the radial distance from the center \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[r\;=\;{\OE}({\mathrm{x}}^{2}\;+\;{\mathrm{y}}^{2})]\end{equation*}\end{document} . The fluorescence averaged in this way is represented versus r and z in Fig. 1 A. The dependence could be fitted as the product of two gaussians: P \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}=\;G(r)\;H(z),\;{\mathrm{where}}\;G\;=\;k\;{\mathrm{exp}}[-0.5\;(r/{\mathrm{{\sigma}}}_{{\mathrm{xy}}})^{2}],\;H\;=\;{\mathrm{exp}}[-0.5(z/{\mathrm{{\sigma}}}_{{\mathrm{z}}})^{2}],\;{\mathrm{{\sigma}}}_{{\mathrm{xy}}}\;=\;0.20\;{\mathrm{{\mu}m,\;and\;{\sigma}}}_{{\mathrm{z}}}\;=\;0.614\;{\mathrm{{\mu}m}}\end{equation*}\end{document} . The two-gaussian description, which is used later in the analysis, is documented in B and C. Fig. 1 B contains data as a function of the axial coordinate z . The fluorescence values represented are averages within each section, over a 0.1425-μm circle centered on the \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{x}}\;=\;0,\;{\mathrm{y}}\;=\;0\end{equation*}\end{document} line (•), or in a ring region 0.285 < r < 0.4275 μm (○). The lines are best fit gaussians with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}\;=\;0.608\;{\mathrm{and}}\;0.736\;{\mathrm{{\mu}m}}\end{equation*}\end{document} , respectively, indicating some deviation from the product of gaussians at high values of r . In Fig. 1 C, the fluorescence is represented as a function of distance r at constant z . The three sets of data are from the central section (| z | < 0.18 μm; •), an intermediate region (0.18 < | z | < 0.54 μm; dotted symbols), and an outlying region (0.72 < | z | < 0.98 μm; ○). The best fit gaussians shown have very similar spread \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\sigma}}}\;=\;0.196,\;0.2,\;{\mathrm{and}}\;0.207\;{\mathrm{{\mu}m,\;respectively}})\end{equation*}\end{document} . In the analysis of data, we neglect the deviations and approximate the PSF as a product of a gaussian function of r, with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{xy}}}\;=\;0.2\;{\mathrm{{\mu}m}}\end{equation*}\end{document} (corresponding to a full width at half magnitude [FWHM] of 0.47 μm) and a gaussian function of z , with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{z}}}\;=\;0.615\;{\mathrm{{\mu}m}}\end{equation*}\end{document} (FWHM of 1.44 μm). Similar values were obtained with a second C-apochromat 1.2 N.A. objective in our microscope, and with either objective in a LSM 410 (Carl Zeiss, Inc.) confocal scanner. The xy or focal spread measured was close to the expected value for this objective and wavelengths of excitation and emission , but the axial spread was worse than expected. To rule out trivial errors in scanner alignment, we carried out extensive comparisons of axial PSFs, using the front surface reflection method and different objectives, after aligning the system for the objective in use. With the C-apochromat, the FWHM z was 1.50 μm with a 2.0-mm iris and 1.15 μm using a 0.7-mm iris. With the same technique, a plan-apochromat, 63×, 1.4 N.A., oil immersion objective (Carl Zeiss, Inc.) gave a FWHM z of 0.98 μm with a 2-mm iris, and 0.63 μm using the 0.7-mm iris. An 60×, 1.4 N.A., oil objective (Olympus Corp.) gave slightly higher resolution in the focal plane and approximately the same in the axial direction as the plan-apochromat. These results indicate that the wider axial spread is a characteristic of the C-apochromat objective and is increased substantially by the larger iris. Even though this seems to imply that the oil immersion objective is better for recording sparks, the spark morphology did not change significantly with the two objectives. On the other hand, the water immersion objective has three advantages, it collects ∼2.5× greater fluorescence intensity at comparable magnification, this fluorescence intensity is nearly independent of vertical position of the focal plane within the fluorescent object or solution, and its working distance is substantially greater. For these reasons, it was used in most of the experiments reported here. Imaging of fluorescence sparks was carried out in segments of singly dissected semitendinosus muscle fibers of Rana pipiens (which were anesthetized in a 15% ethanol solution, and then killed by pithing), using two conventional techniques for eliciting sparks. In one, fiber segments were voltage clamped in a two-vaseline gap chamber, while their cytoplasmic medium was equilibrated with an “internal” solution containing (mM): 125 Cs-glutamate, 10 Cs-HEPES, 0.5 MgCl 2 , 1 EGTA, nominal [Ca 2+ ] set to 100 nM, 0.1 fluo-3, 5 creatine-phosphate, 5 Mg-ATP, 5 glucose, pH 7.0. The “external” solution in the middle pool of the chamber contained (mM): 131.5 tetraethylammonium (TEA)–methanesulfonate, 10 TEA-HEPES, 10 Ca-methanesulfonate, 10 −6 g/liter tetrodotoxin. In this case, sparks were elicited by voltage clamp depolarization to low voltages (between −70 and −50 mV). In the other technique, similar to that described by Lacampagne et al. 1998 , the fiber segment was mechanically fixed to the coverslip bottom of a single compartment chamber. Its membrane was permeabilized by several large cuts, at distances of 100–150 μm, or by brief exposure to an internal solution with 0.01% saponin, and immersed in internal solution with fluo-3. Because one of the purposes of using the permeabilized fiber was to reach higher dye concentrations more rapidly inside the cells, the internal solution in this case contained 0.2 mM fluo-3. To elicit sparks, the internal solution contained lower nominal [Mg 2+ ] , and total ATP was increased for a nominal [Mg ATP] of 5 mM. All solutions were titrated to pH 7.0. The experiments were carried out at 17°C. Ca 2+ sparks were imaged in line scan mode. Images are formed by juxtaposition of 768 line scans of 512 pixels obtained at 2-ms intervals and stored as arrays F ( x j , t i ), [sometimes referred to as F ( x,t )], where j and i are integers varying between 1 and 768 and 1 and 512, respectively. Experimental images are presented normalized to the resting fluorescence F 0 ( x ), calculated by averaging F ( x,t ) over time. In the voltage-clamp experiments, this averaging is restricted to the interval before the depolarizing pulse. In the experiments with permeabilized fibers, it is done after removing the spark regions located automatically by a simple amplitude criterion applied to a filtered version of the image . Once the events were located, all subsequent analysis was carried out on subsets of the line scan, 45 × 45 arrays, centered at the peak of the located spark. Four morphological parameters were determined first: amplitude ( a ) full width at half magnitude, full duration at half magnitude (FDHM), and rise time. a was determined as the difference between the peak value of the normalized fluorescence and its baseline value at the same spatial position immediately before the event (the average of 10 values between 40 and 20 ms before the peak). FWHM was determined on the spatial distribution of fluorescence at the time of maximum change. FDHM was determined on the time dependence at the spark center. Rise time as the interval between time to 10% increase from pre-event baseline and time to peak, determined on a spline interpolate of the time-dependent fluorescence, averaged over three spatial pixels at the center of the spark. Even though the event locator uses filtration steps, the morphometric measurements were carried out on the unfiltered image, normalized as described. In some cases, averages were made of the events located automatically. Fluorescence line scan images were analyzed to derive [Ca 2+ ]( x , t ) and release flux, following a procedure modified from that of Blatter et al. 1997 . When the dye concentration is low, so that autofiltration can be neglected, and the dye is in equilibrium with Ca 2+ , fluorescence is 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F=M\;B{\mathrm{^{\prime}}}_{{\mathrm{min}}} \left \left( \left \left[{\mathrm{dye}}\right] \right +R{\mathrm{^{\prime}}} \left \left[{\mathrm{dy}} \right {\mathrm{eC}} \left {\mathrm{a}}^{{\mathrm{2+}}}\right] \right \right) \right \;=M\;B{\mathrm{^{\prime}}}_{{\mathrm{min}}}\frac{K{\mathrm{^{\prime}}}_{{\mathrm{d}}}+R{\mathrm{^{\prime}}} \left \left[{\mathrm{Ca}}^{{\mathrm{2+}}}\right] \right }{K{\mathrm{^{\prime}}}_{{\mathrm{d}}}+ \left \left[{\mathrm{Ca}}^{{\mathrm{2+}}}\right] \right }dye_{{\mathrm{T}}}{\mathrm{,}}\end{equation*}\end{document} where K ′ d is the dye's dissociation constant in the cytoplasmic medium, R ′ is the ratio between the fluorescence of Ca 2+ -bound and free dye, M is the adjustable gain of the scanner, B ′ min is a constant and dye T is total dye concentration. (As follows from , the minimum and maximum fluorescence were derived as \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F_{{\mathrm{min}}}\;=\;B^{\prime}_{{\mathrm{min}}}\;M\;dye_{{\mathrm{T}}}{\mathrm{and}}\;F_{{\mathrm{max}}}\;=\;R^{\prime}\;F_{{\mathrm{min}}}\end{equation*}\end{document} ). The parameters of fluo-3 were given values within the range estimated by Harkins et al. 1993 for the cytoplasmic medium. Specifically, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{\prime}_{{\mathrm{d}}}\;=\;1.03\;{\mathrm{{\mu}M\;and}}\;R^{\prime}\;=\;100\end{equation*}\end{document} . Dye T , a function of position x along the scanned line, was calculated replacing F in by F 0 ( x ), the fluorescence averaged over time during the periods of rest before the sparks of interest (or voltage pulse), and assuming the resting concentration [Ca 2+ ] 0 to be equal to the concentration in the internal solution, 100 nM. When [Ca 2+ ] 0 is much lower than K ′ d (for instance at 100 nM), a simplification applies 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F=B{\mathrm{^{\prime}}}_{{\mathrm{max}}}\frac{ \left \left[{\mathrm{Ca}}^{{\mathrm{2+}}}\right] \right }{K{\mathrm{^{\prime}}}_{{\mathrm{d}}}}M\;dye_{{\mathrm{T}}}{\mathrm{,}}\end{equation*}\end{document} where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}B^{\prime}_{{\mathrm{max}}}\;=\;R^{\prime}\;B^{\prime}_{{\mathrm{min}}}\end{equation*}\end{document} . For the calibration situation applies, with B max and K d substituted for the corresponding values inside the cell. From this calibration, an approximate formula follows for the dye concentration: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}dye_{{\mathrm{T}}}/100\;{\mathrm{{\mu}M}}\;=\;(F_{0}/F_{100})\;([{\mathrm{Ca}}^{2}+]_{0}/100\;{\mathrm{nM}})\;(K^{\prime}_{{\mathrm{d}}}/K_{{\mathrm{d}}})\;(B_{{\mathrm{max}}}/B^{\prime}_{{\mathrm{max}}})\end{equation*}\end{document} , where F 100 is the fluorescence in cuvette at 100 μM [Ca 2+ ] and 100 μM dye T . K d is 0.48 μM in our cuvette calibrations. Assuming that the resting [Ca 2+ ] is equal to 100 nM and that B max /B ′ max ≈ 1 , the equation simplifies further to dye T ≈ 2 × 100 μM ( F 0 / F 100 ). Because F 0 is in general a function of x , dye T is also a function of x . If, instead of 1.03 μM, we used the upper estimate of Harkins et al. 1993 for K ′ d , 2.53 μM, the estimate of dye T would increase by a factor of ∼2.5. In this sense, we used a low estimate of dye T . When [Ca 2+ ] is changing steeply in space and time, we evaluate it as that needed to produce the observed distribution of dye :Ca 2 + , the concentration of which is proportional to the observed changes in fluorescence. Specifically, [ dye :Ca 2+ ] was derived from the spatially resolved fluorescence F ( x,t ), using \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[dye:{\mathrm{Ca}}^{2}+](x,t)\;=\;{\mathrm{dye}}_{{\mathrm{T}}}(x)\;[F(x,t)\;-\;F_{{\mathrm{min}}}(x)]/[F_{{\mathrm{max}}}(x)\;-\;F_{{\mathrm{min}}}(x)]\end{equation*}\end{document} . Then [Ca 2+ ]( x,t ) was obtained numerically solving the diffusion–reaction equation that governs the evolution of [ dye :Ca 2+ ]: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{{\partial} \left \left[dye{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right }{{\partial}t}= \left \left[dye\right] \right \left \left(x,t\right) \right \left \left[{\mathrm{Ca}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right k_{on}- \left \left[dye{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right k_{off}+D_{{\mathrm{dyeCa}}}{\mathrm{{\Delta}}} \left \left[dye{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right {\mathrm{,}}\end{equation*}\end{document} where k on and k off are the rate constants of the fluo-3:Ca 2+ reaction, D dyeCa is the diffusion coefficient, and Δ is the laplacian operator (∂ 2 /∂ x 2 + ∂ 2 /∂ y 2 + ∂ 2 /∂ z 2 ). The line scan only provides the partial derivative in the x direction. Blatter et al. 1997 used 3 ∂ 2 /∂ x 2 as an approximation to Δ. M. Cannell (University of Auckland, New Zealand, personal communication) suggested assuming that the fluorescence increase is spherically symmetric, a function of time and the distance (ρ) to its center. In that case, the dependence of F with x gives all the information needed to calculate the laplacian correctly, as ∂ 2 /∂ x 2 + 2 (∂/∂ x )/ x . This approximation does not work when the sources of release are spatially complex , but seems better in the present case, when there are few evidences of spatial complexity. The approximation has some drawbacks: the first is that the operation applied to finite functions F leads to a singularity at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}x\;=\;0\end{equation*}\end{document} . In actual work, this is avoided by using interpolated arrays that do not include \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}x\;=\;0\end{equation*}\end{document} . Still, at the smallest x values, the operation increases local noise greatly, which may result in the appearance of spurious sources when working on noisy data. Additionally, the source may not be spheric if it is constituted by multiple channels. An estimation of the errors expected if the source was not spheric is presented in results . Calculation of release flux was done as described by Blatter et al. 1997 . The flux density of the source, represented as Ṙ, satisfies the equation 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\dot {R}}={{\partial} \left \left[{\mathrm{Ca}}^{2{\mathrm{+}}}\right] \right }/{{\partial}t-D_{{\mathrm{Ca}}}{\mathrm{{\Delta}}} \left \left[{\mathrm{Ca}}^{2{\mathrm{+}}}\right] \right }+{{\partial} \left \left[dye{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right }/{{\partial}t}-D_{dye{\mathrm{:Ca}}}{\mathrm{{\Delta}}} \left \left[dye{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right +{{\partial}rem}/{ \left \left({\partial}t\right) \right }{\mathrm{,}}\end{equation*}\end{document} where D Ca and D dye :Ca are the diffusion coefficients of Ca 2+ and its complex with the dye. ∂ rem /∂ t is a sum of terms of the form 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{{\partial} \left \left[b{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right }/{{\partial}t-D_{b{\mathrm{:Ca}}}{\mathrm{{\Delta}}} \left \left[b{\mathrm{:Ca}}^{2{\mathrm{+}}}\right] \right }\end{equation*}\end{document} for each intrinsic buffer b of the cell (parvalbumin, ATP, nondiffusible sites in troponin and the sarcoplasmic reticulum, SR), plus the flux density of sinks (a positive function of space and time). Buffers are assumed to be homogeneously distributed. The terms of are calculated from the function [Ca 2+ ] ( x,t ), solving for [ b :Ca 2+ ] the diffusion–reaction equation of form 3 for the corresponding buffer. In general, this requires simultaneously solving an equation for the free buffer . This is avoided in the present calculations assuming that the free buffer and its Ca 2+ complex have the same diffusion coefficient, or equivalently that total buffer concentration is constant everywhere. In the case of parvalbumin and ATP, buffers that react with both Ca 2+ and Mg 2+ , diffusion–reaction equations of form 3 still apply, but [ b :Ca 2+ ] is in both cases equal to [ b ] T − [ b ] − [ b :Mg 2+ ] (where [b] T is the total concentration of buffer), and [ b :Mg 2+ ], a function of space and time, is calculated from its own diffusion reaction equation 6 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{{\partial} \left \left[b{\mathrm{:Mg}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right }{{\partial}t}= \left \left[b\right] \right \left \left(x,t\right) \right \left \left[{\mathrm{Mg}}^{2{\mathrm{+}}}\right] \right k_{{\mathrm{on}}}- \left \left[b{\mathrm{:Mg}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right k_{{\mathrm{off}}}+D_{b}{\mathrm{{\Delta}}} \left \left[b{\mathrm{:Mg}}^{2{\mathrm{+}}}\right] \right \left \left(x,t\right) \right {\mathrm{,}}\end{equation*}\end{document} assuming [Mg 2+ ] to be constant. Parameter values are given in Table . The SR pump contribution to removal was proportional to the fractional occupancy of pump sites of dissociation constant 1 μM, with maximum (saturation) pump rate given in the table. An exploration of the effects of changing the assumed values of the parameters is presented later. Numerical processing of data was done in the IDL environment (Research Systems Inc.). When specifically stated, the records of fluorescence were first subjected to three-point smoothing (replacing each value by the “boxcar” average of the three-element–wide array that surrounds it). Differentiation with respect to spatial coordinates was carried out by convolution with a 17-element Kaiser window kernel, with a corner frequency of either 2.45 or 1.75 μm −1 . Smoothing and the heavier filtering differentiator were used when processing individual sparks, but not when analyzing averages. Reaction–diffusion equations were written to describe the movements of Ca 2+ , Ca 2+ -bound, and Ca 2+ -free fluo-3 in the presence of an isotropic myoplasm containing endogenous buffers (troponin, SR pump, parvalbumin, ATP) and EGTA. EGTA, ATP, and parvalbumin species were treated as diffusible. In most of the simulations illustrated, the spark was modeled as the fluorescence increase resulting from a 5-ms square wave release of calcium (of current intensity that varied between 0.1 and 25 pA) deposited uniformly into a sphere (the diameter of which varied between 100 and 1,500 nm). Additional simulations were carried out with other time dependencies or with cylindric sources. The reaction–diffusion equations were solved by the Galerkin finite element method with adaptive gridding, using the program PDEASE (Macsyma Inc.). The resulting function of spatial position is spherically symmetric (a function, f (ρ), of radial distance to the center of the release sphere), and was generated as a two-dimensional array for suitable values of ρ and time. To make it comparable with the experimental results, these arrays were spaced at increments of 0.15 μm and 2 ms. The simulation approach, including the assumption of a homogeneous medium, isotropic for removal and diffusion, was similar to that of Smith et al. 1998 , who did it for a specifically cardiac removal system. The resulting sparks were of smaller normalized amplitude for the same release current in the present work, because we assumed a briefer release in a medium that included higher [fluo-3], diffusible buffers (EGTA, ATP, and parvalbumin) and a higher concentration of troponin. Additionally, we used a broader PSF when blurring the simulated sparks. Blurring, the effect of the limited resolution of the microscope when imaging fluorescence sparks, was simulated assuming a point spread function similar to that determined experimentally. Let F represent the detected fluorescence, or fluorescence in image space. The object fluorescence, f ( x,y,z ), is always assumed to be a spherically symmetric function (of radial distance to the center of the spark). In general , the image of this object will be the convolution of f with the PSF P , which for a given time results in a function of three space coordinates. 7 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F \left \left(X,Y,Z\right) \right ={\int }{\int }{\int }f \left \left(x,y,z\right) \right P \left \left(x-X,y-Y,z-Z\right) \right dx\;dy\;dz{\mathrm{.}}\end{equation*}\end{document} Because in all cases we processed or simulated line scans, we only concerned ourselves with the dependence of F on X . Additionally, we only processed sparks assumed to be centered on the scanning line. Therefore, the function F above only had to be evaluated at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}Y\;=\;0\;{\mathrm{and}}\;Z\;=\;0\end{equation*}\end{document} . Representing F ( X ,0,0) as F( X ), simplifies to 8 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F \left \left(X\right) \right ={\int }{\int }{\int }f \left \left(x,y,z\right) \right P \left \left(x-X,y{\mathrm{,}}\;z\right) \right dx\;dy\;dz{\mathrm{.}}\end{equation*}\end{document} We further simplified because the PSF, as shown in results , is approximately separable as the product of two gaussian functions: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}G(x,y)\;=\;{\mathrm{exp}}\{-[(x^{2}\;+\;y^{2})/2{\mathrm{{\sigma}}}_{{\mathrm{xy}}}^{2}]\}/2{\mathrm{{\pi}{\sigma}}}_{{\mathrm{xy}}}^{2}\end{equation*}\end{document} , a function of the radial distance to the center of the scanned spot in the focal plane, and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}H(z)\;=\;{\mathrm{exp}}\{-[(z^{2})/2{\mathrm{{\sigma}}}_{{\mathrm{z}}}^{2}]\}/{\mathrm{{\sigma}}}_{{\mathrm{z}}}\;{\sqrt{}}2{\mathrm{{\pi}}}\end{equation*}\end{document} , a function of the axial distance to the focal plane, in the z axis direction. The simulated fluorescence spark is spherically symmetrical [ f ρ (ρ)]. It is well fitted by a gaussian function of polar radius (as shown with an example in discussion ), namely 9 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{{\mathrm{p}}} \left \left(0\right) \right {\mathrm{exp}} \left \left[{- \left \left(x^{2}+y^{2}+z^{2}\right) \right }/{2{\mathrm{{\sigma}}}^{2}}\right] \right =f_{{\mathrm{p}}} \left \left(0\right) \right {\mathrm{exp}} \left \left({-x^{2}}/{2{\mathrm{{\sigma}}}^{2}}\right) \right {\mathrm{exp}} \left \left({-y^{2}}/{2{\mathrm{{\sigma}}}^{2}}\right) \right {\mathrm{exp}} \left \left({-z^{2}}/{2{\mathrm{{\sigma}}}^{2}}\right) \right =f_{{\mathrm{p}}} \left \left(0\right) \right \;g \left \left(x\right) \right \;g \left \left(y\right) \right \;g \left \left(z\right) \right {\mathrm{.}}\end{equation*}\end{document} With these approximations, the blurred function F ( X ) , , simplifies to: 10 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}F \left \left(X\right) \right =f_{{\mathrm{{\rho}}}} \left \left(0\right) \right {\int }g \left \left(x\right) \right G \left \left(x-X\right) \right {\int }g \left \left(y\right) \right G \left \left(y\right) \right dy{\int }g \left \left(z\right) \right H \left \left(z\right) \right dz{\mathrm{,}}\end{equation*}\end{document} where G ( x − X ) ⊸ G ( x − X , 0) and G ( y ) ⊸ G (0, y ). This is done for every point in time and presented to the release calculation algorithm as a line scan image of a spark. A simulated spark before and after blurring is illustrated . The analysis of fluorescence sparks, real or simulated, starts with a deblurring step to recover the object spark. We assume, in agreement with our analysis of simulated sparks, that the object spark can be put as the spherically symmetric function f ρ (0) g(x)g(y)g(z) . The form of the function g is deduced deriving from the expression 11 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{{\mathrm{{\rho}}}} \left \left(0\right) \right {\int }g \left \left(x\right) \right G \left \left(x-X\right) \right dx=\frac{F \left \left(X\right) \right }{JL}{\mathrm{,}}\end{equation*}\end{document} where F ( X ) is the line scan image, and J and L are constants, the integrals over y and z in . Therefore, g ( x ) is the deconvolution of F and the spatial spread G, scaled so that \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}g(0)\;=\;1\end{equation*}\end{document} . Evaluating at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}X\;=\;0\end{equation*}\end{document} , it follows that \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f_{{\mathrm{{\rho}}}}(0)\;=\;F(0)/J\;^{2}L\end{equation*}\end{document} . In more intuitive terms, deblurring changes the spatial shape of the spark from F to g , making it sharper, and rescales it by the factor 1/ J 2 L , which “undoes” the three-dimensional averaging and consequent loss in amplitude, due to the spread of the microscope. J and L quantify these averaging effects and are therefore smaller than 1. The final expression of the deblurred line scan is F (0) g ( x )/ J 2 L , a function that is calculated at every time t j in the experimental arrays, or at arbitrary increments in simulations. The deblurring operation is tested in Fig. 2A and Fig. B . The simulated spark before blurring is within 5% of that recovered after successive blurring and deblurring. When applied to noisy data, deblurring may lead to uninterpretably noisy results. In such cases, we used either no or partial deblurring (assuming a sharper PSF). The effects of partial deblurring, evaluated for simulated and experimental data, are illustrated . The basic self-consistency of the method is established by first applying it to simulated sparks. Then tests are presented on simulated sparks plus noise, which give a more realistic picture of the method's value and limitations. The algorithm is then applied to experimental sparks. The study ends with a consideration of lower bounds of the release estimate, crucial to evaluate the possibility that a spark may be caused by a single channel. Sparks were simulated as described in methods , as the increase in fluorescence determined by release of Ca 2+ from a spherical source into a homogeneous medium whose removal properties copied those of the skeletal myoplasm. It includes components with the properties of the most important removal processes in the cell; namely, parvalbumin, troponin, the SR pump, the extrinsic buffer EGTA, which in the experiments analyzed later is present at 1 mM, and ATP, a rapidly diffusing low affinity buffer . The model parameters had values taken from the literature, listed in Table . Shown are simulations with a release current of 20 pA and 5-ms duration, originating from a sphere of 0.2-μm radius. These simulated sparks were similar to an average experimental spark shown later. To reproduce the conditions of this average, they were generated assuming a dye concentration of 50 μM. Simulated sparks were first blurred (according to ) with half widths \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{FWHM}}_{xy}\;=\;0.4\;{\mathrm{{\mu}m\;and\;FWHM}}_{{\mathrm{z}}}\;=\;1.4\;{\mathrm{{\mu}m}}\end{equation*}\end{document} , to produce the effect of detection by the confocal microscope in its usual configuration. The final result of the simulation, with or without blurring, is represented in Fig. 2A and Fig. B , as normalized fluorescence profiles that pass through the maximum, at constant t and x . In thin lines is the spark before blurring (“simulated”). In thick trace is the spark after imaging (“blurred”). It is broader and of approximately half the amplitude of the object spark. The release calculation algorithm starts with a deblurring step ( ), which seeks to reconstruct the object spark. Its result is represented by the dashed curves (“deblurred”), and is similar to the original simulated spark. In Fig. 2C and Fig. D , the release flux density used in the simulations is compared with that reported by the release algorithm. The release flux density used in the simulations was calculated as I /(2 F )/ v s (where F is the Faraday constant and v s is the volume of the source, a sphere of radius 0.2 μm) and is represented by the thin trace. The curves in the thick trace represent release flux density calculated by the algorithm (“derived”), in Fig. 2 C as a function of position ( x ) at the maximum of release, and in D as a function of time at the center of the source. As seen in both representations, the derived release is a reduced and spread out version of the release used in the simulation. The peak of the derived flux is 1,750 mM/s rather than the theoretical 3,100 mM/s. The full duration at half magnitude, 4.7 ms, is in excellent agreement with release duration. The half width of the derived release is somewhat greater (0.46 μm) than that used in the simulation (0.4 μm). Total release flux was computed by volume integration of the spherically symmetric flux density. Expressed as a current and compared with the current of 20 pA used in the simulation. The current initially lags behind the correct value and later overshoots it, but yields a reasonable estimate. This is shown in Fig. 2 F, where the peak of the derived release current is plotted against the simulation current. A good correspondence is found in the range explored, with some evidence of saturation at the high end. Fig. 2 C illustrates that release typically undershoots, going negative at the edges of the releasing volume. This error is due in part to the use of a wide kernel for spatial differentiation. It is also due to the inherent imprecision of an analysis that samples a continuous physical phenomenon at spatial and temporal frequencies that are comparable with those in the sampled object. Though small in amplitude relative to the positive peak of release, the error becomes proportionally greater when total flux is calculated by integration over the volume of the spark. For this reason, when calculating total flux, the volume integration is stopped at the first negative value of the computed release waveform. The method was also tested with simulations that specifically violated the assumption of spherical symmetry at the source, in which release originated from a cylinder of 1-μm length and 0.2-μm radius (not shown). The current derived by the algorithm in this case overestimated the simulation current when the simulated spark was scanned along the axis of the cylinder and underestimated it when scanning transversely. The errors, however, were within 50% in either direction. The conclusion from this section is that the release calculation algorithm is self-consistent—it recovers approximately the input current used in simulations. More realistic tests required adding noise to the simulated sparks. Fig. 3 and Fig. 4 illustrate performance of the algorithm in the presence of noise. In Fig. 3 , the algorithm operates on (A) the simulated fluorescence spark of Fig. 2 (corresponding to a release current of 20 pA and 5 ms), to which pseudo-random noise has been added, with normal distribution of standard deviation 3 in fluorescence units. Such noise amounts to ∼10% of F 0 , which is comparable with that of averaged experimental records. The main problem resulting from the presence of noise at this level is that the deblurring correction, which increases the higher frequency components more, may give uninterpretably noisy results. In the example, we used a partial deblurring correction for a less severe microscope spread \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{FWHM}}_{{\mathrm{z}}}\;=\;0.88\;{\mathrm{instead\;of}}\;1.4\;{\mathrm{{\mu}m}})\end{equation*}\end{document} . The consequences of partial deblurring are explored later. Fig. 3B and Fig. C , plots multidimensionally the resulting release flux waveform; quantitative aspects are in D–F. Due to the partial correction, the resulting release flux is a worse underestimate than in the case of Fig. 2 , and the “footprint” of release is wider than the actual source. The half duration of release was 6 ms, and the half width 0.55 μm, or 35% greater than the diameter of the simulation source. The underestimation of flux density and overestimation of spatial width tend to compensate, so that the integrated current peaked at 18.4 pA in the example shown, close to the 20 pA used to generate the spark. In summary, if the noise level prevents application of the deblurring operation, then the release flux density is broadened and more severely underestimated, while the source current is still evaluated fairly well. Because it was not possible in general to use the full deblurring correction with experimental records, we evaluated in detail the effect of deblurring under different assumptions for the system's PSF. Results are represented in Fig. 4 A, where the peak release current derived for simulations analogous to those in Fig. 2 and Fig. 3 is plotted against “deblurring length,” the FWHM z assumed for correction. The values of deblurring length spanned the range from 0 (i.e., no correction), to 1.4 μm (“full correction”). In every case, FWHM xy was equal to FWH z /3.5. • represents simulations without noise and ♦ records with noise . It can be seen that increasing the blurring correction increases the estimate of release current, which tends to the correct values as the deblurring length increases. The changes are moderate and vary continuously with deblurring length. When noise is present, the dependence of the parameters on deblurring length changes abruptly at or near 1 μm, indicating that the deblurring correction does not work properly beyond this point (another evidence, not shown, is that the results become variable with each different realization of pseudo-random noise). This test indicates that the algorithm applied with partial deblurring still evaluates source current acceptably, to some extent underestimating it. This issue will be considered further when processing experimental sparks. Additional tests evaluated the ability of the algorithm to monitor current when spatial aspects of the source were varied. Simulations were similar to those shown before (20 pA, 5 ms, noise at 10% of baseline fluorescence, deblurring at 0.88 μm), but the source diameter varied between 0.2 and 1.5 μm. • in Fig. 4B and Fig. C , plots peak current and FWHM of the derived release against source diameter. It can be seen that the method evaluates current well at every diameter, but only follows the changes in diameter at values beyond 0.5 μm (C). The spatial dimension of the source is overestimated at diameters below 0.6 μm, an evidence of limited spatial resolution, due to noise and the optical spread of the microscope. ○, in Fig. 4B and Fig. C , illustrates results of applying the algorithm to simulations with noise at 30% of F 0 , a level characteristic of individual experimental sparks. Under these conditions, the algorithm can be used provided that fluorescence images are smoothed first, that a more heavily filtering differentiator is used (see methods ), and that no deblurring correction is applied. Given the high level of noise, the results change with different noise realizations. Fig. 4 , ○, represents averages of six realizations, bars are ±SEM. In this form, which can be applied to individual experimental sparks, the algorithm still evaluates release current within 50% error (B), but fails to report changes in source diameter under 1 μm (C). In summary, the algorithm evaluates release current well under realistic conditions of noise, within a wide range of source parameters. It is able to detect changes in source diameter above 0.7 or 0.8 μm when used with averages, or above 1 μm when used with individual sparks. The characteristics of sparks obtained with two different experimental techniques were somewhat different. Fig. 5 A contains a line scan image obtained from a fiber under voltage clamp, which had been exposed for 45 min to an internal solution with a nominal free [Ca 2+ ] of 100 nM and 610 μM [Mg 2+ ]. In all experiments prepared this way, almost no sparks were observed at rest, but small pulse depolarizations elicited many, as illustrated. The plot at bottom represents the relative fluorescence averaged in a 0.43-μm wide region at the position marked by the line a–a′ in Fig. 5 A. The large spark there peaked at 6.0× the resting fluorescence \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\Delta}}}F/F_{0}\;=\;5.0)\end{equation*}\end{document} . Under voltage clamp, repeated depolarizations can be applied and large numbers of sparks collected in a short period of time within a small region of the fiber. We illustrate below the application of the release algorithm to an average of such sparks. The average was made with the events identified and measured by an automatic routine in Fig. 5 A and in nine other images obtained during a brief period. A total of 301 sparks were identified whose amplitude was >1.0 U resting fluorescence. Their morphology is illustrated in Fig. 5B and Fig. C , which plot, respectively, FWHM and rise time versus spark amplitude. The events of amplitude >2.0 U are marked with circles in A and identified with ○ in the scatter plots. The graphs demonstrate several aspects of spark morphology that were similar in five experiments studied in the same detail (listed in Table ). Events span a wide range of amplitudes, between seven and the limit of detection. There does not seem to be any discrete segregation in morphology among sparks; that is, no systematic difference appears between the morphological parameters of large versus small amplitude sparks. Accordingly, the correlation between amplitude and the other variables is weak or nonexistent. The lines represent first order regressions to the open symbols. The regression coefficient r 2 was 0.102 for the spark width (range 0.012–0.127 in six experiments) and 0.008 (0.000–0.017) for the rise time. This low correlation suggests that the observed spread in amplitude was not a consequence of the existence of multiple spatially resolved sources, which could collectively generate a large spark , nor was it the trivial result of exceptionally long rise times in the largest sparks. The amplitude with which a spark is recorded in a line scan depends on the actual (object) amplitude of the spark and on its position relative to the scanned line, reaching a maximum (image) amplitude when it is centered on the scanned line. Events that appear large in a line scan image are likely to be closer to the scanned line, hence more suitable to our release analysis (which assumes centered sparks). Of course, sparks that are larger as objects are also likely to appear larger in line scans. In the following, we present the results of the release analysis applied to the average of the 157 events with amplitude >2.0 identified in the experiment illustrated. For the considerations above, this average selects a subset of events that are larger as objects and/or originated closer to the scan line. The application of the release algorithm to the average spark is illustrated in Fig. 6 . Parameter values are listed in Table ; [Mg 2+ ] and [Ca 2+ ] were assumed to have the nominal internal solution values. The dye concentration was set at the average of the estimates for each event (which ranged between 38 and 42 μM). The average was centered at the peaks of the sparks and is shown in Fig. 6 A. Because of the low level of noise in the average, no smoothing or other filtering was applied. Partial deblurring was used with deblurring length = 0.88 μm; results with other degrees of deblurring are represented in Fig. 4 A, ○. The release flux density calculated by the algorithm is represented in Fig. 6B–E . The derived release current is plotted in F. The algorithm applied to this average spark results in flux that arises from a region 0.5 μm wide, and has a half magnitude duration of 6.3 ms and an amplitude of 16.9 pA. It is demonstrated later that duration and width are quite insensitive to assumptions in the method. The calculated current, however, depends steeply on [Ca 2+ ] 0 . When, instead of 100 nM (the concentration set in the cut-end solutions), [Ca 2+ ] 0 was assumed to be 50 nM, peak current was reduced by ∼35% . The dependence is explicable because [fluo-3] is derived from resting fluorescence, which is approximately proportional to both [fluo-3] and [Ca 2+ ] 0 , hence a change in assumed [Ca 2+ ] 0 alters [fluo-3] and the derivation of [Ca 2+ ]( x,t ). In every fiber prepared the same way, several hundred sparks were identified (100 or more of which were >2.0 in amplitude). The average morphological parameters, listed in Table , and the calculated dye T , were very similar in all cases, hence the release must have been similar. It was possible to reduce the uncertainty in the derivations by using the permeabilized fiber preparation, described in methods , in which the fiber membrane is cut at multiple locations and the cell is immersed in the internal solution. Intracellular fluorescence increases with a time constant of 10–20 min. For analysis of these experiments, it is assumed that free [Ca 2+ ] has the value present in the internal solution . dye T is derived from the internal resting fluorescence as described in methods . Sparks occur spontaneously in this preparation, and their frequency can be controlled by varying free [Mg 2+ ] . A line scan image of a permeabilized fiber is in Fig. 7 . Shown is the fluorescence normalized to its resting value F 0 ( x ). This image is representative of several experiments in which dye T reached a value close to 400 μM (that is, F 0 reached a value close to that in the 200 μM dye internal solution in which the fiber was immersed). Unlike the situation with voltage-clamped fibers, the sparks obtained with permeabilized fibers were fewer and did not allow for massive averaging. Limited averaging could be done occasionally, as in the case of Fig. 7 . The image shows several sparks arising at the same triad in a field that was of very low activity elsewhere. Therefore, it is likely that all those sparks were generated at the same release unit . The fluorescence profile in a 0.39-μm region along Fig. 7 , a–a′, plotted at bottom, reveals seven sparks whose morphological parameters are represented in B and C. Note the brevity of rise times, of 4 ms or less, their poor correlation with amplitude , and their large width, close to 2 μm for all but the smallest event. These seven sparks, unusual for originating at the same spot, are not unlike other large sparks observed in permeabilized fibers. Compared with the events under voltage clamp (whose average parameter values are in Table ), large sparks in permeabilized fibers were often wider, briefer, and of shorter rise times. A detailed comparison of events under different conditions is currently in progress (G. Pizarro, A. González, W.G. Kirsch, N. Shirokova, and E. Ríos, manuscript in preparation). Fig. 8 illustrates the application of the release algorithm to an average of the four largest sparks in Fig. 7 . No deblurring was used. The parameter values were the same as in the previous case, except for [Mg 2+ ], which was set to the concentration applied, 0.15 mM. Fig. 8 A represents the spark average, after smoothing, and B–E represent the calculated release flux density. The current ( F ) peaked at 15.8. Half width of the release source was 1.1 μm and half duration was 7.3 ms. Release was calculated in the same way for large sparks in 16 images from 12 similarly prepared fibers. The results are listed in Table . The average parameters of the release source include a current of 14.4 pA, duration of 7.5 ms, and half width of 0.89 μm. As illustrated above, the algorithm produces adequate estimates of release current when applied to sparks that are generated by the same process modeled in the algorithm and using the same parameter values. Neither condition applies with real data; there may be differences in parameter values, or other differences (for instance lumped components, diffusion barriers, etc., not considered in the present homogeneously distributed model of removal). As a way to gauge the likely range of the first class of errors, we imposed parameter changes in the calculation of release for the average of 157 sparks studied in Fig. 6 . The changes and the resulting morphological measures of derived release current are listed in Table . Changes in parameters were introduced one at a time. For those parameters expected to have a major effect, two alternative values were used, defining a reasonable range that should comprise the actual fiber values. This was done for the diffusion coefficients of Ca 2+ , the dye, and ATP, and for the concentrations of ATP, EGTA, and Mg 2+ . For maximum pump rate and the concentrations of troponin and parvalbumin sites, only one change was explored, sufficient to demonstrate a relative lack of consequences. Changing properties of the dye:Ca 2+ reaction had the greatest