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10444016 | Involvement of the uterine cervix by nerve sheath tumors, particularly benign nerve sheath tumors, is very uncommon. Such masses are difficult to diagnose as this is an unusual finding. We present, to the best of our knowledge, the first case of a benign nerve sheath tumor resected hysteroscopically. A 69 year-old white female presented for evaluation of post menopausal bleeding. The patient had been bleeding for one week prior to presentation. She had no prior history of post menopausal bleeding and was not on any hormone therapy. Initial evaluation consisted of an endometrial biopsy, which showed inactive endometrium and blood. Due to morbid obesity, the pelvic exam was suboptimal, and a pelvic ultrasound was ordered. The ultrasound findings showed a 4 cm cervical mass, the etiology of which was uncertain, as well as endometrial stripe thickening . Magnetic resonance imaging (MRI) of the pelvis was then obtained for further characterization. The MRI showed a large cervical mass, which was felt to be suspicious for malignancy . She then had a consultation with the Gynecologic-Oncology division who felt that this was most likely a cervical myoma and recommended no further intervention. The patient then returned with further, heavier vaginal bleeding and was taken to the operating room for a dilatation and curettage with hysteroscopy. At surgery, the uterine cavity sounded to a depth of 7 centimeters. Upon hysteroscopically inspecting the cavity, multiple small fundal polyps were noted, as well as what appeared to be a large cervical myoma . The continuous flow resectoscope was used to resect the pathology found . Blood loss and fluid deficit were minimal. The patient had an uneventful initial postoperative course. Pathology of the endometrial polyp showed a hyperplastic polyp without atypia. The cervical mass showed a sharply circumscribed tumor of low cellular density partially covered on its surfaces by stretched-out endocervical epithelium. The pattern had a neural appearance with intertwining bundles of stroma with widely spaced spindle cells with elongated nuclei, many with a wavy S-shaped pattern commonly seen in neural sheath tumors. The individual cells did not show any significant pleo-morphism and no mitotic activity. The overall appearance was that of a benign nerve sheath tumor. Nerve sheath tumors of the uterine cervix appear to be a very rare entity. It is clinically difficult to distinguish them from myomas. Tumors that are part of a more generalized condition may be neurofibromas. 1 , 2 Other masses may exhibit characteristics that identify them as pri-mary malignant Schwannomas. These are more frequently found in the extremities, the head and neck region, the trunk, and the para vertebral areas. 3 Certain light and electron microscopic findings, 4 as well as the use of special stains, can confirm the suspected diagnosis. Management of these masses will vary depending on the patient, the suspected preoperative diagnosis, and the endoscopie expertise of the surgeon. Hysteroscopic resection can be accomplished safely in the outpatient setting, eliminating the need for hysterectomy. | Clinical case | biomedical | en | 0.999997 |
10444017 | Approximately 260,000 cases of pulmonary embolism (PE) are clinically diagnosed each year in the United States; and, in the absence of prophylaxis, the incidence of fatal PE ranges from 0.1% - 0.8% in patients undergoing elective general surgical procedures. 1 Since the introduction of laparoscopic cholecystectomy (LC), laparoscopic surgery has become the preferred therapy for patients with gallstone disease. Significant complications following LC are uncommon compared to open cholecystectomy (OC). 2 Three prospective randomized trials comparing LC to OC suggest that the morbidity following LC is equal to or less than after OC. 3 , 4 Deep venous thrombosis (DVT) and PE as a complication of laparoscopic surgery have been reported in multiple case reports and individual series, but incidence of DVT and PE following laparoscopic surgery is unknown. 5 , 6 In this report, a case of pulmonary embolism after laparoscopic repair of a paraesophageal hernia is described, and a review of the literature is summarized with respect to the incidence of PE after laparoscopic surgery. Recommendations regarding DVT prevention after laparoscopic surgery are provided. A 75-year-old obese male presented with a history of multiple episodes of severe postprandial coughing and epigastric pain over the previous several months. His past medical history was only significant for a history of cardiac arrhythmia. On physical examination, his abdominal exam was benign without organomegaly. As part of his workup by his internist, he underwent a computer tomography (CT) scan of his abdomen. The CT scan demonstrated an intrathoracic stomach with organo-axial volvolus without complete obstruction. Subsequently, the patient underwent a laparoscopic repair of the hiatal hernia (type III). The operation was uneventful, with an operative time of 5 hours and 14 minutes. Elastic stockings, intermittent pneumatic compression, and heparin (5,000U BID) were used as DVT prophylaxis. His immediate postoperative course was uneventful. He started ambulating on postoperative day (POD) 3 and was discharge on POD 6. The patient returned to clinic on POD 15 for a follow-up visit with a complaint of shortness of breath, low grade fever and poor appetite. A chest X-ray revealed a left-sided pleural effusion. An arterial blood gas demonstrated a pO 2 of 57 on room air. A venous doppler study revealed left common femoral, superficial femoral and popliteal vein thrombosis. The patient was started on a heparin drip to maintain the activated partial thrombin time at one-anda-half time normal. A ventilation/perfusion scan revealed almost no perfusion to the right upper lobe consistent with a high probability of PE. The patient was treated with anticoagulation, supportive care and bed rest with gradual improvement of his O 2 saturation and bilateral lower extremity edema. He was then switched to coumadin prior to discharge. At two-year follow-up, he is doing well, off all anticoagulation. DVT and PE following major surgical procedures remain significant causes of major morbidity and mortality. The incidence of DVT as demonstrated by fibrinogen scanning in surgical patients receiving no prophylaxis is 25%, and clinically significant PE occurs in 1.6% of surgical patients. 7 The use of prophylactic regimens can significantly reduce the incidence of postoperative DVT and PE. Although laparoscopic surgery now composes a major part of general surgery, very little is known of the relative risk of developing complication of venous thrombosis after laparoscopic procedures. Furthermore, specific recommendations regarding prophylactic measures against DVT formation after laparoscopy are not widely appreciated. It is important to determine the incidence of DVT and PE following this emerging procedure. In addition, it is important to determine the risk factors in laparoscopic surgery, which can contribute to the development of DVT and the use of appropriate prophylactic measures in the prevention of this dreaded complication. Factors predisposing patients to thromboembolism can be grouped into inherited and acquired factors. Inherited risk factors include antithrombin III deficiency, protein C deficiency, protein S deficiency, and dysfibrinogenemia. Acquired risk factors include malignancy, age over 40 years, immobilization, prior thromboembolism, stasis, obesity, pregnancy, sepsis, stroke, inflammatory bowel disease and prior major surgical procedures. 7 Each individual surgical patient may have any number of these risk factors, which may cumulatively increase their risk. Apart from these well recognized predisposition conditions, several factors specific to laparoscopic surgery may increase the risk of developing DVT. Others factors specific to laparoscopy may actually decrease the risk of DVT formation. Factors which increase the risk of DVT development include CO 2 pneumoperitoneum, reverse Trendelenburg position, and prolonged operative time. The pressure effects of pneumoperitoneum impair lower extremity venous return leading to venous stasis. Ido et al. demonstrated a significant decrease in femoral blood velocity and an increase in femoral vein cross-sectional area in human patients after abdominal insufflation to 10 mm gh. 8 Millard et al. similarly showed reduction in peak systolic velocity in the femoral vein in patients undergoing pneumoperitoneum and demonstrated that these effects can be reversed with the use of intermittent sequential pneumatic compression. 9 The frequent use of reverse Trendelenburg position during laparoscopic surgery can also enhance venous stasis. Moneta et al. demonstrated a decrease in peak velocity of femoral vein and an increase in common femoral vein diameter when proceeding from a 10-degree head-down position to a 30-degree head-up position. 10 The increase in operative time associated with advanced laparoscopic procedures (ie, laparoscopic Nissen fundoplication and laparoscopic colorectal surgery) can increase the duration and, thus, effect all the above-mentioned operative factors that may promote thrombosis. Conversely, early ambulation and the potential reduction in postoperative hypercoagulation after laparoscopic surgery may decrease the risk of DVT development. LC, for example, is associated with early ambulation, while it may take several days for patients to become fully ambulatory after an open cholecystectomy. The enhanced mobility may reduce venous stasis and decrease the risk of venous thrombosis. The hypercoagulable state normally presents after major surgery has been shown to play a major role in DVT formation. Theoretically, laparoscopic surgery may blunt the hypercoagulable response due to the reduced tissue injury and stress response associated with these procedures. Initial studies show conflicting data on whether the laparoscopic methods may to some degree attenuate the hypercoagulable response. Caprini et al. demonstrated a significant postoperative hypercoagulable state in patients undergoing LC as seen by an increase in the thromboelastography (TEG) index and a significant reduction in a PTT level in the postoperative period. 11 Velpen et al. demonstrated no statistical differences in plasma concentration of interleukin-6 or various mediators of coagulation-fibrinolysis between evenly matched patients undergoing open or laparoscopic cholecystectomy. 12 Dexter et al also demonstrated similar perioperative changes in the coagulation and fibrinolytic pathway for patients undergoing laparoscopic and open cholecystectomy. 13 Our initial results, however, provided evidence of a blunted hypercoagulable state after laparoscopic surgery. 14 We measured TEG in 21 pigs selected to undergo LC (N=10) or OC (N=11). A profound hypercoagulable state was evidenced by changes in 3 of 4 TEG parameters (R, K, and MA parameters) to reflect a state of hypercoagulation compared to preoperative values following OC. Swine undergoing LC, however, developed a blunted hypercoagulable state with hypercoagulable changes in only one of the TEG parameters (MA parameter). Further investigation will be necessary to clarify the importance of laparoscopy on postoperative hypercoagulation We present in this report a case of pulmonary embolism following laparoscopic repair of 67 paraesophageal hernias at our institution. The risk factors for venous thrombosis in this patient included age over 40 years, obesity, pneumoperitoneum, reverse Trendelenburg position, and lengthy operative time. DVT prophylaxis in this patient included the use of elastic compression stockings, intermittent pneumatic compression boots intraoperatively and postoperatively, and low-dose sub-cutaneous heparin. Despite these measures, he developed DVT and subsequently pulmonary embolism on POD 15. This thromboembolic complication after an advanced laparoscopic procedure raises the issue regarding the risk of DVT/PE formation after laparoscopy. Multiple reports of DVT and PE have been described in the literature following laparoscopic cholecystectomy, laparoscopic Nissen fundoplication and laparoscopic colorectal surgery. Table 1 lists all reported series in a single institution with DVT or PE described as a complication of LC. The mean incidence of PE among these single institutional series that report PE as a complication is 0.139%. This value is only an estimate, and caution is necessary in interpreting these data since there is tremendous variability in describing the types and range of complications among different studies. Some studies provide an exhaustive list of complications, while others only provide a limited list. In addition, it is difficult to know if DVT and PE occurred in a series that did not report these complications in their report. We only considered studies that specifically stated DVT or PE as a complication of the procedures—thus excluding studies in which no DVT or PE was reported. The incidence of PE following laparoscopic cholecystectomy is comparable to other reported incidences of open cholecystectomy. Berci et al. reported a 0.167% incidence of PE following 1,200 open cholecystectomies, and Shea et al. reported an incidence of 0.31% following l,6ll open cholecystectomies. 15 , 16 Table 2 lists the incidence of PE/DVT as reported in a multiple institution series of laparoscopic cholecystectomy. This incidence of PE ranged between 0.004% -0.329%, with a mean incidence of 0.018%. Table 3 lists all studies with DVT or PE reported as a complication following laparoscopic Nissen fundoplication and laparoscopic paraesophageal hernia repair (PEH). The incidence of PE is estimated to be 1.76%. This number is significantly higher than the described incidence for patients undergoing LC. This may be due, in part, to the prolonged operative time associated with this advanced laparoscopic procedures. Table 4 lists studies that report DVT and PE as a complication following laparoscopic colorectal surgery. The number of reported laparoscopic colorectal procedures is small compared to LC and laparoscopic antireflux surgery. The incidence of PE in this group ranged from 0.00% - 3.39%, with a mean incidence of 0.68%. Additional risk factors for development of DVT in this group of patients are the presence of malignancy, advanced age and prolonged operative time. Data from randomized control trials have shown that prophylaxis in general surgical patients reduces the incidence of DVT at least by half compared to control patients. Randomized trials of low-dose heparin showed a 68% reduction in DVT and a 49% reduction in PE. 1 There was also a reduction in the mortality associated with PE. Currently, the recommended modality of DVT and PE prevention in patients undergoing general surgery and laparoscopic surgery are categorized according to the risk classification. 17 Caprini et al. developed a Risk Assessment System to determine criteria for instituting prophylaxis and the appropriate regimen for a given patient. 18 This system is based on a scoring system containing 20 clinical risk factors. For low risk general surgery patients (0-1 factor), early ambulation and elastic stocking are recommended. For moderate risk patients (2-4 factors), either low-dose heparin or a combination of elastic stocking and intermittent pneumatic compression is advocated. For the high risk group (>4 factors), a combination of heparin, elastic stocking and pneumatic compression is recommended. According to the Caprini DVT Risk Assessment System, most patients undergoing laparoscopic surgery will fall into the category of moderate or high risk and, therefore, will require DVT prophylaxis in the form of low-dose heparin or pneumatic stockings. Although these guidelines for DVT prophylaxis are not validated by prospective studies, they constitute a rational approach that is currently applicable until further investigation yields alternative recommendations. Conditions specific to laparoscopic surgery may profoundly impact normal physiologic mechanisms of coagulation and thrombosis. Based on the current literature, it is unclear whether laparoscopic surgery results in an increase or decrease in the rate of DVT formation. Retrospective studies show similar incidences of DVT/PE in both open and laparoscopic cholecystectomy but slightly higher incidences following more advanced laparoscopic procedures, like laparoscopic Nissen fundoplication and paraesophageal hernia repair. Well-controlled prospective studies are lacking. Until reliable data regarding DVT incidence after laparoscopy are available, it is prudent to assume that laparoscopy carries an added risk. Liberal application of DVT prophylaxis for laparoscopic procedures thus seems warranted. The prevention and management of thromboembolic complications in this rapidly expanding era of modern laparoscopic surgery constitutes an important issue that requires more attention and investigation. | Clinical case | biomedical | en | 0.999998 |