effects. When the ON rate constant was reduced by a factor of 2.5, to bring K ′ d to the upper value in the range estimated in vivo by Harkins et al. 1993 , the derived release current increased by ∼50% (because the change increased the estimated Ca 2+ concentration transient). Similar changes in current were produced by increasing threefold the dye diffusion coefficient. Because the model considers multiple removal processes, even drastically changing one removal parameter alone had little effect. The only substantial change in the estimate of current (increase by ∼50%), occurred when the concentration of EGTA was increased fivefold. The spatial and temporal characteristics of the derived release source were even more robust. Other than changing the properties of the dye, the only changes with major consequences on the derived release width and duration were large changes in [ATP] and the reduction of [Mg 2+ ] to 0.15 μM (which increases the free concentrations of ATP and parvalbumin). Changes in troponin and the SR pump had little effect on the release estimate. To elicit a “low estimate” of release current, the method was applied with all buffer concentrations reduced threefold. The derived release parameters are in the last row of Table . With such low values, the release current was 8.05 pA. A minimum estimate of another sort was reached by simply integrating the dye-binding term (∂[ dye :Ca 2+ ]/∂ t ) in the representation of flux density given by . The meaning of this estimate is illustrated in Fig. 9 , where it is developed for both the average of four sparks in Fig. 8 and a simulated spark copying the experimental conditions. Fig. 9 , top, shows the concentration of dye :Ca 2+ , which for the experimental spark is calculated from the fluorescence following . Fig. 9 , middle, shows the rate of change of [ dye :Ca 2 + ], which in both cases reaches a pronounced maximum right before the peak of fluorescence. The bottom graphs plot this rate of change (∂[ dye :Ca 2+ ]/∂ t ) at the time of its maximum, versus x . Intuitively, the Ca 2+ release flux at this time should be greater than the total flux of Ca 2+ binding to the dye, which can be calculated by volume integration of the rate of change shown. The result of the volume integration, expressed as a current intensity, is plotted in the bottom graphs as a function of position, starting from zero at the center of the spark. The limiting value, reached far from the spark center, is ∼15 pA (0.074 fmol/s) in the experimental case. The simulated spark reaches a similar maximum rate of change, but has lesser spatial spread. Accordingly, the total flux binding to the dye in the simulation corresponds to <4 pA. In every case tested, the integrated rate at early times during a spark was less than the maximum release flux, in agreement with the idea that the other removal and diffusion processes are largely positive at these early times. The value of this dye-related estimate is listed for all events analyzed in permeabilized fibers, in the last column of Table . Its average is 8.26 pA. To test the possibility that release evaluations were affected by the less than ideal axial resolution determined in our conditions, we compared the spark morphology and release flux with our preferred C-apochromat objective and the 63×, 1.4 N.A., oil immersion plan-apochromat (the PSFs of both objectives are compared in methods ). Pooled averages and their standard errors, for 1,850 sparks obtained with the C-apochromat and 1,591 in five experiments in which the system was aligned and all images were acquired with the plan-apochromat were, respectively: amplitude, 1.09 (0.04) and 1.10 (0.05); FWHM (μm), 1.40 (0.07) and 1.57 (0.09); FDHM (ms), 9.10 (0.4) and 9.87 (0.4). None of these differences were significant. (The average amplitudes were smaller than in Table due to the use of a lower detection criterion.) In one experiment, the system was aligned and initial images were acquired with the C-apochromat, and then the fiber was imaged with the plan-apochromat and additional images were acquired. Again, no significant differences were observed between averages of 239 and 198 events obtained with the two objectives. Because sparks were similar, the release algorithm applied to the largest sparks or to spark averages gave similar results in both types of experiments with either objective. The conclusion is that the better axial resolution provided by the oil-immersion objective does not result in a spark of greater amplitude, which may be an indication of differences in the PSF that require more than the FWHM for their description. We continue to prefer the water immersion C-apochromat because the morphology of events imaged with it does not appear to depend on depth within the cell, which allows one to obtain many more images from the same fiber. An algorithm introduced for the evaluation of release flux in cardiac Ca 2+ sparks was modified and applied to sparks of skeletal muscle. The algorithm yields release flux density (Ṙ, a function of space and time), which can then be integrated to obtain total flux (or current). We first tested the consistency of the algorithm (or “backward” calculation) on sparks simulated as the result of Ca 2+ diffusion from a finite source (the “forward” calculation). The backward calculation retrieved approximately the same total current used in the forward construction, over a wide range of release current. There were some deviations, namely the flux density in the backward calculation was ∼55–60% of that in the simulation, and the half width of the region where the back-calculated source resided was slightly greater than the simulation source. These errors appear to be due to the finite spatial and temporal resolution of the experimental methods, which requires discrete and necessarily coarse approximations to steeply space- and time-dependent variables. They approximately compensated each other, however, resulting in a nearly correct determination of total current. A set of more realistic tests, which characterized the performance and limitations of the method when applied to experimental data, included noise in the records. Because Ca 2+ removal and the monitoring dye reaction are mass action-driven processes that occur at finite rates, the release algorithm involves differentiation stages that increase noise. Additionally, it starts with a deblurring step, to undo the blurring introduced by imaging. Deblurring is basically a deconvolution resulting in a greater enhancement of the high (spatial) frequency components of the signal, and a disproportionate increase in noise when applied to real data. To counter these problems, we altered the method in two ways, we used less than full deblurring (that is, deblurring for a sharper PSF than actually measured) and we used smoothing and filtering digital differentiation. The effects of changing the deblurring length (FWHM of the PSF assumed in the correction) was explored on simulated sparks. This study, illustrated in Fig. 4 , shows that increasing the deblurring length gradually increases the estimate of release current. The change is gradual, albeit sizable (in going from no deblurring to full deblurring the current estimate may increase by as much as 40%). In the same tests, noise, added to the simulations at the levels found in spark averages, results in an erratic morphology of derived release beyond a certain point. This translates to a limiting value of ∼0.9 μm for the deblurring length. Using this practical level of deblurring correction, we then asked how would the algorithm depend on the spatial aspects of the source, namely the diameter (because the source is assumed to be spheric). As shown in Fig. 4 , the algorithm underestimated source current by ∼30%, regardless of source diameter. The source radius reported by the algorithm was rather insensitive to changes in actual source geometry below 0.7 μm. This limits the ability of the technique to distinguish between extended and point sources. In summary, the algorithm slightly underestimates source current, slightly overestimates source duration, and reflects source diameter beyond ∼0.7 μm. Its performance is degraded when used with digital low-pass filtering, but release current is never overestimated. The method was applied to an average including every spark of amplitude above 2 F 0 obtained under voltage clamp in the same cell. Because the images were acquired over a short period, the dye concentration varied <10%, which makes it legitimate to average the events and then process the average. There was a poor correlation between amplitude and rise time for these sparks, as well as a low positive correlation between amplitude and half width. This suggests that the selected sparks were not “special”, other than by surpassing the amplitude criterion. Their large amplitude indicates that the sparks originated near the scanning line, were large as objects (i.e., before imaging), or both. The derived source current is represented in Fig. 4 (○) for various values of deblurring length. Comparing with the values obtained with simulations, it appears that the average spark originated from a source of at least 20 pA (the release current in the simulation). The range of source widths derived for the average spark, 0.47–0.7 μm, does not allow one to tell whether the flux originated at a point or an extended source. The algorithm was also applied to sparks from permeabilized fibers immersed in internal solution. This technique had two advantages. Dye concentrations in the few hundred micromolar were achieved rapidly inside the cells, leading to a more robust determination of release flux. An even more important advantage of the permeabilized preparation was the better determination of resting [Ca 2+ ], which should have been close to that in the equilibrating internal solution. The method was applied to individual events or to averages of just a few, which typically required low-pass filtering of the images and did not allow for deblurring. For 19 large sparks from 11 permeabilized cells, the average release current was 14.4 pA, and half width 0.89 μm. Considering the conditions under which the algorithm was applied, the release current is probably an underestimate, and therefore not inconsistent with the estimate of 20 pA obtained under voltage clamp. The large source width derived under these conditions should not be overinterpreted because the algorithm applied to simulations with high noise levels was quite insensitive to source width in the range below 1 μm. Nevertheless, it is interesting that in several experiments source diameters >1 μm were derived. In all those cases, fluorescence sparks had large width. One way in which sparks of such width could be simulated in forward calculations was by assuming source diameters >1 μm. These observations therefore raise the possibility that some wide sparks of high amplitude may originate from sources of resolvable size. One problem with this interpretation is that scanning was perpendicular to the Z disks (and the long axis of the transverse tubules), hence it is difficult to imagine a structural substrate for such extended release sources. The large width of some sparks therefore remains without a convincing explanation or simulation. The present estimates of release current underlying a spark should be compared with single channel currents recorded in bilayers, in conditions as close as possible to physiological. Mejía-Alvarez et al. studied heavy SR cardiac release channels carrying Ca 2+ current in the lumenal-to-cytoplasmic direction, driven by gradients of 2–10 mM, and in the presence of high concentrations of Cs + (as a substitute for the inhibitory effects of K + on Ca 2+ current). At 2 mM lumenal Ca 2+ , and in the presence of symmetric 150 mM Cs + , the single channel current was 0.35 pA. This is not the best possible representation of the physiological situation because Mg 2+ was not included. It is, however, a good representation of our permeabilized fibers, in which [Mg 2+ ] was 0.15 mM. The comparison implies that ∼60 fully open channels are necessary to account for release in the sparks studied here. This number, however, is subject to multiple possible errors in parameter values or in the structure of the model itself. For this reason, we provide two lower estimates of current. One is 8.09 pA, obtained when concentrations of all the removal molecules of the muscle cell model were reduced to one third their consensus values. Another low estimate, not requiring any assumptions about removal processes, was obtained by computing the rate of change of dye-bound Ca 2+ before the fluorescence peak. This estimate is equivalent to the rate of change of “signal mass,” defined by Sun et al. 1998 to evaluate Ca 2+ release underlying InsP 3 -triggered signals in Xenopus oocytes. Its average for the large sparks of Table is 8.28 pA. Using these low estimates, 20 or 25 channels are required to account for the release current. The unitary current could be greater if [Ca 2+ ] SR was higher than the 2 mM used in the bilayer experiments. The range of possible values of [Ca 2+ ] SR is limited, however. In cardiac muscle, [Ca 2+ ] SR has been found to vary within a narrow band , about a decade lower than its thermodynamic limit of 10–20 mM. This agrees with evidence that the SR pump stops working, for kinetic rather than thermodynamic reasons, at a [Ca 2+ ] SR of ∼5 mM . Assuming 5 mM as the maximum possible concentration would increase at most by a factor of 2.5 the single channel current, resulting in a lower limit of eight for the number of channels simultaneously open during the rise time of a large spark. Three recent reviews consider the question of number of channels underlying a spark. Sparks were originally proposed to arise from one or a few channels in cardiac muscle , and one or “one or two” in skeletal muscle. Evidence for a many-channel mechanism has increased with the observation of release in events smaller than sparks and of multifocal sparks . Similarly, it is believed that “calcium puffs” produced by release through InsP 3 receptors are due to activation of multiple channels . Part of the evidence for one- or two-channel mechanisms was the observation of multiple modes in the distribution of event amplitude , but the existence of modes has been contested on the basis of simulations of confocal sampling , and on theoretical grounds . In favor of single channel mechanisms, it has been shown that release under a spark starts and ends abruptly and that Imperatoxin A, which induces prolonged openings of release channels in bilayers, causes the appearance in muscle fibers of long duration events of sizable amplitude . In a model of Stern et al. 1997 , closely packed arrays of release channels interact for activation and inactivation, by virtue of their sensitivity to Ca 2+ . These functionally interacting arrays, termed “couplons,” correspond structurally to all the release channels on one side of one junctional segment of transverse tubule . In the simulations of Stern et al. 1997 , a couplon can activate rapidly, in full or in part, and then inactivate, to generate an event with properties similar to sparks. The above estimate of 60 channels in a large spark is in reasonable agreement with the number of channels that fit on one side of a junctional unit (the units of largest size, 0.9 μm, Protasi and Franzini Armstrong, personal communication, fit a double row of 60 channels at 30-nm spacing). The minimum estimate of eight channels per spark suggests instead partial couplon activation. The present conclusions apply to sparks that were selected for their large amplitude. Examination of the amplitude distribution of sparks in view of the artifacts introduced by the process of line scanning , indicates that there is a wide, perhaps severalfold range of spark currents. The conclusion that the large sparks studied here require at least eight open channels must therefore be accompanied by the realization that other, smaller sparks of the same fibers must involve fewer simultaneously open channels, perhaps leading to a reconciliation of the different views, as sparks would then be produced by the opening of variable numbers of channels. Ca 2 + Release in Sparks | Study | biomedical | en | 0.999998 |
10398692 | 10 yr ago, the gene that is mutated in the hereditary disease cystic fibrosis was cloned and sequenced and named cystic fibrosis transmembrane conductance regulator (CFTR) 1 . In accordance with the reduced chloride conductance of epithelial tissue from cystic fibrosis patients described earlier , CFTR was shown to be a chloride channel that can be activated by cAMP-dependent PKA . Early on, it was recognized that CFTR belongs to a large family of membrane transport facilitators that were called TM6-NBF , traffic ATPases , or ATP-binding cassette transporters . The common feature is a twofold repetition of a transmembrane domain (TMD), often comprised of six hydrophobic sequences, followed by a nucleotide binding fold or domain (NBF or NBD) with the characteristic Walker A and Walker B consensus sequences . Unique to the CFTR is an additional large cytoplasmic domain inserted in the sequence between NBF1 and TMD2 with many consensus sites for phosphorylation by PKA or PKC. This domain was termed the R domain . Although it is undisputed that nucleoside triphosphates are necessary to open CFTR channels after phosphorylation by PKA, the mechanism of action of ATP is the topic of some controversy. In some studies, it was suggested that ATP is hydrolyzed during CFTR gating, based on the inability of the nonhydrolyzable ATP-analogue adenylylimidodiphosphate (AMP-PNP) to open CFTR channels , whereas others concluded nonhydrolytic binding of ATP was sufficient because of a perceived lack of binding of AMP-PNP to CFTR in single-channel studies with heterologously expressed CFTR , or because mixtures of ATP and AMP-PNP stimulated endogenous CFTR . The stimulatory effect of AMP-PNP on CFTR channels opened by ATP was confirmed later and a model was presented where ATP hydrolysis at one NBF (termed NBD-A) leads to channel opening, and ATP binding at another NBF (termed NBD-B) prevents channel closing . The effect of AMP-PNP was interpreted as reflecting its ability to bind but not be hydrolyzed at NBD-B, where ATP is normally hydrolyzed . Most studies of CFTR gating have investigated single-channel or multichannel nucleotide-dependent gating under steady state conditions. Under these conditions, it may be difficult to separate the effects of a nucleotide interacting in more than one way with the two NBDs of the CFTR. Therefore, we designed experiments under pre–steady state conditions and followed the relaxation of large populations of CFTR channels to a new steady state. To achieve fast access to the cytosolic membrane surface, and to improve the signal-to-noise ratio, we used the giant patch technique , using pipettes with a tip opening diameter of >20 μm. This allowed recording of an initial PKA and ATP-induced chloride current corresponding typically to ∼4,000 simultaneously open CFTR channels . We employed flash photolysis of ATP-P 3 -[1-(2-nitrophenyl)ethyl]ester or fast solution changes to generate rapid changes in the nucleotide concentration. In this manner, the relaxation current seen upon ATP addition to closed channels as well as the current decay upon ATP removal could be analyzed, providing new information on the nucleotide dependence of CFTR opening. An up to now not observed binding of AMP-PNP to NBD-A could be clearly demonstrated, which clarifies further the requirements for CFTR channel opening. The newly observed effect of ADP-induced acceleration of channel closing leads to a revision of current gating cycle schemes. cRNA was prepared from the plasmid, pCFTR(SP), containing the human CFTR cDNA as described . Intact ovary lobes were removed from female Xenopus frogs anaesthetized by immersion into a tricaine solution (0.2%) for 5–10 min. Oocytes were prepared and injected with the CFTR cRNA as described previously . Oocytes were kept in a modified Ringer's solution containing 110 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.6, and incubated at 18°C for 2–4 d before measurement. Oocytes were shrunken by brief immersion in a hypertonic solution containing 200 mM potassium aspartate, 20 mM KCl, 2 mM MgCl 2 , 5 mM EGTA, 10 mM HEPES, pH 7.4. Thereafter, the vitelline membrane could be removed using sharpened watchmaker's forceps. The oocyte was placed in a small petri dish (35 mm) filled with 60 mM N -methyl-glucamine-Cl, 40 mM NaCl, 20 mM tetraethylammonium-Cl, 2 mM MgCl 2 , 5 mM EGTA, 10 mM HEPES, pH 7.4, which was mounted on the stage of an inverted microscope (Zeiss Telaval 31; Carl Zeiss Jena). Seals were formed using borosilicate glass pipettes with tip openings of 18–24 μm. The pipette solution contained 150 mM N -methyl-glucamine (NMG)-Cl, 2 mM BaCl 2 , 2 mM MgCl 2 , 0.5 mM CdCl 2 , 10 mM HEPES, pH 7.4. To accelerate seal formation, slight suction was applied to the pipette. Withdrawal of the pipette yielded large inside-out membrane patches having seal resistances of 2–20 GΩ. The excised patch was then transferred to a temperature controlled chamber . The recording chamber was continuously perfused at a rate of 0.8–1.2 ml/min with a standard solution composed of 140 mM NMG, 20 mM tetraethylammonium-OH, 5 mM glutathione (reduced form), 5 mM EGTA, 2 mM MgCl 2 , 10 mM HEPES, titrated to pH 7.4 with aspartate, resulting in a chloride gradient between pipette and bath of ∼40:1. To this solution, additions were made from concentrated stock solutions of nucleotides and other drugs. Electrical valves (General Valve, modified by us) were employed for solution switching. The bath volume was maintained at a low level (10–15 μl) by continuous vacuum-driven solution drain from the chamber. To perform time-resolved relaxation measurements after a concentration jump, the system time constant for the solution exchange in the measuring chamber had to be determined. To do this, the chloride current through the oocyte's endogenous calcium-activated chloride channels was recorded. When these channels were opened by elevating the calcium concentration in the bath, subsequent changes in the chloride concentration of the bath produced current steps, the time course of which depended on the speed with which the solution in the measuring chamber was exchanged . When a logarithmic dependence of the current amplitude on the chloride gradient across the membrane and a continuous dilution of the buffer in the chamber was assumed, a time constant for the solution exchange could be obtained from the current record . This system time or solution exchange time constant was expected to vary depending on the position of the pipette within the chamber, the perfusion rate, as well as the geometry of the pipette tip, and was found to be in the range of 15–150 ms in most cases. The calibration for the solution exchange time constant was done at least once during each patch clamp experiment. Measurements of nucleotide-induced relaxations were rejected for kinetic analysis if the observed relaxation time constants were not significantly (i.e., by a factor of five or more) larger than the solution exchange time constant. The experimental conditions also necessitated a delay (system delay or solution exchange delay) between the switching of the valve and the arrival of the solution at the pipette tip. This solution exchange delay could be measured as the time between valve switching and the onset of the current response. It was determined to 0.5 ± 0.1 s for the chloride concentration changes and 0.7 ± 0.2 s for changes from 0 to 500 μM ATP, whereas it was 1.6 ± 0.5 s for changes from 500 to 0 μM ATP \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;11)\end{equation*}\end{document} . We therefore concluded that the delay observed with ATP addition is not significantly different from the “solution exchange delay” (see also caged ATP photolysis experiments, below), whereas the delay observed with ATP removal is significantly longer. Although this delay time is considered to reveal a valuable parameter of CFTR gating, it was not included in the analysis of this study. Under the conditions used to measure CFTR currents, the calcium-activated chloride channels did not contribute to the current signal, as intracellular calcium was kept low by the addition of the chelator EGTA. The chamber was further equipped with a duct through which a quartz fibre serving as a light guide could be positioned in close proximity (100–200 μm) to the pipette tip . 10-ns light pulses of 308-nm wavelength generated by a XeCl excimer laser (Lambda Physik) were fed into the light guide. The UV light was employed to generate rapid ATP pulses by photolysis of the photolabile NPE-ATP. Due to the short pulse duration of the excimer laser, it was not usually possible to liberate more than one third of the NPE-ATP during a single laser pulse, as the energy densities required for near-complete photolysis would cause the patch membrane to disintegrate. An example of a current induced by incomplete photolysis of 450 μM NPE-ATP is shown in Fig. 1 C. The rise in current resulting from the ATP liberation was not instantaneous, but showed a short delay of several milliseconds that may be attributed to the time required for the photolysis reaction to generate ATP. More importantly, the signal rise time was rather slow with time constants of 500–1,500 ms if an exponentially rising signal was assumed. As was obvious from the slow rise times seen with a 10-ns pulse, the pulse duration could safely be extended to several milliseconds using a low-power continuous wave He/Cd laser of 325-nm wavelength (Kimmon Electric Co., Ltd.) without compromising the time resolution of the current signal. The 325-nm continuous laser beam was pulsed by means of an electrical shutter device (Uniblitz; Vincent Associates) with a response time of ∼1 ms. Pipette current was measured with an EPC-7 patch clamp amplifier (List Medical) at a holding potential of 0 mV. Seal resistance was checked before and after each experiment. All experiments were carried out at 25°C. Membrane currents and solution switching pulses were routinely recorded on a chart recorder (Kipp & Zonen). Currents were filtered online at 20 Hz and continuously recorded at 100 Hz on the hard disk of a personal computer using KAN1 software (MFK; Michael Friedrich, Neidernhausen, Germany). In addition, currents filtered at 100 Hz were recorded at 500 Hz on a second personal computer using PCLAMP 6 software (Axon Instruments), which was also employed to trigger the laser pulses and for electric valve switching. Relaxation currents were fitted using either the PCLAMP simplex fitting algorithm or the Levenberg-Marquardt fitting tool from the ORIGIN 5.0 analysis program (Microcal Software). Mg-ATP (from equine muscle), ADP, and AMP (free acid) were from Sigma Chemical Co., NPE-ATP was from Molecular Probes, Inc., AMP-PNP was from Boehringer Mannheim, and PKA catalytic subunit was from Promega Corp. Buffer chemicals were from Merck or Sigma Chemical Co. When a giant patch was excised from an unstimulated oocyte expressing human CFTR channels, activity in response to the channel opener ATP was negligible, indicating a low basal PKA activity in the resting oocyte. Application of exogenous PKA catalytic subunits in conjunction with ATP gave rise to a substantial chloride current in the range of hundreds of picoamperes to a few nanoamperes , recorded at a holding potential of 0 mV and with a chloride gradient of 150 mM:4 mM between pipette and bath. Upon removal of the kinase, the signal underwent first a rapid and subsequently a slow rundown that was partly reversible by readdition of the PKA . In the absence of PKA and in the presence of millimolar concentrations of magnesium ions, the prephosphorylated channels could be opened by ATP alone. The application of an ATP pulse thus provided a means to analyze the kinetics of channel gating from the multichannel current signal. To judge whether the observed relaxation kinetics were determined by the interaction of ATP with CFTR or by the solution exchange at the patch membrane, the solution exchange speed was always calibrated with chloride concentration jumps under conditions when endogenous chloride channels in the oocyte membrane were open (see the description in methods ). This provided a CFTR-independent estimation of the solution exchange time and of the delay between valve switching and arrival of the solution at the patch membrane. When ATP was added to closed channels, the current followed an exponential time course in approaching the new steady state level. Usually, relaxation kinetics could best be approximated by the sum of two exponentials with a fast time constant in the range of several hundreds of milliseconds that accounted for 60–95% of the signal amplitude and a slow time constant ∼10× greater that was often difficult to determine because of its small amplitude . The slow time constants, as well as the above mentioned delay times, were not systematically evaluated. In most cases, therefore, our analysis is restricted to the rates of the fast-relaxing current component. When ATP pulses were applied consecutively, the resulting currents exhibited a time-dependent change in both signal amplitude and relaxation rates. The relative amplitude of the slowly relaxing current always diminished with time and the signal could then be approximated reasonably well by a monoexponential function. This is shown in Fig. 2 for the current rise upon ATP addition, but was similarly observed for the current decay upon ATP withdrawal. For the kinetic analysis, the slowly relaxing amplitude of the current signal was neglected and the signal was fitted with a simple monoexponential function using only the initial part of the record. In all cases (68 of 68 experiments) the rundown of the current signal was accompanied by an acceleration of the fast relaxation rate. Presumably because of differing phosphatase activity in different patches, the relaxation rates exhibited considerable variance between measurements (see below). This necessitated the use of a standard ATP jump (between 0 and 500 μM ATP) as an internal reference, which was applied at regular intervals during the course of an experiment. The average value of all these standard rates was 1.2 ± 0.4 s −1 (means ± SD, n = 189) for the current rise after ATP addition and 0.8 ± 0.4 s −1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{means}}\;{\pm}\;{\mathrm{SD}},\;n\;=\;183)\end{equation*}\end{document} for the current decay after ATP removal. Referencing relaxation rates to the standard rate enabled us to compare normalized relaxation rates obtained from different patches. Because ATP jump experiments were performed after much of the rundown had already taken place , the change in the kinetics over time was not large. To confirm the physiological significance of the observed relaxation rates, a second approach was followed to complement the calibration via chloride concentration jumps (see materials and methods ). It has been shown for CFTR reconstituted into a black lipid membrane that the photolabile ATP analogue NPE-ATP can be used to open the CFTR after photolytic cleavage to ATP and 2-nitrosoacetophenone. The photolysis reaction proceeds with a rate of ∼40 s −1 at pH 7.4 due to a [H + ]-dependent rate-limiting step in the liberation of ATP from its caged precursor . Since the unphotolyzed NPE-ATP caused inhibition of the ATP-induced current in mixtures of ATP and NPE-ATP (data not shown, but see below), near complete photolysis of the caged ATP seemed necessary to avoid a distortion of the relaxation signal. The energy density and thus the efficiency of the photolysis reaction could be regulated by the length of the light pulse employed to liberate the ATP. As shown in Fig. 3 A, a photolysis time of 50 ms was necessary for a release ratio of >90%. The relaxation rates after a photolytically engineered ATP jump were compared with the relaxation observed after an ATP jump by rapid solution change and were found to be virtually identical . Thus, by means of flash photolysis of NPE-ATP, we were able to ascertain that the fastest component in the current rise after an ATP jump had a time constant of several hundred milliseconds and could thus readily be resolved by rapid solution switching. Because removal of ATP from the membrane was only possible by perfusing the bath with an ATP-free solution, a comparison between ATP jumps by solution exchange and by photolysis was limited to ATP addition. Nevertheless, because the kinetics of ATP removal showed relaxation rates in the same time range as those of ATP addition and because these were slower than the routinely performed chloride concentration jumps (yielding the solution exchange time), we are confident that the rates obtained with rapid solution exchange reflect the true kinetics of the channels. The observed inhibition of the ATP-induced steady state current by NPE-ATP was investigated by means of relaxation experiments. When channels were incubated with NPE-ATP and the NPE-ATP was subsequently replaced by ATP, an additional slow component was introduced into the relaxation kinetics . This slowly relaxing current was not seen in the absence of NPE-ATP or when the NPE-ATP was converted into ATP by photolysis . The rate of the fast relaxing component was not affected by the NPE-ATP nor was the relaxation when ATP was replaced by NPE-ATP. This shows that NPE-ATP may bind to the CFTR in the absence of ATP; i.e., when all channels are closed. Although the concentration dependence of the effect of NPE-ATP was not investigated, the slowly relaxing component in the current after ATP addition most likely is caused by those channels that have bound NPE-ATP, which prevents ATP binding until it has dissociated. The fast relaxing component then represents those channels without bound NPE-ATP that can open normally once ATP is applied. As shown for NPE-ATP, the comparison of a standard ATP-induced relaxation current with the relaxation signal after preincubation with other substances allows detection of interactions with the closed channel. When this is done using the nonhydrolyzable ATP analogue AMP-PNP instead of NPE-ATP, a slow component is again introduced into the relaxation after ATP addition , indicating binding of AMP-PNP to the closed channel (at NBD-A) resulting in competitive inhibition of ATP binding. If the presumed competitive binding of AMP-PNP and ATP at NBD-A was the only AMP-PNP effect on CFTR gating, no alteration of the relaxation after ATP removal is to be expected. This is true for NPE-ATP, but not for AMP-PNP, as shown in Fig. 5 . When ATP was removed in the presence of 500 μM AMP-PNP, an additional slow component with a relaxation rate of 0.033 ± 0.008 s −1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;4)\end{equation*}\end{document} was observed. The amplitude of this slow component varied and appeared to be higher if AMP-PNP was applied early after phosphorylation; i.e., before most of the rundown had occurred. In all cases, however, more than half of the total amplitude was accounted for by a fast relaxing current with a relaxation rate indistinguishable from the rate in the absence of AMP-PNP, indicating that the greater part of the channels was not affected in its kinetics by AMP-PNP. Next, the dependence of the ATP jump relaxation rates on the ATP concentration was determined. There was a pronounced dependence of the relaxation rate after ATP addition ( k ad ) on the ATP concentration in the range from 25 μM to 2.5 mM. The ATP dependence could be reasonably well fitted by a simple saturation function: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{{\mathrm{ad}}}\;=\;k_{{\mathrm{max}}}\;*\;[{\mathrm{ATP}}]/(K_{1/2}\;+\;[{\mathrm{ATP}}])\end{equation*}\end{document} , with a K 1/2 of ∼100 μM ATP . An even better fit could be obtained by a saturation function including a basal rate k 0 : k ad = k 0 + k max * [ATP]/( K 1/2 + [ATP]), with a K 1/2 of 190 μM ATP, a k max of 1.35 s −1 , and a basal rate k 0 (at 0 ATP) of 0.22 s −1 , leading to a maximal rate (at saturating ATP) of ∼1.6 s −1 . The fitted basal rate k o seems high. It is difficult to determine it more accurately by further lowering the ATP concentration as the resulting amplitudes are usually very small and lead to a greater error. It is, however, an important observation that this basal rate is significantly lower than the relaxation upon ATP removal. This observation already excludes certain simple gating models similar to ligand-gated channels. The relaxation rate obtained after ATP removal was not sensitive to the ATP concentration present before ATP removal . This relaxation rate was always the same when normalized to the standard rate (which was itself not a constant but subject to a time-dependent acceleration, as described earlier). Such a dependence is to be expected if only ATP-independent events contribute to the relaxation. The ATP-invariant fast closing rate is therefore an additional proof that the rate of solution exchange is not limiting for the observed relaxation rates. If the solution exchange rate were limiting for the observed current decay, then the removal of high ATP concentrations would result in a slower current response than the removal of low concentrations of ATP. Finally, we investigated the interaction of ADP with the CFTR, which is known to inhibit ATP-induced opening . The inhibitory effect of ADP on the ATP-induced steady state current was measured at 500 μM ATP and yielded an apparent K I of 215 μM . This translates to a K I of 31 μM, at a K m for ATP of 84 μM , assuming a competitive binding of ATP and ADP to the CFTR, as suggested by Anderson and Welsh 1992 and Schultz et al. 1995 . When an ADP to ATP jump was performed and the resulting relaxation current compared with that of a standard ATP jump, a single relaxation rate was found, which was significantly reduced . This is at variance with the NPE-ATP to ATP or AMP-PNP to ATP jumps, where a biphasic relaxation was observed. Increasing the ADP concentration from 0.5 to 1 mM caused only a small reduction in the normalized relaxation rate from 0.43 ± 0.08 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document} to 0.40 ± 0.09 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;5)\end{equation*}\end{document} , indicating that at 500 μM the ADP-induced effect on the relaxation rate had almost reached saturation. Given that a competitive binding of ADP to the ATP binding site responsible for channel opening occurs, preincubation of the channels with ADP should result in a delayed opening and therefore a lower relaxation rate. Since no biphasic relaxation was seen, it must be postulated that under the conditions employed here all channels have bound ADP at the time the ATP is applied. This is to be expected with 500 μM ADP and a K I of 31 μM, although further experiments should determine the exact K I of ADP binding to closed channels under these pre–steady state conditions. The inhibitory effect of ADP on channel opening should be even more pronounced if ADP is present throughout the entire ATP pulse. In this case, ADP that dissociates from the binding site may be replaced by either ATP or ADP, depending on the concentration ratio of the two nucleotides, thereby reducing the rate of ATP binding. This reduced relaxation rate was indeed observed as shown in Fig. 9 . When an ATP jump in the presence of 500 μM ADP was performed, the relaxation rate of ATP-induced opening was by a factor of 4.0 lower than in the absence of ADP and by a factor of 1.7 lower than the relaxation rate after an ADP to ATP jump . The steady state amplitude of the chloride current was also reduced, as expected from the ADP titration shown in Fig. 7 B. When ADP was replaced by AMP, which was reported to have no effect on CFTR gating , the relaxation was not altered , indicating that the observed effects were specific for ADP. Interestingly, besides reducing the relaxation rate after ATP addition, ADP enhanced the relaxation rate of channel closing by a factor of ∼1.7 without introducing additional components into the current signal. This effect seemed to saturate at high ADP concentrations since a doubling of the ADP concentration from 0.5 to 1.0 mM lead only to a 25% increase in the relative relaxation rate from 1.6 ± 0.4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document} to 2.0 ± 0.3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;6)\end{equation*}\end{document} . Again, equally high concentrations of AMP did not exhibit any effect on the relaxation kinetics. The accelerating effect of ADP on the relaxation rate was the same regardless of whether or not ADP was present before ATP removal , providing further evidence that only channel closing events contribute to the current signal after ATP removal. The significance of this finding, which to our knowledge has not been reported thus far, will be discussed later with respect to a possible CFTR gating cycle. This study introduces a new experimental approach to efforts to gain insight into the complicated regulation of CFTR channel gating by nucleotides. We applied fast changes of nucleotide concentrations and measured the relaxation of many CFTR channels to a new equilibrium. This pre–steady state experiment is a different and complementary approach to single channel studies that measure rate constants under steady state conditions. In our discussion, we use the terms NBD-A and NBD-B to differentiate between two nucleotide binding sites as observed in this and previous studies and introduced by Hwang et al. 1994 . These do not necessarily correspond to the sequence-based nucleotide binding folds 1 and 2 . Our experimental approach could only provide meaningful results if the concentration changes are faster than the observed relaxations that were tested by two independent procedures. The time constant of solution exchange was tested for each individual patch and was faster than 150 ms in all experiments used for analysis. The observed relaxation rates were at least five times slower than this solution exchange rate. An additional independent test of the validity of the relaxation rates obtained by solution exchange was provided by flash photolysis of caged ATP to generate a fast increase in ATP concentration. The observed relaxation was indistinguishable from the one obtained by a fast solution change . Further support for the validity of the observed relaxation rates comes from the [ATP] dependence of the relaxation rates upon ATP withdrawal. We interpret these data under the assumption that the observed relaxations of the transmembrane chloride current are caused by conformational changes of CFTR from one or several closed states to one or several “activated states” with a certain open probability or vice versa. The open probability of this activated state might be 1 (i.e., the activated state is an open state) or it might be >0 and <1 (i.e., a fluctuation between an open and a closed state). By stepping the ATP concentration from zero to a given value and back to zero again, we were able to generate two different types of relaxation currents. The current recorded after ATP addition is governed by the kinetics of channel opening and closing, since ATP is present to support the complete gating cycle. The current relaxation recorded after sudden ATP withdrawal might only depend on the kinetics of channel closing or, if ATP-bound closed channels open equally fast or faster than ATP dissociates, might again depend on the kinetics of channel opening and closing. Although determination of rate constants for channel opening and closing from single channel measurements have produced a wealth of valuable information about nucleotide-dependent CFTR gating , at present stage it is not at all clear if one ATP-binding/hydrolysis event leads to only one channel opening or better opening burst. It is well established that enzymes that catalyze an ATP-driven transport cycle can undergo a shuttle between intermediary states that leads, for example, to Na i + /Na o + exchange in the case of the energized (phosphorylated) Na/K-ATPase . Similarly we cannot a priori exclude that, in the ATP hydrolysis cycle of CFTR, energized CFTR might shuttle between an open and a closed state before finally relaxing to the (low energy-) closed state that strictly needs ATP to open again. We imagine