10444018 | Traditionally, peritoneal dialysis catheters have been placed using mini-laparotomy. With the advances in minimally invasive surgery, techniques have been described for the placement and management of these catheters using less invasive techniques. Blind catheter placement after peritoneoscopic exploration of the abdomen has been utilized. 1 This technique uses a peritoneoscope for identification of a clear site for placement, and then a modified Seldinger technique for introduction of the catheter. Laparoscopic techniques using three or four port sites also have been described, 2 , 3 which have the advantage of direct visualization while the catheter is positioned. A two-puncture laparoscopic technique has been described that has simplified the laparoscopic approach somewhat, but this still requires dissection of the catheter entrance site with suturing of the catheter to the anterior rectus sheath and creation of a subcutaneous tunnel. 4 Laparoscopy is also being used to salvage nonfunctioning catheters with good success rates and low morbidity. 5 , 6 A new laparoscopic approach for placement of peritoneal catheters, which can be performed under local anesthesia with brief operative times, is described in this report. This technique was performed using a 3 mm needleoscopic port and 2.9 mm laparoscope, or with a standard 10 mm port and 10 mm laparoscope. The patients were followed prospectively for type of anesthesia and operative time for the procedure, whether the procedure was performed as an inpatient or an outpatient, and for any complications or leakage that occurred following the procedure. A total of 12 patients underwent placement of 13 needleoscopic peritoneal catheters between November 1995 and May 1998. Six patients were male and seven were female. Seven catheters were placed for peritoneal dialysis, and six catheters were placed for palliation of malignant ascites. Six patients underwent laparoscopic placement of a peritoneal catheter utilizing a 10 mm laparoscope, and additional 5 mm ports in some cases. These patients had an average age of 72 years. Two were male and four were female. Two patients underwent catheter placement for peritoneal dialysis and four for malignant ascites. The patient is placed in the supine position and undergoes either general anesthesia or IV sedation and local anesthesia. The abdomen is sterilely prepped and draped in the usual fashion. A small skin nick is made in the superior midline , and a 3 mm needleoscopic port (LifeQuest - Atlanta, GA) is inserted into the abdomen over a Veress needle. The abdomen is insufflated with carbon dioxide to a maximum pressure of 12 mm of Mercury. The Veress needle is removed, and a 2.9 mm laparoscope is then inserted into the abdomen. For the standard laparoscopic technique, the abdomen is insufflated using a Veress needle, and a 10 mm port is inserted for a 10 mm laparoscope. The abdomen is inspected for adhesions or other pathology. A small incision is made over the lateral portion of the rectus sheath on either the left or right side of the abdomen. An 8 mm trocar is inserted into the incision and is advanced until it passes through the anterior rectus sheath. Pressure on the trocar is released so that the shield is deployed covering the trocar tip. The port is passed bluntly through a submuscular tunnel to the lower abdomen. The trocar shield is reset so that the trocar can penetrate the peritoneum and enter the peritoneal space. A double-cuffed, pigtail peritoneal dialysis catheter (Tenckhoff catheter) is placed over a stylette, inserted through the 8 mm port and into the pelvis. The port is removed, leaving the catheter in place. The interior cuff on the catheter is positioned just exterior to the peritoneum with the external cuff in the subcutaneous space . The stylette is removed, and the catheter is secured to the skin with suture. Pneumoperitoneum is released, the 3 mm trocar is removed, and its small skin nick is closed with an adhesive strip. Out of the 13 patients undergoing needleoscopic peritoneal catheter placement, three (23%) were placed under local anesthesia, and ten (77%) under general anesthesia ( Table 1 ). Five patients (38%) were treated as outpatients and eight (62%) were treated as inpatients. The operative time for the procedure averaged 24 minutes. Two patients were converted to either laparoscopic or open procedure. Excluding these two patients, the operative time averaged 12 minutes. One patient had additional ports placed and the procedure converted to standard laparoscopy while having a catheter placed for malignant ascites in order to clear adhesions for catheter placement. A second patient, who also had the procedure performed for malignant ascites, had previously undergone an omentectomy and had dense adhesions of the small intestine to the underside of the anterior abdominal wall, which resulted in an enterotomy made by the Veress needle. This patient's procedure was converted to an open procedure. The needle hole in the small intestine was sutured and the catheter placed in a different location using open technique. Additional complications included a mild catheter site infection, which resolved with oral antibiotics. One catheter had to be removed for chronic peritonitis over a year after its placement. After clearing of the infection, a catheter was replaced. Two catheters developed late occlusion due to fibrin, one of which was able to be cleared laparoscopically. There was no leakage from any of the catheters, and there was no resistance to flow of dialysate instillation. Of the six patients who underwent laparoscopic peritoneal catheter placement, two (33%) were carried out using local anesthesia and four (67%) general anesthesia ( Table 2 ). No patients were treated as an outpatient. All of the patients undergoing catheter placement for malignant ascites also required biopsy of intraperitoneal tumor at the time of their catheter placement. Operative time averaged 41 minutes. There were no complications or catheter leakage. It was previously believed that peritoneal catheter placement requires purse string suturing and/or tunnel creation to prevent leakage. This report shows that catheter placement through a tunnel created by the tangential insertion of a laparoscopic port is sufficient for a secure catheter placement without leakage. A standard 10 mm laparoscopic approach was chosen if biopsy of intraperitoneal tumor was required, so that a 10 mm operative laparoscope could be used, which allowed passage of a biopsy forceps through the same port. If adhesiolysis was required, the wider field of view of the 10 mm laparoscope was desirable. Peritoneal inflation with carbon dioxide was well tolerated in awake patients with local anesthesia, largely due to the fact that the procedure can be performed with short operative times and minimal dissection of the abdominal wall. This was useful in patients who presented a significant medical risk for general anesthesia. Advantages of the technique include that the catheters are placed under direct visualization so optimal placement of the catheter in the pelvis is assured. The catheter traverses a straight path through the abdominal wall so there is minimal chance of kinking, which may contribute to catheter occlusion. The 8 mm trocar creates a tract primarily by dilation, which allows for a snug fit for the catheter. This, along with the incorporation of the catheter cuffs into the surrounding tissue, produces a very secure catheter placement with no leaking. The needleoscopic technique described here is very simple to perform. It requires no special peel-away sheaths or other specialized instruments. There is no dissection of the abdominal wall required. It can be performed under local anesthesia with minimal discomfort. The patient recovery has been very good with no operative mortality, minimal morbidity, and no catheter problems related to the technique. | Review | biomedical | en | 0.999997 |