that it might be difficult to discern these hypothetical intermediary (energized) closed states from the “final” (low energy-) closed state. The ability to discern or to dismiss this possibility depends on the individual rate constants and on the ability to collect enough single channel data under stable phosphorylation conditions. To keep CFTR channels stably phosphorylated for a long enough time is a big problem not only in these experiments, as outlined below, but even more so for single channel measurements when considering the much smaller data base. As reported earlier and shown in Fig. 2 , CFTR channel activity undergoes a continuous rundown after the withdrawal of PKA. At least a part of this rundown can be explained by dephosphorylation of CFTR as it can be reversed (to a great part at least) by addition of PKA . Since both the amplitude of the steady state chloride current and the time constants for the relaxation to the activated state are changing, we can conclude that dephosphorylation is not an all or nothing event, but that gradual dephosphorylation leads to graded changes in CFTR gating as already described earlier . The rundown accompanying the change from a two- to a mono-exponential relaxation can be explained on the basis of the postulated phosphorylation-dependent involvement of two nucleotide binding folds in channel gating, suggesting ATP binding to only one of the two NBDs (NBD-A) in CFTR when there is a low degree of phosphorylation. Binding of ATP to the second NBD (NBD-B) may stabilize the channel in its open conformation, thereby slowing the gating cycle, which might lead to the observed slow relaxation. This binding of ATP to NBD-B, however, was found possible only if CFTR was sufficiently highly phosphorylated . Early on, it was found that the hydrolysis-resistant ATP-analogue AMP-PNP is unable to open prephosphorylated CFTR . From this observation, it was concluded that ATP hydrolysis is necessary for ATP-induced opening of CFTR channels. This was later disputed because single-channel measurements with mixtures of AMP-PNP and ATP led to the conclusion that AMP-PNP does not bind to CFTR . In contrast to these equilibrium measurements, our relaxation measurements enable detection of AMP-PNP binding to closed CFTR channels even though this binding does not lead to channel opening. The observed slowing of ATP-induced opening of CFTR channels when ATP was applied concomitant with AMP-PNP removal is a clear indication of AMP-PNP binding to closed CFTR channels via NBD-A, similar to ADP binding (see below). Although such an inhibitory binding of AMP-PNP to NBD-A should be observed also under steady state conditions, so far it has not been, presumably because of the strong stimulatory effect of AMP-PNP on NBD-B. Such an inhibitory binding of AMP-PNP to NBD-A was postulated by Mathews et al. 1998 to explain slowed locking into the open state caused by AMP-PNP. Therefore, Fig. 5 presents the first direct demonstration of inhibitory binding of AMP-PNP to NBD-A. From the amplitude and time dependence of recovery from the AMP-PNP–induced inhibition, an apparent binding constant in the range of several 100 μM and an off rate of ∼0.05 s −1 can be estimated. Similarly, preincubation with NPE-ATP slowed activation by ATP, suggesting binding of NPE-ATP to NBD-A. On the other hand, NPE-ATP does not seem to bind to NBD-B because the current relaxation when switching from ATP to NPE-ATP was very similar to the one observed when switching to no nucleotide . The slight difference in the observed slow relaxations with small amplitude might either reflect residual ATP in the caged ATP solution (typically 0.5%), or indeed very low affinity binding of NPE-ATP to NBD-B. This seems unlikely, however, because we found inhibition of the steady state ATP-activated current in mixtures with NPE-ATP, in accordance with its competitive binding to NBD-A (data not shown). A stimulating effect of AMP-PNP on CFTR activity under certain conditions was reported for endogenous CFTR in sweat glands , for endogenous cardiac CFTR , and for black lipid membrane–reconstituted human CFTR . In apparent contrast to these observations are studies by Schultz et al. 1995 , Schultz et al. 1996 , who found neither an inhibiting nor a stimulating effect of AMP-PNP. One explanation for these conflicting results is the strong temperature dependence of the AMP-PNP effect, as noted already by Schultz et al. 1995 and shown in more detail by Mathews et al. 1998 . An important modulator of the action of AMP-PNP on CFTR is the degree of its phosphorylation. It was shown that higher phosphorylation corresponds to a higher open probability of CFTR and is a prerequisite of the “open-locking” action of AMP-PNP . We found that a portion of the channels showed a much delayed closing rate in the presence of AMP-PNP, whereas other channels did not show an altered closing rate when AMP-PNP was present, resulting in a biphasic relaxation current . Obviously, only a subpopulation of channels had the potential to become locked by the binding of AMP-PNP to NBD-B, which might have several causes. Probably not all channels are sufficiently phosphorylated to enable binding of ATP or AMP-PNP to NBD-B. Sufficiently phosphorylated channels, on the other hand, can only bind AMP-PNP on NBD-B if this site is not already occupied by ATP and if the concentration of AMP-PNP is high enough to allow fast binding before the channel relaxes to the closed state, which needs ATP to open. As outlined in methods , at least three time constants might be found in the current relaxation upon ATP withdrawal: a delay time constant, a fast relaxation with a large amplitude, and a slow relaxation with a small amplitude. For this study, we analyzed only the fast relaxation. It is interesting to compare this rate of ∼0.8 s −1 to closing rates obtained from single channel studies. The closing rate is the inverse of the mean open (burst) time obtained from a single channel experiment. Published mean open burst times range from 200 ms to ∼1 s at 25°C or room temperature, corresponding to closing rates in the order of 1–5 s −1 . These mean open burst times are probably influenced by the degree of phosphorylation of CFTR as it is observed for the fast relaxation rate of closing (0.8 s −1 ) in this study. The tentative agreement between these rates might allow the speculation that ATP hydrolysis leads to one open burst that is a priori assumed in most single channel studies. So far, however, we have no real proof for this assumption. The main impediment to a careful comparison of closing rates and the closing relaxation rate is the uncertain phosphorylation status of CFTR in this study, as well as in all previous single channel studies. The relaxation upon ATP addition on the other hand appears to have no delay time constant, as evidenced by experiments with photolysis of caged ATP (NPE-ATP). It can be fitted by a fast relaxation with a large amplitude and a slow relaxation with a small amplitude. Again, we only analyzed the fast relaxation. The inverse time constant of the fast relaxation shows a saturating dependence on the ATP concentration that could be fitted best by a model with a maximal value of ∼1.5 s −1 , a K 1/2 of ∼200 μM, and a basal value of ∼0.2 s −1 . The maximal rate at saturating ATP concentrations corresponds quite well to the opening rate (i.e., the inverse of the long and ATP-dependent mean closed time) derived from single-channel measurements at saturating ATP in several single channel studies . If only ATP binding and dissociation would gate CFTR like proposed in a simple three-state model , the basal rate of opening and the closing rate should have the same value. The clear deviation therefore is evidence for irreversible steps in the gating cycle, as expected from the contribution of ATP hydrolysis. A recent publication by Ramjeesingh et al. 1999 questions the assumption of a tight coupling between ATP hydrolysis and channel gating. In fact, their time constants for open time bursts and interburst duration for wild-type (WT) CFTR (roughly 150 ms) are too fast to agree with our observed relaxation time constants and would suggest several openings and closings during one ATP hydrolysis cycle. This would be contrary to present models where channels are opened by ATP binding or hydrolysis , and close after dissociation of the last hydrolysis product. Their conclusions were derived from studying the gating (temperature not specified) and the ATPase activity (at 30°C) of purified CFTR protein, either WT or the mutants K464A or K1250A. However, in contrast to their open burst duration for the mutant K1250A of 265 ms (temperature not specified) are dramatically increased open burst durations found by other groups . Carson et al. 1995 found an open burst duration for K1250A at 34–36°C of ∼1 s. Further experiments are needed to establish the “tightness” of coupling between ATP hydrolysis and CFTR gating. Therefore, we use in our cyclic models of CFTR gating an activated state A instead of an open state O in order to not exclude a priori the possibility of several openings and closings during the ATP hydrolysis cycle. The term “activated” was also used in the model proposed by Gunderson and Kopito 1995 . In their model, CFTR gets activated by ATP hydrolysis at NBF1, but the channel can only open after ATP binding at NBF2. With activated, we mean that the CFTR channel is able to open once or several times. ADP was recognized as an inhibitor of CFTR soon after the activating role of ATP was detected . Its inhibitory mechanism could be modeled by competitive binding to the nucleotide binding site. Studies with mutants suggested that ADP binds to NBF2 . Our results confirm the strong inhibitory action of ADP with an apparent K I of 215 μM at an ATP concentration of 500 μM, which for competitive inhibition corresponds to a K I of 31 μM compared with a K m for ATP of 84 μM. Our pre–steady state experiments when activation by ATP is initiated concomitant with ADP removal showed a clear reduction in the relaxation rate, suggesting competitive binding of ADP and ATP to the nucleotide binding site involved in channel opening (NBD-A). Contrary to our expectations, however, we also found a significantly increased relaxation rate upon ATP removal when the ATP-free solution contained ADP. It is obvious that this finding cannot be explained by a competition between ATP and ADP at an empty NBD-A site on the closed (not activated) channel. In the absence of ATP, only interactions with the activated channel can contribute to a change of the relaxation rate because a shift from closed channels to closed channels with bound ADP is electrically “silent” under these conditions. We have to conclude, therefore, that ADP may also bind to the activated channel. We propose two possible mechanisms for the observed accelerated closing by ADP, depending on whether ADP acts at NBD-A or NBD-B. The two models are distinguished by the order of release of the two hydrolysis products; i.e., ADP or P i first. We will first discuss a possible explanation of the ADP effect on the basis of a single hydrolysis cycle (at NBD-A). This requires ADP binding to NBD-A after the channel has been opened by ATP interaction at NBD-A. In this case, ADP may only bind after ADP generated by hydrolysis of ATP has been released from NBD-A. ADP would then act via product inhibition, with the channel driven back to the closed state by reversion of ATP hydrolysis, in this way rebuilding ATP. This is thermodynamically feasible since the process takes place at a very low ATP:ADP ratio (during the relaxation, [ATP] is essentially zero, while [ADP] is 0.5 mM). Also, to explain the observed acceleration of channel closing by ADP, the rate of closing by reversal of activation must be about the same as the rate of closing by completion of the ATP hydrolysis cycle. This explanation for the effect of ADP on the rate of channel closing requires that during the ATP hydrolysis cycle ADP is released before P i , because otherwise reversion of ATP hydrolysis is impossible under our conditions where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{P}}_{{\mathrm{i}}}]\;=\;0\end{equation*}\end{document} . This is illustrated by the gating model shown in Fig. 11 . In Fig. 11 A, a general model of a gating cycle driven by ATP hydrolysis at a single site is presented. The channel is enabled to open simultaneous with or after ATP hydrolysis and loses this ability when the two hydrolysis products, ADP and P i , are released. This general model does not specify at which step in the hydrolysis cycle the closed-to-open and open-to-closed transitions are located, nor does it specify the order of release of the two hydrolysis products. Included in the model is the competitive binding of ADP to the closed channel (state C 1 ), which explains the slowing of opening upon a change from ADP to ATP, but cannot explain the acceleration by ADP of the relaxation upon ATP withdrawal. To incorporate the binding of ADP to the activated channel into the model, X 2 must be an activated state and must be able to bind ADP. This implies that ADP is released before the phosphate. If one assumes that the closed-to-activated transition is coupled to ATP hydrolysis, rather than to ADP release, X 1 is also an activated state. This results in the model in Fig. 11 B with two activated states, A 1 and A 2 , and three closed states, C 1 , C 2 , and C 3 . The ratio of A 1 to A 2 is dependent on the ADP concentration. If [ADP] is low, the channels will close via the A 2 –C 1 transition. At high [ADP], however, the A 1 :A 2 ratio will be higher, allowing the channels at low [ATP] to close via the A 1 –C 2 transition, which may have a similar or higher rate than the A 2 –C 1 transition, resulting in the accelerated relaxation rate observed in our measurements. Although attractive on the grounds that only one NBD has to be invoked (in accordance with our observation of a single-exponential accelerated decay), this explanation is in contrast to previously published models about the hydrolysis cycle of CFTR or Pgp, another ATP-binding cassette transport protein , all of which argue for the phosphate release to precede the ADP release. To explain the observed effect of ADP on the “closing” relaxation with a model where phosphate release precedes ADP release, we therefore had to consider ADP binding to a second site. Previously, it was shown that binding of nucleotides (specifically AMP-PNP) to a second site, NBD-B, modulates channel closing . Therefore, ADP binding to NBD-B could accelerate one of the steps in the hydrolysis cycle at NBD-A, and consequently lead to a higher channel closing rate as seen in our relaxation experiments. A model that explains the closing of CFTR channels as dependent on nucleotide binding to NBD-B was already proposed by Hwang et al. 1994 . According to this model, ATP binding to NBD-B is only possible for highly phosphorylated CFTR, whereas no data were available for ADP binding to NBD-B. As can be inferred from the only partial effect of AMP-PNP on channel closing, our data were obtained from partly dephosphorylated channels, but show ADP-induced acceleration of closing for all CFTR channels. Assuming binding of ADP to NBD-B, this could indicate that NBD-B is accessible to ADP regardless of the phosphorylation state. Recently, Senior and Gadsby 1997 proposed for highly phosphorylated channels that NBD-B always has bound nucleotides (ATP or ADP) similar to a G-protein. Although this model was further modified by an additional pathway, closing without ATP binding at the second site , it cannot explain all of our data. Therefore, we have adapted it by inserting an additional alternative pathway into the cycle and by eliminating the need for constantly bound nucleotide at NBD-B. In the presence of ATP, if NBD-B is accessible, the gating cycle is driven by ATP hydrolysis at both NBDs. NBD-B becomes accessible to nucleotides only after ATP is bound and hydrolyzed at NBD-A, when the channel is activated. In addition to the complete cycle via S4 and S5, which requires ATP binding to NBD-B, two shortcuts in the cycle may occur. One of these bypasses nucleotide binding to NBD-B and allows the channel to close from the S3 state. This corresponds to the closing in the absence of any nucleotide as observed in the relaxation after ATP removal. Although this is the shortest cycle in the scheme , it is not the cycle with the shortest open duration, see below. In the model proposed by Gadsby and Nairn 1999 , the corresponding states S1, S2, and S3 have bound ADP on NBD-B, which we did not include for simplicity and because we have no experimental evidence for it. NBD-B–bound ADP in states S1 and S2 is not in contrast to our experimental findings; however, NBD-B–bound ADP in S3 is in contrast as ADP led to the shortest open duration (i.e., fastest closing relaxation) in our experiments. Therefore, we postulate that a third pathway allows ADP binding to S3, thereby bypassing state S4. The observed acceleration of the closing relaxation by ADP could then be explained if S3 were long-lived compared with S5 so that the transition S3–S5–S1 becomes faster than S3–S1. This implies that ADP bound to NBD-B speeds the rate of release of hydrolysis products from NBD-A. If, as in our model, S1 has no bound ADP on NBD-B (which we cannot determine presently, see above), the release of ADP from NBD-B must also occur rapidly during the S5–S1 transition. In the presence of ATP, ATP binding to NBD-B might occur in the state S3 leading to state S4, and the transition rate from S4 to S5, which may be slow, will determine the time the channel remains activated. If this explanation is correct, competition between ATP and ADP not only regulates the likelihood of channel opening, but the rate of channel closure as well. Based on our relaxation experiments, we cannot yet discriminate between the two possible modes of ADP interaction with the activated channel. It is clear, however, that the inhibitory action of ADP is more complicated than previously thought, since a competition with ATP for binding at the activating site (NBD-A) alone cannot explain all our observations. By observing the relaxations of large numbers of CFTR channels under pre–steady state conditions, we have shown that the time course of channel opening and closing is modulated by the presence of different nucleotides. The nonhydrolyzable ATP analogue AMP-PNP delays closing of CFTR channels, previously opened by ATP. This is in agreement with earlier findings and the suggestion that AMP-PNP (like ATP) prevents channel closing by binding to NBD-B. Our new finding of inhibitory binding of AMP-PNP to NBD-A is further strong support for the requirement of ATP hydrolysis at NBD-A in channel opening. In addition to inhibitory competing with ATP, we found that ADP regulates the closing rate of the channel as well. This constitutes a new twist to the mechanism by which the energy charge of the cell (i.e., the [ADP]:[ATP] ratio) regulates CFTR activity, as proposed by Reddy and Quinton 1996 . The findings lead to a modified model for the regulation of CFTR gating by nucleotides. The results presented in this study were obtained from partly dephosphorylated channels with a lower than maximal open probability. We present evidence that at higher phosphorylation nucleotide-dependent gating is additionally modified, leading to slower relaxations. The characterization of channels with better defined phosphorylation status, as well as of CFTR with mutant NBDs, as discriminating between interactions with either or both NBDs awaits further study. | Study | biomedical | en | 0.999998 |
10398693 | Changes in pH at the intracellular or extracellular face of an ion channel under physiological or laboratory-defined conditions can have strong effects on gating or permeation properties. pH-dependent ion channel behavior, controlled by the binding of protons to important functional regions of the channel protein, has been observed for a wide variety of channel types, including voltage-dependent Na + , Ca 2+ , K + , glutamate receptor , and cyclic nucleotide–gated ion channels . In some cases, the effects of protons on ion permeation can provide insight into the detailed structure of the pore. One example is the promotion of subconductance states by the binding of H + to the pore of the cardiac L-type voltage-dependent Ca 2+ channel. Unitary Ca 2+ channel recordings in cardiac ventricular myocytes showed that the binding of protons to a single extracellular site having a pK a of ∼7.5 caused the channel to switch from a high-conductance state to a state with threefold lower conductance . More recent investigations of this phenomenon using Xenopus oocyte expression of the cloned cardiac L-type Ca 2+ channel have revealed that the proton-binding site is formed by an asymmetric cluster of four glutamate residues in the pore that also play a fundamental role in the high-affinity binding of permeant Ca 2+ ions . A similar phenomenon has been observed in the cloned catfish olfactory cyclic nucleotide-gated (CNG) 1 channel, a nonselective cation channel having homology to voltage-gated potassium channels . Fig. 1 A shows a single-channel record collected at −80 mV with 130 mM NaCl, pH 7.6, on both sides of the membrane; the recording was made in the presence of deuterium oxide ( 2 H 2 O) instead of H 2 O to slow the transition rate between the three conductance states (see materials and methods ). The amplitude histogram in Fig. 1 B, calculated from the activity of the channel shown in Fig. 1 A, shows three peaks, one for each of the conductance states (∼70, ∼42, and ∼18 pS). In the model proposed by Root and MacKinnon 1994 to explain this behavior, diagrammed in Fig. 1 C, the conductance states were controlled by protonation at two independent and equivalent sites of pK a ∼7.6: the high-conductance state occurred in the absence of bound H + , the intermediate conductance state occurred when one or the other site was occupied by a proton, and the low conductance state occurred when both sites were occupied. This behavior was found to depend on the presence of a pore glutamate residue (Glu333) analogous to the glutamates in the pore of the cardiac L-type Ca 2+ channel; mutation of Glu333 to glycine gave a proton-insensitive channel with a single conductance state. It was proposed that the four Glu333 residues in the pore might form two bi-symmetrical and independent carboxyl-carboxylate pairs, in each of which a H + ion may be shared equally between two carboxyl groups. Carboxyl-carboxylates are well described and can have high pK a values . The purpose of this study was to test the plausibility of the idea that the two protonation sites in the pore of the catfish olfactory CNG channel consist of two physically separate carboxyl-carboxylate pairs. The strategy we used was to create hybrid CNG channels containing a mixture of wild-type (WT) and Glu333Gly (E333G) subunits. If the sites are separate carboxyl-carboxylates, we reasoned, it should be possible to isolate a hybrid channel containing one, but not both, of the original protonation sites intact. By expressing a mixture of WT and E333G subunits, we formed functionally WT channels, pure mutant channels, and four novel channel types, a result consistent with the postulated tetrameric structure of CNG channels. One of the novel channels, which we named Type B, had two pH-dependent conductance states whose occupancy was governed by protonation at a single site having a pK a of 6.8. Expression of tandem dimer constructs enabled us to determine the number of WT and E333G subunits in all of the hybrid channels and revealed that the Type B channel contained two WT and two E333G subunits. We conclude that we were able to isolate, in the Type B hybrid channel, a single protonation site formed by two glutamate carboxyl groups with properties very similar to those in the native channel. Our results corroborate the hypothesis that the protonation sites in the native channel are structurally independent carboxyl-carboxylates. The catfish olfactory CNG channel α subunit was carried in the pGEMHE plasmid . The Glu333Gly mutation was generated in a MluI–ClaI pore cassette as previously described . RNA for oocyte expression was synthesized from SphI-linearized DNA using T7 RNA polymerase. Dimer constructs were made as follows. The catfish olfactory CNG channel α subunit (WT or E333G) was subcloned into the pRSET plasmid (BamHI to HindIII) just 3′ to a 102-bp sequence (NcoI to BamHI) encoding a 34 amino acid peptide linker. The linker, originally designed for antibody studies of the channel, contained a polyhistidine stretch followed by a T7 epitope tag and an aspartate-rich sequence. The entire linker plus channel construct (NcoI to HindIII) was excised from pRSET and inserted into a pGEMHE-CNG channel construct that had been mutated to give an NcoI site at the 3′ end of the α subunit coding sequence. The result was a construct in pGEMHE (BamHI to HindIII) consisting of two complete catfish olfactory CNG channel α subunits separated by the 102-bp linker sequence. To reduce the likelihood of contamination of dimer DNA or RNA by monomers, the following measures were taken: (a) only recA − strains of Escherichia coli were used to carry the dimer plasmids; (b) after the dimer ligation reaction, single colonies were picked and shown to contain a single dimer-sized species by agarose gel electrophoresis; and (c) after in vitro RNA synthesis, dimer RNA was compared side-by-side with monomer RNA on an agarose gel and shown to be free from contamination by the monomer band, which ran at a distinct position. Dimer constructs were made from the WT and E333G subunits in all possible combinations: WT:WT, WT:E333G, E333G:WT, and E333G:E333G. Dimer RNA was synthesized using T7 RNA polymerase after linearization with SphI. Xenopus laevis oocytes were prepared and injected with CNG channel RNA as previously described . For experiments in which WT and E333G subunits (or WT:WT and E333G:E333G dimers) were coexpressed, a 2:1 ratio of WT:E333G RNA was injected because of the tendency of mutant subunits to express to higher levels than WT subunits. Inside-out or outside-out patches were obtained with electrodes fabricated from capillary glass (Drummond Scientific), coated with beeswax, and fire polished to a resistance of 1–5 MΩ. Single-channel currents were recorded 1–3 d after RNA injection using an Axopatch 200 amplifier (Axon Instruments). The amplifier output was filtered at 2 kHz and sampled at 10 kHz using a DAP data collection board (Microstar Laboratories). All-points amplitude histograms were constructed off-line using an analysis program written in Microsoft QuickBASIC. The bin-width of the histograms was 0.05 pA, and at least 500,000 sample points (50 s) of data were used to construct each histogram. Histograms were constructed primarily from open-channel activity, with a small amount of baseline activity included as a reference. The internal and external solutions were made using 2 H 2 O (deuterium oxide; Sigma Chemical CO.) to slow the kinetics of protonation and deprotonation events, as previously described . Both solutions contained (mM): 130 NaCl, 3 HEPES, and 0.5 Na 2 EDTA. To activate the CNG channels, 1 mM Na-cGMP (Sigma Chemical Co.) was added to the internal solution. For experiments in which the extracellular pH was varied, outside-out patches were perfused with solutions of varying pH using a linear array of microcapillary tubes (1 μl, 64 mm special length; Drummond Scientific). Solutions were adjusted to the appropriate pH (6.0–8.5) using concentrated NaOH and HCl solutions prepared with 2 H 2 O. Our primary objective was to test the independence of the two protonation sites found in the WT catfish olfactory CNG channel. To do this, we asked whether it was possible to use a mixture of WT and E333G subunits to create a hybrid channel containing one but not both sites. A 2:1 mixture of WT and E333G subunit RNA was injected into Xenopus oocytes, and single CNG channels were recorded from inside-out patches at a holding voltage of −80 mV, with 130 mM NaCl, pH 7.6, on both sides of the membrane. The varieties of single channels found are shown in Fig. 2 A. In the first two columns, individual examples of current traces are presented alongside amplitude histograms calculated for the same channels. Pure mutant channels with a single conductance state of ∼25 pS were observed , as were WT channels having the usual three conductance states of 65–70, 35–40, and 15–20 pS . In addition, four novel types of hybrid channels were found, which we have called Types A, B, C, and D. The Type A channel had no single well-defined conductance state, spending most of its time at low (∼30 pS) conductances, but also displaying brief spikes to higher conductances (which gave rise to the long tail in the amplitude distribution). The Type B channel appeared to have two well-separated conductance states of ∼50–65 and ∼25–40 pS, with the higher conductance state favored at pH 7.6. The Type C channel appeared to jump rapidly between poorly distinguished conductance levels and visited both lower and higher conductances from its main conductance level of ∼50–60 pS, behavior that gave both high- and low-conductance tails in the amplitude histogram. The Type D channel appeared in single-channel records to show behavior similar to that of the WT channel, with transitions among three conductance states. Its amplitude histogram, however, always showed only two recognizable peaks (∼70–75 and ∼25–30 pS), perhaps because the lowest-conductance state was so infrequently and briefly occupied at pH 7.6. The third column shows average amplitude histograms calculated from all of the individual histograms assigned to each category (pure mutant, A, B, C, D, or WT). The shapes of the group average histograms were similar to those of the individual examples, underscoring the uniqueness of the hybrid channel types. The broader peaks seen in the group average Type B and WT histograms reflect the variation we observed in the absolute amplitude (although not in the shape) of these channel types. Fig. 2 B shows the number of each channel type found, out of a total of 65 single-channel patches pulled from WT and E333G coinjected oocytes. As expected, the least common species were the homomultimeric WT channels (two observed) and pure mutant channels (six observed). Of the hybrid channels, Types A and B were found more than twice as frequently as Types C and D. Fig. 3 shows amplitude histograms compiled from examples of the four hybrid channel types at a range of extracellular pH values. Recordings were made from outside-out patches, and pH changes were made by moving the patches between microcapillary perfusion tubes. While the behavior of Type A channels did not change significantly between pH 8.5 and 6.0 (aside from a slight flattening of the high-conductance tail of the amplitude histogram), the behavior of Types B, C, and D was strongly pH dependent. Type B channels appeared to undergo a smooth transition from a well-defined high-conductance state to a well-defined low-conductance state as the pH was lowered. Type C channels also shifted to lower conductances at lower pH values, although it was not possible to discern two clearly separable conductance states. For the Type D channel, a transition between two well-defined conductance states at high pH was followed by a more gradual shift to lower conductances at low pH. While the amplitude distributions of Type B channels kept their distinctive two-peaked shape even at pH 6.0, the Type A, C, and D distributions all seemed to converge at low pH on a common shape having a low-conductance hump with a long, high-conductance tail. Of the four hybrid channels, the Type B channel, exhibiting two distinct conductance states over a wide pH range, seemed like the best candidate for a hybrid having one of the WT protonation sites. Amplitude histograms were compiled from Type B channel activity in outside-out patches at various values of extracellular pH and fitted to a sum of three Gaussian functions corresponding to the closed state and two open states of the channel. Fig. 4 A shows amplitude histograms from the same channel at three pH values; the top (pH 8.0) histogram appears with its three-Gaussian fit (solid line). The relative area under the Gaussian functions for the nonzero conductance states, a measure of the relative occupancy of the states, was calculated for three channels and plotted as a function of pH . The dotted lines in Fig. 4 B show the predictions of a model in which transitions from the high to the low state are governed by protonation at a single site . The good agreement with the data indicates that the steepness of the transition between states in the Type B channel is consistent with a single-site mechanism. The best fit to the data gives a pK a for the single site of 6.8. To gain a better understanding of the subunit composition of the hybrid CNG channels, we constructed and expressed tandem dimers of WT and E333G subunits. Our hypothesis was that the subunit composition of channels formed from dimers, instead of coexpressed monomers, would be more tightly constrained. In the optimal case, in which dimers associate in one orientation to form full tetrameric channels, one would expect expression of each single type of dimer to give rise to a single type of channel. The dimers used in these experiments consisted of two complete CNG subunits joined by a 34 amino acid linker (see materials and methods ). Fig. 5 shows the behavior of channels observed in inside-out patches after several types of dimers were expressed. Expression of the dimer consisting of a WT subunit linked in tandem to an E333G subunit (the E:G dimer, named after the residues at position 333 in each subunit) gave rise to two and only two of the hybrid channel types, Types B and C. These were identified on the basis of the shapes of their amplitude histograms at pH 7.6 and their pH dependence in outside-out patches (data not shown). Expression of the opposite dimer, G:E, gave the same two types of hybrid channels, as did expression of a 2:1 mixture of the E:E and G:G dimers (which also gave WT and pure mutant channels, not shown). The fact that expression of the E:G or G:E dimer alone produced more than one type of channel—as well as the fact that coexpression of the E:E and G:G dimers produced two different hybrid channels—suggested that the dimers were associating in more than one way. One explanation for the data of Fig. 5 is that the CNG dimers can assemble into tetrameric channels in all three possible dimer–dimer configurations, illustrated in Fig. 6 , top. In the first configuration (i), dimers associate “head-to-tail,” with the A protomer of one dimer contacting the B protomer of the other. In the second configuration (ii), dimers associate “head-to-head,” with the A and B protomers of the two dimers contacting each other. In the third configuration (iii), the dimers interlock to form channels, and the A and B protomers of each dimer sit at opposite corners of the channel. This scheme depends on the assumptions that CNG channels are fourfold symmetric tetramers and that all of the dimers associate as dimers, contributing both of their subunits to each of the channel species formed. As shown in Fig. 6 , bottom, the first two configurations (i and ii) are sufficient to give two channel structures when the E:G or G:E dimer is expressed alone. These structures are each predicted to have two WT and two E333G subunits, with the WT subunits lying across the channel from each other (i) or adjacent to each other along a side of the channel (ii). When the E:E and G:G dimers are coexpressed, association in the first two configurations would give rise to only one of these two structures (the structure having adjacent WT subunits), and it becomes necessary to invoke the third configuration (iii) to produce the structure having WT subunits at opposite corners of the channel. As indicated in the figure, coinjection of the E:E and G:G dimers would also be expected to give rise to WT and pure mutant channels. If the scheme shown in Fig. 6 is correct, then the Type B and Type C channels each have two WT and two E333G subunits, with one of them having adjacent and the other opposite WT subunits; the data in Fig. 5 do not indicate with certainty which channel type corresponds to which arrangement. Regardless of their orientation, however, the presence of exactly two WT subunits, and hence two pore glutamates, in the Type B channel is a further indication that this channel could contain a single carboxyl-carboxylate that is similar to those formed in the pore of the WT channel. Fig. 7 shows the types of channels observed in inside-out patches when the E:G dimer was coexpressed with either the G:G (top) or the E:E (bottom) dimer. In both cases, coexpression gave rise to the channels expected from expression of the dimers individually: pure mutant, Type B, and Type C channels for the coexpression of the E:G and G:G dimers; WT, Type B, and Type C channels for the coexpression of the E:G and E:E dimers . In both cases, an additional hybrid channel type was also found. For the E:G + G:G coexpression, this additional hybrid channel was the Type A channel, while for the E:G + E:E coexpression, the additional channel was the Type D channel. As before, the hybrid channels were identified based on the shapes of their amplitude histograms at pH 7.6 and their pH dependence in outside-out patches (not shown). The additional hybrid channel produced in each case, not expected from expression of the dimers individually, most likely arose from the two coexpressed dimers coming together to form a unique channel. This implies that the Type A channel contained one WT subunit and three E333G subunits, while the Type D channel contained three WT subunits and one E333G subunit. Only one unique hybrid is expected in each case, since coassociation of the coexpressed dimers in all three of the configurations shown in Fig. 6 , top, would give rise to the same subunit arrangement. Types A and D channels were never seen when the E:G, G:E, or E:E + G:G dimers were expressed , consistent with the conclusion that they have a one WT, three mutant or three WT, one mutant subunit arrangement. The central goal of this study was to test the idea, proposed by Root and MacKinnon 1994 , that the two proton-binding sites in the pore of the catfish olfactory CNG channel are physically independent carboxyl-carboxylate structures. In the WT channel, these sites are formed by four glutamate residues in the pore, one contributed by each of the channel subunits. Our strategy was to create hybrid channels containing varying numbers of pore glutamates and determine whether it was possible to isolate a hybrid species containing one, but not both, of the original protonation sites. By expressing a mixture of WT and E333G monomers and various combinations of dimers, we formed four easily distinguished hybrid types of CNG channels. One of the hybrid channels (Type B) had the properties expected of a channel containing a single proton acceptor site similar to the two in the WT channel: (a) it had two conductance states whose occupancy was controlled by protonation at a single site of pK a 6.8, and (b) it contained two WT and two E333G subunits. Because our dimer constructs assembled into tetrameric channels in several ways, we were not able to determine whether the two glutamate residues in the Type B channel were positioned on adjacent or opposite subunits. Based on the equivalence and independence of the two sites in the WT channel , we might have expected that the Type B site would have a pK a of exactly 7.6, not 6.8. This apparent inconsistency, however, is not very surprising. Independence of the two sites in the WT channel means that protonation of the one site is independent of protonation of the other site. However, that does not mean that protonation of one of the sites would be insensitive to complete removal of the glutamate side-chains comprising the other site. Side-chain removal is a much larger perturbation of the environment of the channel pore than addition of a proton. Therefore, these data strongly suggest that we were able to reconstitute individually in the Type B hybrid channel one of the carboxyl-carboxylate sites in the pore of the WT channel, which supports the notion that the sites in the WT channel are made from structurally independent glutamate pairs. The present results, taken together with the earlier results of Root and MacKinnon 1994 , suggest an arrangement of glutamates in the pore of the catfish olfactory CNG channel that is much different from the arrangement of the glutamates in the pore of the cardiac L-type Ca 2+ channel. In the Ca 2+ channel, the four pore glutamates are proposed to form an asymmetric cluster giving rise to a single protonation site, with each glutamate making a distinct contribution to the site and two of the glutamates, those in domains I and III of the channel, acting in concert to coordinate a proton . In the catfish olfactory CNG channel, the four glutamates are apparently present in a symmetric ring such that they can form two identical pairs (upon the breaking of symmetry at the level of individual side chains). In light of these different arrangements, it is interesting to consider the different types of ion binding sites formed by the sets of glutamates in the two types of channels. The glutamates in the pore of the bovine retinal CNG channel form a high-affinity binding site for divalent cation blockers in the outer part of the channel pore , a role perhaps suited to their arrangement as a symmetrical ring of negative charge at a fixed position in the channel mouth. In contrast, the glutamates in the L-type Ca 2+ channel form the essential sites for permeant Ca 2+ ions in the pore, and therefore must, in accordance with the prevailing models of Ca 2+ channel permeation, be able to accommodate two Ca 2+ ions at a time—a function aided by the fact that they presumably constitute an asymmetrical glutamate cluster with mobile carboxylate moieties positioned at a range of depths in the tightest part of the pore . Given the homology between the channel under study and potassium channels, which have been shown to have a stoichiometry of four subunits per channel , it is tempting to postulate a tetrameric structure for the catfish olfactory CNG channel. The present results lend credence to this idea. Expressing a mixture of WT and E333G subunits gave WT channels, pure mutant channels, and four varieties of hybrid channels having easily distinguished, qualitatively different properties. Assuming that subunits contribute symmetrically to the channel, and assuming that the order of the glutamates around the channel pore is important, this number of hybrid channels is most simply explained if four subunits come together to form a complete channel. A symmetric three-subunit channel would be expected to give just two different hybrid structures, while a symmetric five-subunit channel would be expected to give six hybrid structures. Of course, the present results could arise from a channel structure with five or more subunits if some of the hybrid structures are degenerate, having different configurations of WT and E333G subunits but identical behavior as a function of pH, but a tetrameric channel provides the simplest explanation. Our conclusion closely parallels the findings of Liu et al. 1996 , who coexpressed the 30-pS WT bovine retinal CNG channel and an 85-pS chimeric bovine retinal channel containing the catfish olfactory CNG channel P region and counted the number of hybrid CNG channels, identified by their different conductances, that were produced. The authors observed four different intermediate conductance levels and concluded both that the channels formed were tetrameric and that the order of subunits around the channel pore was important. When these authors expressed dimer constructs, they found that association of the dimers primarily occurred in one configuration, the head-to-tail configuration shown in Fig. 6 , (i), whereas our dimers appeared to associate in three different ways. The simpler behavior observed by Liu et al. 1996 could reflect the fact that the dimers used in those experiments did not include an extra peptide between the COOH terminus of the A protomer and the NH 2 terminus of the B protomer, or could reflect inherent differences in the association properties of retinal versus olfactory CNG channel subunits. The idea that bovine retinal CNG channel dimers might constrain subunit order better than catfish olfactory CNG channel dimers is also suggested by the experiments of Gordon and Zagotta 1995 exploring the intersubunit coordination of Ni 2+ ions by retinal CNG channels. The authors found quantitative evidence that subunit order was well constrained in these experiments, even though a 21 amino acid linker was used between retinal CNG subunits (comparable with the 34 amino acid linker used in our experiments). Expression of CNG channel dimers made it possible to draw conclusions about the subunit makeup of the four types of hybrid channels we observed when WT and E333G monomers were coexpressed. Fig. 8 shows the possible WT and E333G subunit combinations expected for a tetrameric channel and correlates these combinations with the hybrid channels that were observed. The Type A channel could be unambiguously assigned to a specific structure since coexpression of the E:G + G:G dimer combination was not sensitive to the variability of dimer association. Since the Type A channel arose uniquely when the E:G and G:G dimers were coexpressed, we conclude that this channel has only one WT subunit and therefore only one pore glutamate. This is consistent with this channel's lack of a strong pH dependence between pH 8.5 and 6.0, since one would expect a lone carboxyl group to have a pK a several units lower. The Type B and Type C channels each contain two WT and two E333G subunits, although the flexibility of dimer association prevented us from determining which of these had two adjacent WT subunits and which had two opposite WT subunits. This leaves doubt as to the precise configuration of the carboxyl-carboxylate interaction in the Type B channel—it is possible that it occurs across the channel between opposite subunits or along a side of the channel between adjacent subunits. Which of these possibilities is more plausible depends critically on the position and angle at which Glu333 projects into the channel pore. The fact that the Type C channel does show pH dependence between pH 8.5 and 6 presents the possibility that its two glutamates can combine to form a protonation site of fairly high pK a . Fig. 3 suggests that in this channel, protonation at a site with a pK a of ∼6.25 causes a gradual shift to a lower conductance state of ∼20–25 pS. The Type C channel resembles the EIIQ L-type Ca 2+ channel mutation studied by Chen et al. 1996 in two respects: (a) it flickers from its main conductance level to higher and lower conductance levels ; and (b) as the pH is lowered, the lower conductance state becomes populated, but the flickers between the middle- and high-conductance states remain, suggesting that the distribution between the high and middle states does not change with increasing [H + ]. This analogous behavior is intriguing, especially considering that the Type C channel is expected to contain a twofold symmetric arrangement of two glutamates, while the EIIQ Ca 2+ channel is expected to contain an asymmetric arrangement of three glutamates. It will be interesting to compare further the structure and ion conduction properties of these two channels in search of a common mechanism underlying their similar single-channel behavior. The Type D channel, which arose uniquely when the E:G and E:E dimers were coexpressed, could be unambiguously assigned to a structure having three WT subunits (and hence three pore glutamates). Consequently, its conductance shows strong pH dependence. Since this channel has glutamates positioned next to each other and across the channel from each other, it should accept protons both like a Type B and like a Type C channel. This is consistent with the pH dependence shown in Fig. 3 , which shows a clear Type B–like transition between two conductance states at high pH followed by a less well-resolved Type C–like transition to a lower conductance state at lower pH. The WT channel, containing all four glutamates, would also be expected to show both Type B and Type C behavior. Protonation in the Type B mode (i.e., via independent carboxyl-carboxylate interactions) would give the transitions among three clear conductance states that are the hallmark of this channel. Protonation in the Type C mode, causing a poorly resolved transition to an intermediate conductance state, might be expected to affect the behavior of the WT channel more subtly. We modeled this effect by adding a fourth conductance state to the channel, as depicted in Fig. 9 A. In the model, Type B protonation at the two independent and equivalent carboxyl-carboxylates causes transitions along the top row of states, from the high-conductance state s1 (65–70 pS) to the middle- and low-conductance states, s2 (40–45 pS) and s3 (15–20 pS) . Type C protonation shifts the channel to an intermediate state s4 whose conductance, estimating from Fig. 3 , is ∼20–25 pS, between the conductances of s2 and s3, an estimate that depends on the assumption that protonation in the Type C mode has the same effect on the WT channel, which has four pore glutamates, as on the Type C channel, which has only two glutamates. In theory, a second Type C protonation is possible in the WT channel, which would be expected to give rise to a fifth conductance state in which both Type C sites are occupied. However, since we could only measure one Type C transition in the Type C channel (which contains only two glutamates), and since we have no way of predicting the pH range of the second Type C protonation or its effect on the channel conductance, we limit our model to only one Type C transition. This model is therefore incomplete and should be regarded as a first approximation. Because Type C, like Type B, protonation should occur in the WT channel at either of two sites and is assumed to be diffusion limited, we take the transition rate from s1 to s2 or s4 to equal 2α[H + ], where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\alpha}}}\;=\;6.4\;{\times}\;10^{9}{\mathrm{M}}^{-}1*{\mathrm{s}}^{-}1\end{equation*}\end{document} . While β, the proton off rate from the carboxyl-carboxylates, must be set at 200 s −1 to give a pK a of 7.6 for these sites, β′, the off rate of protons from the Type C site, must be adjusted to 1,920 s −1 to account for the lower pK a of the Type C site, estimated from Fig. 3 to be ∼6.25. The model allows concerted transitions between s2, in which a single proton is bound in the Type B configuration, and s4, in which it is bound in the Type C configuration. The rates of such transitions are assumed to be of the same order of magnitude as the rates of exit from s2 and s4 to s1; this, along with the assumption of microscopic reversibility around the s1–s2–s4 loop, sets γ + at 1,920 s −1 and γ − at 200 s −1 . Fig. 9 B compares a WT amplitude histogram recorded at pH 7.6 and −80 mV with amplitude histograms generated from the model under the same conditions and suggests that Type C protonation in the WT channel might explain a subtle but consistent feature of WT behavior. As indicated by the arrow in Fig. 9 B, the WT channel consistently tends to visit current levels between the low- and middle-conductance states more often than expected; the extra density in this region is greater than can be explained by overlap of the Gaussian functions for the two neighboring states. This feature was observed in every WT channel recorded in this study and is evident in the original data of Root and MacKinnon 1994 . Fig. 9 B shows how inclusion of the second type of protonation, which allows the channel to make brief transitions to s4, can explain this extra density between 20 and 30 pS (∼1.5–2.5 pA at −80 mV). Simulations of the WT channel including s4 were also better than simulations without s4 in predicting the behavior of the WT channel at other pH values (not shown). Thus, protonation at the two equivalent and independent Type B sites, while necessary to explain transitions among the three major conductance states, is not sufficient to explain WT channel behavior in its full detail. To capture all of the nuances of the channel's behavior, we must invoke a second, lower-affinity type of configuration in which the pore glutamates can pair to accept protons. | Study | biomedical | en | 0.999995 |
10398694 | In Xenopus laevis , the olfactory organ is compartmentalized into two independent subregions, the medial and lateral diverticulums. Anatomical observation has indicated that the olfactory sensory epithelium in the medial diverticulum comes into contact with air, while the epithelium in the lateral diverticulum is in contact with water . The latter, therefore, is called the water nose. The two olfactory subregions have a different affinity to lectins; the epithelium of the medial diverticulum was slightly labeled with soybean agglutinin–horseradish peroxidase, which has been shown to selectively label a portion of olfactory neurons, whereas the epithelium of the lateral diverticulum was not labeled . Xenopus laevis possesses a gene repertoire encoding two distinct classes of olfactory G-protein–coupled receptors; one class is related to the olfactory G-protein–coupled receptors of fish, and the other is related to the olfactory G-protein–coupled receptors of mammals . An in situ hybridization study of the fish-like olfactory G-protein–coupled receptors resulted in labeling of the sensory epithelium in the lateral diverticulum, whereas the medial diverticulum was devoid of any hybridization signals. All the probes representing mammalian-like receptor signals yielded results only in the medial diverticulum. On the assumption that these are odor receptors, the results suggest that olfactory neurons in the medial diverticulum are expected to respond to volatile odorants and neurons in the lateral diverticulum to water-soluble odorants (e.g., amino acids). Until recently, however, physiological functions including the odor selectivity of olfactory neurons in these two diverticula have remained unclear. In the present study, we recorded the responses to water-soluble odorants as well as to volatile odorants from Xenopus laevis lateral olfactory receptor neurons under whole-cell voltage-clamp conditions. The results obtained indicated that the lateral olfactory neurons responded to both water-soluble and volatile odorants. Many single olfactory neurons responded to a variety of amino acids, including acidic, basic, and neutral amino acids. The method of preparation was essentially the same as that described in a previous paper . Frogs, Xenopus laevis , were obtained from commercial suppliers and maintained at 15°C. Animals were fed porcine liver ad libitum. For the staining of olfactory epithelial slices with methylene blue (Merck), animals were bathed in 0.1% methylene blue solution for 6 h. For the preparation of olfactory epithelial slices, animals were cooled to 0°C to anesthetize completely, and then decapitated. Olfactory epithelium was quickly removed from the decapitated frogs. The epithelia were cut into slices ∼120-μm thick with a vibrating slicer in normal Ringer solution at 0°C and stored at 4°C. Epithelial slices were fixed on the glass at the bottom of a recording chamber, permitting access by patch pipette to neurons on the surface of the slice. The preparations were viewed under an upright microscope (OPTIPHOT; Nikon Inc.) using a 40× water immersion lens. The conventional whole-cell patch clamp method was used to measure transmembrane currents . Patch pipettes with resistances of 5–10 MΩ were made from borosilicate glass capillaries with an inner filament (GD-1.5; Narishige Co.) using a two-stage electrode puller (PP853; Narishige Co.), and then heat-polished. Gigaohm seals were obtained by applying negative pressure (−30 to −100 cm H 2 O). The whole-cell configuration was attained through the application of additional negative pressure. Membrane currents (holding potential, −70 mV) and voltages were recorded in the whole-cell configuration. Data were recorded continuously using an EPC-7 patch clamp amplifier (List Electronic) and stored on video cassette via a digital audio processor (PCM-501; Sony Corp.). All recordings were carried out at room temperature. Membrane currents were low-pass filtered with two-poles, Bessel filter of EPC-7. The filter frequency was 10 kHz. We estimated the magnitude of inward currents from just before the response to the peak. Analysis was carried out on a personal computer using pCLAMP software (Axon Instruments). All values are given as a mean ± SEM. A liquid junction potential was measured as described by Neher 1992 . The liquid junction potential never varied more than several mV in the altered solutions (−2.6 ∼ 2.0 mV). All data have been corrected for the liquid junction potential. Lucifer yellow CH was dialyzed intracellularly by using patch pipettes filled with 1% Lucifer yellow solution as described previously . After the measurement of voltage-activated currents, the pipettes were pulled back from the surface of the cells. The specimens were then transferred to the stage of a fluorescent microscope (OPTIPHOT) and observed. Olfactory neurons were stimulated with extracellular solutions by bath application from the outlet of the stimulating tube. Ringer solution, delivered by gravity, was alternated with odorant solutions by means of eight electrically actuated valves. The volume of the solutions in the chamber was ∼90–350 μl. A stimulating tube with a lumen 160–200 μm in diameter was placed under visual control ∼3 mm from the cell to eliminate the mechanical effects of alternating among solutions. The flow rate, which was adjusted with needle valves, was 12.3–23.3 μl/s. The delay, due to dead space and to exchange solution in the chamber, was 3–25 s, depending on the flow rate. Our attempt to eliminate deviations in the delay was unsuccessful because the precision of the needle valves was not sufficient to regulate the flow of small amounts of the solutions. To examine the extent of the dilution during stimulation, 50 mM 1-anilinonaphtalene-8-sulfonate (ANS) was applied from the stimulating tube to the recording chamber, and the fluorescence intensity of ANS at the point where the epithelium was usually placed was measured with an inverted microscope (Axiovert 135; Carl Zeiss, Inc.). The fluorescence intensity reached a saturated level within 4–18 s. This saturated fluorescence intensity was similar to that of 50 mM ANS. The extracellular Ringer solution consisted of (mM): 116 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES-NaOH, ph 7.4. Patch pipettes were usually filled with a normal internal solution containing (mM): 115 KCl, 2 MgCl 2 , 2 EGTA, 10 HEPES-KOH, pH 7.4. Further details on the solutions are shown in the individual figure legends. Lucifer yellow was dissolved in the internal solution at a concentration of 1%. The odorant cocktails were vigorously stirred with the magnetic stirrer for more than 30 min at room temperature. We confirmed that the solutions of odorant cocktails were completely clear. The final concentrations of each odorant in volatile odorant cocktail I, which induced inositol 1,4,5-trisphosphate accumulation in rat olfactory cilia, were 40 μM lilial, 40 μM lyral, and 20 μM ethyl vanillin . The final concentration of each odorant in volatile odorant cocktail II (citralva, hedione, eugenol, l -carvone, and cineole), which induced cAMP accumulation in the rat and frog olfactory cilia, was 200 μM . Stock solutions of amino acids were prepared at 0.1 M and stored at 4°C. These solutions were added to normal Ringer solution to give the concentrations of odorants indicated. The final concentration of each amino acid in the amino acid cocktail ( l -alanine, l -arginine, l -glutamic acid, and l -methionine) was 1 mM. The deviations in pH in the stimulating solutions were within 0.2. Tetrodotoxin (TTX) 1 and Lucifer yellow CH were obtained from Sankyo and the Aldrich Chemical Co., respectively. Amino acids ( l -alanine, l -arginine, l -glutamic acid, and l -methionine) and N -methyl- d -glucamine were obtained from Wako. All volatile odorants were kindly supplied by Takasago International. All chemicals used were of the highest grade available. Fig. 1A and Fig. B , show micrographs of the olfactory epithelia of the lateral diverticulum and medial diverticulum of Xenopus laevis bathed in water containing 0.1% methylene blue. The surface of the lateral diverticulum was clearly stained with methylene blue, while that of the medial diverticulum was not stained at all. These results support a previous finding that the lateral and medial olfactory epithelia demonstrate differentiated contact with water-soluble odorants and volatile odorants, respectively . Fig. 1 D shows a fluorescent micrograph of an olfactory neuron dialyzed with Lucifer yellow in the olfactory epithelium of the lateral diverticulum. The cell bodies are elliptical with dimensions on the order of 20 × 12 μm. The cells have dendritic processes that reach the surface of the epithelium. Initial studies of the whole-cell clamp technique were made using the current-clamp mode. With K + and Na + as the main internal and external membrane-permeable cations, respectively, the mean resting membrane potential for these neurons was −46.5 ± 1.2 mV \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;68)\end{equation*}\end{document} ; a value similar to the value measured in the Xenopus olfactory receptor neuron . The injection of current (1–3 pA) elicited action potentials from a conditioning voltage at −60 mV . The threshold for action potential generation in these neurons was commonly between −35 and −55 mV. A large current injection generated brief trains of action potentials. The impulse trains typically consisted of up to 30 spikes. The firing frequency linearly increased with increasing current injections from 1 to 30 pA and reached a plateau at 30 pA . The action potential latency was reduced with increases in the amplitude of the current injection. The spike induced by current (10 pA) was blocked by 0.3 μM TTX , suggesting that a voltage-sensitive Na + current was involved in its generation. Voltage-clamp recordings confirm that the current responsible for the rising phase of the current-induced action potential is carried by Na + . Fig. 3 A shows the two major currents elicited by the depolarizing steps of voltage from a holding potential of −70 mV. A transient inward current appeared at greater than −40 mV, and an outward current was observed after the rapid inward current. The current–voltage curves taken at the peak of the inward and outward currents are shown in Fig. 3 B. To examine the transient inward currents, internal KCl was substituted with CsCl. Replacing Na + with choline in the external solution reversibly and completely abolished the transient inward current in all five neurons . The addition of 1 μM TTX to the external solution also completely inhibited this current in all four neurons . These results suggest that the current is probably carried by Na + . To study the outward K + currents, 25 mM tetraethylammonium (TEA) was substituted with an equivalent amount of NaCl, and 1 μM TTX was added to the external medium. The outward current was partially blocked by the addition of 25 mM TEA to the external solution in all six neurons. The magnitude of the outward currents was reduced to 32.3 ± 4.4% \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;6)\end{equation*}\end{document} by 25 mM TEA. The presence of a Ca 2+ -dependent K + current in the neuron was studied by measuring the outward current in an external solution with and without Ca 2+ , respectively. The outward current was partially attenuated by the elimination of Ca 2+ into the external solution , suggesting the presence of a Ca 2+ -activated K + channel. Recently, K + currents mediated with RELK1 channels insensitive to 100 mM TEA have been shown to be blocked by Ba 2+ . As shown in Fig. 3 G, TEA-insensitive outward currents in Xenopus water nose olfactory receptor neurons were also blocked by Ba 2+ . To evaluate the odor selectivity of the olfactory neuron of the lateral diverticulum, an amino acid cocktail and two volatile odorant cocktails were separately applied to neurons. The application of these three odorant cocktails induced inward currents in 46 of 238 neurons . In 7 of 32 neurons responding to the amino acid cocktail, the magnitude of the inward current was reduced rapidly but not completely during continuous stimulation . The remaining responses to the amino acid cocktail did not show sharp peaks . The peak amplitude of inward currents in response to the amino acid cocktail ranged, typically, from 0 to 126 pA. The mean amplitude of inward currents in response to odorant cocktails was calculated using neurons that responded to any one of the three odorant cocktails. The volatile odorant cocktails I and II induced inward current responses in 17 (37%) and 14 (30%) neurons, respectively . The peak amplitudes of inward currents induced by the odorant cocktails I and II ranged, typically, from 0 to 100 and 0 to 164 pA, respectively. There are single neurons that responded to both cocktails. Some single neurons responded to all three cocktails (i.e., the amino acid cocktail as well as the two volatile odorant cocktails). One example of plural responses is shown in Fig. 4 E. In this neuron, the response to the amino acid cocktail is larger than that to the volatile odorant cocktail I or the volatile odorant cocktail II. Fig. 5 shows the response profiles of individual olfactory neurons to the amino acid cocktail, the volatile odorant cocktail I, and the volatile odorant cocktail II. The population of neurons responding to the amino acid cocktail was larger than that responding to either of the two volatile odorant cocktails. 5 of 46 single olfactory neurons responded to all three odorant cocktails. Seven neurons responded to two odorant cocktails. Thus, ∼26% of the single olfactory neurons responded to plural odorant cocktails. More than 19% of the neurons responded to both the amino acid cocktail and the volatile odorant cocktail. Alanine of varying concentrations was applied to the receptor neurons . The magnitude of the inward current induced by alanine increased with increases in alanine concentrations. Fig. 6 B shows the magnitudes of the responses to alanine of various concentrations. The currents appeared at 100 μM and increased with increases in concentration. The application of arginine also induced inward current responses in a dose-dependent manner . To explore the selectivity of single olfactory neurons with neutral, basic, and acidic amino acids, alanine, arginine, and glutamic acid were applied to the olfactory neuron of the lateral diverticulum . The application of 100 μM of individual amino acids induced inward current responses in the single sensory neuron, as shown in Fig. 7 A. The magnitude of the response to arginine was larger than that to alanine or glutamic acid. The neurons shown in Fig. 7 B responded to either 1 mM alanine or 1 mM glutamic acid, but not to 1 mM arginine. Thus, although single olfactory neurons responded to plural amino acids, the response selectivity was differentiated among neurons. Fig. 7 C shows the response profiles of individual olfactory neurons to 100 μM or 1 mM alanine, arginine, and glutamic acid, respectively. These amino acids induced responses in 13 of 152 neurons. More than 50% of the sensory neurons responded to plural amino acids. In particular, four neurons (31% of neurons stimulated by three odorants) responded to all three amino acids. Lilial and citralva at different concentrations were applied to single olfactory neurons. The neuron shown in Fig. 8 A responded to 1 and 10 μM, and to 1 mM alanine. Six of nine cells responded to alanine, and to lilial, and/or citralva. Fig. 8B and Fig. C , shows the magnitudes of response to lilial and citralva of varying concentrations. Distinct responses appeared at 1 μM for the odorants, indicating that the olfactory receptor neuron of the water nose responds sensitively to volatile odorants. In this study, we examined the electrophysiological properties of receptor neurons in the lateral diverticulum of the olfactory sensory epithelium of Xenopus laevis using the whole-cell voltage-clamp technique, and showed that olfactory receptor neurons in the water nose respond to both amino acids and volatile odorants. Odorants are considered to be received by olfactory G-protein–coupled receptors, which are linked to adenylyl cyclase or phospholipase C via the G-proteins . In situ hybridization experiments to identify olfactory G-protein–coupled receptors in the rat, mouse, and catfish have suggested that a single olfactory G-protein–coupled receptor was expressed in only 0.1–2% of olfactory neurons, and that each olfactory neuron might have only a single particular type of olfactory G-protein–coupled receptor . In the Xenopus laevis olfactory system, olfactory G-protein–coupled receptors have been cloned by Freitag et al. 1995 . Probing for individual receptor mRNA suggests that only a small percentage of the Xenopus laevis olfactory neurons (<1%) express any particular olfactory receptor protein. In the present study, we showed that >50% of single olfactory neurons of Xenopus laevis respond to a variety of amino acids, including acidic, neutral, and basic amino acids. Single olfactory neurons in catfish also responded to plural amino acids . Single olfactory neurons in other animals in the rat, frog, and turtle respond to many species of odorants with diverse structures . An olfactory G-protein–coupled receptor (OR5) has a broad specificity for volatile odorants , hence each olfactory neuron carrying a single type of olfactory G-protein–coupled receptor may respond to many odorants. It is also possible that single olfactory neurons have multiple receptors for various odorants. A cross-adaptation experiment of single olfactory receptor neurons in the turtle and the bullfrog has directly indicated that multiple odorant receptors exist in single olfactory neurons . For example, the magnitude of inward currents induced by the application of hedione to a single neuron after desensitization of the current in response to lyral or citralva is similar to that induced by hedione applied alone, and vice versa. An in situ hybridization study of the olfactory G-protein–coupled receptors of Xenopus laevis has shown that G-protein–coupled receptors similar to those in fish exist not in the epithelium of the medial diverticulum, but in the epithelium of the lateral diverticulum, while G-protein–coupled receptors similar to those in mammals exist in the epithelium of the medial diverticulum, but not in the epithelium of the lateral diverticulum . These results suggest that olfactory neurons in the medial and lateral diverticula demonstrate differentiated responses to volatile odorants and water-soluble odorants such as amino acids, respectively. Volatile odorants induce inward current responses in the olfactory neuron in the medial diverticulum of the Xenopus laevis , supporting this idea. In the present study, we showed that olfactory neurons in the lateral diverticulum respond to acidic, neutral, and basic amino acids. Therefore, it is likely that olfactory neurons having G-protein–coupled receptors in the lateral diverticulum similar to those in fish will respond to amino acids. It should be noted, however, that many olfactory neurons in the lateral diverticulum respond not only to amino acids, but also to volatile odorants. The sensitivity of the neurons to volatile odorants is similar to the olfactory neurons of salamanders . It is possible that olfactory G-protein–coupled receptors similar to those in fish receive both water-soluble odorants and volatile odorants, or that unknown olfactory G-protein–coupled receptors similar to those in mammals, which were not observed by Freitag et al. 1995 , exist in the lateral diverticulum olfactory neurons. In addition, it is also possible that volatile odorants induce responses in the lateral diverticulum olfactory neurons in an olfactory G-protein–coupled receptor-independent manner. | Study | biomedical | en | 0.999994 |
10398695 | Large-conductance Ca 2+ -activated K + (BK) 1 channels, which are activated by micromolar concentrations of intracellular Ca 2+ (Ca 2+ i ) and by depolarization are present in a wide variety of tissues . Activated BK channels reduce membrane excitability by allowing K + efflux through their opened pores, which drives the membrane potential more negative. Hence, BK channels form a link in a negative feed-back loop that decreases excitability in response to both increased Ca 2+ i and depolarization. Information about the gating mechanism of BK channels has accumulated from kinetic studies on native and heterologously expressed channels. BK channels are homotetramers, formed from four alpha subunits , with one or more Ca 2+ -binding sites per subunit . BK channels typically display a steep relation between Ca 2+ i and open probability, P o , with Hill coefficients usually in the range of 2–4 . Hill coefficients >1 suggest a cooperative action of Ca 2+ on P o , and are often associated with allosteric modulation of activity . Models drawn from the Monod-Wyman-Changeux (MWC) model describing the transitions of tetrameric allosteric proteins have often been used to describe the Ca 2+ -dependent gating of BK channels. For example, McManus and Magleby 1991 found that a model with three open and five closed states drawn from the MWC model could account for many of the basic features of the Ca 2+ dependence of the single-channel gating kinetics from low to intermediate levels of activity, and Cox et al. 1997b found that the full 10-state MWC model (and also an extended 12-state model) could account for macroscopic conductance–voltage relations over a wide range of Ca 2+ i . Despite these successes, MWC-type models appear too simple to account for certain details of BK channel gating. Cox et al. 1997b found that such a model would not account for the details of the macroscopic activation and deactivation kinetics at large positive or negative potentials, and a number of studies have suggested that closed states in addition to those in the MWC model may contribute to the gating . For example, Rothberg and Magleby 1998 found that gating mechanisms drawn from the MWC model underpredicted the numbers of brief closed intervals (flickers) adjacent to the various open intervals. Single-channel analysis then suggested that there were additional closed states, either intermediate (in the activation pathway) or secondary (beyond the activation pathway) that contributed to the generation of the flickers. We now test further whether the above extensions of the MWC model are consistent with the underlying gating mechanism. Our approach is to examine the activity of single BK channels from low (4 μM) to very high (∼1 mM) intracellular concentrations of Ca 2+ (Ca 2+ i ). By driving the channel toward its fully liganded states, we estimated the numbers of open and closed states involved in the gating at high Ca 2+ i , and through analysis of two-dimensional dwell-time distributions and dependency plots, we determined the minimum number of independent transition pathways connecting the fully liganded open and closed states. We found that at high Ca 2+ i , the BK channel gates among three to four open and four to five closed states, with two or more effective transition pathways (gateway states) connecting the open and closed states. Neither the general model of Eigen 1968 nor the 55-state extension of Eigen's model by Cox et al. 1997b appear consistent with these findings for the gating at high Ca 2+ i . Extensions of Eigen's model to a two-tiered gating mechanism, in which the upper tier was comprised of closed states and the lower tier was comprised of open states, provided a model that would allow gating in the necessary numbers of open and closed states at high Ca 2+ i , together with the required multiple transition pathways between open and closed states. In the two-tiered model, each of the closed conformations of the channel can undergo a direct (possibly concerted) transition to an open conformation. Models drawn from the general two-tiered model could describe the single-channel kinetics from low to high Ca 2+ i . Since the general two-tiered model (and simple extensions of this model) contain previous models that have been used to account for gating under more limited conditions , it can serve to unify descriptions of gating. A simpler model that assumed the Ca 2+ -dependent transition rates approached a maximum in high Ca 2+ i could also approximate many features of the single-channel kinetics from low to high Ca 2+ i . The considered models can serve as working hypotheses to further study the gating mechanisms of BK channels. Currents flowing through single BK channels in patches of surface membrane excised from primary cultures of rat skeletal muscle (myotubes) were recorded using the patch clamp technique . Cultures of rat myotubes were prepared from fetal skeletal muscle as described previously . All recordings were made at +30 mV using the excised inside-out configuration of the patch clamp technique in which the intracellular surface of the patch was exposed to the bathing solution. Analysis was restricted to patches containing a single BK channel. Single-channel patches were identified by observing openings to only a single open channel conductance level during several minutes of recording in which the P o was >0.4. Experiments were performed at room temperature (22–24°C). Data are presented as mean ± SD. The solutions bathing both sides of the membrane contained 150 mM KCl and 5 mM TES ( N -tris(hydroxymethyl)methyl-2-aminoethane sulfonate) pH buffer, with the pH of the solutions adjusted to 7.0. Contaminant Ca 2+ i was determined by atomic absorption spectrometry. The solution at the intracellular side of the membrane also contained added Ca 2+ (as CaCl 2 ), to bring the Ca 2+ concentration at the intracellular surface (Ca 2+ i ) to the indicated levels. (The solutions did not contain Ca 2+ buffers.) No Ca 2+ was added to the extracellular (pipette) solution. Solutions were changed through the use of a microchamber . Single-channel currents were recorded on a digital data recorder (DC-37 kHz; Instrutech Corp.), low-pass filtered with a four-pole Bessel filter to give a final effective filtering of 6–10 kHz (−3 dB), and sampled by computer at a rate of 125–200 kHz. The effective filtering is expressed in terms of dead time, which is the duration of an underlying interval before filtering that would just reach 50% of the single-channel current amplitude with filtering . Five channels in which stable single-channel data were obtained over a wide range of Ca 2+ i were analyzed in detail. The channels and their dead times were: B04, 16 μs; B06 and B16, 28.5 μs; B12, 22.9 μs, and B14, 17.9 μs. The sampled currents were analyzed using custom programs written in the laboratory. The methods used to set the level of filtering to exclude false events that could arise from noise, measure interval durations with half-amplitude threshold analysis, test for stability, and identify activity in the various modes using stability plots, have been described previously, including the precautions taken to prevent artifacts in the analysis . The analysis in the present study was restricted to channel activity in the normal mode, which typically involves ∼96% of the detected intervals . Activity in modes other than normal, including the low activity mode and transitions to isolated long shut intervals, were removed before analysis. The low activity mode is readily distinguished from normal activity over the range of low to high Ca 2+ i , as detailed in Rothberg et al. 1996 . The isolated long shut intervals are also readily identified at high Ca 2+ i , and appear to contribute an insignificant number of the long shut intervals at lower Ca 2+ i . The mean frequency and duration of the isolated long shut intervals are Ca 2+ independent, indicating that isolated long shut intervals do not arise from discrete Ca 2+ block . In spite of their low frequency, appreciable time can be spent in the isolated long shut intervals at high Ca 2+ i because most of the other closed intervals are so brief. For higher Ca 2+ i s that give P o s > 0.8, BK channels can spend >30% of their time in the low activity mode that has a P o of ∼0.001 . Because of the potential effects of the low activity mode and isolated long shut intervals on the single-channel record, it is essential to identify and exclude these (and other modes), as we have done, when studying normal activity over a wide range of Ca 2+ i . Both one- (1-D) and two-dimensional (2-D) dwell-time distributions were analyzed. The 1-D distributions of open and closed interval durations were log-binned as described previously at a resolution of 25 bins per log unit for fitting with mixtures (sums) of exponential components. Details of estimating the numbers of significant exponential components are in McManus and Magleby 1988 , including the use of the likelihood ratio test for significance. The 2-D distributions of adjacent open–-closed interval pairs were log-binned at a resolution of 10 bins per log unit, as described previously . The 2-D distributions were fitted with mixtures (sums) of 2-D exponential components to estimate the numbers of underlying 2-D exponential components, and hence the minimum number of kinetic states, as detailed in Rothberg et al. 1997 . With filtering, detected intervals with durations less than about twice the dead time are narrowed . For the plotting of 2-D dwell-time distributions, the plotted intervals have not been corrected for this narrowing. For the fitting of kinetic models using 2-D dwell-time distributions, the measured durations of these intervals were corrected to their estimated true durations before binning and fitting, using the numerical method in Colquhoun and Sigworth 1995 . Even with correction, the data were typically fit starting at ∼1.5 dead times for two reasons: (a) intervals whose underlying (true) durations are less than the dead time can be detected when data are filtered, placing an excess of intervals in the first few bins , and (b) intervals that are undetected due to filtering (missed events) can introduce extra (phantom) components with time constants typically less than one-half the dead time , which can also add excess intervals to the first few bins. Depending on the gating mechanism, noise, and filtering, the excess intervals arising from either phenomena can lead to the detection of excess brief exponential components if the fitting includes intervals less than ∼1.5–2 dead times. Dependency plots were constructed from the 2-D dwell-time distributions as detailed in Magleby and Song 1992 and Rothberg and Magleby 1998 . In brief, the dependency for each bin of open–closed interval pairs with mean durations t O and t C is: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{dependency}} \left \left(t_{{\mathrm{O}}},t_{{\mathrm{C}}}\right) \right =\frac{N_{{\mathrm{Obs}}} \left \left(t_{{\mathrm{O}}},t_{{\mathrm{C}}}\right) \right -N_{{\mathrm{Ind}}} \left \left(t_{{\mathrm{O}}},t_{{\mathrm{C}}}\right) \right }{N_{{\mathrm{Ind}}} \left \left(t_{{\mathrm{O}}},t_{{\mathrm{C}}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where N Obs ( t O , t C ) is the experimentally observed number of interval pairs in bin ( t O , t C ), and N Ind ( t O , t C ) is the calculated number of interval pairs in bin ( t O , t C ) if adjacent open and closed intervals pair independently (at random). The method of calculating expected frequencies for observations that are independent (contingency tables) is a common statistical procedure . The expected number of interval pairs in bin ( t O , t C ) for independent pairing is: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}N_{Ind} \left \left(t_{{\mathrm{O}}},t_{{\mathrm{C}}}\right) \right =P \left \left(t_{{\mathrm{O}}}\right) \right {\times}P \left \left(t_{{\mathrm{C}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where P ( t O ) is the probability of an open interval falling in the row of bins with a mean open duration of t O , and P ( t C ) is the probability of a closed interval falling in the column of bins with a mean closed duration of t C . P ( t O ) is given by the number of open intervals in row t O divided by the total number of open intervals in all rows, and P ( t C ) is given by the number of closed intervals in column t C divided by the total number of closed intervals in all columns. Since open and closed intervals are paired, the total number of open intervals is equal to the total number of closed intervals. Each open and closed interval forms two pairs: one with the preceding interval and one with the following interval. Hence, the number of interval pairs in a 2-D dwell-time distribution is equal to the number of open plus closed intervals minus one. The most likely rate constants for the examined kinetic schemes were estimated from the simultaneous fitting of the 2-D dwell-time distributions obtained at six different Ca 2+ i using the iterative maximum likelihood fitting procedure described in Rothberg and Magleby 1998 with Q-matrix methods to calculate the predicted 2-D dwell-time distributions . Corrections for missed events were applied during the fitting using the method described in Crouzy and Sigworth 1990 . Since the gating of native BK channels in rat skeletal muscle appears consistent with microscopic reversibility , microscopic reversibility was maintained during the fitting. The number of underlying 2-D exponential components that sum to form a 2-D dwell-time distribution is given by the product of the numbers of open and closed states . Thus, a single 2-D dwell-time distribution fitted with, for example, three open and five closed 2-D exponential components can potentially define 22 parameters: three open and five closed time constants and the volumes of 14 of 15 (3 × 5) underlying 2-D components. One of the volumes is not free since the volumes must sum to 1.0. Simultaneous fitting of three to six 2-D dwell-time distributions obtained at different Ca 2+ i further increased the information available to estimate the fixed number of rate constants that defined the models. In spite of all this information, rate constants can remain ill-defined in complex models , which is why the analysis in this paper has been restricted to models with the fewest numbers of states that can approximate the examined kinetic features of the data. Rather than estimating the uncertainty in the rate constants for any single experiment, the uncertainty was estimated by comparing rate constants obtained from fitting data from different channels. For a given model and rate constants, the equilibrium occupancies of the states could be calculated as described in Colquhoun and Hawkes 1995b . The frequencies of entry into each state could then be calculated by dividing the equilibrium occupancy of a state by its mean lifetime. Normalized likelihood ratios have been used to indicate how well any given kinetic scheme describes the 2-D dwell-time distributions when compared with a theoretical best description of the data. Normalization corrects for the differences in numbers of interval pairs among experiments so that comparisons can be made among channels. The normalized likelihood ratio per 1,000 interval pairs is defined as 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{NLR}}_{1000}={\mathrm{exp}} \left \left[ \left \left({\mathrm{ln}}S-{\mathrm{ln}}T\right) \right \left \left({1,000}/{n}\right) \right \right] \right {\mathrm{,}}\end{equation*}\end{document} where ln S is the natural logarithm of the maximum likelihood estimate for the observed 2-D dwell-time distributions given the kinetic scheme, ln T is the natural logarithm of the likelihood of the theoretical best description of the observed distributions, and n is the total number of fitted interval pairs (events) in the observed dwell-time distributions . The NLR gives a measure of how well different kinetic schemes describe the distributions, but it cannot be used to directly rank schemes since no penalty is applied for the numbers of free parameters. To overcome this difficulty, models were ranked using an information criteria approach , which has the limitation that the significance level is not known. If 