10444019 | Laparoscopic cholecystectomy is the standard method of performing cholecystectomy in both the elective and the acute setting. Removal of the gallbladder through the abdominal wall either at the epigastric or subumbilical port site is often aided by enlargement of the fascia, which is required either because of the large size of the gallbladder itself or because of large stones. The enlargement is performed by manually spreading and tearing the fascia with an instrument such as a Kelly forceps or by cutting the fascia with a scalpel. Only rudimentary descriptions exist for the technique of fascial enlargement. 1 , 2 Even when the gallbladder has been evacuated of all liquid bile, the stones, if multiple and/or single but large, can result in difficulty delivering the gallbladder. The stones can be crushed within the gallbladder but this risks perforation of the gallbladder with bile spillage and often does not eliminate the need for fascial enlargement. 3 The extraction of the gallbladder component of laparoscopic cholecystectomy has suffered from lack of similar technological advances that have occurred in the laparoscopic technique. The incidence of gallbladder perforation during removal of the gallbladder through the abdominal wall as opposed to intra-abdominal laceration secondary to dissection has not been reported in any large series. However, experience dictates that methods to enlarge the fascia and excessive traction of the gallbladder do result in laceration with bile and stone spillage into the peritoneal cavity and the trocar wound resulting in the potential for port-site wound infection. The infection rate is reported to be 0.5–1.0% in large series; however, the cause of the wound infection is not identified. 4 , 5 Closure of the fascia is the final step in a successful operation. A late port-site hernia may develop in 10 mm trocar sites in which the fascia has not been closed with sutures. Some large series do not even report this complication, 6 while others report complication rates for trocar-site herniae of 0.12–0.5%. 4 , 5 This is often difficult to adequately visualize, particularly in the obese patient, and the risk of including intra-abdominal structures such as small bowel exists during the fascial closure. This report describes a simple, safe, cost-effective, reusable instrument and technique for fascial enlargement and closure. The grooved spoon-shaped director SM1N\0497 MAKAR Director (Southmedic Co) was developed to aid in fascial enlargement without injury to the gallbladder or intra-abdominal viscera . The director is placed between the gallbladder wall and the fascia to be incised. The spoon shape allows for this instrument to be placed under the fascia with ease. The groove allows the scalpel to follow a path along the spoon shape in a safe, secure manner precisely incising the fascia . Other aids in removing the gallbladder can be used in addition to fascial enlargement with the director, including evacuation of bile and crushing of stones within the gallbladder. The blunt forceful tearing of the fascia with an instrument such as a Kelly forceps is avoided, thereby avoiding inadvertent injury of the gallbladder wall as well as uncontrolled, excessive tearing of the fascia. This instrument can be used equally as well in either the epigastric or umbilical trocar sites. In more than 30 laparoscopic cholecystectomies, the grooved director has been used without injury to the gallbladder wall. This was found to be particularly useful in obese patients in whom the fascia was deep to a thick layer of abdominal wall fat and not easily visualized. In these individuals, the director was easily placed beneath the fascia and confirmed by tactile sensation of “lifting” against the overlying fascia. The fascia was then easily incised with a scalpel, avoiding risk to the underlying viscera. Fascial closure is aided with the grooved director by placing the director beneath the fascia. The fascia is again easily identified by tactile sensation or by direct vision with the aid of skin and subcutaneous tissue retractors. A #1 suture needle will follow the groove of the director through the fascia while all intra-abdominal viscera are protected by the spoon . In the patients, both the epigastric and umbilical ports were closed utilizing this technique without injury to the underlying viscera and with accurate apposition of the fascial edges. In obese patients, the needle follows the groove beneath the fascia without direct visualization of the fascia and is placed safely knowing that the underlying viscera are protected. The grooved director is a safe, effective instrument, which aids both the fascial enlargement and closure. It is an instrument that allows for safe extraction of the gallbladder through the abdominal wall without laceration of the gallbladder wall and consequent bile spillage. It also provides a means to perform a safe, secure fascial closure. This instrument and technique improves the “low tech” components of laparoscopic cholecystectomy, ie, gallbladder extraction and fascial closure. The reusable grooved director can also be used in other advanced laparoscopic procedures for fascial enlargement for removal of other organs and closure of any 10 mm port site. | Review | biomedical | en | 0.999997 |
10444020 | As a general surgeon in the 1960s, Phillipe Mouret rotated on a gynecology service during which he had his first contact with laparoscopy. 1 “At that time we used the instruments constructed by Palmer,” notes Mouret. “Since then I have brought my interest in that particular endoscopie technique into surgery.” 2 Mouret shared his surgical practice with a gynecologist and thus had access to both laparoscopic equipment and, importantly, to patients requiring laparoscopy. “I could not understand why surgeons were not using laparoscopy, but still laparotomy. The patients clearly preferred laparoscopy,” 3 stated Mouret in 1994. Mouret was using diagnostic laparoscopy in the 1970s, and, by the early 1980s, was able to sharpen his skills as instruments became more sophisticated. “Originally it [laparoscopy] had a strictly diagnostic purpose,” remarks Mouret, “then it was used to obtain a better topographic overview before operating and finally, a small therapeutic-operative endoscope was developed, to complete endoscopie use.” 4 According to Mouret, in 1983, unaware of Semm's efforts, he carried out his first laparoscopic appendectomy (LA). “The first step was to expose the appendix laparoscopically and then to pull it out of the abdomen,” notes Mouret. “After that, I cut it off extracorporeally.” 5 Mouret never attempted to publish his initial work on LA. In the late 1980s, laparoscopy was still mainly a gynecologist's tool, so the gynecologists were the first to be offered electronic laparoscopes. With his close connections to the gynecological field, Mouret was exposed to these new developments. In 1987, he began to use an electronic laparoscope. As Mouret recalls, in March 1987 he operated on a woman suffering from both a gynecological disorder and gallstones. 6 Pointing the laparoscope upward, he removed the gallbladder. A skilled laparoscopist, Mouret managed the technical side of the operation. “That time the technique was not fully developed,” he explains. “In particular, the clips were inadequate, which led to desufflation …” 7 He excised the gallbladder extracorporeally. He never published this experience. “I did not see any chance for publishing in a surgical journal,” he stated in 1994 . 8 He opted to present only a video tape with laparoscopic cholecystectomy at a gynecological meeting in Paris. 9 Cholecystectomy by “mini-laparotomy” under local anesthesia represented a trend toward reducing the surgical incision. This technique was developed in the early 1970s and became popular in France. 10 The small incision and absence of drainage catheters led to a very short postoperative hospitalizaron; in many cases, a so-called “one-day cholecystectomy” was possible. 11 By the early 1990s, surgeons at the Hospital International of the University of Paris had performed more than 1,500 such operations. 12 One of those surgeons, Francois Dubois, advocated the “mini-cholecystectomy,” convinced it offered the best possible surgical access to gallbladder disorders. He was particularly proud of its patient-centered aspect. As Dubois remembers it, in the fall of 1987 he was closing an operative incision when he noticed a new nurse on the operating team. 13 He called her attention to “the smallest scar in the world after gallbladder removal.” 14 But Claire Jeaupitre remained unimpressed. “There is another way of removing the gallbladder by an even smaller opening,” she responded. 15 Dubois was astounded. She continued, “I mean the laparoscopic cholecystectomy.” “Removing a gallstone by means of a laparoscope is not possible,” returned Dubois. Jeaupitre explained that a surgeon in Lyon named Mouret was using a laparoscope to remove gallbladders. Dubois could only repeat (a bit angrily), “Laparoscopic cholecystectomy is not possible!” But the nurse held her ground and suggested that he call Mouret, with whom she had worked before moving to Paris. 16 Mouret had never met Dubois before and was surprised by his call. The Paris surgeon stated he would be interested in obtaining more information about Mouret's experiments with laparoscopic surgery. Mouret pointed out that laparoscopic removal of the gallbladder was not in the experimental, but rather in the clinical, stage. He offered to show Dubois a video tape of the technique. Since he happened to be coming to Paris for a gynecological meeting, the two surgeons agreed to meet there in a couple of weeks. 17 It was December 1987 18 19 when Dubois and Mouret met in the Paris Hilton, a stone's throw from the Eiffel Tower. Mouret recalls that Dubois said little, his attention concentrated on the video with an occasional “hm, hm” tossed in. “Dubois then said ‘thank you very much, very interesting, good bye.’ That was all,” noted Mouret seven years later. 20 Dubois had watched a tape of two cases. Perhaps he was reserved in his reaction that evening, but Mouret's demonstration impressed him. The tape of a step-by-step laparoscopic cholecystectomy (LC) convinced Dubois that endoscopie removal of the gallbladder was possible. Dubois was an open-minded surgeon. Along with his work on “mini-laparotomy” cholecystectomy, he decided to devote some of his energy to Mouret's method. Dubois acted immediately, collecting literature on laparoscopic surgery. “I had no prior laparoscopic experience,” he recalled in 1995 . “Especially the books of Semm drew my attention.” 21 He borrowed laparoscopic instruments from gynecologists. Interestingly, Dubois used a monocular endoscope of 10 mm with a cold-light optic; the video camera had to be attached to the eye piece outside the patient's body. 22 Dubois soon accumulated his first experiences with basic laparoscopic technique on animals and moved on to clinical tests. He describes these first cases: At the very beginning I was not skilled enough to finish this procedure in the endoscopie way. The first operations I always began laparoscopically, but I finished the procedure using laparotomy. Finally it worked! My first fully laparoscopic cholecystectomy was done at the end of April 1988. 23 During this time, Jacques Perissat of Bordeaux, France, was working on extracorporeal shock wave lithotripsy (ESWL) for gallbladder disease. Disappointed with ESWL results, Perissat decided to modify the method by putting a device inside the patient's body. An endoscopie tool was necessary to produce intracorporeal shock waves in the gallbladder utilizing a percutaneous approach. 24 Perissat came up with the idea of using laparoscopy, a technique already familiar to him. His first experience with laparoscopy dated back to 1965, when he mastered the laparoscopic technique with Palmer's instruments. Perissat did laparoscopy to look for an ectopic pregnancy or to make explorations for liver disease. “In the 1970s, ultrasonography and CT came and we forgot laparoscopy,” notes Perissat. 25 In the late 1980s, Perissat experimented with intracorporeal lithotripsy. With laparoscopic assistance and under general anesthesia, Perissat introduced an ultrasonic lithotriptor through a 5 mm trocar into the gallbladder. The gallbladder was constantly irrigated, and the jacket around the laparoscope (or “cholecystocope,” as Perissat called it) prevented leakage into the peritoneal cavity. The vibrating bar of the lithotriptor was connected into a suction pump to evacuate the fragments of the destroyed stones. 26 One of Dubois' lectures gave Perissat the idea of carrying out cholecystectomy in place of cholecystotomy. As Perissat remembers the event, it was in Paris, probably July 1988. He was participating in an academic meeting and heard Dubois speak on laparoscopic appendectomy (not laparoscopic cholecystectomy). “I thought, this fellow has a good idea, remove the organ laparoscopically rather than leave it after lithotripsy,” recalls Perissat . 27 In October 1988, the new procedure was introduced in the Center Hospitalier of the University of Bordeaux. After clearing the gallbladder of stones, Perissat dissected the empty gallbladder under laparoscopic vision and removed it through a lithotripser channel. “It was a more complicated procedure,” recalled Perissat in 1995, “because one had to enter the gallbladder to reveal the stones. And it lasted very long.” 28 Occasionally, some of the stones were so small that it was unnecessary to use lithotripsy. In such cases Perissat performed only laparoscopic cholecystectomy. In early 1989, however, he treated most gallbladder cases with a combination of intracorporeal lithotripsy and cholecystectomy. 29 According to Troidl, Perissat called in the spring of 1989 to invite him to Bordeaux. Perissat sketched his concept, and Troidl immediately decided to go to France. Troidl recounts his experience: I personally do not like X-ray techniques and Jacques was performing lithotripsy under radiological vision. When I was in Bordeaux, Jacques told me about Dubois' work. Because I was so interested, Jacques called Dubois and the next day I observed LC in Paris. Even at that time, Dubois was carrying out LC in an elegant way. I liked it very much. 30 With his ties to gynecologists, Mouret had given a new impulse to laparoscopic applications in surgery. His knowledge of technological developments in that field led him to introduce electronic videolaparoscopy into general surgery. Dubois, a surgeon with broad expertise in biliary surgery, was the second link in the French connection. Although he became aware of Mouret's work more or less by accident, he was quick to respond to that message. Despite his lack of experience with laparoscopy, Dubois appreciated the new technique and devoted his laboratory time to learning abdominal endoscopy. For his part, Perissat tried to solve the problem of cholelithiasis by using ESWL; he initially saw the laparoscope as a way to control the entry into the gallbladder. Personal contact and exchange provided the catalyst to the spread of laparoscopic cholecystectomy in France. The first connection (Mouret-Dubois) was oral communication, buttressed by information recorded on video tape. The second connection (Dubois-Perissat) came about in a more traditional way — Perissat modified his work, spurred by Dubois' lecture. News of the French experience in laparoscopic cholecystectomy soon swept beyond the country's borders. It reached Cuschieri in Scotland, Katkhouda in southern France, Klaiber in Switzerland, Phillips in Los Angeles, Troidl in Germany, and others. Many surgeons came to Paris or Bordeaux (or both) to assist LC. In the early phase of the laparoscopic breakthrough in Europe, Paris and Bordeaux hosted frequent visitors. Perissat also presented his video tape at the April 1989 SAGES meeting in Louisville . 31 Three weeks later, Dubois published the results of his 36 cases in La Presse Medicale. 32 Since that particular publication was in French, it was not accessible to many American surgeons. Dubois' second paper, “Coelioscopic Cholecystectomy,” published in Annals of Surgery, had a wider audience. 33 It appeared in January 1990, three months after the American College of Surgeons meeting and two months before the Second World Congress on Surgical Endoscopy in Atlanta. Dubois and Perissat spoke enthusiastically about their work at the meetings and were largely responsible for establishing what is today called the French technique. Regarding the laparoscopic breakthrough in Europe, two things more must be stressed. First, it is important to note that the climate for laparoscopy had changed radically and rapidly from the time of Muehe's first reports on laparoscopic cholecystectomy. French and North American medicine in the late 1980s was ripe for the introduction of laparoscopic techniques into general surgery. Second, it is important to note that many of the early workers in Europe were not aware of the activities of the others. The French surgeons were working independently from Mühe (Böblingen, Germany), McKernan and Saye (Marietta, GA), Olsen and Reddick (Nashville, TN), Ko and Airan (Chicago, IL), and some others. For the first time, around the Second World Congress on Surgical Endoscopy in Atlanta , most of the pioneers came into touch with each other. By that time, however, the laparoscopic “revolution” was already in full swing. 34 | Other | biomedical | en | 0.999996 |