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{log}}_{{\mathrm{e}}} \left \left({m_{{\mathrm{g}}}}/{m_{{\mathrm{f}}}}\right) \right > \left \left(k_{{\mathrm{g}}}-k_{{\mathrm{f}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} then model g is ranked above model f, where m g and m f are the maximum likelihood estimates for models g and f, and k g and k f are the number of free parameters for each scheme. We also compared the Akaike rankings to those of the Schwarz 1978 criterion and found that the Akaike rankings for different schemes were more consistent among channels and also were in better agreement with rankings based on the visual perception of the ability of models to describe major features of the data. To evaluate models, it was useful to have an estimate of the theoretical best descriptions of the dwell-time distributions for comparison to the distributions predicted by the various examined gating mechanisms. For gating consistent with a discrete state Markov process (the rate constants remain constant in time for constant experimental conditions), as appears to be the case for BK channels , the log likelihood for a theoretical best description of the 2-D dwell-time distribution could be obtained by fitting the distribution with an uncoupled (generic) scheme . Uncoupled schemes have direct transition pathways between every open and closed state, and no transition pathways between open states or between closed states . The log-likelihood value for the theoretical best fit for the simultaneous fitting of dwell-time distributions obtained at different Ca 2+ i was given by the sum of the log-likelihood values obtained by fitting data at each Ca 2+ separately with the uncoupled scheme. To make comparisons between the observed distributions and those predicted by the kinetic models, simulated single-channel current records were generated with filtering equivalent to that used in the analysis of the experimental current records and with noise similar to that in the experimental current record. The simulated single-channel currents were then analyzed in the same way that the experimental currents were in order to obtain the predicted 2-D distributions, dependency plots, and numbers of exponential components observed in the predicted dwell-time distributions. The method used to simulate single-channel currents with filtering and noise is detailed in Magleby and Weiss 1990a . Visual comparison between observed and predicted distributions and dependency plots served to guide the analysis, indicate where models were inadequate, and give a sense for how well the models described the data. The critical assessment of the models was based on quantitative comparisons using maximum likelihood and Akaike rankings ( and ). Figure S1 presents the equilibrium occupancy of the states in Fig. 7 at low (5.5 μM) and high (1,024 μM) Ca 2 + i . At low Ca 2 + i , the channel spent most of its time (91.7%) in closed states C8–C11, considerably less time (7.5%) in open states O1–O6, and even less time (0.8%) in the intermediate closed states C12–C17. At high Ca 2 + i , the channel spent essentially all (99.4%) of its time in the fully liganded open and closed states, with most (96.6%) of the time spent in open states O1 and O4, considerably less time (2.8%) in closed states C7, C12, and C15. A very small amount of time (0.5%) was spent in open states O2 and O5, with negligible time (0.1%) spent in all of the remaining open and closed states. Thus, at high Ca 2 + i , Fig. 7 gates as though there were one effective transition pathway between open and closed states (C15–O4), inconsistent with the observed significant dependencies at high Ca 2 + i . Available at http://www.jgp.org/cgi/content/full/114/1/93/DC1 Figure S2 presents the estimated rate constants for the three channels examined in detail for Fig. 4 Fig. 5 Fig. 6 Fig. 7 , Fig. 12 , and Fig. 13 . Available at http://www.jgp.org/cgi/content/full/114/1/93/DC1 The findings presented in this paper are based on a complete single-channel analysis of data obtained from three different BK channels, each studied at six different Ca 2+ i , from low to high (4–1,024 μM). In addition to these three channels, two additional channels were analyzed at three Ca 2+ i , including high Ca 2+ i , and an additional four channels were also examined, with findings consistent with the channels analyzed in more detail. All of the data, analysis, and figures presented in this paper are restricted to data collected during normal (mode) activity, which typically includes ∼96% of the intervals . All experiments were performed at +30 mV. Currents flowing through a single BK channel in an inside-out patch of membrane excised from a cultured rat skeletal muscle cell are shown in Fig. 1 A. These traces are representative of BK channel gating during normal activity over a range of Ca 2+ i , and illustrate a small fraction of the ∼25 min of continuous recording from this channel. The current traces are excerpts of data obtained with 5.5, 12.3, 132, and 1,024 μM Ca 2+ i that gave mean open probabilities ( P o ) during normal activity of 0.061, 0.50, 0.97, and 0.97, respectively. Thus, while the 2.2-fold increase in Ca 2+ i from 5.5 to 12.3 μM gave an 8.2-fold increase in P o , the 7.8-fold increase in Ca 2+ i from 132 to 1,024 μM resulted in no increase in P o , indicating that the channel had reached a maximum level of activation. The saturation in P o at high Ca 2+ i is readily apparent in the current traces in Fig. 1 . The effect of Ca 2+ on P o for five different single BK channels during normal activity is illustrated in Fig. 2 A. For these channels, increasing Ca 2+ i in the 4–20 μM range increased P o from ∼0.02 to 0.85, while further increases in Ca 2+ i to either 132 or 1,024 μM led to a maximum P o ranging from 0.93 to 0.98. Maximum P o s within this range have been observed previously . Fitting P o versus Ca 2+ i with the Hill equation for data from these five channels gave a K 0.5 (the Ca 2+ i for a P o of 0.5) of 11.1 ± 0.7 μM, with a Hill coefficient of 3.5 ± 0.6, consistent with at least four Ca 2+ -binding steps contributing to maximal channel activation. The mean of the maximal fitted P o was 0.95 ± 0.03, and the fitted line indicated that a Ca 2+ of ∼40 μM was sufficient to drive the channel to within 1% of the maximum P o . Hill coefficients ranging from 2–4 are a common feature of BK channels (see introduction ), and higher slopes have been reported , consistent with the channel binding at least four Ca 2+ to become fully activated. The P o did not reach 1.0 during normal activity at high Ca 2+ i because of frequent sojourns to brief closed states that generated flickers, and also because of much less frequent sojourns to longer closed intervals with durations of typically 1–10 ms. Examples of the frequent flickers and of the less frequent longer closed intervals during normal activity at high Ca 2+ i (>100 μM) are presented in Fig. 1 B for data obtained at 132 and 1,024 μM Ca 2+ i . Analysis of the current records obtained at 132 and 1,024 μM Ca 2+ i indicated that only 2.1 ± 1.0% of the closed intervals had durations >1 ms, and even fewer, 0.026 ± 0.023%, had durations >10 ms. (The estimates obtained at both 132 and 1,024 μM Ca 2+ i from each of five channels were combined, as the percentages of longer closed intervals at the two different high Ca 2+ i were not significantly different.) The increase in P o with increasing Ca 2+ i was associated with increases in the mean open interval durations and decreases in the mean closed interval durations , consistent with previous reports for BK channels from skeletal muscle and mSlo BK channels . The increase in mean open interval duration was much less Ca 2+ sensitive than the decrease in mean closed interval duration , suggesting that the major effect of Ca 2+ i is to decrease the durations of the closed intervals. The decreased filtering for channel B04 would contribute to the briefer observed open times for this channel, as more of the flickers would be captured. Just as P o saturated at high Ca 2+ i , the mean durations of the open and closed intervals also saturated in high Ca 2+ i at ∼3.6 and ∼0.15 ms, respectively . Thus, any viable mechanism for the gating of the BK channel must account for a saturation in mean open times, mean closed times, and in P o at high Ca 2+ i . To further characterize the effect of high Ca 2+ i on gating, the open and closed interval durations during normal activity were measured and plotted as 1-D dwell-time distributions in Fig. 3 . The thick lines in the open and closed distributions in Fig. 3 are the fits with mixtures (sums) of three open and five closed exponential components, respectively. Increasing Ca 2+ i from 5.5 to 132 μM shifted the open intervals to longer durations (note rightward shift of the major peak for the open times) and the closed intervals to briefer durations (note leftward shift and decreasing amplitude of the peak describing the longer closed times.). A further increase in Ca 2+ i from 132 to 1,024 μM had little additional affect on either the open or closed distributions, as seen in Fig. 3D and Fig. H , where the fits to the distributions at 132 μM Ca 2+ i are plotted as thin lines on the fits to the distributions at 1,024 μM Ca 2+ i . The thin lines essentially superimpose the thick lines, indicating essentially unchanged gating kinetics at high Ca 2+ i . Fig. 3 H, inset, where the ordinate is plotted on a log scale to present the tails of the distributions at high gain, shows that the thin line also superimposes the thick line (within the range of the data) at the longer intervals where the frequency of occurrence of intervals is low. Results consistent with those in Fig. 3 were observed for four additional channels. Table presents the time constants and areas of the three open and five closed exponential components fitted to the 1-D distributions in Fig. 3 . For increases in Ca 2+ i up to 132 μM, the time constant of the briefest open component remained relatively unchanged, the time constants of the two longer open components increased with increasing Ca 2+ i , and the area from the briefest open component shifted into the longer open components. In contrast, the time constants of the longer closed components became briefer and their areas shifted from the longer to the briefer closed components. Increasing the Ca 2+ i from 132 to 1,024 μM then had little additional effect on the open and closed components, when compared with the large effects observed for lower concentrations of Ca 2+ i . Table presents the mean ± SD of the time constants and areas of the exponential components for data obtained from five channels at two levels of high Ca 2+ i . For four of the channels, the data were obtained at 132 and 1,024 μM Ca 2+ i , and for the fifth channel the data were obtained at 100 and 1,000 μM Ca 2+ i . The data from the fifth channel was pooled with the data from the other four since there was no apparent difference in the findings. Increasing the Ca 2+ i from 132 to 1,024 μM had no significant effect on either the time constants or areas of any of the open or closed components ( Table , P > 0.05; paired t test). The measured increase in the time constant of the slowest closed component in Table for channel B06 would be consistent with stochastic variation, as there were <20 intervals contributing to this component at each of the two levels of high Ca 2+ i . The apparent lack of effect of an eightfold increase in Ca 2+ i on the dwell-time distributions at high Ca 2+ i suggest that the rates for the transitions between states that dominate the gating at high Ca 2+ are either Ca 2+ independent or saturate at high Ca 2+ . These possibilities will be considered later. It is well established that Ba 2+ produces discrete block of BK channels . If Ca 2+ produces a similar block, then a potential difficulty with conducting experiments in high Ca 2+ i is the possibility of discrete (slow) channel block by Ca 2+ . Closed intervals arising from discrete Ca 2+ block could then be mistaken for closed intervals arising from sojourns to closed states during the gating. The observations in Fig. 1 and Fig. 3 at high Ca 2+ i are consistent with our previous findings of a lack of evidence for discrete Ca 2+ block of native BK channels from rat skeletal muscle . Increasing Ca 2+ i eightfold from 132 to 1,024 μM had little effect on the closed dwell-time distributions . If appreciable closed intervals arose from discrete Ca 2+ block, then increasing Ca 2+ i eightfold might have been expected to have a noticeable effect on the distributions, which was not observed. As a more critical test for discrete block, we tabulated the frequency of occurrence of closed intervals >1 and >10 ms at the two different Ca 2+ i . For the five examined BK channels, the frequency of closed intervals with durations >1 ms was 7.4 ± 3.6 s −1 for 132 μM Ca 2+ i and 5.6 ± 2.5 s −1 for 1,024 μM Ca 2+ i , values that were not significantly different \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(P\;=\;0.098,\;{\mathrm{paired}}\;t\;{\mathrm{test}})\end{equation*}\end{document} . The frequency of closed intervals with durations >10 ms was 0.12 ± 0.11 s −1 with 132 μM Ca 2+ i and 0.076 ± 0.086 s −1 with 1,024 μM Ca 2+ i , values that were also not significantly different \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(P\;=\;0.44,\;{\mathrm{paired}}\;t\;{\mathrm{test}})\end{equation*}\end{document} . Thus, the lack of effect of Ca 2+ on the closed dwell-time distributions from brief to long times, and also on all closed intervals >1 and >10 ms, suggests that discrete Ca 2+ block did not contribute to the closed intervals. As expected, increasing Ca 2+ i to 1,024 μM did decrease the conductance of the channel by ∼10%, presumably due to a screening (fast blocking) effect . Cox et al. 1997a studying mSlo BK channels also found that high Ca 2+ i (1,000 μM) reduced single-channel conductance through a fast block, but did not induce discrete block. Since high Ca 2+ i does not introduce closed intervals arising from discrete block, it is possible to estimate the numbers of kinetic states that contribute to the gating at high Ca 2+ i . Assuming that the gating is consistent with a discrete-state Markov model , the number of significant exponential components required to describe the dwell-time distributions gives an estimate of the minimum number of states entered during the gating . Estimates of the numbers of significant exponential components were made from fitting both 1-D and 2-D dwell-time distributions (see methods ). These estimates were then plotted against Ca 2+ i in Fig. 4 . In general, the 1-D and 2-D methods gave similar estimates of the numbers of exponential components, but there were some differences. 2-D fitting can have an increased ability to detect components over 1-D fitting when the same numbers of intervals are analyzed . However, with 2-D fitting, when very brief intervals pair with longer intervals, both intervals in the pair are excluded from the fitting to avoid potential artifacts arising from fitting intervals with durations <1.5–2 dead times (see methods ). The combination of the greater ability to detect components together with the fitting of fewer intervals for 2-D fitting, when compared with 1-D fitting, would contribute to the minor differences in the numbers of significant components estimated by the 1- and 2-D fitting methods. At the lower concentrations of Ca 2+ i , the distributions were typically described by three to four open and four to seven closed components for fitting with either 1- or 2-D distributions , consistent with previous observations . At the high concentrations of 132 and 1,024 μM Ca 2+ i , the open distributions were still typically described by three to four open components, and the closed distributions were typically described by four to five closed components. Thus, at kinetically saturating levels of Ca 2+ i , the gating typically involved transitions among at least three to four open and four to five closed kinetic states. Functional BK channels can be formed by four alpha subunits , and the BK channels in skeletal muscle, as studied here, are unlikely to be associated with the auxiliary beta subunit that increases the Ca 2+ sensitivity of the channel . If one or more Ca 2+ ions bind to each alpha subunit to fully activate the channel , then this would give a basis for the high Hill coefficients of 2–4 that are typically observed for activation (see introduction ). Although relatively simple in concept, with four subunits and at least one Ca 2+ -binding site per subunit, theoretical models for the gating of a ligand-activated homotetramer, such as the BK channel, can be highly complex, with 35–55 potential states . The 55-state model is given by Fig. 1 , where each subunit can assume either of two conformations, indicated by squares or circles, and each subunit in either conformation can either be free of Ca 2+ (open symbols) or bound with a Ca 2+ (shaded symbols). Many of the states are isoforms, in which subunits with diagonal and adjacent conformational changes and Ca 2+ bindings have potentially different functional properties. For the gating of the channel, the states in the top row of Fig. 1 are assumed to represent closed states of the channel, and the states in the bottom row are assumed to represent open states. The conductance of the states in the middle three rows is less clear, but may be open, closed, or partially conducting . Fig. 1 reduces to the 35-state model of Eigen 1968 if it is assumed that diagonal and adjacent subunits in the same conformation have identical properties. Because of the complexity of the 55- and 35-state schemes, Fig. 1 is often reduced further to the 25-state model described by Fig. 2 . If it is further assumed that each collection of isoforms has the same properties, and that conformational changes are concerted so that all four subunits undergo conformational changes simultaneously or that the lifetimes of the middle three rows of states in Fig. 1 and Fig. 2 are very brief, then Fig. 1 and Fig. 2 reduce to the 10-state Monod-Wyman-Changeux model for allosteric proteins described by Fig. 3 . It has been shown previously for low to intermediate levels of Ca 2+ i that the Ca 2+ dependence of the single-channel kinetics of BK channels in rat skeletal muscle can be approximated by the gating mechanisms described by Fig. 4 Fig. 5 Fig. 6 . Thus, we first examined whether these schemes might also account for the single-channel kinetics through high Ca 2+ i . Fig. 4 is drawn from the MWC model. Fig. 6 is an expansion of Fig. 4 because it includes closed states beyond the activation pathway. Fig. 5 can be viewed as a condensed version of Fig. 1 and Fig. 2 if it is assumed that the intermediate states in Fig. 1 and Fig. 2 are too brief to be detected or that only a subset can be detected. It is the additional brief closed states C9, C10, and C11 that generate most of the flickers (brief closings) in Fig. 5 and Fig. 6 , whereas flickers for Fig. 4 are generated mainly by sojourns between states O2 and C5. Flickers are highly characteristic of single-channel currents and can be seen in Fig. 1 . Before examining whether Fig. 4 Fig. 5 Fig. 6 could account for the gating from low through high Ca 2+ i , we first examined whether they could describe the 1-D dwell-time distributions from low to intermediate levels of Ca 2+ i , as reported previously . Rate constants for each scheme were estimated by the simultaneous fitting of 2-D dwell-time distributions obtained at three different Ca 2+ i of 5.5, 8.3, and 12.3 μM (see methods ). The most likely rate constants were then used to simulate single-channel data with noise and filtering equivalent to that of the experimental data. The simulated current records were then analyzed in the same manner as the experimental data to determine the predicted 1-D dwell-time distributions. As expected, the predicted distributions gave excellent descriptions of the observed 1-D dwell-time distributions from low to intermediate Ca 2+ i for the three channels examined in detail. For example, for the data in Fig. 3A , Fig. B , Fig. E , and Fig. F , at 5.5 and 12.3 μM Ca 2+ i , the predicted distributions typically overlapped or were within a line width of the thick lines describing the dwell-time distributions in these figures (not shown). We next examined whether Fig. 4 Fig. 5 Fig. 6 could predict the distributions from low to high Ca 2+ i . Rate constants for each scheme were estimated by the simultaneous fitting of 2-D dwell-time distributions obtained at six different Ca 2+ i of 5.5, 8.3, 12.3, 20.3, 132, and 1,024 μM. These rate constants were then used to predict the observed distributions over the full range of Ca 2+ i . As shown in Fig. 5 , Fig. 4 could not simultaneously describe the dwell-time distributions from low to high Ca 2+ i . Fig. 5 gave a better description than Fig. 4 , but still could not describe the distributions. The predictions of Fig. 6 were very slightly better than those of Fig. 5 , and are not shown. Table shows the schemes rankings: Fig. 6 > Fig. 5 > Fig. 4 (Akaike rankings, ). The inability of these schemes to account for the single-channel kinetics from low to high Ca 2+ i indicates that the models described by Fig. 4 Fig. 5 Fig. 6 are too simple. Such a finding is, perhaps, not surprising since Fig. 4 with eight states and Fig. 5 and Fig. 6 with 11 states include only a small subset of the minimal 55 potential states for the gating of a ligand-activated homotetrameric channel based on theoretical considerations . Nevertheless, it is possible that the channel does not gate as described by the theoretical 55-state model or, if it does, that only a fraction of the potential 55 states actually contribute to the gating. Consequently, we examined the differences between the observed and predicted responses for these schemes to determine how the minimal schemes might be expanded to be more consistent with the gating of the channel. For high Ca 2+ i , Fig. 4 Fig. 5 Fig. 6 predicted fewer brief openings than were observed in the single-channel data . Brief openings in Fig. 5 typically arose from sojourns such as -C11-O3-C11-, and in Fig. 6 from sojourns such as -C11-O3-C11- or -C6-O3-C6-, as O3 was the open state with the briefest lifetime in both schemes. For these schemes, the channel would be unlikely to reach O3 or the associated closed states at high Ca 2+ i , as the high Ca 2+ i would drive the gating towards the fully liganded states. Thus, to generate more brief open intervals at high Ca 2+ i , there needs to be a means for the gating to reach directly one or more brief open states from the fully liganded closed states. In addition, the underprediction of the long closed intervals at high Ca 2+ i suggests that the channel may also gate among additional closed states at high Ca 2+ i not included in Fig. 4 Fig. 5 Fig. 6 . Further evidence that the channel gates among additional states at high Ca 2+ i arises from the observation in Fig. 4 that the channels typically entered at least three to four open and four to five closed states at high Ca 2+ i , while analysis of the dwell-time distributions predicted by Fig. 4 Fig. 5 Fig. 6 at high Ca 2+ i indicated that the distributions were described by only one open and two to three closed components. We next examined how Fig. 5 might be expanded to provide the required access to more open and closed states at high Ca 2+ i . Fig. 5 is contained within the general Fig. 1 and Fig. 2 if it is assumed that the three rows of intermediate states in the general schemes are closed states that can be collapsed into one row of brief lifetime intermediate closed states. However, since it is possible that the last two rows of states in Fig. 1 and Fig. 2 are open states and that the remaining intermediate closed states do not collapse into one row of closed states, we investigated whether simplified schemes drawn from Fig. 2 , with an assumption of three rows of closed states and two rows of open states, could account for the data. Fig. 7 presents a gating mechanism of this type, where open states O4-O5-O6 (with brief lifetimes) and closed states C12-C13-C14 are the additional rows of open and closed states when compared with Fig. 5 . With high Ca 2+ i , the channel could now make sojourns from closed states to brief open states, such as -C15-O4-C15-, giving brief open intervals, and also make sojourns from closed states to longer open (and compound open) states, such as -C15-O4-O1-O4-C15-, giving longer open intervals. The extra row of closed states should also allow the generation of longer closed intervals at high Ca 2+ i by allowing more sojourns among closed states between openings. The most likely rate constants for Fig. 7 were determined from the simultaneous fitting of 2-D dwell-time distributions at six different Ca 2+ i (5.5, 8.3, 12.3, 20.3, 132, and 1,024 μM). The thick lines in Fig. 5 show that the additional states allowed Fig. 7 to describe the 1-D open and closed dwell-time distributions from low to high Ca 2+ i . In this scheme, the mean lifetimes at 5.5 μM Ca 2+ i of states O4, O5, and O6, of 0.11, 0.09, and 0.03 ms, tend to be brief compared with the lifetimes of the final row of open states O1, O2, and O3 of 0.50, 0.18, and 0.06 ms. The improved ability of Fig. 7 to describe the single-channel gating when compared with Fig. 4 Fig. 5 Fig. 6 is also reflected in the greatly improved likelihood ratios in Table . We also examined whether a scheme like Fig. 7 , but with one less row of intermediate closed states, could account for the data. For the three channels examined, the likelihood estimates were two to four orders of magnitude less than for Fig. 7 , and the reduced scheme ranked below Fig. 7 for all three channels (not shown). Fig. 7 accounts for the 1-D open and closed dwell-time distributions from low to very high Ca 2+ i . Such a description would be sufficient to predict P o as a function of Ca 2+ i over a wide range of activity, but the 1-D distributions do not take into account the correlation information between adjacent intervals, which can give insight into the connections (transition pathways) among the various states . To examine the correlation information, we determined whether Fig. 7 could account for the kinetic structure of the single-channel data. The kinetic structure is described by 2-D dwell-time distributions and dependency plots. The 2-D distributions indicate the frequency of occurrence of pairs of adjacent open and closed intervals , and the dependency plots convey information about the correlations of adjacent interval durations . The kinetic structure for the same channel featured in the previous figures (channel B06) is shown in Fig. 6 at four different Ca 2+ i . The 2-D dwell-time distributions are plotted on log–log coordinates with the logs of the durations of adjacent open and closed intervals locating the position of the bin on the x and y axis, respectively. The z axis plots the square root of the numbers of intervals in each bin. These 2-D dwell-time distributions thus extend the Sigworth and Sine 1987 transform used in the previous figures to two dimensions. From the 2-D dwell-time distributions it can be seen that pairs of long open intervals adjacent to brief closed intervals (flickers) occur most frequently of all the interval pairs, and this is the case from low to high Ca 2+ i . It is these interval pairs that give rise to the characteristic longer openings separated by flickers in the experimental data . At the lowest Ca 2+ i of 5.5 μM, there were also large numbers of longer open intervals adjacent to longer closed intervals and brief open intervals adjacent to longer closed intervals (left, position 3). As the Ca 2+ i was raised, the longer closed intervals shifted to briefer durations. The peak at position 6 with 5.5 μM Ca 2+ i shifted towards position 5 at 12.3 μM Ca 2+ i , and position 4 at higher Ca 2+ i . The dependency plots in Fig. 6 (right) present the fractional excess or deficit of interval pairs of specified durations over that expected if the intervals paired at random. Dependencies of +0.5 or −0.5 would indicate a 50% excess or 50% deficit of interval pairs over the number expected if open and closed intervals paired independently. The thick lines indicate a dependency of zero. Because the dependency plots present magnified representations of excesses and deficits in the numbers of observed interval pairs relative to the numbers expected for independent pairing, they must be interpreted with some caution, as the estimates of dependency can be unreliable where the numbers of observed interval pairs per bin in the 2-D dwell-time distributions are small. Consequently, references to dependency will only be made when the referenced dependencies are known to be significantly different from zero. Such dependencies will be referred to by numbers on the dependency plots. Examples of which areas of the dependency plots are significantly different from zero are presented in Rothberg and Magleby 1998 for low to intermediate levels of Ca 2+ i and will be presented in a later section for data obtained at high Ca 2+ i . The dependency plots in Fig. 6 (right) indicate that over the wide range of examined Ca 2+ i there was a deficit of brief open intervals adjacent to brief closed intervals (position 1), an excess of brief open intervals adjacent to both intermediate and long closed intervals (positions 2 and 3), and a deficit of long open intervals adjacent to long closed intervals (position 6; not clearly visible in the presented orientation, but visible when the plots were rotated). At the lower Ca 2+ i of 5.5 and 12.3 μM, there was also an excess of longer open intervals adjacent to brief closed intervals (position 4). These specific excesses and deficits of interval pairs give rise to the characteristic saddle shape of the dependency plots for BK channels , and were consistently seen for all the examined channels. While the kinetic structure at intermediate levels of Ca 2+ i for six different BK channels has been presented previously , it was important to determine whether the kinetic structure at high Ca 2+ , as shown in Fig. 6 , was consistently observed. (It will be shown in a later section that the presence or absence of significant dependencies at high Ca 2+ i is a key factor in distinguishing gating mechanism.) Fig. 7 presents such plots for data obtained at 132 and 1,024 μM Ca 2+ i for two additional channels. Although there were some obvious differences in the magnitudes of the dependencies, the general shape of the kinetic structure in these plots was the same as for the channel shown in Fig. 6G and Fig. H , in that there were obvious deficits of brief open intervals adjacent to brief closed intervals and obvious excesses of brief open intervals adjacent to intermediate duration closed intervals (right, position 2). Dependency significance plots were made for the data obtained at high Ca 2+ i to estimate which dependencies were significantly different from zero. A paired t test was used to compare the number of interval pairs in each bin of the observed 2-D dwell-time distribution with the number expected if adjacent open and closed intervals paired independently, by using a moving 3 × 3 bin array as detailed in Rothberg and Magleby 1998 . Results are shown in Fig. 8 for channel B12. The plots in Fig. 8A and C , present the dependency significance for the front and back views, respectively, of the dependency plot in Fig. 7 E obtained at 132 μM Ca 2+ i , and the plots in Fig. 8B and Fig. D , present the dependency significance for the front and back views of the dependency plot in Fig. 7 F obtained at 1,024 μM Ca 2+ i . The dependency significance plots present the logarithm of the estimated P value, which is multiplied by the sign of the dependency to indicate whether the paired intervals are in excess or deficit. The thick lines on the plots at −1.3 and 1.3 indicate a significance level of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{P}}\;=\;0.05\end{equation*}\end{document} . Absolute values of dependency significance > 1.3, 2, 3, and 4 would indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively. From Fig. 8 it can be seen that the dependencies at the numbered positions 1, 2, 4, and 5 were significantly different from zero at both 132 and 1,024 μM Ca 2+ i . Importantly, there was a significant excess of long open intervals adjacent to brief closed intervals (position 4), even though the fractional excess of these interval pairs was small in the dependency plots . The reason for this apparent discrepancy is that most of the interval pairs fall at position 4 in high Ca 2+ i , as can be seen from the 2-D dwell-time distributions , so that even an appreciable excess of interval pairs at position 4 would still appear small when plotted as dependency, which plots the fractional excess of intervals ( ). In six of six dependency significance plots that were examined at high Ca 2+ i , the dependencies at positions 1, 2, and 5 were significant, and the dependency at position 4 was significant in five of six plots. The one plot where significance was not observed at position 4 had fewer numbers of analyzed intervals. The observations that the numbers of detected kinetic states remained relatively unchanged from low to high Ca 2+ i and that the general shapes of the dependency plots also remained relatively unchanged from low to high Ca 2+ i raise the possibility that the basic gating mechanism remains relatively unchanged from low to high Ca 2+ i . To determine whether Fig. 7 could account for the kinetic structure, the most likely rate constants for Fig. 7 , determined from the simultaneous fitting of 2-D dwell-time distributions at six different Ca 2+ i (5.5, 8.3, 12.3, 20.3, 132, and 1,024 μM), were used to simulate single-channel data for Fig. 7 with noise and filtering equivalent to that of the experimental data. The simulated current records were then analyzed in the same manner as the experimental data to determine the predicted kinetic structure shown in Fig. 9 . Fig. 7 captured the basic features of the kinetic structure at 5.5 and 12.3 μM Ca 2+ i . Fig. 7 also captured the basic features of the 2-D dwell-time distributions at the high Ca 2+ i of 132 and 1,024 μM . However, Fig. 7 predicted that little or no dependence would be observed at high Ca 2+ i , in contrast to the significant dependencies observed in the experimental data . Thus, Fig. 7 predicted that open and closed intervals would pair randomly at high Ca 2+ i , in contrast to the dependent pairing observed in the data. These observations indicate that Fig. 7 is too simple to capture the features of the gating at high Ca 2+ i . To explore why Fig. 7 did not predict the dependency at high Ca 2+ i , we calculated the equilibrium occupancies of the various open and closed states for this scheme. At low to intermediate Ca 2+ i (5.5–12.3 μM), the channel readily entered all the states in Fig. 7 , with occupancy biased towards the closed states C8–C11 at the low Ca 2+ i . At high Ca 2+ i (132 and 1,025 μM), the channel spent 99.6% of its time in the fully liganded states, with 96.6% in open states 1 and 4 and 2.8% in closed states 7, 12, and 15. The reason that Fig. 7 predicted a lack of dependence between adjacent open and closed intervals at high Ca 2+ i is now readily apparent. Because the channel spent 99.4% of its time in the fully liganded column of states at high Ca 2+ i , the channel would essentially gate as Fig. 8 . Fig. 8 has a single effective transition pathway between the open and closed states, given by C15-O4. A single effective transition pathway gives a single gateway state, which would lead to independent pairing of open and closed intervals and lack of significant dependencies . If Fig. 7 does effectively gate in the fully liganded column of states at high Ca 2+ i , as indicated by Fig. 8 , then a maximum of two open and three closed components would be detected in the dwell-time distributions predicted by Fig. 7 at high Ca 2+ i . This was found to be the case. Fitting exponentials to dwell-time distributions simulated with Fig. 7 at high Ca 2+ i gave two open and three closed components, compared with the typically three to four open and four to five closed states detected in the experimental data. Fig. 7 also predicted a lack of dependence and too few components at high Ca 2+ i for the two other channels analyzed in detail. The above findings indicate that Fig. 7 can thus be rejected as a model for gating, as it cannot describe the dependencies at high Ca 2+ i . By analogy, Fig. 1 and Fig. 2 and all schemes based on subsets of states drawn from these schemes, such as Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 , can also be rejected as such schemes would also not describe the dependencies at high Ca 2+ i , provided that the forward rate constants for binding of Ca 2+ are sufficiently rapid at high Ca 2+ to effectively keep the gating in the fully liganded column of states. The above results suggest that models for gating at high Ca 2+ i must: (a) allow effective transitions among at least three to four open and four to five closed states at high Ca 2+ i to generate the required numbers of exponential components in the dwell-time distributions , (b) have two or more independent transition pathways between open and closed states (two or more gateway states) at high Ca 2+ i to generate a dependent relationship between the durations of adjacent intervals , and (c) gate among the three to four open and four to five closed states at high Ca 2+ i in a manner that is essentially independent of Ca 2+ i for Ca 2+ i > 100 μM to account for the observed lack of effect of Ca 2+ on the gating at high Ca 2+ i . Since Fig. 1 and Fig. 2 are theoretical schemes based on a ligand-activated homotetramer, it might be useful if these schemes could be modified to account for gating at high Ca 2+ i . As pointed out by Cox et al. 1997b , it is not necessarily clear which of the states in Fig. 1 are open and which are closed. Perhaps all 55 or 25 states are closed, each with the potential of opening through a concerted conformational change of the subunits. Concerted conformational changes leading to opening have been suggested previously for the gating of Shaker channels . If each closed state can open for BK channels, then Fig. 1 and Fig. 2 would become two-tiered 110- and 50-state models, respectively, with the upper tiers composed entirely of closed states and the lower tiers of an equal number of open states. Preliminary data using macroscopic ionic and gating currents from mSlo are consistent with such models . The extension of Fig. 2 to a two-tiered model is given by Fig. 9 , where in the graphic representation all 25 closed states in the upper tier are visible and only the first two rows of the 25 open states in the lower tier are visible A difficulty with Fig. 9 , as was also the case for Fig. 1 and Fig. 2 , is that Fig. 9 has so many rate constants that it would be difficult if not impossible to determine unique rate constants for this scheme, even by the simultaneous fitting of 2-D dwell-time distributions over a range of Ca 2+ i (see methods ). Consequently, we examined whether a reduced version of Fig. 9 might be sufficient to approximate the kinetic structure. The number of rows of closed states in the upper tier was reduced from five to three, and the number of rows of open states in the lower tier was reduced from five to two to obtain the reduced Fig. 10 . Some of the open and closed states with zero and one bound Ca 2+ were also omitted in Fig. 10 to reduce further the number of rate constants because, over the range of Ca 2+ i examined in this paper, it might be expected that these states would contribute little to the gating . Fig. 10 is like Fig. 7 , except that Fig. 10 has a total of six independent transition pathways between the open and closed states, compared with three in Fig. 7 . Two of the independent transition pathways connect fully liganded open and closed states, which would then allow at least two functional gateway states for gating with high Ca 2+ i . Two or more gateway states would be needed to generate the dependencies observed in high Ca 2+ i . Fig. 10 was tested by determining the most likely rate constants from the simultaneous fitting of 2-D dwell-time distributions obtained at six different Ca 2+ i ranging from 5.5 to 1,024 μM. The most likely rate constants were then used with Fig. 10 to obtain the predicted kinetic structure in Fig. 10 . The predicted kinetic structure shows that Fig. 10 captured the major features from low to high Ca 2+ i , including the general shapes of the dependency plots at high Ca 2+ i . Fig. 10 predicted the excess of brief open intervals adjacent to the longer closed intervals (position 2) and the deficits of brief open intervals adjacent to brief closed intervals (position 1) that were not predicted by Fig. 7 at high Ca 2+ i . For intervals with durations less than ∼0.05 ms, Fig. 10 predicted too great of a deficit at position 1. This could reflect an inadequacy of the model or it could reflect the fact that dwell times <0.05 ms were not fitted, so that the predictions of the model were not constrained below this time. As would be expected from the reasonable descriptions of the kinetic structure, Fig. 10 also described the Ca 2+ dependence of the 1-D dwell-time distributions. The distributions predicted by Fig. 10 essentially superimposed the thick lines in Fig. 5 . Fig. 10 also described the Ca 2+ dependence of P o and of the mean open and closed interval durations . Fig. 10 could also describe the kinetic structure obtained from the two other channels examined over a wide range of Ca 2+ i . The rankings of the various kinetic schemes for the three channels together with the NLR 1000 are presented in Table . The NLR 1000 , which gives a measure of how well the schemes describe the data (see below and methods ), indicated that Fig. 10 was more likely than the other examined schemes for all three channels . While the NLR 1000 can indicate which schemes are most likely, it does not apply any penalties for additional free parameters. Consequently, the schemes were ranked by the Akaike criteria, which applies a penalty for additional free parameters ( ). The general rankings were: Fig. 10 > Fig. 7 > Fig. 4 Fig. 5 Fig. 6 ( Table ). The Akaike test ranks schemes, but does not give the significance of the rankings. The likelihood ratio test can be used to estimate the significance of rankings for nested models . Fig. 7 and Fig. 10 ranked significantly above Fig. 4 Fig. 5 Fig. 6 and Fig. 5 -sat ( P < 0.001) for all three channels. Interestingly, Fig. 10 ranked above Fig. 7 for only two of three channels, and this ranking was significant for only one channel (B06, P < 0.001) in spite of the fact that Fig. 10 gave better likelihoods than Fig. 7 for all three channels. The apparent discrepancy between visual observations and the significance of some of the rankings may reflect the necessarily conservative nature of statistical tests. Alternatively, the discrepancy may reflect that Fig. 10 is still too simple, so that obvious improvements in some aspects of the gating, such as in the dependency plots, are countered by minimal improvements or even small detrimental changes in other aspects of the gating. Thus, Fig. 10 may have to be expanded into Fig. 9 to obtain sufficiently improved descriptions of the data to outweigh the heavy penalty imposed by the ranking tests. Consistent with this possibility, Fig. 10 predicts only two open and three closed exponential components at high Ca 2+ i . It will be shown in a later section that the gating of the fully liganded channel is described better by models that more closely approximate Fig. 9 . The NLR 1000 values in Table give a numerical measure of how well the various schemes describe the experimental data. A NLR of 1.0 indicates that a kinetic scheme describes the 2-D dwell-time distributions as well as the theoretical best description for a discrete state Markov model (see methods ). The thick lines in Fig. 3 show the theoretical best description of the 1-D distributions. For channel B06, the values of NLR 1000 (normalized to 1,000 interval pairs) ranged from 3.15 × 10 −33 for Fig. 4 to 3.25 × 10 −4 for Fig. 10 . These values give likelihood ratios per interval pair of 0.93 [(3.15 × 10 −33 ) 0.001 ] for Fig. 4 and 0.99 [(3.25 × 10 −4 ) 0.001 ] for Fig. 10 . Such values suggest an average likelihood difference per interval pair