10444063 | Three human cell lines (WI-38, NHF-1, and HeLa S3) were used for these studies. WI-38 and HeLa S3 cells (American Type Culture Collection) were grown as monolayers in MEM (GIBCO BRL) plus 10% FBS (Summits Biotechnology) supplemented with sodium pyruvate acid (GIBCO BRL) and MEM nonessential amino acids (GIBCO BRL). NHF-1 (normal human fibroblast) cells were grown as monolayers in MEM (GIBCO BRL) plus 10% FBS (Summits Biotechnology) supplemented with sodium pyruvate acid (GIBCO BRL) and MEM nonessential amino acids (GIBCO BRL), 1% l -glutamine (GIBCO BRL), and 50 μg/ml gentamicine (Sigma Chemical Co.). For in situ nuclear matrix preparation, cells plated on coverslips were permeabilized with CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl 2 , 10 μM leupeptin, 1 mM EGTA, 1.2 mM PMSF, 0.5% Triton X-100, with or without 4 mM vanadyl complex) followed by extraction with 0.25 M (NH 4 ) 2 SO 4 , 0.65 M (NH 4 ) 2 SO 4 , or 2.0 M NaCl, with or without RNase A (200 μg/ml) digestion for 1 h on ice . Human chromosome painting probes, which were directly labeled with Spectrum green or orange, were obtained from GIBCO BRL and Vysis. Experiments were performed after standard fluorescence in situ hybridization (FISH) procedures, modified with instructions from GIBCO BRL and Vysis. In brief, the cells were fixed with 4% formaldehyde and denatured with 50% formamide/2× SSC by heating in an 80°C waterbath for 30 min. 1 μl chromosome painting probe with 7 μl hybridization buffer and 2 μl double distilled H 2 O was denatured by heating in an 75°C waterbath for 10 min, and then placed immediately on ice. The final concentration of hybridization buffer was 50% formamide, 2× SSC, and 10% dextran sulfate. The probe in hybridization buffer was added to the coverslips, placed cell-side down on a glass microscope slide, and sealed with rubber cement. After incubation in a 37°C incubator overnight, the cell samples were washed three times (10 min each) in 50% formamide in 2× SSC at 45°C, once for 10 min in 2× SSC at 45°C, once for 5 min in 2× SSC with 1% NP-40, and then air dried. DNA replication sites were labeled after a 5 min in vivo incorporation of 5-bromo-2-deoxy-uridine (BrdU; 10 μM) in NHF-1 cells grown on coverslips, according to the instructions of the BrdU labeling and detection kit I (Boehringer Mannheim Corp.) as previously described with mouse anti-BrdU mAbs and Texas red-conjugated anti-mouse IgG secondary antibodies (Boehringer Mannheim Corp.). Double labeling of nuclear matrix associated proteins was performed in NHF-1 cells after appropriate extractions and fixation with 4% formaldehyde. Antibody reactions were carried out successively with mAb antimatrin 250, also known as nuclear matrix associated RNA polymerase II 0 , and rabbit antilamin A/C polyclonal antibodies (diluted 20-fold with PBS) for 1 h at 37°C. FITC and Texas red secondary antibodies (both diluted 50-fold with PBS), respectively, were used for detection. In experiments involving propidium iodide staining of the DNA, the nuclear lamins were detected with FITC-conjugated antibodies. Samples from HeLa S3 cells were prepared for thin section EM . Cells were fixed in 2.5% glutaraldehyde, 100 mM sodium cacodylate, pH 7.4, and 3 mM MgCl 2 for at least 2 h at 4°C. The cells were rinsed with cacodylate buffer without glutaraldehyde, post-fixed with 1% OsO 4 for 30 min on ice, dehydrated (graded 30–100% ethanol and 100% acetone), and infiltrated with Epon-Araldite resin (Electron Microscopy Sciences). Cured blocks were thin sectioned and stained with lead citrate and uranyl acetate. Sections were examined on a Hitachi H-500 electron microscope. The confocal imaging was performed on a Bio-Rad MRC-1024 three channel laser scanning confocal imaging system equipped with a Nikon Optiphot 2 microscope, a Nikon 60×, 1.4 NA objective, and an argon/krypton laser (λ = 488/565 nm). Optical sections of 512 pixels × 512 pixels × 8 bits/pixel were collected through the samples at 0.5 μm intervals. For biochemical studies, HeLa S3 cells were used. Nuclei were isolated from disrupted cells using the syringe technique in Belgrader et al. 1991 . Our strategy of identifying proteins released in correlation with chromosome disruption is shown in Fig. 4 . It involves consecutive extraction of isolated HeLa cell nuclei with CSK buffer, RNase A digestion, 0.65-M (NH 4 ) 2 SO 4 and 2.0-M NaCl extractions. For total nuclear matrix proteins, HeLa S3 cells were permeabilized by CSK buffer, followed by DNase I digestion and 0.25-M (NH 4 ) 2 SO 4 extraction. Protein was determined using the BCA kit (Pierce Chemical Co.). DNA was measured after incorporation of [ 3 H]thymidine (1 μCi/ml) into HeLa S3 cells for 48 h of culture and RNA by alkaline hydrolysis methods . The data were averaged from four individual experiments. For two-dimensional PAGE, proteins were run on a nonequilibrium pH gradient (first dimension) and on SDS-PAGE in the second dimension . The proteins were stained with Coomassie blue (0.2%). Total nuclear matrix protein was prepared after treatment of isolated nuclei with DNase I and 0.25-M (NH 4 ) 2 SO 4 extraction . Previous studies have shown that human interphase chromosomes are confined to discrete regions in the cell nucleus termed chromosome territories . To study the possible role of the nuclear matrix in chromosome territory organization, we extracted normal diploid human fibroblasts cells (NHF-1 or WI-38) grown on coverslips for DNA-rich nuclear matrix . In DNA-rich nuclear matrix preparations, the nuclear DNA is not cleaved by either endogenous or exogenously added nucleases. The protein components in DNA-rich nuclear matrix, however, are virtually identical to DNA-depleted matrix in recovery of total nuclear protein and overall polypeptide composition on SDS-PAGE . Moreover, the DNA in such structures is maintained predominantly inside the nuclear structures in a highly supercoiled state . We find that the chromosomes are maintained in the DNA-rich nuclear matrices as separate territories that are indistinguishable from those in intact cells . Similar observations were made with all human chromosomes examined (numbers 1, 2, 4, 7, 9, 11, 14, and 22) and different two paint combinations. There was no chromosome hybridization signal in DNA-depleted nuclear matrix (results not shown). These experiments were performed using moderate ionic strength for salt extraction (0.25 M (NH 4 ) 2 SO 4 ; Belgrader et al., 1991). We next used a higher salt concentration (2.0 M NaCl and the same ionic strength 0.65 M (NH 4 ) 2 SO 4 ) to extract the cells. The chromosome territories remain intact after these higher salt extractions , and were indistinguishable from those observed in nuclear matrix prepared with moderate salt levels or intact cells . Greater than 90% of the total histone proteins and other soluble nuclear proteins are removed under these salt extraction conditions . Chromosome territories are, therefore, maintained in association with the nuclear matrix without the bulk of the histone proteins. Since the structural integrity of the nuclear matrix is dependent on RNA and intermolecular disulfide bonds , salt extractions were also performed after 200 μg/ml RNase A digestion, in the presence of 20 mM DTT or a combination of these two treatments. Surprisingly, chromosome territories are highly disrupted after RNase A and 2.0-M NaCl treatment , but extraction with similar ionic strength 0.65 M (NH 4 ) 2 SO 4 after RNase treatment had no visible effect . Identical results were obtained in eight separate experiments using eight different human chromosome paints (numbers 1, 2, 4, 7, 9, 11, 14, and 22) examined in three human cell lines (WI-38, NHF-1, and HeLa S3). 82% of the cells (>500 cells counted in each experiment) had disrupted chromosome territories when averaged among all the experiments (results not shown). In contrast, >90% of the cells had intact territories after all other nuclear matrix preparations , including the cells extracted with 20 mM DTT (results not shown). This compares with intact cells, in which nearly 100% of the chromosome territories were intact even under conditions where a small percentage of the nuclei (∼20%) showed visible breakage or leakage of chromatin. We conclude that intermolecular disulfide bonds, which form between nuclear matrix proteins , do not play a significant role in maintaining chromosome territory organization. RNA and/or RNP interactions, however, are crucial. While disrupted chromosome territories display diffuse staining inside the nucleus , it could be argued that the individual chromosomes maintain a degree of their territorial organization despite unraveling into more diffuse structures. Double labeling experiments , however, clearly show a high degree of mixing (yellow coloration) of the two chromosome pairs and confirm the loss of territorial arrangement. The difference in chromosome territory organization between 2.0-M NaCl extraction and its same ionic strength 0.65 M (NH 4 ) 2 SO 4 after RNase A digestion led us to study the nuclear matrix organization after these salt extractions. In the same preparations, matrin 250 and lamin A/C were used as markers for the internal nuclear matrix and the peripheral nuclear lamina, respectively, by double immunofluorescence labeling (see Materials and methods). Cells extracted with 0.65 M (NH 4 ) 2 SO 4 had a matrin 250 staining intensity and pattern identical to intact cells . Extraction with 2.0 M NaCl, however, resulted in significant aggregation of the structures . Matrix preparations obtained with RNase A after 0.65 M (NH 4 ) 2 SO 4 are depleted in matrin 250, but it is still detectable . There is no detectable matrin 250 staining inside of the nuclei after RNase A digestion and 2.0-M NaCl extraction . Virtually identical results were obtained when the internal nuclear matrix was decorated with matrin CYP instead of matrin 250 (results not shown). In contrast, there is no detectable effect on the nuclear lamina structure by any of the extraction conditions . The overall nuclear morphology of the extracted cell samples were then studied by thin sectioning EM. The results are in close agreement with the double immunofluorescence analysis of nuclear matrix components. An elaborate structure is seen in the nuclear matrix after 0.65-M (NH 4 ) 2 SO 4 extraction . Nuclear matrix structure is significantly aggregated by 2.0-M NaCl extraction . After RNase A and 0.65-M (NH 4 ) 2 SO 4 treatments, the DNA-rich nuclear matrices are depleted of internal structure, although it is still detectable . The interior of the nuclear matrix, however, is virtually devoid of structure when extracted by 2.0 M NaCl after RNase A digestion . Extraction of internal nuclear matrix components with RNase and 0.65 M (NH 4 ) 2 SO 4 had no visible effect on the relative intactness of the resulting nuclear matrix structures, as evaluated by fluorescence microscopy or EM . Similarly, the majority of nuclear matrix structures observed after chromosome disruption (RNase and 2-M NaCl extraction) were indistinguishable in terms of overall nuclear shape from those following other extractions . Significantly, chromosome territories were routinely disrupted irrespective of the degree of intactness of overall nuclear shape . When DNA-rich nuclear matrices are prepared under conditions where cleavage or nicking of DNA is avoided, the DNA remains inside the nuclear matrix structure and is highly supercoiled . Progressive relaxation of this supercoiled DNA with intercalating agents, such as ethidium bromide, leads to the corresponding formation of a halo of relaxed DNA loops surrounding the overall nuclear matrix structure . Some preparations of DNA-rich nuclear matrix, however, are designed for studying DNA halos and include the ethanol dehydration and air drying steps that are used in electron microscopic spreading techniques for visualizing DNA loops in halos . These differences in DNA halo formation led us to examine this property more closely in our preparations. Using the nuclear lamina as a marker for the periphery of the overall nuclear structure , we determined whether or not significant levels of DNA were detected in regions that extended beyond the nuclear lamina border (i.e., a DNA halo). Under conditions where chromosome territories remained intact, virtually all of the DNA was detected inside the nuclear matrix structure in a manner indistinguishable from unextracted cells. This is demonstrated in Fig. 3D and Fig. E , for untreated cells and RNase A–0.65-M (NH 4 ) 2 SO 4 extracted cells, respectively. In contrast, in DNA-rich nuclear matrix, where the chromosome territories are routinely disrupted (RNase A–2-M NaCl treated), faint DNA halos were observed surrounding the nuclear lamina . Although a vast majority of the DNA was still contained inside the nuclear matrix structure , small amounts of both total DNA and chromosome specific territories extend beyond the border of nuclear matrices with disrupted chromosome territories , but not in whole cells or other nuclear matrix preparations where the chromosome territories remain intact . Since the formation of a faint DNA halo in these DNA-rich nuclear matrices with disrupted chromosome territories might be the result of the unpacking of multiple regions of DNA loops from within the nuclear matrix structure, we decided to examine DNA replication sites under these conditions. Individual sites of replication in mammalian cells are discrete and contain an average of ∼1 mbp DNA per site during early S-phase . Thus, each early S replication site likely contains at least several typically sized DNA loops (replicons) of 50–200 kbp packaged together . The question we could then address is whether chromosome territory disruption leads to a corresponding disruption at the level of clusters of DNA loops that compose each replication site. We found that replication sites characteristic of early S phase in unextracted cells are maintained in DNA-rich nuclear matrix, as long as the chromosome territories are intact . After territory disruption, however, the replication sites were still identifiable but highly diffuse in structure . This indicates a change in the organization (relaxation) of DNA loops or their higher order packing in correlation with territorial disruption. The differential effect of 2.0-M NaCl vs. 0.65-M (NH 4 ) 2 SO 4 salt extraction after RNase A treatment suggests a strategy for identifying proteins that may play a role in chromosome territory organization. In brief, the bulk of nuclear matrix proteins are released under conditions where the chromosome territories are maintained intact (0.65 M (NH 4 ) 2 SO 4 after RNase A, nuclear matrix 1 of Fig. 4 ). Then, a second salt extraction treatment (2.0 M NaCl) results in disruption of the chromosome territories . Proteins released into the second extraction are candidates for chromosome territory anchor proteins (CTAPs) and, therefore, we term this fraction the CTAPs extract. Under these described fractionation conditions, 4.7% of the total nuclear protein (∼0.56% of total cellular protein) was released in conjunction with the chromosome territory disruption. 97.3% of the total nuclear DNA, and nearly one third of the nuclear RNA, remain associated with the final extracted nuclear matrix pellet . The CTAPs extract was composed predominantly of protein (>95%) with only trace amounts of RNA (1.5% of total nuclear RNA) and DNA (0.4% of total nuclear DNA). Two-dimensional SDS-PAGE was then performed in an attempt to identify the specific proteins released in conjunction with chromosome territory disruption. The CTAPs 2-M NaCl extract was highly depleted in the major proteins that constitute a typical nuclear matrix fraction . As anticipated, the major nuclear matrix proteins were released after the 0.65-M (NH 4 ) 2 SO 4 extraction, along with numerous other proteins . Over 95% of the lamin proteins remain in the final pellet after extraction for CTAPs . Small amounts of lamins are occasionally found in the CTAPs extract and likely represent contamination from the final pellet fraction. A relatively simple constellation of polypeptides was found in the CTAPs extract that migrated predominantly between 40 and 90 kD in the acidic region of the gel . As indicated by the corresponding boxed areas in Fig. 5 , these proteins were found in trace amounts or were not detectable in the two-dimensional gel of total nuclear matrix proteins , the 0.65-M (NH 4 ) 2 SO 4 extract , or the final pellet . The final pellet, however, contained numerous proteins in the boxed region corresponding to lamin B (arrow) and a cluster of presumptive cytokeratin proteins that do not correspond to those in the CTAPs extract. Taken together, our results indicate that the proteins released in conjunction with chromosome territory disruption are a minor acidic subset of the total nuclear matrix proteins. In this paper we have examined the possible role of the nuclear matrix in the territorial organization of human chromosomes in the interphase cell nucleus. DNA-rich nuclear matrix are prepared by in situ extraction of human cells grown on coverslips. Previous studies have demonstrated that nuclear DNA anchored to these structures is highly supercoiled and present predominantly inside the nuclear structures . Since DNA halos can be observed after relaxation of the supercoiled DNA loops or after spreading techniques on electron microscopic grids , it is likely that recent studies demonstrating DNA halos in cells extracted for DNA-rich nuclear matrix are due to unwinding of the supercoiled DNA during the preparative steps and/or spreading induced by ethanol dehydration and air drying of the specimens . We reasoned that specific nuclear matrix proteins likely interact with chromatin at matrix attachment sites. Moreover, there may be higher levels of organization of these interactions ranging from the chromatin loop clusters to the level of the chromosome territory. We report a striking maintenance of chromosome territory organization despite the extraction of >90% of the histones and other soluble nuclear proteins in these DNA-rich nuclear matrix preparations . Our results further suggest that nuclear matrix components are involved not only in the anchoring of chromatin in repeating domains, but also in constraining the