between the predicted and theoretical best fits of 7% for Fig. 4 and only 1% for Fig. 10 . The 7% difference per interval pair is readily seen , while the small 1% difference is still visually apparent as less than perfect descriptions of the data . In spite of its relative success, Fig. 10 is still too simple. Analysis of simulated data indicated that Fig. 10 predicted only two significant open and four significant closed components at high Ca 2+ i , compared with the three to four open and five closed components in the experimental data. This underprediction is not surprising, since for Fig. 10 the high Ca 2+ i would effectively drive the gating towards the two open and three closed fully liganded states. Since Fig. 10 predicts too few components at high Ca 2+ i , we explored what types of gating mechanisms might be consistent with the gating at high Ca 2+ i . In theory, Fig. 9 could be examined directly, but the data would be insufficient to constrain the large numbers of rate constants for the 50-state model. Consequently, we explored the reduced models given by Fig. 11 Fig. 12 Fig. 13 , which are all composed of fully liganded states. Fig. 11 is drawn from the 10-state model describing the fully liganded states in Fig. 9 (the rightmost column of states) with two open states excluded because they had no significant effect on the likelihood estimates. Fig. 12 expands Fig. 11 so that transitions to the open states pass through intermediate states, and Fig. 13 has closed states beyond the activation pathway. Fig. 12 and Fig. 13 were examined because BK channels gate with large numbers of brief closings (flickers) at high Ca 2+ i , just as they do at lower Ca 2+ i . Intermediate and/or secondary states are associated with the generation of flickers at lower Ca 2+ i . Additional support for possible intermediate closed states between the closed and open states comes from the observation of Cui et al. 1997 that there is a 50–150-μs delay in the activation of BK channels by voltage steps. Indirect support for possible secondary closed states comes from observations on Shaker K + channels, where secondary states appear to contribute to the gating . Homology between Shaker channels and the core region of BK channels then suggests the possibility of considering such secondary states for BK channels. Fig. 11 Fig. 12 Fig. 13 were fitted to the 2-D dwell-time distributions obtained at a single high Ca 2+ i of 1,024 μM for each of three channels. All three of these schemes gave reasonable descriptions of the kinetic structure at high Ca 2+ i , and all three schemes gave detected numbers of open and closed states within the range observed in the experimental data at high Ca 2+ i . Fig. 11 gave three open and four closed components, and Fig. 12 and Fig. 13 each gave three open and five closed components. For purposes of comparison, a simpler Fig. 14 with only two open and three closed states, which describes the fully liganded states in Fig. 10 , was also examined. Both the NLR 1000 and Akaike criteria ranked the schemes in the order: Fig. 12 ∼ Fig. 13 > Fig. 11 >> Fig. 14 ( Table ). The likelihood ratio test was applied to the nested Fig. 13 , Fig. 11 , and Fig. 14 , and gave highly significant ( P < 0.001) rankings of: Fig. 13 > Fig. 11 > Fig. 14 . The values of the NLR 1000 for Fig. 11 , Fig. 12 , and Fig. 13 ( Table ) indicated that the fits given by these schemes were considerably better than for the simpler Fig. 14 , and approached the theoretical best descriptions of the single data sets for discrete state Markov models. The values of the NLR 1000 for these schemes ranged from 0.061 to 0.959, giving likelihood ratios per interval pair ranging from 0.9972 (0.061 0.001 ) to 0.9999 (0.899 0.001 ), suggesting little difference in likelihood per interval pair between the observed and theoretical best descriptions of the data. These findings indicate that the gating of BK channels at high Ca 2+ i can be approximated by models based on the fully liganded states in Fig. 9 . Fig. 11 was drawn from the fully liganded states in Fig. 9 . Adding three additional brief states as either intermediate states or secondary states to generate additional flickers significantly improved the description of the data ( Table ). Whether the intermediate or secondary states are needed, or whether these additional states simply provide a means to compensate for the fact that fitting only the fully liganded states excludes potential contributions to the gating from transitions back to the states with fewer than four bound Ca 2+ is not yet clear. What is clear, however, is that at least three open and five to eight closed states, as described by Fig. 11 Fig. 12 Fig. 13 , are required to describe the gating at high Ca 2+ i equivalent to the theoretical best description. These schemes lack Ca 2+ -dependent rate constants and apply only for Ca 2+ i > ∼100 μM, where the gating kinetics are little affected by Ca 2+ i . We also explored an alternative explanation to account for the lack of effect of Ca 2+ i on the gating at high Ca 2+ i . In all of the above considered schemes, the binding rate of Ca 2+ i was assumed to be a first order reaction, increasing linearly with Ca 2+ i . Thus, the effective rate constants for binding are given by the product of Ca 2+ i times the rate constants expressed per micromole per second. An upper limit for the rate constant for such a diffusion-controlled process is ∼10 9 M −1 s −1 . There is, however, no a priori reason to think that the effective binding rate would necessarily increase linearly with Ca 2+ i at high Ca 2+ i . The physical structure of the Ca 2+ -binding sites is not yet known, but if the binding sites are in a vestibule with some additional negative charged groups, then the local concentration of Ca 2+ at the binding sites at lower Ca 2+ could be greater than that in the bulk solution , so that the local concentration could reach a maximum as the concentration of Ca 2+ in the bulk solution is raised. This could give an apparent saturation in the Ca 2+ -dependent rate constants. Alternatively, if the binding, which is represented by a one-step process in the kinetic schemes, is actually a two-step process that involves binding followed by a conformational change, then the apparent binding rate would saturate if the second step becomes rate limiting at high Ca 2+ i . Since the physical details involving Ca 2+ binding and action are not known, we explored these two saturation models by using an approach that was independent of a detailed physical model. As both processes would have the effect of reducing the effective concentration of Ca 2+ i at high Ca 2+ i , we examined whether the kinetic structure from low to high Ca 2+ i could be described by letting the effective Ca 2+ i at high Ca 2+ i be less than the actual Ca 2+ i , to mimic apparent saturation of the binding step. Since it is not known what the value of the effective Ca 2+ i would be at high Ca 2+ i , this value was estimated by iterative fitting. The 2-D dwell-time distributions obtained at six different Ca 2+ i (5.5, 8.3, 12.3, 20.3, 132, and 1,024 μM) were simultaneously fitted to estimate the most likely rate constants for Fig. 5 , and also the most likely effective concentrations of Ca 2+ i for the data obtained at 132 and 1,024 μM Ca 2+ i . When fitting, the Ca 2+ i used for the data obtained at the four lower Ca 2+ i was fixed to the experimental values. Fig. 5 with effective Ca 2+ i of 56.9 and 60.0 μM for the data obtained at 132 and 1,024 μM Ca 2+ i , respectively, and the actual Ca 2+ i for the other four data sets obtained at lower Ca 2+ i could approximate the basic features of the kinetic structure from low to high Ca 2+ i . The predicted kinetic structure at 1,024 μM Ca 2+ i is shown in Fig. 11 and was visually indistinguishable from the predicted kinetic structure at 132 μM Ca 2+ i . Comparison of the predicted kinetic structure in Fig. 11 to that in Fig. 6C , Fig. D , Fig. G , and Fig. H , showed that Fig. 5 with an assumption of saturation could approximate the data at high Ca 2+ i . Fig. 5 with saturation also described the data at lower Ca 2+ i , with the predicted structure similar to that in Fig. 10 (not shown). For the other two channels studied in a similar manner, the effective Ca 2+ i s at 132 and 1,024 μM Ca 2+ i were 67.8 and 114 μM (channel B12) and 35.2 and 39.4 μM (channel B14). The likelihoods indicated that Fig. 5 with an effective saturation in the binding rate (Scheme V-sat) described the kinetic structure from low to high Ca 2+ i slightly less well than Fig. 7 and Fig. 10 ( Table ). The observation that the effective Ca 2+ i was greater for all three channels for the data collected at 1,024 μM than for the data collected at 132 μM suggests that increasing Ca 2+ i from 132 to 1,024 μM may have some additional effects on the gating, but any effects would be small since the exponential components describing the dwell-time distributions at 132 and 1,024 μM Ca 2+ i were not significantly different. Cox et al. 1997b have also found (for mSlo ) that high Ca 2+ i may have additional effects on gating. The results in this section show that a relatively simple gating mechanism with the added assumption of apparent saturation in the Ca 2+ binding steps at high Ca 2+ i can approximate the gating from low to high Ca 2+ i . It will be discussed later that saturating models may be less appropriate than two-tiered models. Fig. 12 presents the estimated rate constants for Fig. 10 and Fig. 11 for the three channels examined in detail. Online supplemental Figure S2 (http://www. jgp.org/cgi/full/114/1/93/DC1) presents estimated rate constants for these same three channels for most of the other examined schemes. The rate constants for the examined schemes typically ranged from ∼0 to 45,000/s, indicating a large range in the height of the energy barriers between the various states. Rate constants were limited so as not to exceed 45,000/s, as letting them go higher gave little improvement in the fits. Estimated rate constants for the simpler models (Schemes IV–VI and XI–XIII) were relatively consistent from channel to channel. For the most complex gating mechanism examined , there could be considerable variability in the estimates, depending on the specific rate constants. In those cases where there was considerable variability in estimates of the rate constants among channels, the variability was typically associated with poorly defined rate constants, as these rate constants could be fixed to various values with little effect on the likelihood values after refitting. The rate constants for Fig. 10 will be used in the discussion to describe how the channel gates at low and high Ca 2+ . This study used detailed single-channel analysis to examine the Ca 2+ -dependent gating of native BK channels in cultured rat skeletal muscle. We have extended previous studies by examining the effects of high Ca 2+ i to obtain critical information about mechanism when the gating is driven towards the fully liganded states. Maximum likelihood fitting together with comparisons of the observed and predicted 2-D dwell-time distributions and dependency plots (the kinetic structure) were used to evaluate gating mechanisms. For low to intermediate levels of Ca 2+ i , the gating was highly Ca 2+ i dependent, with a Hill coefficient of ∼3.5, within the range of 2–4 typically observed for BK channels (see introduction ). In contrast, the gating was Ca 2+ independent for high levels of Ca 2+ i (>100 μM). Increasing Ca 2+ i 8–10-fold to 1,024 μM had little effect on P o (∼0.97), the mean open and closed times, the 1- and 2-D dwell-time distributions, and the dependency plots . Estimates of the numbers of significant exponential components in the dwell-time distributions indicated that the channel entered at least three to four open and four to five closed states during normal activity at high Ca 2+ i , and that the estimated numbers of states did not change when Ca 2+ i was increased from 100 to 1,024 μM . Significant dependencies (correlations) between the durations of adjacent open and closed intervals in the dependency plots indicated that transitions between the open and closed states occurred over at least two independent transition pathways (two or more gateway states) at high Ca 2+ i . Thus, as a first approximation, gating at high Ca 2+ i involves Ca 2+ -independent transitions among at least three to four open and four to five closed states, with two or more independent transition pathways among the open and closed states. Models with these characteristics (Schemes XI–XIII) gave excellent descriptions of the kinetic structure for data limited to high Ca 2+ i ( Table ). The MWC model for allosteric proteins predicts that high Ca 2+ i would drive the gating towards the two fully liganded open and closed states, resulting in a simple two-state gating mechanism at high Ca 2+ i with a single transition pathway between the states. These predictions of the MWC model are inconsistent with our observations at high Ca 2+ i of multiple open and closed states connected by two or more independent transition pathways. The MWC model or extensions of the MWC model (Schemes IV–VI) could not describe the gating from low to high Ca 2+ i . Clearly, the MWC model can be rejected for the gating of BK channels in skeletal muscle. Since models based on the MWC model were inconsistent with the data at high Ca 2+ i , we examined more complex models based on Eigen 1968 general 35-state allosteric model for tetrameric proteins and the 55-state extension of Eigen's model shown in Fig. 1 . Since both the 35- and the 55-state models have too many rate constants to estimate practically, we condensed these models to a 25-state model by assuming that the isoforms of each state were kinetically indistinguishable , and then examined models drawn from the 25-state model. Fig. 7 could describe the 1-D dwell-time distributions from low to high Ca 2+ i , but predicted no dependence between adjacent intervals at high Ca 2+ i , in contrast to the significant dependencies observed in the experimental data . The lack of dependence for Fig. 7 at high Ca 2+ i results because the high Ca 2+ i drives the gating towards the fully liganded states, as summarized by Fig. 8 , where there is a single effective transition pathway between the open and closed states. The observation that Fig. 7 predicted no dependence at high Ca 2+ i suggests by analogy that the more complex 25–55-state models , and all schemes in which there would be a single effective transition pathway between open and closed states at high Ca 2+ i , can be rejected. Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 can be classified as one tiered because the open and closed states can be contained within a single plane. Since the examined one-tiered models were inadequate to describe the data at high Ca 2+ i , we considered what type of model would be required. If all the states in Fig. 1 and Fig. 2 are closed states, then there would be a sufficient number of fully liganded closed states at high Ca 2+ to generate the four to five observed closed exponential components observed at high Ca 2+ . If each of the closed states in Fig. 1 and Fig. 2 can make a direct transition to an open state, then this would give enough fully liganded open states to generate the three to four observed open components at high Ca 2+ . If the gating is effectively confined to the fully liganded states at high Ca 2+ i , then the gating at high Ca 2+ i would be described by Fig. 15 , where all the subunits are bound with Ca 2+ . Each subunit in this scheme can exist in two conformational states, and a concerted conformational change of all subunits is required for opening. Fig. 15 has five independent transition pathways between the fully liganded closed and open states (upper and lower tiers, respectively) that would allow dependence to be generated between open and closed intervals at high Ca 2+ i . Fig. 11 , consistent with Fig. 15 , gave excellent descriptions of the gating in high Ca 2+ i ( Table ) Extending Fig. 15 to include states with zero to three bound Ca 2+ would give the general two-tiered model described by Fig. 9 , with 25 closed states on the upper tier and 25 open states on the lower tier. Fig. 10 , a reduced version of Fig. 9 , could describe the kinetic structure of the channel from low to high Ca 2+ i . Fig. 10 also generated single-channel current records that closely mimicked, except for stochastic variation, the experimental current records, as can be seen by comparing the simulated (predicted) records in Fig. 13 to the experimental records in Fig. 1 . The Ca 2+ -independent gating kinetics at high Ca 2+ i together with the few longer closed intervals are present in the simulated records. Although the more complex Fig. 9 was not tested directly, this general scheme should give an even better description of the gating than Fig. 10 , as Fig. 10 is contained within Fig. 9 . Hence, Fig. 9 can serve as a working hypothesis for the Ca 2+ -dependent gating of BK channels. Horrigan and Aldrich (personal communication), based on analysis of macroscopic ionic and gating currents from mSlo , have also found evidence for two-tiered gating mechanisms. In the context of Fig. 9 , it can be seen that, for any fixed number (0–4) of Ca 2+ bound to the channel, the channel could gate among at least five open and five closed states. Thus, Fig. 9 can be viewed as being comprised of five subschemes . The subunit conformations of the analogous states in each of the subschemes are the same, as are the transition pathways among the various open and closed states. Hence, both the conformations of the subunits and the connections among states are identical for each subscheme, independent of the number of bound Ca. Ca 2+ i acts by driving the gating from the subscheme comprised of the states with zero bound Ca 2+ towards the subscheme comprised of the states with four bound Ca 2+ . The binding of Ca 2+ stabilizes the open states. The dynamics of this Ca 2+ -dependent shift will be presented in a later section. Additional support for Fig. 9 comes from the observation that BK channels gate at very low Ca 2+ i , among multiple closed and open states (Nimigean, Rothberg, and Magleby, unpublished observations). The unliganded states in Fig. 9 have five gateway states, suggesting that there would be a dependent relationship among the durations of open and closed intervals at zero Ca 2+ i , consistent with unpublished observations (Nimigean, Rothberg, and Magleby). The saddle shape of the dependency plots reflects the inverse relationship between the durations of adjacent open and closed intervals. This inverse relationship suggests that more stable (longer duration) open states are effectively connected to less stable (briefer duration) closed states . In terms of Fig. 9 , the general saddle shape of the dependency plots remains the same from low through high Ca 2+ i because the subschemes that are entered from low to high Ca 2+ i are comprised of the same numbers of open and closed states with the same subunit conformations and connections among the states. If the relative stability of the connected open and closed states depends mainly on the subunit conformations rather than on the numbers of bound Ca 2+ i , then the general shape of the dependency plots would remain the same from low through high Ca 2+ i . Since Fig. 10 could account for the kinetic structure from low to high Ca 2+ i , Fig. 10 was examined to gain insight into how Ca 2+ activates the channel. Fig. 14 presents the equilibrium occupancy, mean lifetime, and frequency of entry for each state for gating at low Ca 2+ i \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(5.5\;{\mathrm{{\mu}M}},\;P_{{\mathrm{o}}}\;=\;0.061)\end{equation*}\end{document} and high Ca 2+ i \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(1,024\;{\mathrm{{\mu}M}},\;P_{{\mathrm{o}}}\;=\;0.97)\end{equation*}\end{document} for Fig. 10 . Notice that bars of considerable height in these plots can have small values due to the logarithmic ordinates. At low Ca 2+ i , the channel readily gates among all of the states in Fig. 10 , as indicated by the frequencies of entry into each state (C), but spends most of its time in the closed states with zero (82.9%) and one (8.2%) bound Ca 2+ i (A). At high Ca 2+ i , the gating of the channel is effectively confined to the fully liganded states with four bound Ca 2+ , where it spends 99.2% of its time. Little time (0.76%) is spent in the states with three bound Ca 2+ , and negligible time (<0.04%) is spent in the states with 0, 1, or 2 bound Ca 2+ i . The high P o in high Ca 2+ reflects that the channel spends 96.6% of its time in the open states with four bound Ca 2+ , where Ca 2+ stabilizes the open states. The flickers (brief closings) in the single-channel current record arise mainly from transitions to the brief closed states C12– C16 at lower Ca 2+ i and C12 and C15 at high Ca 2+ i . As emphasized by Schoppa and Sigworth 1998 , all kinetic modeling is by nature approximate. Although Fig. 10 could give good descriptions of the single-channel kinetics from low to high Ca 2+ i , this model must be considered as only an approximation of the actual underlying gating mechanism. Fig. 10 with 17 states was drawn from the general 50-state model described by Fig. 9 . Additional open and closed states with zero and one bound Ca 2+ i would have to be added to Fig. 10 to allow gating in very low Ca 2+ i , and additional rows of open and closed states would have to be added to Fig. 10 to generate the observed numbers of open and closed components at high Ca 2+ i . The addition of these states would bring Fig. 10 to the general 50-state model described by Fig. 9 . However, even the general 50-state Fig. 9 is a reduced model compared with what the actual gating mechanism is likely to be. Fig. 9 excludes the isoforms of the various open and closed states. Including all the isoforms of each state would expand Fig. 9 from 50 to 110 states. BK channels may have additional closed states beyond the activation pathway (secondary states). Rothberg and Magleby 1998 found that models with such secondary states were consistent with the gating from low to intermediate Ca 2+ i , and Fig. 13 with such secondary states gave excellent descriptions of gating in high Ca 2+ i ( Table ). Such secondary states, if present, would add a third tier to Fig. 9 and Fig. 10 consisting of closed states beyond the open states. Secondary states may also contribute to the gating of other K + channels . The skeletal muscle BK channel often passes through a brief lifetime subconductance state upon opening and closing . It is not clear whether these subconductance states would arise from some of the states in Fig. 9 and Fig. 10 , from isoforms of the states that were not included in Fig. 9 and Fig. 10 , or whether additional states would have to be added to Fig. 9 and Fig. 10 to account for the subconductance levels. For example, the concerted conformational changes that occur between closing and opening may occur in two steps, rather than the one indicated in Fig. 9 and Fig. 10 ( Schoppa and Sigworth 1998 . Changing Ca 2+ i more than two orders of magnitude, as was done in our experiments, would be expected to alter surface charge and hence gating . Since the considered gating mechanisms did not take surface charge into consideration, the question arises as to what effects this omission might have on the conclusions of our study. Changing the membrane potential ±20 mV (to mimic possible surface charge effects) did not alter the observed numbers of exponential components or the general saddle shape of the dependency plots (our unpublished observations). Since the rejection of previous models in favor of two-tiered gating mechanisms in our study was based on factors that relate to the observed numbers of states and dependency at high Ca 2+ i , surface charge effects would be unlikely to alter the conclusion of two-tiered gating mechanisms reached in this study, but could alter some of the rate constants. Although Fig. 10 could describe many features of the data, this does not exclude the possibility that other rather different mechanisms might also account for the data. A model with fewer states than Fig. 10 , but with apparent saturation in Ca 2+ binding rate could also give reasonable descriptions of the Ca 2+ dependence of the single-channel kinetics from low to high Ca 2+ i . Nevertheless, we prefer the general two-tiered approach based on Fig. 9 to the more ad hoc saturation models, as two-tiered models provide a means to account for the complexity of the gating in zero Ca 2+ i and are consistent with a tetrameric protein. BK channels can gate in a number of different modes, with 96% of the intervals occurring during activity in the normal mode . The gating mechanisms developed in this study apply only to normal mode activity and would have to be expanded to account for activity in other modes. The gating mechanisms would also have to be expanded or modified to account for the effects of permeant ions on channel activity , the different gating properties of other BK channels such as dSlo (from Drosophila) where mean open times are relatively Ca 2+ independent , and the gating effects of various beta subunits not present in skeletal muscle . The voltage dependence of BK channels is an intrinsic property of the channel and does not appear to arise through voltage-dependent increases in Ca 2+ binding . BK channels show homology to the superfamily of voltage-dependent K + channels, including an S4 voltage sensor . In the context of the general two-tiered Fig. 9 , depolarization could increase P o through voltage-dependent transitions of two general types. Depolarization could drive the concerted conformational changes that occur when states on the closed tier open to states on the open tier, or depolarization could drive the conformational changes of the individual subunits for transitions among states on each tier. Recent preliminary observations on large multistate models using analysis of macroscopic ionic and gating currents and single-channel recordings (Rothberg and Magleby, unpublished observations) suggest that the voltage dependence lies mainly in the Ca 2+ -independent closed–closed and open–open steps with the concerted closed–open step being less voltage dependent. Consistent with this hypothesis, gating charge movement can precede channel opening, and charge movement can also occur after the pore is open . These observations suggest that it may only be necessary to add voltage dependence to the rate constants in the models that we have considered in order to account for the major features of the voltage dependence of the single-channel kinetics. Nevertheless, Shaker K + channels gate as if each subunit undergoes three transitions in sequence followed by two final concerted transitions for opening , and movement of the S2 segment may precede that of the S4 segment , consistent with multiple conformations of each subunit. If the subunits in BK channels also have multiple conformations, then the potential numbers of states would be greatly increased over the models considered here. This study develops gating mechanisms that can describe the Ca 2+ dependence of the kinetic structure of BK channels from low to kinetically saturating levels of Ca 2+ i . These models are drawn from a general 50-state two-tiered model in which each closed state in the upper tier can make a direct transition to an open state in the lower tier. Our previous models that describe the Ca 2+ -dependent gating over more limited conditions are contained within the general 50-state model. Thus, the 50-state model serves to unify previous studies, and can provide a framework for further studies on mechanism through single-channel analysis of gating at very low Ca 2+ i and of the voltage dependence of the gating. | Study | biomedical | en | 0.999996 |
10398696 | Toxins from the carnivorous marine cone snails have been useful tools for the study of voltage-activated calcium and sodium channels . Recently, we reported the electrophysiological effects of κ-conotoxin PVIIA, the first member of a new family of conotoxins that interact with voltage-gated potassium channels . By using the Xenopus oocyte expression system, it was shown that PVIIA inhibits Shaker K + currents, but not the currents mediated by the rat homologues of the Kv1 family tested so far . The same toxin has been shown to act also on one of the two splice variants of the Shaker homologue of lobster . Like scorpion toxins of the charybdotoxin (CTX) 1 family , PVIIA appears to bind to the extracellular mouth of the ion pore because its interaction with the channel protein is strongly modified by mutations of some of the amino acid residues that are believed to shape the outer pore vestibule . Nuclear magnetic resonance spectroscopy studies reveal strong similarities between the folding of PVIIA and CTX, but differences in the residues that most strongly affect the binding of the two toxins make PVIIA an interesting tool for additional information on the molecular structure of potassium channels. Most notably, the mutation F425G that increases the CTX-binding affinity of Shaker channels by more than three orders of magnitude has the opposite effect of making the channels PVIIA insensitive . In this paper, we study the mechanism of inhibition by PVIIA of Shaker channels and Shaker Δ6-46 channels lacking fast N-type inactivation. Our analysis shows that the voltage-dependent modification of the partially inhibited currents cannot be attributed to a change in the gating of toxin-modified channels; e.g., as observed for the binding of the spider venom toxin Hanatoxin . Instead, our findings are consistent with the hypothesis that the toxin blocks the permeation of the channel via a 1:1 binding to the pore, and owes its apparent properties of gating modifier to the dependence of its binding on the state of channel conductance. We shall describe block relaxations during activating pulses showing that the binding of PVIIA to open channels is characterized by a strongly voltage-dependent off rate and a voltage-insensitive on rate, and tonic-block recoveries during resting periods showing that the binding to closed channels has voltage-independent and much slower kinetics. We find that the properties of PVIIA block of Shaker Δ6-46 and Shaker wild-type channels are similar, except that inactivation protects partially the latter from the depolarization-induced unblock, in agreement with the assumption that the toxin senses only the conductive state of the channel. In analogy to CTX block of Ca-activated K + channels and Shaker channels , our data are consistent with the idea that the voltage dependence of PVIIA binding to open channels is mainly an indirect effect of the interaction with potassium ions occupying an outer site in the pore. We shall also argue that the binding to closed channels, particularly its sensitivity to external K + , can be described by the same type of toxin–K + interaction. A consistent molecular model shows that, apart from its use as a structural probe of the outer pore vestibule, PVIIA might also be a valuable tool for investigating more intimate properties of the ion-conduction pathway. The solid-phase peptide synthesis of PVIIA was performed as described in Shon et al. 1998 . cRNA for Shaker H4 and Shaker –Δ6-46 was obtained by a standard protocol using a template with a T7 promoter. Oocytes from Xenopus laevis were prepared as described previously . RNA was injected into stage V–VI oocytes and currents were recorded 1–7 d after injection. Whole-cell currents were recorded under two-electrode voltage clamp control using a Turbo-Tec amplifier (npi Electronic). The intracellular electrodes were filled with 2 M KCl and had a resistance between 0.6 and 1.0 MΩ. Current records were low-pass filtered at 1 kHz (−3 dB) and sampled at 4 kHz. For outside-out patch-clamp recordings , the aluminum silicate glass pipettes had resistances between 0.8 and 1.2 MΩ. The pipette solution contained (mM): 115 KCl, 1.8 EGTA, 10 HEPES, pH 7.2 with KOH. Currents were measured with an EPC-9 patch clamp amplifier driven by the Pulse+PulseFit software package (HEKA Elektronik). Current records were low-pass filtered at 3 kHz (−3 dB) and sampled at a rate of 10 kHz. The bath solution in the electrophysiological experiments was either normal frog Ringer's (NFR) containing (mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES, pH 7.2, with NaOH, or K + -Ringer containing (mM): 115 KCl, 1.8 CaCl 2 , 10 HEPES, pH 7.2 with KOH. Leak and capacitive currents were corrected on-line by using a P/n method. In all experiments, the vitelline membranes of the oocytes were removed mechanically with fine forceps. Toxin solution was added to the bath chamber with a Gilson tip pipette. The indicated toxin concentrations correspond to the final concentration in the bath chamber. As shown by Terlau et al. 1996 , the main effect of PVIIA on Shaker -H4 channels is a reversible reduction of the peak voltage-clamp currents with a dose dependence that is consistent with toxin binding to a single site of the channel protein where it blocks ion permeation. Fig. 1 A shows superimposed current responses to three different step depolarizations, recorded from the same oocyte before and after the addition to the bath of 500 nM PVIIA. All peak amplitudes are reduced roughly 10-fold under toxin, corresponding to an IC 50 of PVIIA block of ∼50 nM. A closer inspection reveals, however, that the time course of the currents is sensibly modified. Fig. 1 B shows that the ratio, U (for unblock probability), of toxin to control responses for the same voltage increases after the time to peak approaching a late steady state value that, for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}\;=\;60\;{\mathrm{mV}}\end{equation*}\end{document} , is about five times larger. Although developing during the onset of inactivation, this effect cannot be attributed to an unblock of inactivated channels because it increases strongly with V in a range where the steady state probability and the time constant of inactivation are fairly constant, ∼0.9 and ∼3.5 ms, respectively. The solid lines in Fig. 1 B are single-exponential fits of U( t ), yielding asymptotic values that increase from 0.21 at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}\;=\;0\;{\mathrm{mV}}\end{equation*}\end{document} to 0.46 at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}\;=\;60\;{\mathrm{mV}}\end{equation*}\end{document} , while the time constants decrease from 27 to 6.3 ms. These data could be related to some average relaxation of toxin binding to open and inactivated channels, but the unfolding of binding and inactivation processes would not be straightforward since their kinetics appear to occur in a similar time range. Nevertheless, the fitted asymptotic values of U( t ) can be used to estimate an apparent dissociation constant, K (O) app , of PVIIA binding to open (noninactivated) channels. K (O) app estimates in the voltage range −20 to +60 mV were fairly well fitted by: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{({\mathrm{O}})}_{{\mathrm{app}}}({\mathrm{V}})\;=\;K^{({\mathrm{O}})}_{{\mathrm{app}}}(0)*{\mathrm{exp}}({\mathrm{V}}/{\mathrm{v}}_{{\mathrm{s}}})\end{equation*}\end{document} . Data from the experiment in Fig. 1 are shown in Fig. 4 C as □ and fitted with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{({\mathrm{O}})}_{{\mathrm{app}}}(0)\;=\;115\;{\mathrm{nM\;and\;v}}_{{\mathrm{s}}}\;=\;45\;{\mathrm{mV}}\end{equation*}\end{document} . Mean values of K (O) app (0) and v s from four different oocytes tested with toxin concentrations between 100 and 500 nM were 125 nM and 46 mV ( Table , columns 2 and 5). Evidence that the inactivation of Shaker -H4 channels is a damping factor rather than the cause of the above effects is provided by the study of the deletion mutant Shaker –Δ6-46 ( Sh -Δ) that lacks fast inactivation . In agreement with Scanlon et al. 1997 , we find that the currents mediated by Sh -Δ channels appear even more strongly modified by the presence of partially blocking concentrations of extracellular PVIIA. Fig. 2 A shows voltage-clamp currents for steps to −10, +10, and +30 mV, recorded from an oocyte expressing Sh -Δ channels before and after the bath addition of 200 nM PVIIA. It is seen that the toxin reduces strongly the early phase, but has much less effect on the steady state of the currents. The currents measured at the half-activation time of the normal responses are reduced at all voltages slightly more than fivefold, indicating a PVIIA dissociation constant from the blocking site of ∼50 nM, close to that estimated for Shaker -H4 channels from the reduction of peak currents. However, this early reduction diminishes during the pulse, and the currents approach asymptotic values that are progressively closer to the toxin-free levels at increasing depolarization. As opposed to the case of the gating modifier Hanatoxin from spider venom , we show that these effects can be explained be assuming that PVIIA binding blocks pore conduction without modifying substantially channel gating and that channel opening reduces the toxin-binding affinity entraining a re-equilibration towards a lower block probability. According to this interpretation, the ratio U of toxin to control currents measures the fraction of unblocked channels and is predicted to have several distinctive properties that characterize a simple second-order reaction of channel-toxin association , where {U} and {B} represent, respectively, an unblocked channel or a channel blocked by the association with the toxin, and where k (O) off and k (O) on are, respectively, the first-order dissociation rate constant and the second-order association rate constant of toxin binding during the step depolarization. A first prediction is that, after a step-like change in the binding parameters, U(t) should follow a single-exponential relaxation from the resting value towards the equilibrium value set by the new binding conditions. Fig. 2 B shows this property for sample voltage steps to 0, 20, 40, and 60 mV. The smooth lines are fits of U(t) with single exponentials rises from the same initial value, U (C) ∼ 0.19, towards asymptotic values, U (O) , and with time constants, τ (O) , that depend on the step voltage. For V ≥ −20 mV, the open-channel probability is close to its maximum and we find that τ (O) is always much larger than the half-activation time of normal currents. Therefore, these relaxations develop almost exclusively while the channels are fully activated, and this justifies the use of the superscript (O) for the parameters that characterize them. A second expectation is that the dependence of both U (C) and U (O) on the toxin concentration, [T], should follow simple Langmuir isotherms: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{U}}^{ \left \left({\mathrm{C}}\right) \right }=\frac{{\mathit{K}}^{ \left \left({\mathrm{C}}\right) \right }}{ \left \left[{\mathrm{T}}\right] \right +{\mathit{K}}^{ \left \left({\mathrm{C}}\right) \right }}{\mathrm{;U}}^{ \left \left({\mathrm{O}}\right) \right }=\frac{{\mathit{K}}^{ \left \left({\mathrm{O}}\right) \right }}{ \left \left[{\mathrm{T}}\right] \right +{\mathit{K}}^{ \left \left({\mathrm{O}}\right) \right }}{\mathrm{,}}\end{equation*}\end{document} where K (C) and K (O) are the dissociation constants characterizing, respectively, the equilibrium binding of PVIIA to resting (closed) or activated (open) channels. Fig. 3 illustrates a single experiment in which [T] was changed progressively from 0 to 10, 20, 50, 100, 200, 500 nM, 1 μM, and back to 0. At each [T], a standard series of current–voltage responses to various pulse potentials, V p , was recorded with 5-s stimulation intervals at a holding potential of −100 mV. Fig. 3 A shows the [T] dependence of the responses to \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}_{{\mathrm{p}}}\;=\;20\;{\mathrm{mV}}\end{equation*}\end{document} . Notice that the record obtained after washing-out the 1-μM PVIIA solution shows a small “run up” of the preparation and a very small residual unblock that we attribute to the remaining presence of a few nanomolar PVIIA around the oocyte. Ratios of the toxin to the initial control responses of Fig. 3 A are shown in B, together with single-exponential fits that are almost indistinguishable from the data at all times after 1 ms from the half-activation time of the control response. Estimates of U (C) and U (O) from these fits are plotted in Fig. 3 C as a function of [T]. The solid lines show that the data are indeed well fitted by Langmuir isotherms, yielding in this case \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{({\mathrm{C}})}\;=\;36\;{\mathrm{nM\;and}}\;K^{({\mathrm{O}})}\;=\;330\;{\mathrm{nM}}\end{equation*}\end{document} . A third expectation is that τ (O) should depend on [T] according to: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}^{ \left \left({\mathrm{O}}\right) \right }={1}/{ \left \left(k_{{\mathrm{off}}}^{{\mathrm{O}}}+k_{{\mathrm{on}}}^{{\mathrm{O}}} \left \left[{\mathrm{T}}\right] \right \right) \right }{\mathrm{.}}\end{equation*}\end{document} Estimates of τ (O) from the single exponential fits of Fig. 3 B are plotted as a function of [T] in D. The solid line is the least-squares fit of the data according to the above relationship, which is clearly well obeyed. Finally, we expect a simple relationship between the estimates of k (O) off and k (O) on from the above fit of the [T] dependence of τ (O) and the equilibrium dissociation constant K (O) estimated from the fit of U (O) data: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{({\mathrm{O}})}\;=\;k^{({\mathrm{O}})}_{{\mathrm{off}}}/k^{({\mathrm{O}})}_{{\mathrm{on}}}\end{equation*}\end{document} . The values of k (O) off and k (O) on fitting the τ (O) data of Fig. 3 D, respectively, 29 s −1 and 88 μM −1 s −1 , yield indeed the same estimate of K (O) ∼ 330 nM as the fit of the U (O) data of Fig. 3 C. In most experiments, only two toxin concentrations were tested on the same oocyte, or experiments were done for a single [T] value in the range of 100–500 nM. The consistency of the results of these experiments with the bimolecular character of the toxin-block reaction was shown indirectly by the agreement of the estimates of K (O) , k (O) off , and k (O) on obtained from single measurements of U (O) and τ (O) in any given condition according to the inverse relationships: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{ \left \left({\mathrm{O}}\right) \right }=\frac{ \left \left[{\mathrm{T}}\right] \right *{\mathrm{U}}^{ \left \left({\mathrm{O}}\right) \right }}{1-{\mathrm{U}}^{ \left \left({\mathrm{O}}\right) \right }}{\mathrm{;}}k_{{\mathrm{off}}}^{ \left \left({\mathrm{O}}\right) \right }=\frac{{\mathrm{U}}^{ \left \left({\mathrm{O}}\right) \right }}{{\mathrm{{\tau}}}^{ \left \left({\mathrm{O}}\right) \right }}{\mathrm{;}}k_{{\mathrm{on}}}^{ \left \left({\mathrm{O}}\right) \right }=\frac{1-{\mathrm{U}}^{ \left \left({\mathrm{O}}\right) \right }}{ \left \left[{\mathrm{T}}\right] \right *{\mathrm{{\tau}}}^{ \left \left({\mathrm{O}}\right) \right }}{\mathrm{.}}\end{equation*}\end{document} The most interesting feature of PVIIA-block relaxations is their strong voltage dependence. In the experiment of Fig. 2 , U (O) increased from 0.51 to 0.8 and τ (O) decreased from 23 to 8.5 ms as V was increased from 0 to 60 mV. Converting these data according to yields the voltage dependencies of the binding parameters shown in Fig. 4 as ○. The figure shows also plots of similar data from a representative experiment in K + -Ringer to be described later . Fig. 4A and Fig. C , shows that both k (O) off and K (O) increase with voltage according to a simple exponential law. The straight lines fitting the semilogarithmic plots of k (O) off and K (O) were drawn according to the expressions: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{O}})}_{{\mathrm{off}}}({\mathrm{V}})\;=\;k^{({\mathrm{O}})}_{{\mathrm{off}}}(0)*{\mathrm{exp}}({\mathrm{V}}/{\mathrm{v}}_{{\mathrm{s}}}),\;[k^{({\mathrm{O}})}_{{\mathrm{off}}}(0)\;=\;22\;{\mathrm{s}}^{-}1;\;{\mathrm{v}}_{{\mathrm{s}}}\;=\;42\;{\mathrm{mV}}],\;{\mathrm{and}}\;K^{({\mathrm{O}})}({\mathrm{V}})\;=\;K^{({\mathrm{O}})}(0)*{\mathrm{exp}}({\mathrm{V}}/{\mathrm{v}}_{{\mathrm{s}}}),\;[K^{({\mathrm{O}})}(0)\;=\;200\;{\mathrm{nM}};\;{\mathrm{v}}_{{\mathrm{s}}}\;=\;44\;{\mathrm{mV}}]\end{equation*}\end{document} . Consistently, Fig. 4 B shows that k (O) on has no systematic trend with a mean value of 110 s −1 μM −1 . Mean values of k (O) on , k (O) off (0), K (O) (0), and v s \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;10)\end{equation*}\end{document} are given in columns 2–5 of Table . Fig. 4 C shows also that the apparent dissociation constant, K (O) app , estimated from the unblock of Shaker -H4 channels, is systematically lower than K (O) . This is consistent with the idea that the reduction of toxin block occurs in the open state, so that the effect is strongly reduced if the channels visit frequently, and for relatively long periods, the inactivated (closed) state. PVIIA has obviously free access to the site of block also when the channels are closed during resting hyperpolarizations. The dissociation constant of PVIIA binding to closed channels, K (C) , can be easily measured by the reduction with [T] of the early responses to pulse stimulations under resting conditions. Our estimates of K (C) in NFR, in the range of 35–80 nM, were not significantly different for Shaker -H4 or Shaker -Δ channels ( Table , column 6). These estimates are approximately fourfold lower than those of K (O) (0) and it is important to know what changes in the kinetic parameters of toxin-binding contribute to this difference. The kinetics of PVIIA binding to closed channels cannot easily be measured from wash-in/wash-out experiments because testing toxin block at any time grossly upsets the block itself and changes the meaning of later tests. A correct wash-in/wash-out experiment should be performed repetitively with a fast-perfusion system testing in each trial PVIIA block at a different time from wash-in. However, the marked change of PVIIA block caused by a pulse depolarization allows us to perform a conceptually identical experiment, by testing at different times the after-pulse re-equilibration of PVIIA binding to closed channels. Fig. 5 shows the results of a double-pulse experiment on an oocyte expressing Sh -Δ channels. Each stimulation consisted of two successive pulses of 40 ms to 40 mV separated by a variable resting period, T i , at −100 mV. Fig. 5 , top, shows the responses for T i increasing from 5 to 200 ms, recorded before and after the addition of 200 nM PVIIA to the bathing NFR solution. It is seen that the two successive responses in the control experiment are always virtually identical, indicating that the channels recover completely their resting state after 5 ms at −100 mV. With PVIIA, however, the second response reveals a long memory of the effects induced by the first pulse: for short interpulses it is dominated by a fast rising phase much as the toxin-free response, and its “tonic” characteristics are not fully recovered even after 200 ms. Fig. 5 , bottom left, plots as a function of T i the ratio of the early currents elicited by the second and first pulses at the half-time of the toxin-free response. This ratio, which is virtually unity in control measurements at all T i , with PVIIA is ∼4 for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{T}}_{{\mathrm{i}}}\;=\;5\;{\mathrm{ms}}\end{equation*}\end{document} , and is still ∼2 for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{T}}_{{\mathrm{i}}}\;=\;200\;{\mathrm{ms}}\end{equation*}\end{document} . We interpret this phenomenon as due to a relatively slow reequilibration of PVIIA binding to closed channels, and we take the early amplitude of the second response, normalized to control, as the fraction of toxin-free channels at the time of onset of the second pulse. As shown in Fig. 5 , bottom right, this quantity decays with T i as a single exponential, from \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{U}}^{({\mathrm{O}})}\;=\;0.86\;{\mathrm{to\;U}}^{({\mathrm{C}})}\;=\;0.22\end{equation*}\end{document} , with a time constant, τ (O) , of 190 ms. We interpret U (O) as the toxin-free probability of open channels at the end of the conditioning pulse, U (C) as the equilibrium toxin-free probability of closed channels, and τ (C) as the relaxation time of PVIIA binding to closed channels. Accordingly, the last two quantities yield estimates of the association and dissociation rate constants, k (C) on and k (C) off , of PVIIA binding to closed channels. From the experiment of Fig. 5 , we obtain: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{C}})}_{{\mathrm{on}}}\;=\;21\;{\mathrm{{\mu}M}}^{-}1\;{\mathrm{s}}^{-}1,\;k^{({\mathrm{C}})}_{{\mathrm{off}}}\;=\;1.2\;{\mathrm{s}}^{-}1,\;K^{({\mathrm{C}})}\;=\;56\;{\mathrm{nM}}\end{equation*}\end{document} . Mean estimates \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document} are given in Table . Compared with open-channel properties, both the on and off rates of PVIIA binding to closed channels are much slower. The approximately fourfold higher value of K (O) (O) relative to K (C) results from the combination of an increase of the rate of PVIIA dissociation by a factor of ∼24 and an increase of the association rate by a factor of ∼6. The most important conclusion from these measurements is that, while the low value of k (C) off could be thought as an extrapolation to hyperpolarized potentials of the voltage dependence of k (O) off , the sixfold lower value of k (C) on is incompatible with the voltage independence of k (O) on and indicates that closed channels indeed have different toxin-binding properties. In two oocytes, the above double-pulse protocol was applied using variable holding potentials between −60 and −120 mV and we observed no significant change in the estimates of the binding parameters (data not shown). Thus, unlike for open channels, the interaction of PVIIA with closed channels appears to be voltage insensitive and this conclusion is also qualitatively consistent with the above reported observation that even at large positive potentials Shaker -H4 channels that are closed by the inactivation gate appear protected from toxin unblock. Due to inactivation, the recovery of the tonic binding of PVIIA to Shaker -H4 channels after a conditioning stimulus appears rather peculiar, as illustrated by Fig. 6 . The control recordings from a double-pulse protocol follow a classical pattern showing that the second response increases with T i as more channels recover from the inactivation produced by the conditioning pulse: the ratio of second to first peak current approaches 1 as a single exponential with a time constant of 28 ms . The recordings with 100 nM PVIIA added to the bath show instead a marked overshoot of the second response that subsides very slowly. This effect has a simple explanation if we assume that the recovery from the unblock induced by the conditioning pulse is much slower than recovery from inactivation. Consistently, as shown in Fig. 6 , bottom right, the fraction of toxin-free channels, estimated from the toxin to control ratio of the second peak response, decreases monotonically with T i . Fitting this decay towards the steady state ratio of the first peak responses with a single exponential yields \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}^{({\mathrm{C}})}\;=\;270\;{\mathrm{ms}}\end{equation*}\end{document} . Combining this estimate with that of the asymptotic toxin-free probability, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{U}}^{({\mathrm{C}})}\;=\;0.44\end{equation*}\end{document} , we estimate in this experiment: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{C}})}_{{\mathrm{off}}}\;=\;1.6\;{\mathrm{s}}^{-}1,\;k^{({\mathrm{C}})}_{{\mathrm{on}}}\;=\;21\;{\mathrm{{\mu}M}}^{-}1\;{\mathrm{s}}^{-}1,\;{\mathrm{K}}^{({\mathrm{C}})}\;=\;78\;{\mathrm{nM}}\end{equation*}\end{document} . Mean estimates of the parameters of PVIIA binding to closed Shaker -H4 channels are given in Table . It is seen that they are not significantly different from those of Shaker -Δ channels, supporting the idea that PVIIA does not distinguish the resting state of the two phenotypes. The voltage dependence of PVIIA dissociation from open channels is strongly reminiscent of the properties of CTX block of Ca-activated potassium channels or Shaker channels , which arise from the interaction of CTX with potassium ions occupying an outer site in the channel pore. To investigate the interaction of PVIIA with potassium ions, we studied the effect of PVIIA on Sh -Δ channels using high extracellular concentrations of potassium ions, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;115\;{\mathrm{mM}}\end{equation*}\end{document} . For more reliable measurements in the voltage range of negative-resistance characteristics of this preparation, few experiments were performed on excised outside-out patches, but very similar results were also obtained outside of this range from experiments on whole oocytes. Fig. 7 illustrates a representative experiment on an outside-out patch exposed to symmetric 115-mM K + solutions. Fig. 7 A shows superimposed current records from standard current–voltage stimulation protocols applied before (left) and after (middle) the addition to the external bath of 1 μM PVIIA. The right diagram gives plots of the late currents at the end of 100-ms pulses as a function of pulse voltage. It is seen that 1 μM PVIIA blocks most of the inward currents, but has a much smaller effect on the outward currents at large depolarizations. This is consistent with the results obtained with NFR \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}([{\mathrm{K}}]_{{\mathrm{o}}}\;=\;2.5\;{\mathrm{mM}})\end{equation*}\end{document} in the external bath. By interpreting the steady state ratios of toxin to control currents as toxin-free probabilities, we obtain for the open-channel dissociation constant, K (O) , similar estimates and the same voltage dependence as in NFR . The comparison of the whole time course of test and control responses also reveals that toxin-block relaxations have properties similar to those observed in low K + solutions, although starting from a different resting-block equilibrium. Fig. 7 B shows three of the control records in A \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{for\;V}}\;=\;-40,\;+20,\;{\mathrm{and}}\;+60\;{\mathrm{mV}})\end{equation*}\end{document} superimposed with the respective records under toxin scaled by a constant factor of 3.3. It is seen that this scaling makes the currents recorded at +20 mV match almost exactly for the whole duration of the pulse, whereas the match for the other records is good only for the initial rising phase. At later times, the inward currents at −40 mV are further depressed approximately fourfold and the outward currents at +60 mV undergo an approximately twofold increase. The most obvious interpretation of these results is that two thirds of the channels are tonically blocked at 1 μM PVIIA according to a toxin dissociation constant from closed channels, K (C) ∼ 430 nM, which is about equal to K (O) at +20 mV, whereas depolarizations below or above +20 mV, leading to lower or higher K (O) values, cause an increase or decrease of PVIIA block. In agreement with this interpretation, the current ratios after almost complete activation follow single-exponential relaxations , as expected from the reequilibration of a toxin-block reaction. The fitting parameters of these relaxations can be used to estimate the rate constants characterizing the binding of PVIIA to open channels. These estimates for the experiment of Fig. 7 are plotted in Fig. 4A and Fig. B , •. The comparison with the data obtained in NFR shows that both the association and dissociation rate constants of PVIIA binding to open channels are insensitive to changes of [K] o . Mean estimates of k (O) on , k (O) off (0), K (O) (0), and v s from four different experiments are given in columns 2–5 of Table . From the data of Fig. 7 B, we estimate a tonic block of 66% in 1 μM PVIIA, which corresponds to a PVIIA dissociation constant of 430 nM and shows that, at variance with the open channel properties, the binding of PVIIA to closed channels is very sensitive to [K] o . Our mean estimate of K (C) from five oocytes at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;115\;{\mathrm{mM}}\end{equation*}\end{document} was ∼400 nM, about a factor of 8 higher than at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;2.5\;{\mathrm{mM}}\end{equation*}\end{document} . We can ask whether the lower affinity of PVIIA at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;115\;{\mathrm{mM}}\end{equation*}\end{document} is due to a smaller rate of association or to a larger rate of dissociation by analyzing a double-pulse experiment such as that illustrated in Fig. 8 . The experiment was done on a whole oocyte in the presence of 1 μM PVIIA and the protocol consisted of several double stimulations, with pulses to −20 mV separated by a variable interpulse interval (T i ) allowing resting periods of at least 3 s between successive stimulations. Fig. 8 A shows a sample of the responses obtained for T i values between 30 and 420 ms. In agreement with what we described above, we observe that the conditioning pulse induces an increase of toxin block that appears as a reproducible peak in the first response of each successive double stimulation. However, for small T i values, the second response has a more normal appearance and no peak, indicating that the fraction of blocked channels is near the equilibrium for open-channel block at −20 mV that was already achieved by the end of the conditioning pulse. We can judge qualitatively the slow recovery of the lower equilibrium probability of PVIIA binding to closed channels by the reappearance of a peak in the second response, which is clearly seen only in the records with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{T}}_{{\mathrm{i}}}\;=\;180\;{\mathrm{or}}\;420\;{\mathrm{ms}}\end{equation*}\end{document} . A more quantitative analysis is shown in Fig. 8 B by plotting, as a function of T i , the ratio of the early amplitudes of test and conditioning responses taken as means in the time interval of 25–75% rise of the toxin-free response. These data are well fitted by a single exponential increase with T i with a time constant of 430 ms. Assuming this value as an estimate of τ (C) and using the value of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{U}}^{({\mathrm{C}})}\;=\;0.28\end{equation*}\end{document} measured from the early fraction of unblocked currents (data not shown), we estimate in this experiment: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K^{({\mathrm{C}})}\;=\;380\;{\mathrm{nM}},\;k^{({\mathrm{C}})}_{{\mathrm{off}}}\;=\;0.65\;{\mathrm{s}}^{-}1,\;k^{({\mathrm{C}})}_{{\mathrm{on}}}\;=\;1.7\;{\mathrm{{\mu}M}}^{-}1\;{\mathrm{s}}^{-}1\end{equation*}\end{document} . Mean estimates obtained from three experiments of this type are k \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}^{({\mathrm{C}})}_{{\mathrm{off}}}\;=\;0.79\;{\mathrm{s}}^{-}1\;{\mathrm{and}}\;k^{({\mathrm{C}})}_{{\mathrm{on}}}\;=\;1.76\;{\mathrm{{\mu}M}}^{-}1\;{\mathrm{s}}^{-}1\end{equation*}\end{document} (see Table ). The value of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{K}}^{({\mathrm{C}})}_{{\mathrm{off}}}{\mathrm{at}}\;[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;115\;{\mathrm{mM}}\end{equation*}\end{document} appears equal to that in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;2.5\;{\mathrm{mM}}\end{equation*}\end{document} within the experimental error. On the contrary, increasing [K] o from 2.5 to 115 mM decreases by more than one order of magnitude the apparent value of k (C) on , which seems to be the only parameter of PVIIA binding to Shaker channels that is very sensitive to [K] o . Notice that we studied the effect of increasing [K] o by substituting Na + with K + , so that this effect is not comparable to the large decrease of toxin association rates observed at increasing ionic strength for CTX block of Ca 2+ -activated channels or Shaker channels . We have described in this paper the dose, voltage, and time dependence of PVIIA effects on the currents mediated by Shaker channels. Our results are consistent with the hypothesis that the most prominent effect of toxin-channel association is the block of the channel conductance and rule out a substantial modification of channel gating as observed, e.g., when the channels bind the spider venom toxin Hanatoxin . In fact, there is no way to account for the [T] dependence of the currents shown in Fig. 3 A as a change in the fraction of channels that have a toxin-modified activation, whereas the analysis illustrated in Fig. 3B–D , shows that the toxin-dependent modification of the currents has all the characteristics expected for the relaxation of a bimolecular binding reaction after a step change of the reaction parameters. An important result of our study for mechanistic interpretations is that the different binding of PVIIA to closed or open channels cannot be attributed solely to the voltage dependence of the reaction rates. First of all, while the low value of k (C) off could be thought as the extrapolation of k (O) off (V) to hyperpolarizing potentials, the independence of K (O) on on voltage and [K] o is incompatible with the 6- or 90-fold lower estimates of K (C) on at \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;2.5\;{\mathrm{or}}\;115\;{\mathrm{mM}}\end{equation*}\end{document} . Secondly, our observation that the relaxations of closed-channel block are independent of the holding potential in the range of −60 to −120 mV shows that, in sharp contrast to the case of open channels, the binding of PVIIA to closed channels is fairly insensitive to the transmembrane voltage. Indeed, the fact that inactivation protects significantly depolarized Shaker -H4 channels from toxin-unbinding is consistent with a similar binding of PVIIA to any nonconducting state of the channels at any voltage. An important consideration that justifies a posteriori most of our experimental analysis is that the relaxations of toxin binding to open or closed channels occur on very different time scales. The mean properties that we expect from flickering between open and closed states are weighted for each state in direct proportion to the probability of that state and in inverse proportion to the time constant of the binding relaxation in that state. Since the binding kinetics for closed channels is at least 10× slower than for open channels, the relative weight of the open state in Sh -Δ channels that do not inactivate would be >0.9 for any open probability, P (O) > 0.5. Therefore, it is fair to assume that, during depolarizations of noninactivating channels, we are essentially measuring the binding to open channels. The situation is less favorable for Shaker -H4 channels, where P (O) ∼ 0.1. In this case, we expect that the relative weight of the open state is ∼0.53, and this is indeed fairly consistent with our finding that the K (O) app measured for Shaker -H4 channels is ∼0.6 K (O) . The structural similarity of PVIIA with the scorpion toxins of the charybdotoxin family and the strong sensitivity of PVIIA efficacy to mutations of some of the amino acid residues that are believed to shape the outer pore vestibule indicate that PVIIA acts as CTX by simply blocking the extracellular mouth of the ion pore. The results presented in this paper demonstrate that CTX and PVIIA also share strong similarities in their mechanism of block. As for the case of CTX block of Ca 2+ -activated K + channels or Shaker channels , we find that PVIIA block is strongly decreased during channel opening with a voltage dependence that is afforded exclusively by the dissociation rate constant and not by the on rate of toxin binding. Also the state dependency of PVIIA binding is qualitatively very similar to that reported for the binding of CTX to Ca 2+ -activated channels, which occurs with a seven- to eightfold lower rate of association when the channels are fully closed . The only clear difference between CTX and PVIIA appears to be that the latter binds with much faster kinetics, due to a much higher dissociation rate constant. However, several CTX variants also block open Shaker channels with relatively fast kinetics that have been observed by fast perfusion of macropatches during long depolarizing sweeps . It is interesting to notice that nearly all of the more than 60 variants of CTX characterized by Goldstein et al. 1994 have second-order association rate constants close to the wild type, and in the same range (20–100 μM −1 s −1 ) of our estimates for PVIIA. None of the CTX variants was studied in a way that would allow a clear separate characterization of closed- and open-channel binding, because slow toxins were studied with multipulse protocols that characterize the binding to closed channels, whereas fast toxins were tested for open-channel block with wash-in/wash-out measurements during long depolarizations. Therefore, we have no direct evidence that CTX block of Shaker channels is state dependent. However, one variant, CTX-R25Q, was studied with both protocols, although with different [K] o conditions (2 vs. 100 mM), and the results of the two measurements were quite different, the fast protocol yielding a 5× larger k on , and 10× larger k off . Although the authors suggest a different explanation related to the different [K] o conditions, we notice that this result is in fact consistent with CTX block having the same state dependency shown here for PVIIA block. While we cannot exclude that a PVIIA molecule bound to an open channel senses a significant fraction of the transmembrane electric field, the above discussed analogies with CTX and the antagonizing action of high-K + solutions on tonic PVIIA block suggest that the unblock observed upon opening the channels in low external K + may largely arise from the destabilization of toxin binding by internal K + , as in the case of CTX . The results reported by Garcia and Naranjo 1999 support to a large extent this hypothesis by showing a significant reduction of the absolute value and voltage dependence of the rate of toxin dissociation upon putatively complete removal of intracellular K + . We discuss below that our findings of a marked difference between the toxin-binding properties of open versus closed channels and of a strong [K] o dependence of closed-channel binding can also have a simple explanation in the context of a model similar to that proposed by MacKinnon and Miller 1988 , in which the major factor influencing toxin binding is the state of occupancy of the outermost K + -binding site within the channel permeation pathway. From now on, we shall refer to this site as if it was the only K + -binding site. The general scheme that we can use to interpret our data is shown in Fig. 2 . Fig. 2 assumes that the toxin (Tx) can bind to whatever state of the channels with the same second-order–association rate constant, k 1 , whereas its first-order–dissociation rate constant is k (K) −1 or k (0) −1 , depending on whether or not the K + -binding site is occupied, and k (K) −1 >> k (0) −1 . All the rate constants are otherwise assumed to be voltage independent. The scheme assumes also that K + binding is in fast equilibrium and is governed by different dissociation constants, K (Tx) d or K (0) d , depending on whether or not there is a bound toxin in the outer pore vestibule. While a bound toxin is blocking the extracellular access, K + binding to the pore is assumed to be in equilibrium only with the cytoplasmic K + concentration, [K] i , and the parenthesis in the transition C:Tx ↔ C:K + :Tx indicates that this transition can occur only when the channel is open. A possible molecular picture of the states C:Tx and C:K + :Tx is shown in the cartoon of Fig. 9 . When the channel is closed and not blocked by PVIIA [K] stands for the extracellular K + concentration, whereas when the channel is open [K] is determined by the flux of ions in the pore. In the case of open channels, it is easily seen that the above scheme reduces to the simple two-state Fig. 1 , provided we identify k (O) on with k 1 , and k (O) off with an average of k (K) −1 and k (0) −1 weighted according to the relative probabilities of states C:K + :Tx and C:Tx. Assuming for K (Tx) d a voltage dependence associated with the charge translocation occurring along the pore in the transition C:Tx ↔ C:K + :Tx, we can express k (O) off as: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{{\mathrm{off}}}^{ \left \left({\mathrm{O}}\right) \right }=k_{-1}^{ \left \left(0\right) \right }+\frac{ \left \left[k_{-1}^{ \left \left({\mathrm{K}}\right) \right }-k_{-1}^{ \left \left(0\right) \right }\right] \right \left \left[{\mathrm{K}}\right] \right _{{\mathrm{i}}}}{ \left \left[{\mathrm{K}}\right] \right _{{\mathrm{i}}}+K_{{\mathrm{d}}}^{ \left \left({\mathrm{Tx}}\right) \right } \left \left(0\right) \right {\mathrm{exp}} \left \left(-{{\mathrm{V}}}/{{\mathrm{v}}_{{\mathrm{s}}}}\right) \right }{\mathrm{,}}\end{equation*}\end{document} where K (Tx) d (0) is the dissociation constant at 0 mV and the voltage dependence is equivalently expressed as if the charge translocation involved the movement of a single K + across a fraction \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\delta}}}\;=\;25\;{\mathrm{mV}}/{\mathrm{v}}_{{\mathrm{s}}}\end{equation*}\end{document} of the transmembrane voltage. It is important to notice that and the condition \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{O}})}_{{\mathrm{on}}}\;=\;k_{1}\end{equation*}\end{document} bear no reference whatsoever to the extracellular solution conditions, a strong prediction of Fig. 2 that was verified in our comparative experiments with low- or high-K + solutions. Notice also that Fig. 2 leads us to conclude that our voltage-independent estimates of k (O) on provide a direct measurement of the true rate constant of PVIIA association to open channels. The expression of k (O) off according to is the same as that used by MacKinnon and Miller 1988 to describe the voltage and [K] i dependence of the dissociation rate constant of CTX from Ca 2+ -activated K + channels and predicts that both dependencies should be sigmoidal. However, it is easily seen that for large values of the ratio k (K) −1 / k (0) −1 the voltage dependence of k (O) off is practically exponential (increasing e-fold/v s ) in the wide voltage range: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{ln}} \left \left(\frac{K_{{\mathrm{d}}}^{ \left \left({\mathrm{Tx}}\right) \right } \left \left(0\right) \right }{ \left \left[{\mathrm{K}}\right] \right _{{\mathrm{i}}}}{\times}\frac{k_{-1}^{ \left \left(0\right) \right }}{k_{-1}^{ \left \left({\mathrm{K}}\right) \right }}\right) \right <\frac{{\mathrm{V}}}{{\mathrm{v}}_{{\mathrm{s}}}}<{\mathrm{ln}} \left \left(\frac{K_{{\mathrm{d}}}^{ \left \left({\mathrm{Tx}}\right) \right } \left \left(0\right) \right }{ \left \left[{\mathrm{K}}\right] \right _{{\mathrm{i}}}}\right) \right {\mathrm{.}}\end{equation*}\end{document} In the study of single channels reconstituted in lipid bilayers, MacKinnon and Miller 1988 could show the tendency to saturation of the K + -binding site by using [K] i values up to 700 mM. Likewise, by adjusting internal Ca 2+ to maintain channel openings at hyperpolarized potentials, they could also approach the finite lower limit of k (O) off . From their data, one estimates K (Tx) d (0) values in the range of 1–3 M, k (K) −1 / k (0) −1 ratios (β max /β min , in their terminology) in the range of 20–50, and a value of δ close to 1. As discussed below, it is practically impossible in the oocyte preparation to study open-channel block by PVIIA outside the range of the exponential rise of k (O) off , so that imposing the consistency of our data with yields only lower estimates for K (Tx) d (0) and k (K) −1 and a good estimate of v s . Our overall mean estimate of v s ∼ 40 mV, corresponding to \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\delta}}}\;=\;0.63\end{equation*}\end{document} , is in good agreement with the estimates of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\delta}}}\;=\;0.55\end{equation*}\end{document} obtained by Scanlon et al. 1997 from the voltage dependence of τ (O) at low [T] values for which τ (O) ∼ 1/ k (O) off . Low estimates of δ, ranging from 0.31 to 0.62, were also found in the study by Goldstein and Miller 1993 of the voltage dependence of Shaker open-channel block by several CTX variants with relatively fast binding kinetics. Our measurements of k (O) off do not show any obvious deviation from an exponential rise up to \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}\;=\;+80\;{\mathrm{mV}}\end{equation*}\end{document} , where k (O) off ∼ 200 s −1 . Thus, for consistency with , these data imply k (K) −1 >> 200 s −1 and K (Tx) d (0) >> [K] i *e 2 ; i.e., K (Tx) d (0) >> 0.8 M, in qualitative agreement with the studies of CTX block. The large lower estimate of k (K) −1 explains why also the low pedestal predicted by at negative voltages is not seen in our data. According to Fig. 2 , k (0) −1 is a lower bound for the dissociation rate constant in any channel configuration, so that k (0) −1 ≤ k (C) off ∼ 1 s −1 . Since \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{O}})}_{{\mathrm{off}}}{\mathrm{is}}\;{\sim}26\;{\mathrm{s}}^{-}1\;{\mathrm{at\;V}}\;=\;0\end{equation*}\end{document} , we expect that k (O) off approaches its low pedestal only for voltages more negative than −80 mV, where open channels are impossible to explore. In conclusion, the predictions of Fig. 2 are consistent with our data on PVIIA binding to open channels according to the following quantitative estimates: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{1}=k_{{\mathrm{on}}}^{ \left \left({\mathrm{O}}\right) \right }\approx 140\;{\mathrm{{\mu}M}}^{-1}{\mathrm{s}}^{-{\mathrm{1}}}{\mathrm{;}}k_{-1}^{ \left \left({\mathrm{K}}\right) \right }{\gg}200\;s^{-1}{\mathrm{;}}K_{{\mathrm{d}}}^{{\mathrm{Tx}}} \left \left(0\right) \right >1\;{\mathrm{M;{\delta}}}=0.63{\mathrm{.}}\end{equation*}\end{document} We discuss now the predictions of Fig. 2 for the case of closed channels. As already anticipated, the most important difference with respect to the previous case is the absence of C:Tx ↔ C:K + :Tx transitions. An additional important difference is the fact that C: ↔ C:K + transitions involve only a very fast equilibration with external K + . With these two conditions, Fig. 2 reduces to Fig. 3 , where {U} lumps together the unblocked states C: and C:K + , and p is the probability that a U state is a C:K + state. A further simplification arises from the above consideration that k (K) −1 is several hundred times larger than k (0) −1 , which implies that the probability of state C:K + :Tx is insignificant relative to that of state C:Tx for p < 0.99. This reduces Fig. 3 to a simple two-state scheme , where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k^{({\mathrm{C}})}_{{\mathrm{off}}}\;=\;k^{(0)}_{-}1\;{\mathrm{and}}\;k^{({\mathrm{C}})}_{{\mathrm{on}}}\;=\;(1\;-\;{\mathrm{p}})*k_{1}\end{equation*}\end{document} . Both our findings that k (C) off is independent of [K] o and k (C) on decreases upon increasing [K] o are fully consistent with this interpretation. In principle, the [K] o dependence of k (C) on could be used to estimate the dissociation constant of K + binding to the outer site, K (0) d . With the questionable assumption that sodium ions do not compete significantly for the same site, we can tentatively use for p the expression: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}p=\frac{ \left \left[{\mathrm{K}}\right] \right _{{\mathrm{o}}}}{ \left \left[{\mathrm{K}}\right] \right _{{\mathrm{o}}}+K_{{\mathrm{d}}}^{ \left \left(0\right) \right }}{\mathrm{.}}\end{equation*}\end{document} Since we have data only for two values of [K] o , 2.5 and 115 mM, our estimates of K (0) d can be only very crude. For \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;2.5\;{\mathrm{mM}}\end{equation*}\end{document} , we estimate k (C) on ∼ 1/6 k (O) on (i.e., (1 − p ) ∼ 1/6) and for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{K}}]_{{\mathrm{o}}}\;=\;115\;{\mathrm{mM}}\;k^{({\mathrm{C}})}_{{\mathrm{on}}}\;{\sim}\;1/90\;k^{({\mathrm{O}})}_{{\mathrm{on}}}\;[{\mathrm{i.e}}.,\;(1\;-\;p)\;{\sim}\;1/90]\end{equation*}\end{document} . These two data are roughly consistent, yielding K (0) d estimates of 0.5 or 1.3 mM, respectively. Notice that these guesses imply that the ratio K (Tx) d (0)/ K (0) d is in the order of 10 3 , close to our lower estimate for the ratio k (K) −1 / k (0) −1 . This is an important verification of the physical consistency of Fig. 2 , which requires that the destabilization of PVIIA binding by K + entrains a reciprocal effect of PVIIA on K + binding. The low δ value accounting for the voltage-dependent destabilization of PVIIA binding to open Shaker channels may appear in contrast with the model underlying Fig. 2 . Finding a value of δ close to 1 was used by MacKinnon and Miller 1988 as evidence that the K + -binding site is located at the extreme outer end of the K + -conduction pore, from where K + destabilizes CTX binding by speeding up CTX dissociation by a factor of 20. Using the same model, we find that PVIIA dissociation is accelerated by K + at least 10× more ( k (K) −1 / k (0) −1 >> 200), so that we might intuitively expect that the site from which K + exerts its repulsion on the toxin is even more to the outer extreme of the pore conduction pathway. However, it may be naive to interpret δ as the true electrical distance traversed by a single K + that leaves the cytoplasmic medium and goes all the way through the pore to reach the outermost binding site in the conduction pathway. A plausible, more realistic interpretation is illustrated by the cartoon in Fig. 9 . The conduction pore can be safely assumed to contain three binding sites, in agreement with classical models proposed to explain single-file diffusion properties of potassium channels and with the structural study of Doyle et al. 1998 . It is also plausible to assume that when the outer pore mouth is occluded by a toxin molecule the pore always has one, but very unlikely all three, of these binding sites occupied. Accordingly, the occupational configurations that most significantly represent the states indicated in Fig. 2 as C:Tx and C:K + :Tx could be those shown in Fig. 9 . If this is the case, the transition from C:Tx to C:K + :Tx would involve the concerted movement of two potassium ions, each traversing only a fraction of the electrical distance across the pore; the necessary push/pull of intercalated water molecules might be eased by a water shunt of the toxin plug. Even assuming no voltage drop from the outermost site to the external vestibule, the transition could then involve for each ion a δ value of 1/3, and this would yield an apparent overall \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\delta}\;of}}\;2/3\;=\;0.66\end{equation*}\end{document} , consistent with our measurements. As a consequence of our study, we propose that the dependence of the PVIIA block on channel conductance does not involve state-dependent changes of the molecular interactions of the toxin with the residues that shape the pore vestibule, but rather the indirect modulation of toxin binding by the interaction with cations occupying nearby sites. For our comprehensive modeling of the voltage, [K] o , and state dependence of PVIIA block, we have used a somewhat natural expansion of the scheme proposed by MacKinnon and Miller for CTX block. Therefore, we believe that a similar analysis could be applied to CTX variants with relatively fast binding kinetics like PVIIA. In general, our analysis suggests an additional potential value of pore-blocking molecules for the study of ion channels, besides their use as probes of the pore-mouth structure. Indeed, a detailed study of the interaction of these substances with permeant ions, by an appropriate evaluation of the effects of such interaction upon the pore blockade, may also provide important information about more intimate properties of the ion-conduction pathway. | Study | biomedical | en | 0.999997 |
10398697 | Conotoxins are a family of small peptide toxins derived from the venom of marine snails of the Conus , a genus composed of ∼500 predator species . These peptides, which bind with high affinity to excitable tissue, are small (8–35 residues) and structurally constrained by intrapeptide disulfide bounds . The pattern of formation of the disulfide bonds yields a characteristic folding in the peptide family that allows for structural subfamily classification. A set having three disulfide bonds that defines a four-loop framework specifically blocks voltage-gated ion channels . Although members of the four-loop toxins may share little amino-acid similarity among the noncysteine residues, their overall folding can be remarkably similar . Thus, specificity for different voltage-gated channels is believed to be conferred solely by the specific sequence of each peptide. For example, ω-, δ-, and κ-conotoxins are specific for Ca, Na, and K voltage-gated channels, respectively. However, detailed knowledge of their mechanism of inhibition on voltage-gated channels is not yet clear. κ-Conotoxin-PVIIA (κ-PVIIA) 1 is a 27-residue peptide component of the Conus purpurascens venom found to inhibit K channels. This peptide acts rapidly on its target, so it is proposed to have an important role in the quick excitotoxic prey immobilization after the venomous sting . The structure has recently been resolved by nuclear magnetic resonance methods and revealed as a member of the four-loop family . Although κ-PVIIA is ∼10-residues shorter, it shows striking similarities to the space distribution of functionally relevant basic residues of charybdotoxin (CTX), a member of a well-characterized group of K channel pore blockers, the α-KTx, . In CTX, the amino group of the side chain of Lys-27 has been proposed to interact intimately with the K-permeation pathway by occluding the pore. Moreover, its structure shows a lysine paired with an aromatic side chain 6–7 Å away, a conserved dyad in peptide blockers of K channels . Savarin et al. 1998 have proposed that the lysine side chain of this dyad interacts with the K channel pore as Lys-27 does in CTX. In addition to this architectural mimicry to other K channel peptide blockers, there are three lines of evidence suggesting that κ-PVIIA binds to the external vestibule of K channels. First, different pore splice variants bind κ-PVIIA with dissimilar affinity . Second, κ-PVIIA competes with other putative pore blocker toxins . Third, point mutations in the external vestibule of the voltage-gated Shaker K channel modify the extent of inhibition by κ-PVIIA, suggesting that this peptide also inhibits K channels by occluding the permeation pathway . However, its microsite specificity is different from that of CTX; the same vestibular mutation, Phe425 → Gly, which enhances ∼2,000-fold affinity for α-KTx , decreases it >20-fold for κ-PVIIA . On the other hand, recent observations are somehow conflicting with a simple pore blocker mechanism. κ-PVIIA seems to act differently whether or not Shaker K channels carries the N-type of inactivation domain, a portion of the protein that inactivates the open channel conformation from the intracellular side . In the inactivating channel, the toxin-induced inhibition seems to be independent of the voltage applied to activate the channels , whereas in the inactivation-removed Shaker , the effect of this toxin is an apparent voltage-dependent modification of the rate of activation . This latter result could be interpreted as if the main toxin effect was to delay the gating mechanism as some spider and scorpion toxins do . In this paper, we attempt to establish the mechanism of action of κ-PVIIA on Shaker K channels. Using the well-known α-KTx inhibition mechanism on K channels as a paradigm for comparison , we made a detailed study of the mechanism by which κ-PVIIA acts on open Shaker K channels. To our knowledge, together with Terlau et al. 1999 , these are the first studies in detail of a four-loop conotoxin inhibition mechanism on a voltage-gated ion channel. Here, we present results indicating that (a) κ-PVIIA binding to Shaker K channel is consistent with a 1:1 stoichiometry and diffusion-limited association, (b) a well-known K-pore blocker, tetraethylammonium, reduces the toxin association, but not the dissociation rate, and (c) the peptide toxin interacts, perhaps electrostatically, with K + from the intracellular side of the channel. We also argue that by binding to the external vestibule, the toxin reduces the occupancy of permeant ions inside the channel pore. Together, these results provide compelling evidence that κ-PVIIA inhibits potassium currents by plugging the pore of K channels in way analogous to scorpion toxins . Boc- l amino acids were obtained from Novabiochem or the Peptide Institute, t-Boc-Val-OCH 2 -PAM-resin (substitution value 0.77 mmol g −1 ) was obtained from Perkin Elmer. 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Richelieu Biotechnologies. Other reagents were of peptide synthesis grade from Auspep. Stepwise synthesis (0.5 mmol scale, 0.649 g resin) was conducted manually using in situ BOC SPPS , starting from Boc-PAM-Val resin. The average coupling was 99.80, as determined by ninhydrin assay . The peptide was cleaved from the resin using HF/ p -cresol/ p -thiocresol (18:1:1) at −10–0°C for 1 h. Peptide was precipitated with cold ether, collected by filtration on sintered glass, washed with cold ether, and dissolved in 50% AcOH, diluted with water and lyophilized. The crude peptide was purified by preparative chromatography (Vydac C18 column, 2.2 × 25 cm), using a 1% gradient \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(100\%\;{\mathrm{A\;to}}\;80\%\;{\mathrm{B}},\;80\;{\mathrm{min}};\;{\mathrm{A}}\;=\;0.1\%\;{\mathrm{trifluoroacetic\;acid}}\;[{\mathrm{TFA}}]\;{\mathrm{in\;H}}_{2}{\mathrm{O,\;B}}\;=\;0.09\%\;{\mathrm{TFA}},\;10\%\;{\mathrm{H}}_{2}{\mathrm{O}},\;90\%\;{\mathrm{CH}}_{3}{\mathrm{CN}})\end{equation*}\end{document} to give reduced peptide in 34% yield. The folded product was obtained by dissolving reduced peptide (10 mg) in aqueous 0.33 M NH 4 OAc/0.5 M GnHCl (154 ml), with pH adjusted to 7.8 using 0.01 M NH 4 OH. The solution was stirred at 4°C for 5 d, in the presence of reduced and oxidized glutathione (molar ratio of peptide:GSH:GSSG was 1:100:10). Lowering the pH to 2–3 with TFA (5 ml) terminated the oxidation. The reaction mixture was loaded onto a preparative HPLC column (Vydac C18 column, 2.2 × 25 cm) (8 ml min −1 ) and washed with 0.1% TFA until all oxidation buffer had eluted. A 1% gradient (100% A to 80% B, 80 min) was applied and pure oxidized κ-PVIIA was isolated in 95% yield. Electrospray ionization mass spectra recorded on a PE Sciex API III triple quadrupole mass spectrometer were used to confirm the purity and molecular weights of synthetic peptides. For electrophysiology, salts of analytical grade were purchased from Baker. Gentamicin, sodium pyruvate, EGTA, HEPES, N -methyl- d -glucamine (NMG), and BSA were from Sigma-Aldrich Química S.A. de C.V. Females Xenopus laevis (Xenopus One) were anesthetized by immersion in ice. Ovarian lobes were surgically removed and collected in ND96 solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 HEPES, pH 7.6, and 50 μg/ml gentamicin. Type II collagenase, 0.9–1.5 mg/ml, was used for digestion of connective tissue (Worthington Biochemical Corp.). After removing the enzyme, stage IV–VI oocytes were isolated and manually defolliculated in a nominally Ca 2+ -free ND96 solution. The inactivation-removed Shaker H4 Δ(6-46) subcloned in vectors under control of either the cytomegalovirus or the SV40 late promoter were a gift from Dr. Christopher Miller (Brandeis University, Waltham, MA) . To expose the nucleus for injection of the nonlinearized plasmids, oocytes were put with the animal pole facing up in a swinging-bucket rotor and spun for 7–12 min at 1,000 g in a Centrific centrifuge (Fisher Scientific). After nuclear injection of 0.05–0.2 ng of cDNA, oocytes were incubated at 18°C in ND96 supplemented with sodium pyruvate (2.5 mM) and bovine serum albumin (0.04%). After 15–72 h incubation, subsequent recordings with two-electrode voltage clamp (TEVC) or outside out patch clamp were made at room temperature (22°–24°C). For whole-cell TEVC recordings, oocytes were positioned in the middle of an ∼500-μl longitudinal recordings chamber . All measurements were made under continuous perfusion of either a standard recording solution (containing [mM]: 96 NaCl, 2 KCl, 1 MgCl 2 , 0.3 CaCl 2 , 10 HEPES-NaOH, pH 7.6, and 25 mg/l BSA) or the same solution plus κ-PVIIA, 30–1,000 nM, added from a stock of 1 mM. Voltage pulse protocols and data acquisition were performed from a personal computer running pClamp 5.5 through a Digidata 1200B acquisition interface (Axon Instruments). Whole oocyte recordings were made with an OC-750C voltage clamp amplifier (Warner Instruments). Recording electrodes, with resistance of 0.3–1 MΩ were made with Ag/AgCl pellet assemblies (Axon Instruments) inside a capillary filled with a solution made of 3 M KCl, 5 mM EGTA, and 10 mM HEPES-KOH, pH 7.0. Voltage pulses of 50–500 ms were applied from a holding potential of −90 mV, and usually ranged from −60 to 50 mV in 10-mV intervals. Because oocytes expressing outward currents >10 μA at 50 mV usually exhibited obvious slower rising times, experiments were performed in oocytes expressing 0.5–8 μA only. For patch clamp recording, the vitelline membrane was removed after a 10-min incubation in a solution containing (mM): 200 K-aspartate, 10 KCl, 10 EGTA, and 10 HEPES, pH 7.4. After vitelline membrane removal, we followed conventional patch-clamp techniques . Outside-out patches were excised from the oocyte membrane and positioned near the outlet of a rapid perfusion system . In such a system, a single solenoid movement (225P071; NResearch) performs rapid exchange between two solution streams converging into the tip of the patch pipette. The solenoid simultaneously opens the path of one solution while it closes the path for the other. Solution exchange rate was complete in <5 ms. For most experiments described, patch pipettes (1–4 MΩ) were filled with solutions consisting of (mM) 80 KF, 20 KCl, 1 MgCl 2 , 10 EGTA, and 10 HEPES-KOH, pH 7.4 (100 -K in ). For the experiments with reduced internal K + concentration (15- K in ), solution was 90 NMG-F, 10 KF, 1 MgCl 2 , 10 EGTA, and 10 HEPES-KOH, pH 7.4. External recording solution was 115 NaCl, 1 KCl, 0.2 CaCl 2 , 1 MgCl 2 , and 10 HEPES-NaOH, pH7.4 (1- K ex ). For all patch clamp experiments shown in this paper, 500 nM κ-PVIIA and 1 mM tetraethylammonium (TEA + ) were added to the external patch clamp recording solution. The small inflection of the K current (∼5%) produced by 1 mM TEA + blockade was used to monitor the position of the patch pipette to obtain the optimal rate of solutions exchange . Because of intrinsic variation of the solenoid latency, the TEA + -produced inflection is not often visible in the averaged records. For experiments in nominally zero internal potassium (0- K in ), all potassium salts were replaced with NMG-F and the membrane patch was formed and pulled out in the 1- K ex recording solution. Then, the patch was moved to the perfusion system running a solution made of 16 NaCl, 100 KCl, 0.2 CaCl 2 , 1 MgCl 2 , and 10 HEPES-KOH, pH 7.6 (100- K ex ). For whole oocyte and outside-out currents, off-line leak subtraction was carried out before performing point-by-point division of current records. Curve fitting, statistics, and figure preparation were carried out with Microcal Origin 3.5 (Microcal Inc.). Fig. 1 describes the 1:1 toxin binding equilibrium, where Sh , T, and Sh ·T correspond to a conducting empty channel, the toxin, and a nonconducting channel–toxin complex, respectively. The rate constants for association and dissociation are k on and k off , respectively. This scheme predicts that in response to a step-like perturbation, the toxin-binding will relax exponentially to a new equilibrium. The relaxation rate, measured as the reciprocal of the relaxation time constant, is a function of the toxin concentration as following: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{1}{{\mathrm{{\tau}}}}=k_{{\mathrm{on}}} \left \left[{\mathrm{T}}\right] \right +k_{{\mathrm{off}}}{\mathrm{,}}\end{equation*}\end{document} which defines a straight line with slope k on , and k off as intercept. Meanwhile, the fraction of unblocked channels in the new equilibrium is given by: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*} \left \left(\frac{I_{{\mathrm{PVIIA}}}}{I_{{\mathrm{control}}}}\right) \right _{{\mathrm{{\infty}}}}=\frac{k_{{\mathrm{off}}}}{k_{{\mathrm{off}}}+k_{{\mathrm{on}}} \left \left[{\mathrm{T}}\right] \right }{\mathrm{.}}\end{equation*}\end{document} Together, and provide a system with two equations and two unknowns, k on and k off . Thus, by fitting the values of τ and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*} \left \left(\frac{I_{{\mathrm{PVIIA}}}}{I_{{\mathrm{control}}}}\right) \right _{{\mathrm{{\infty}}}}\end{equation*}\end{document} to the macroscopic relaxation in response to each voltage step, a pair of values for k on and k off is obtained. The magnitude of effective valence for the voltage dependence of each rate constant, z δ, was calculated from the expression: 3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k=k_{ \left \left(0\right) \right }e^{0.04{\cdot}z{\mathrm{{\delta}V}}}{\mathrm{,}}\end{equation*}\end{document} where V is the membrane potential in millivolts, and k (0) is the rate constant at zero applied potential. Because the voltage dependence of macroscopic relaxation depends nonlinearly on the voltage dependence of both k on and k off to describe it, we preferred to use the expression: 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}={\mathrm{{\tau}}}_{ \left \left(0\right) \right }e^{{-{\mathrm{V}}}/{{\mathrm{V}}_{{\mathrm{s}}}}}{\mathrm{,}}\end{equation*}\end{document} where τ (0) is the time constant at zero applied potential and V s is the membrane voltage, in millivolts, that produces an e-fold increase in τ. Whole cell TEVC recordings in oocytes expressing the inactivation removed Shaker (Δ6-46) K channels were used to examine the effect of κ-PVIIA . In this set of experiments, the toxin was applied continuously to the recording solution superfusing the oocyte. Fig. 1A and Fig. B , shows K currents elicited by 200-ms depolarizing steps between −60 and +50 mV at 10-mV intervals in the presence and absence of 100 nM κ-PVIIA. The apparent effect of 100 nM κ-PVIIA on the K channels is to make activation kinetics more complex by the introduction of a second, and slower, phase in the rising of the currents elicited by voltage steps . Also, the conductance–voltage relationship measured at the end of the pulse is shifted to the right and is less steep in the presence of the toxin . This voltage-dependent effect is in contradiction with the nearly voltage independent affinity of κ-PVIIA on the inactivating Shaker H4 observed previously . Results described in Fig. 1 may suggest that the toxin effect on Shaker K channels is to modify the gating mechanism. The toxin would bind preferentially to the closed channel conformation, delaying the rate of activation. Such a mechanism has been proposed for hanatoxin, a component of the spider venom, on other K channels as Kv2.1 . An alternative explanation could be that the toxin binding to its receptor in the channel is voltage dependent. In this latter view, the positive-going voltage pulse used to activate the channels would also destabilize the binding of the toxin. Thus, the slow apparent activation of the currents would represent the time course by which the toxin binding relaxes from one equilibrium at resting potential to one of lower affinity at a more positive voltage. This kind of mechanism would be reminiscent to the “hook” seen in the tail K currents induced by internal tetra-alkylammonium . Data shown in Fig. 2 Fig. 3 Fig. 4 Fig. 5 are from experiments made to distinguish among these two possible mechanisms. Fig. 2 A shows a point-by-point division of the traces in the presence of the toxin by the control traces at pulse voltages positive to −20 mV shown in Fig. 1 . As done before by Scanlon et al. 1997 , single exponential fits (thin lines on top of each trace) to the normalized traces were extrapolated to the beginning of the voltage pulse. Regardless of the blocking mechanism, a common origin for the curves indicate the level of the blocking equilibrium at the holding potential. As the voltage pulse is made more positive, the time constant of relaxation gets smaller, and the steady state inhibition is reduced . Good descriptions by single exponential of the traces suggest a voltage-dependent first-order process. A simple bimolecular stoichiometry predicts that the rate of relaxation should increase linearly to the toxin concentration. Fig. 3 A shows records obtained with a 100-ms pulse to +40 mV with 0, 30, 100, and 300 nM κ-PVIIA. In Fig. 3 B, the point-by-point divisions of the traces by their controls at zero toxin are shown. With this operation, the concentration dependence of the blockade at holding potential becomes apparent. From these measurements, a dissociation constant at holding potential of 65 ± 11 nM is obtained . This value is in close agreement with the 60 nM measured by Terlau et al. 1996 and with tonic inhibition measured at resting . Thus, we can explain the discrepancy between our results and the nearly voltage-independent affinity measured for κ-PVIIA inhibition to the N-inactivating Shaker H4 by Terlau et al. 1996 . Because N-type inactivation proceeds much faster than the toxin relaxation (milliseconds versus tens of milliseconds), measurement of fractional inhibition, early during the pulse, at the peak of the current in N-inactivating channels should closely resemble the binding equilibrium at holding potential. As is apparent from Fig. 3A and Fig. B , the rate of relaxation increases at higher toxin concentrations. Fig. 3 C shows that the rate of relaxation, measured as the reciprocal of the time constant, increases linearly with the concentration of κ-PVIIA at all voltages. Linear regressions to the data show that the intercept changes with the pulse voltage; meanwhile, the slope is almost invariant. This linearity is consistent with a bimolecular stoichiometry for the interaction between κ-PVIIA and the Shaker K channel as shown by Fig. 1 in methods . For such a scheme, states that the slope is k on and the intercept is k off . Fig. 3 D plots the voltage dependence of both rate constants for the data plotted in C. It is clear that most of the voltage dependence resides in k off (•). At zero voltage, k off is 17 ± 1 s −1 , with a \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}z{\mathrm{{\delta}}}\;=\;0.58\;{\pm}\;0.08\end{equation*}\end{document} . Meanwhile, the apparent second-order rate constant is nearly voltage independent. The rate constant k on has a zero-voltage value of 70 ± 9 μM −1 s −1 with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}z{\mathrm{{\delta}}}\;=\;0.08\;{\pm}\;0.16\end{equation*}\end{document} . This value agrees very well with that of 61 μM −1 s −1 for charybdotoxin, a toxin whose binding to potassium channels is proposed to be diffusion limited . This “on” rate constant seems too fast to be the rate limiting step for a protein conformational change; nevertheless, it is slow enough for a conformational change in the channel promoted by a toxin that is already bound at resting—before the activating voltage pulse is given . Because the use of voltage-step protocols is inherent to the study of Shaker K channels, to differentiate between the effects of the voltage or the open probability of the channels on κ-PVIIA binding, chronic bath application to whole oocytes are inadequate. We compared chronic and rapid applications of the toxin to membrane patches expressing macroscopic currents in the outside-out configuration . Outside-out patches were separated from the oocyte following standard techniques . Holding the pipette voltage at −90 mV, the pipette tip was positioned near the opening of the fast perfusion system and 200-ms test voltage pulses from −60 to 50 mV were applied. Fig. 4A and Fig. D , shows two different patches with typical sets of current records obtained with the control recording solution (1- K ex ) washing the membrane patch, and with the 15- K in solution in the pipette (see methods ). At most test voltages, currents were already fully activated 30 ms after the beginning of the pulse. However, the time courses of the currents are bit more complex than those obtained in whole oocytes recordings. At potentials positives to −20 mV, a time-dependent decrease of the current becomes evident. Such reduction was not apparent in the whole oocyte records. We do not know if this difference from the two-electrode voltage clamp experiments is due to an inactivation process, a local potassium depletion that renders currents smaller, or both. No further attempts to study this phenomenon were made. To separate gating from toxin kinetics in the chosen interval of pipette voltages, toxin was applied ∼40 ms after the beginning of the test pulse. Visual clues are not usually enough to position the pipette tip where solution exchange is optimally fast. To monitor the optimal positioning of the membrane patch in the rapid application system, together with the toxin, we added 1 mM TEA + to the test solution. Because this TEA + concentration produces a fast blockade of a small but measurable fraction of the current, we could monitor the solution exchange rate by monitoring the speed of the inflection produced by the TEA + effect on K currents on individual records . Fig. 4 B shows an average of four runs of κ-PVIIA/TEA + . The rapid TEA + inflection is not visible in averaged records because of inherent variable latency of the solenoid valve (<5 ms). At all voltages, 1 mM TEA + /500 nM κ-PVIIA pulse applications produced a decrease in the current in which the kinetics of the onset and offset of toxin blockade are faster as the voltage is made more positive. As with the whole oocyte recordings, we made point-by-point division of toxin current records by control records at each voltage. As a result of such an operation, we also avoided possible distortions on toxin kinetics resulting from the apparent inactivation, as seen by the complete recovery in the current records taken at more positive voltage pulses . These normalized records show voltage-dependent on and off kinetics in addition to a voltage-dependent steady state inhibition. Single exponential fits applied to each normalized trace are shown as solid lines over some traces. A general result from single exponential fits to the normalized traces is shown in Fig. 5 (open symbols). This figure plots averages of data pooled from a total of 13 outside-out patches with pulse applications of κ-PVIIA. Because we did not find significant difference in the toxin kinetics between experiments obtained with 100 -K in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;7)\end{equation*}\end{document} or 15- K in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;6)\end{equation*}\end{document} solutions in the patch pipette (not shown, see Table and discussion ), these data also represent the merging together of these two experimental conditions. If the toxin inhibition is produced merely by toxin binding instead of inducing, or preventing, a conformational change, the kinetics of the response to a voltage step in the presence of the toxin should be identical to that induced by pulse applications of toxin at a constant voltage. To the patch whose current record is shown in Fig. 4 D, 1 mM TEA + /500 nM κ-PVIIA was chronically applied, a protocol equivalent to that of the whole-oocyte experiments . As in the whole oocyte experiments, the apparent time course of the current activation is also delayed in these conditions. A point-by-point division of these traces by the controls of Fig. 4 D is shown in F. This family of normalized traces shows that the voltage dependence of the relaxation and steady state inhibition is similar to those with the toxin pulse protocol shown in Fig. 4 C. Thin lines in Fig. 4 F correspond to exponential fits to some of the normalized traces. Average time constant and steady state inhibition for 11 pooled patches are plotted as solid symbols in Fig. 5 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;4\;{\mathrm{with}}\;100-K_{{\mathrm{in}}}{\mathrm{and}}\;7\;{\mathrm{with}}\;15-K_{{\mathrm{in}}}{\mathrm{in\;the\;pipette}})\end{equation*}\end{document} . Fig. 5A and Fig. B , summarizes results of the kinetic analysis of the experiments of κ-PVIIA applications to outside out patches. In Fig. 5 A, time constants for the onset of inhibition and recovery after toxin removal for the pulse protocol experiments are plotted as open symbols; meanwhile, plotted as solid symbols are the time constants of the relaxations in the experiments with chronic application as shown in Fig. 4 F. Fig. 5 B plots the steady state inhibition for pulse (○) and chronic (•) applications. Two lines of evidence strongly suggest that the rate-limiting step for the relaxation of the inhibition is the association of the toxin with the channel and not a conformational change from a closed to open state. (a) Although the levels of steady state fractional current reached with both protocols are significantly different, in average, the systematic difference between both curves accounts for <3% of the inhibition. On the other hand, for both protocols of κ-PVIIA applications, no significantly different values for the time constant of onset relaxation are detected, indicating that both the voltage pulse in the presence of κ-PVIIA and the toxin pulse applied to the open channels promote a nearly identical perturbation on the blockade equilibrium, as expected by a most simple voltage-dependent blocking mechanism. Such a mechanism only requires toxin binding with 1:1 stoichiometry to produce inhibition. (b) Although the toxin binds with moderately higher affinity to the closed state , in both protocols, no inflection is observed between values obtained at voltages around 10 mV . At this range of voltages, the open probability of the channels is near its maximum and becomes roughly voltage independent , thus, reinforcing the idea that the toxin inhibits the open K channels mostly by a voltage-dependent binding. Zero-voltage time constant of the onset of chronic application is \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{{\mathrm{on}}}\;=\;29\;{\pm}\;3\;{\mathrm{ms,\;with\;V}}_{{\mathrm{s}}}\;=\;141\;{\pm}\;58\;{\mathrm{mV}}\end{equation*}\end{document} . For pulse application, the zero-voltage onset time constant is \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{{\mathrm{on}}}\;=\;27\;{\pm}\;2\;{\mathrm{ms,\;with\;V}}_{{\mathrm{s}}}\;=\;80\;{\pm}\;17\;{\mathrm{mV}}\end{equation*}\end{document} , while for the recovery it is \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{{\mathrm{off}}}\;=\;73\;{\pm}\;2\;{\mathrm{ms\;with\;V}}_{{\mathrm{s}}}\;=\;56\;{\pm}\;4\;{\mathrm{mV}}\end{equation*}\end{document} . In a bimolecular scheme as presented in methods , the time constant of the macroscopic relaxation is governed by . In the absence of toxin, k off can be calculated directly from the reciprocal of the recovery time constant, τ off in the measurements of toxin removal. Also, we have two additional experimental conditions in which the relaxation in the presence of the toxin is seen, chronic and pulse application onset. For a 1:1 stoichiometry, these two types of macroscopic relaxations should reach a steady state level given by . From the values plotted in Fig. 5 , we calculated both k on and k off for each pipette potential by using and . Results from such calculations are summarized in Fig. 6 . The small open and filled symbols correspond to the calculations of k on and k off , respectively. There is a good agreement in the values of k off measured directly from pulse experiments with those calculated from the two-equation system regardless of the experimental protocol. As in the whole oocyte experiments of Fig. 3 , most of the voltage dependence appears to be in k off . An overall average calculated from all three experimental data sets is plotted as the big circles in Fig. 6 . The solid line is a single exponential fit to the calculated rate constants. At zero voltage, the second order rate constant, k on , is 43 ± 3 μM −1 s −1 with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}z{\mathrm{{\delta}}}\;=\;0.11\;{\pm}\;0.05\end{equation*}\end{document} . This value for k on is only 40% smaller than that measured on the whole-oocyte experiments and is also consistent with a diffusion-limited association . Similarly, showing good agreement with the whole oocyte experiments, k off at zero voltage is 13 ± 2 s −1 with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}z{\mathrm{{\delta}}}\;=\;0.53\;{\pm}\;0.09\end{equation*}\end{document} . The near perfect agreement between the patch clamp and two-electrode voltage clamp experiments reinforce the idea that the κ-PVIIA exerts its inhibition effect on the open K channel by binding to an external site only. Thus, the relaxation of the macroscopic K currents observed in oocytes under chronically applied toxin is analogous to the slow hook seen previously when tetraethylammonium ions dissociate from squid axon potassium channels before rapid deactivation in response to repolarization . In both cases, macroscopic kinetics are determined by a temporal superposition of blocking and gating transitions. Additionally, owing to the noninactivating feature of Shaker -IR, we established that in the relatively slowly perfused whole oocyte preparation we could make high resolution kinetic measurements of a toxin with a resident time in the tens of milliseconds time scale. Although there is no compelling evidence that κ-PVIIA plugs the pore of K channels, there are several lines of evidence that κ-PVIIA interacts with the Shaker external vestibule. Transference of the H5 segment from a κ-PVIIA–sensitive K channel to an insensitive channel also transfers toxin sensitivity . Mutations in the H5 segment alters toxin affinity . Also, externally applied TEA + , a specific pore blocker for K channels, reduces the extent of the blockade, suggesting a competitive interaction with the toxin . As an antecedent to these experiments, TEA + only reduces the association rate of CTX to the large conductance Ca-activated K channels (MaxiK-channel), without an effect on the dissociation rate . Thus if κ-PVIIA binds to the Shaker vestibule, similar effects are expected. With two-electrode voltage clamp, κ-PVIIA binding was studied in the presence of TEA + 0–10 mM, added on top of the recording solution. Fig. 7 shows a summary of such experiments. In the same fashion as we did for the results shown in Fig. 3 , we made a point-by-point division of records taken in κ-PVIIA/TEA + solution from those taken in TEA + only (not shown). Applying and to the time constants and steady state inhibition obtained from the single exponential fits to the relaxations, apparent k on and k off for the effect of TEA + were obtained. TEA + decreases the apparent association rate with little effect on the dissociation rate. This effect seems to be specific for TEA + because raising Na + concentration to 10 mM does not significantly affect either k on or k off . Together, these results indicate that the effect of TEA + on κ-PVIIA binding is not due to the increased ionic strength, but rather to a specific exclusion of the toxin from the external vestibule of Shaker . To determine whether κ-PVIIA inhibit ionic conduction by occluding the ionic pathway, we analyzed the effect of altering permeant ion concentration in the opposite side of the pore. This strategy had shown that the binding of CTX to the MaxiK-channel is very sensitive to the intracellular K + . The rationale for these experiments is that occupancy of the pore by permeant cations coming from the internal side of the channel will repel electrostatically the highly positively charged toxin (+4). In the MaxiK-channel, an approximately fourfold increase in the toxin residence time is expected when the internal K + concentration is lowered from 110 to ∼10 mM. As with the Ca-activated K channel, half saturation concentration for K conductance in Shaker K channels is near 300 mM, suggesting the presence of a low affinity K-binding site . To our surprise, in outside out patches, preliminary experiments showed a minor effect, either on the association or the dissociation rates, produced by the reduction of the intracellular K + concentration from 100 to 15 mM ( Table ). This result suggests that, if κ-PVIIA occludes the conduction pathway, it is not very sensitive to K occupancy in the pore, or the pore occupancy is not very different in these two distinct conditions. As in the MaxiK-channel, it had already been suggested that in the Shaker K channel there are at least one micromolar affinity potassium binding sites inside the K channel pore . Thus, we substituted all the potassium by the nonpermeant cation NMG + in the patch pipette solution, and we formed and pulled outside-out patches from the oocyte membrane in the 1- K ex recording solution. We attempted to reduce contamination of the patch pipette with K + , and thus minimized occupancy of the pore from internal potassium ions. After obtaining a stable outside-out patch, the pipette was positioned in the rapid perfusion system applying an external solution containing 100 mM K + . Under these conditions, 0 -K in //100- K ex and high levels of channel expression, voltage pulses activated inward currents that frequently produced positive feedback responses characteristic of inadequate clamp. This latter effect was avoided by doing recordings with small currents (<200 pA). Fig. 8 A shows 400-ms records with pulse application of a solution that, in addition to the 100 mM KCl, contained 500 nM κ-PVIIA and 1 mM TEA + . Because, at any voltage, this type of record displayed <10% of inactivation (not shown), the rate of dissociation was measured by fitting single exponential functions directly to the time course of the inward currents recovery. Average values (mean ± SEM) for four such experiments are plotted in Fig. 8 B. For the sake of comparison, the best fit to the average dissociation rate made with in whole oocyte TEVC at 100 mM external K + is shown by dotted lines . In agreement with the results that Goldstein and Miller 1993 previously obtained for CTX in the nominal absence of internal K + , both the amplitude of the zero-voltage dissociation rate and its effective valence were reduced by half. Such a trans-pore effect of potassium ions is by itself indicative that the mechanism of inhibition κ-PVIIA on K channel is physically blocking the ion conduction pathway . These results also suggest that, as in CTX, the side chain of a basic residue interacts with permeant ions residing in the narrowest region of the pore. As with CTX , the complete removal of the internal K + does not abolish completely the voltage dependence of κ-PVIIA binding to Shaker . The most economical interpretation for this residual voltage dependence is that a positive charge located in the pore-occluding side chain of the toxin interacts intimately with the narrowest part of the K channel pore, getting located inside the electric field. An electrical distance of 0.2–0.25 coincides with an externally located cationic binding site that is present in the pore of Shaker . A binding site detected at approximately the same electrical distance can accommodate mono- and divalent cations. NH 4 + , Cs + , Rb + , and K + bind to this site with low millimolar or high micromolar affinity . Occupation of this site impeded or delayed C- or P-type inactivation, pore collapsing conformational changes proposed to occur near the external entrance of Shaker pore . On the other hand, Gómez-Lagunas 1997 recently found that the occupation of an external site by the permeant ions or by TEA + protects Shaker K channels from visiting a long-lived nonconducting state christened “defunct” . This state arises when the channels are opened in the absence or presence of submillimolar K + concentration; then they are forced to close after its pore occupancy has diminished . Closing the channel after the occupying ions are allowed to escape away from the pore promotes a collapse of the pore that traps the gating machinery in a altered set of states . Thus, both types of nonconducting states depend largely on pore occupancy. We tested whether we could promote any of these nonconducting states just by blocking the channel with κ-PVIIA in the absence of internal permeant ions . Outside-out patches were recorded in 0 -K in //100- K ex condition. From a holding potential of −90 mV, the channels were opened by a pulse to −40 mV to ensure a strong driving force for K + to flow inwardly. First, we determined that an application of nominally zero K + solution (100 mM NMG + ) to the patch pipette that exposed 5–8 ms before the voltage pulse ended was necessary to observe any measurable amount of current reduction between consecutive pulses . For an interpulse interval of 5 s, this reduction indicates the population of channels visiting any of the occupancy-dependent nonconducting states. Because Shaker K channels show little rectification between +40 and −40 mV, we expected the external binding site to be empty in <500 μs . Thus, the amount of time needed to observe current reduction might represent incomplete wash out of the high K + solution. In any case, this figure represents an upper limit to the time needed to empty the pore, and is 20–30-fold shorter than the residence time of the bound toxin at −40 mV in this experimental condition . After the 40th pulse, <40% of the current remains . On the other hand, Fig. 9 B shows an experiment in which, instead of exposing the open channel to zero potassium, we applied κ-PVIIA to the patch with the intention of occluding the external exit of the pore. In such a case, the K channel pore should be emptied into the internal solution. At 150 ms after channel opening, a 150-ms pulse of 1 mM TEA + /500 nM κ-PVIIA that blocked ∼60% of the channels was applied . While the toxin was still being applied, the channels were forced to close by stepping the voltage back to −90 mV. Thus, given that the toxin blocking time is more than two orders of magnitude longer than the time needed to empty the pore ( Table ), closing the channels while they are still blocked should drive a measurable fraction into any occupancy-dependent nonconducting state. Fig. 9 B shows a set of 10 traces representing one of every four acquisitions from one of such an experiment. If the toxin does not protect the channel, ∼40% of the current is expected to have disappeared at the 40th pulse . However, in four experiments like this, no reduction of the current was detected at even the 60th pulse, suggesting that the channel pore cannot collapse while the toxin is bound. Because both types of nonconducting states require or are facilitated by pore vacancy, while κ-PVIIA is bound to the channel, the pore behaves as if occupancy were preserved. The simplest interpretation to this result is that a part of the toxin impedes pore collapse. Thus, we suggest that a component of κ-PVIIA occupies the most externally located K + binding site of Shaker pore, resulting in a protection of the channel. In normal physiological conditions, such occupancy should reduce the average number of permeant ions residing in the pore. Here we present evidence for a mechanism of inhibition of a peptide component of the venom of the predator marine snail Conus on K channels. The 27-residue κ-PVIIA inhibits K + currents by binding to the external vestibule of the Shaker K channel pore with a 1:1 stoichiometry at a rate consistent with a diffusion-limited manner . It competes with TEA + and unbinds the channel vestibule in a voltage-dependent manner, probably due to electrostatic interaction with permeant ions inside the pore. Such a mode of action is remarkably similar to that proposed for scorpion toxins of the family α-KTx, the best characterized of all peptide toxin specific for K-channels . As with α-KTx, κ-PVIIA voltage dependence of the binding equilibrium to the channel resides in the dissociation rate . For CTX bound to the external vestibule of the MaxiK-channel, the effective valence of the dissociation rate is 1.0 . In the absence of internally present permeant cations, z δ vanishes. Thus, the voltage dependence seems to arise exclusively from the electrostatic repulsion between K + residing inside the pore and the positively charged toxin . By performing extensive mutagenesis of positively charged residues in recombinant CTX, Park and Miller 1992a , Park and Miller 1992b found that neutralization of a single residue, Lys27, also abolished voltage dependence of the dissociation rate, a property not shared with any other residue on the toxin surface. Thus, removal of either the permeant ions or the positive charge of residue 27 produced an equivalent cancellation of z δ, indicating that the ∈-amino group of Lys27 interacts electrostatically with K + residing in the pore . Qualitatively similar, but quantitatively different to the MaxiK-channels, CTX binding to Shaker K channels is less sensitive to the internal K + concentration. Goldstein and Miller 1993 found that, although neutralization of residue 27 eliminates voltage dependence, after replacing internal K + with a nonpermeant ion as Li + , there is still some voltage dependence left for CTX unbinding. These authors interpreted the residual valence of 0.2 as the partial electrical distance that the amino group of Lys27 moves into the pore upon toxin binding; as if it goes deeper into the Shaker pore than into the MaxiK one. Our results with κ-PVIIA/ Shaker agree qualitatively and quantitatively with those of CTX/ Shaker . Replacement of the internal potassium with the nonpermeant NMG + reduces, but not eliminate, the effective valence of the κ-PVIIA unbinding. For κ-PVIIA, the value z δ is reduced from ∼0.55 in 100 mM internal K + to 0.29 in nominally zero internal K + . This trans-pore effect suggests that, as in CTX, a fraction of the voltage dependence of κ-PVIIA unbinding arises from electrostatic interaction with permeant ions in the conduction pathway. Thus, we postulate that, as with α-KTx, a positively charged residue in the surface κ-PVIIA interacts with permeant ions inside the Shaker K channel pore. At pH 7.6, κ-PVIIA net charge is ∼4. It contains two Asp, three Lys, and three Arg residues, and it is not amidated as many conotoxins are . In the absence of experimental data regarding the specific nature of the positively charged amino acid of κ-PVIIA that occludes the pore, we favor a Lys instead of an Arg residue to be the best candidate. First, based on the structural mimicry between CTX and κ-PVIIA on the space position of a Lys associated with a key aromatic residue to conform a functional dyad present at all K channel pore peptide blockers known . Second, for all functionally studied K channel–specific families of peptide toxins, independently of the animal origin, substitution of a single Lys, not an Arg, promotes the most dramatic destabilization effects on binding to K channels . Third, the agreement with CTX in the magnitude of the change in z δ promoted by the internal K + replacement by nonpermeant cations suggests a similar electrostatic interaction. In the MaxiK-channel, the internally accessible binding site that destabilizes CTX binding has an affinity for K + in the 1–3-M range . A simple interpretation for the high effective valence of the toxin destabilization process \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(z{\mathrm{{\delta}}}\;=\;1)\end{equation*}\end{document} is that a K + would have to travel across the whole transmembrane electric field to interact with CTX. In other words, the internal cation has to permeate all the way across the pore to bind a site near the external end to electrostatically repel CTX. In agreement with the idea that equates permeation with occupancy of an external site, half-maximal K + concentration for K conductance of the MaxiK-channel is near 300 mM . However, this K channel can host multiple ions in single-file occupancy , a key feature of ion permeation through K channels . With K + in the millimolar range, three or four binding sites are already occupied . Most probably, in the multiply occupied pore, and with the ionic conduction blocked from the external end by the toxin, a K + cannot permeate through the single-file of resident cations inside the ionic pathway whose occupancies are in equilibrium with the internal side of the channel. Instead, the occupancy of the externally located binding site depends on the ability of the dwelling ions to “transmit” the information that the internally accessible site is occupied by a new K + . This telegraphic effect, via occupied binding sites, would be ultimately responsible for the strong electrostatic repulsion of the bound toxin. A simple mechanism of transference would be that the K + binding to the internally accessible site promotes a rearrangement of the ions residing in the pore in such a way that results in an increased probability of occupancy of the most external binding site. This rearrangement requires of ion translocation among neighboring binding sites in analogous way to permeation. In this scheme, a z δ of 1.0 for the toxin destabilization observed in the MaxiK-channel implies a net translocation of one charge across the permeation pathway. Also, it accounts for the close agreement between half-maximal K + concentration for K conductance and toxin destabilization effect. Thus, in this view, the magnitude z δ is determined by coupling between the binding of K + to the internal site and occupancy of the most external one. Although MaxiK-channel's conductance is ∼20-fold higher than that of Shaker K channels, in addition to their high K + selectivity, they share many ionic conduction properties. Shaker K channel is also a multiple-occupancy channel . As with the MaxiK-channel, at physiological conditions Shaker can simultaneously host up to four K + in single file . Also, its half-maximal K + concentration for conductance is near 300 mM . Based on these similarities, we expected κ-PVIIA off rate to have a similar dependence on the internal K + , as CTX does in the MaxiK-channel. By reducing the internal K + from 100 to 15 mM, we anticipated a fivefold increase in the κ-PVIIA residency time. To our surprise, we found that the toxin dissociation rate was nearly identical to that of 100 K + ( Table ). In contrast to the CTX/MaxiK-channel system, in κ-PVIIA/ Shaker , the toxin-unbinding rate seems to be rather insensitive to the occupation of a internally accessed binding site. To observe any trans-pore effect on κ-PVIIA unbinding rate, we had to replace K + completely with the nonpermeant NMG + . Although the contaminating K + in our internal NMG + solutions may be enough to keep busy a high affinity binding site in the Shaker pore, thus accounting for the residual z δ, the overall voltage dependence of the toxin unbinding is smaller than in the MaxiK-channel. In such an experimental condition, instead of 1.0 as in the MaxiK-channel, only half of the total effective valence of the dissociation rate is accounted for in the replacement of the internal K + . This low voltage sensitivity attributable to the interaction with internal K + may not correspond specifically to charge delocalization in the κ-PVIIA basic residue that occludes the pore. Instead, because in Shaker the interaction with K + also accounts for a small fraction of the total voltage dependence of CTX, this low z δ (together with the low sensitivity to internal K + ) may be a reflection of the manner in which the toxin interacts with permeant ions inside the pore of Shaker . The net charge translocation associated with the occupancy of the externally located binding site is 0.3–0.4 of an electronic charge. Thus, it seems that in the MaxiK-channel the electrostatic repulsion coupling between the occupancy of the most internal and external binding sites is three- to fivefold stronger than in Shaker . This difference could represent an average greater charge separation of the ions inside the Shaker pore. Although in physiological conditions both types of channels seem to be occupied simultaneously by four cations , this greater charge separation could arise from either lower occupancy in the toxin-blocked Shaker or greater electrical separation among neighboring binding sites. This suggestion is also in agreement with Terlau et al. 1999 (in this issue), who, with different arguments, have made the same suggestion. For us, the most economical explanation to the difference in the MaxiK-channel would be that, in Shaker , the blocking ∈-amino group of the toxins occupies the most external site, at an electrical distance of 0.2–0.3, effectively reducing the number of available sites, and then K + occupancy. The experiments outlined in Fig. 9 agree with this interpretation, suggesting that κ-PVIIA occupies the most external binding site in Shaker pore. Thus, the electrostatic repulsion can occur only from a more internally located site in the pore. Consequently, the net charge translocation associated with electrostatic interaction with the toxin is less than in the MaxiK-channel because the toxins, κ-PVIIA or CTX, reduce the net K + occupancy of the pore. In this scheme, the elements for a mechanism of destabilization of a toxin bound to the external vestibule seems to be the same as that for ionic permeation: the exit rate depends on electrostatic repulsion and single-file multiple occupancy. Conus purpurascens preys on teleost fish. It has been proposed that, upon injection of the Conus venom into the prey, κ-PVIIA is a central player in the excitotoxic cabal, which causes a massive depolarization of excitable tissue that quickly immobilizes the prey . In this scheme, activation of Na channels and blockade of K channels by the venom produces a nerve depolarization in the site of the injury that is propagated orthodromically by sensory nerves and antidromically by motor fibers. In the following part of the discussion, we review the suggestion that κ-PVIIA behaves as part of an optimized prescription to promote the excitotoxic paralysis of C . purpurascens prey. We have found that the mode of action of κ-PVIIA is nearly identical to the mechanism of action of distantly related scorpion peptide toxins of the α-KTx family: first, both bind with 1:1 stoichiometry to the pore of K-channels and, second, the only action that the peptide toxin performs is to occlude the permeation pathway with a tethered substrate analogue that interrupts K + transport. The high association rate of κ-PVIIA to Shaker is consistent with a diffusion-limited, but aided by through-space electrostatic effects, protein–protein interaction . A mechanism of such nature seems to be the fastest possible. On the other hand, there are many hints that it is shared with other K channel toxins from distantly related animals as the α-KTx from scorpions and BgK from sea anemone . In the classical example of structural and functional convergence, the catalytic site of the bacterial subtilisin and the mammalian serine proteinase share a similar space topology. Although dissimilar in their overall construction, the side-chain atoms forming the functional catalytic triad, Ser/His/Asp, are in almost identical relative space positions. The breakdown of the peptide bond proceeds by the same basic mechanism . Three structural characteristics of K channel–specific peptide toxins suggest a similarly convergent mechanism of action. First, as noted by Dauplais et al. 1997 , toxin affinity for its receptor is disrupted mostly by mutations in a lysine and in an aromatic residue that are usually ∼7 Å apart . Dauplais et al. 1997 suggested that the Lys-Aromatic residue forms a functional dyad specific for K channels. Second, it has an excess of positive charges that may play an important role in the overall probability of colliding with a negatively charged vestibule . Third, the excess of charge in the surface of the toxin is unevenly distributed in such a manner that it generates a dipole moment oriented perpendicular to the interaction surface of the toxin . Thus, some peptide toxins seem to have had a convergence to a common mechanism of action on K channels. They bind with 1:1 stoichiometry to the most conserved structural locus of the channel protein: the pore. Such a precise mechanism appears to constrain the possible ways in which K channel–specific peptide toxin can be constructed. | Study | biomedical | en | 0.999997 |
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