overall architecture of the chromosomes. We next examined the role of the nuclear lamina versus the internal nuclear matrix components in chromosome territory organization. Previous studies have demonstrated that chromatin is attached to both nuclear lamina and internal nuclear matrix components . Extraction of the bulk of the internal nuclear matrix with RNase treatment followed by 0.65 M (NH 4 ) 2 SO 4 had no effect on the chromosome territory organization. While this might argue for a critical role of the nuclear lamina in territory organization, it was also possible that a minimal internal matrix structure that resisted extraction was also involved. Indeed, examination by immunofluorescence microscopy and EM confirmed a minimal internal matrix structure in these preparations. In contrast, extraction with RNase treatment followed by 2 M NaCl resulted in a dramatic disruption of the chromosome territories and a corresponding complete extraction of the internal matrix as evaluated by the same microscopic criteria. Despite disruption of chromosome territories in conjunction with the removal of the internal matrix, the DNA remained predominantly inside the residual nuclear structures. A small amount of DNA, however, extended past the nuclear lamina boundary to form a faint DNA halo . This phenomenon was also observed with individual chromosome territories: the vast majority of the disrupted territories remained inside the nuclear matrix structure while a small but discrete amount extended past the nuclear border in DNA loop-like fashion . Thus, an important role in the overall anchoring of the chromosomal DNA is attributable to the nuclear lamina, but specific territorial arrangements requires the additional participation of a component(s) of the internal nuclear matrix. To study the possible relationship of chromosome territories to higher order chromatin loop organization further, we examined DNA replication sites under conditions of intact and disrupted chromosome territories. Recent studies suggest that individual replication sites contain ∼1 mbp of DNA arranged in a cluster of repeating DNA loops or replicons . Correlative with the disruption of chromosome territories, we observed a corresponding disruption of individual replication sites from discrete to markedly diffuse structures . These findings support the view that destabilization of DNA loop anchoring sites or their higher order arrangement after extraction of nuclear matrix components leads to a corresponding disruption of the chromosome territories. Of course, we cannot rule out the possibility that these structural relationships are purely coincidental and that other features of higher order chromatin organization other than nuclear matrix association of DNA loop domains are mediating these interactions. The overall intactness of the nuclear matrix shape, however, did not correlate with territorial disruption. It is also possible that while RNase–2-M NaCl extraction has a destabilizing effect on chromosome territories, it is actually the harsh FISH procedure that leads to disruption, rather than the extractions themselves. The effect, however, is specific for RNase–2-M NaCl treatment and, in the absence of FISH, results in the virtual complete emptying of the nuclear matrix and a corresponding formation of a faint DNA halo and dispersion of DNA replication sites. This argues for a direct dispersive effect of the extractions on higher order chromatin architecture. Our findings also provide a potentially powerful approach for elucidating the proteins and other factors that are involved in higher order chromosome territory organization. We have identified a small constellation of proteins whose release from the nuclear matrix correlates with the disruption of human chromosome territories. Work is in progress to further identify these proteins and their possible relationship to S/MARs binding proteins , proteins of the human SWI/SNF complex that associate with both chromatin and nuclear matrix , the mitotic scaffold associated proteins Sc II and XCAP , which are believed to play a major role in maintaining the condensed state of chromosomes. The nuclear matrix associations that we demonstrate in this study further suggest that the chromosome territories may be highly constrained in the cell nucleus. Initial studies of territories by an in vivo labeling approach in living mammalian cells supports this conclusion, but also demonstrates a limited degree of mobility and putative shape changes . Recently, Abney et al. 1997 directly measured chromatin mobility in the nuclei of living cells using the FRAP (fluorescence recovery after photobleaching) technique. They found that the overall chromatin in the nucleus is highly immobile at the level of 0.6–0.8-μm diam spots. This is a size considerably larger than the ∼0.5-μm diam replication sites that have been estimated to contain ∼1 mb DNA . Since recent results suggest that large molecules, up to 500 kD, can freely diffuse in the nucleus of living cells , Abney et al. 1997 concluded that the chromatin in living cells is likely constrained by attachment to a nuclear substructure. On the other hand, several investigations have revealed significant mobility of subchromosomal regions in the interphase nucleus, especially involving the centromeric and telomeric regions of chromosomes . In the interphase nucleus of living HeLa cells, however, centromeres are generally motionless . Taken together, these results suggest that chromatin in the nucleus of living cells may be highly constrained, but there is a degree of plasticity that allows limited motion and potentially dynamic shape changes while maintaining an overall high degree of organization of the chromosome territories . In this regard, it has been proposed that specific positional movements of chromosomal regions are regulated by the transcriptional state of the cell and further postulate that this might be mediated by an underlying dynamic nuclear matrix structure . Indeed, there is growing awareness of the dynamics of nuclear architecture . It is important to stress that disruption of the human chromosome territories, while related to complete extraction of the internal nuclear matrix structure, also requires digestion of RNA with RNase. This is consistent with the previous findings that treatment of HeLa matrices and their intermediate filament-like core filaments with RNase A results in destabilization of internal matrix structure and further implies an important role of RNA and/or RNPs in stabilizing chromosome territories. In this regard, the perichromosomal layer, which is likely involved in chromosome organization , contains several classes of proteins and RNPs, including nuclear matrix associated proteins . The findings of Clemson et al. 1996 demonstrating a role of XIST RNA in maintaining the territorial organization of the inactive X chromosome may be a specialized example of what we report for chromosome territories in general. Significantly, the XIST RNA is a component of the underlying nuclear matrix structure . Moreover, SAF-A, a presumptive MAR binding protein associated with the nuclear matrix, binds both DNA (single and double strands) and RNA, and is identical to hnRNP-U, which is involved in packaging of hnRNA into RNP particles . All of these studies suggest a closer interplay than generally acknowledged between the organization of chromosomes and RNP in the cell nucleus. | Study | biomedical | en | 0.999997 |
10444064 | DNA replication and transcription sites were labeled based on previous procedures and those briefly reported by Wei et al. 1998 . Mouse 3T3 fibroblast cells are grown on coverslips in DME or MEM supplied with 10% FCS for 24–48 h. Cells are washed with ice-cold TBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 ) and further washed with glycerol buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM PMSF) for 10 min on ice. Washed cells were permeabilized with 0.025% Triton X-100 in glycerol buffer (with 25 U/ml of RNasin; Promega Corp.) on ice for 3 min and immediately incubated at room temperature for 30 min with nucleic acid synthesis buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 150 mM NaCl, 25% glycerol, 0.5 mM PMSF, 25 U/ml of RNasin, 1.8 mM ATP) supplemented with 0.5 mM CTP, GTP, and BrUTP (Sigma Chemical Co.) for labeling transcription sites (nascent RNA), 0.1 mM dATP, dCTP, dGTP, and 25 μM digoxigenin-11-dUTP (Boehringer Mannheim) for labeling DNA replication sites (nascent DNA), or both for simultaneously labeling transcription and replication sites. After incorporation, the cells were fixed with 3% freshly made paraformaldehyde in PBS on ice for 5 min, washed with ice-cold TBS-Tween buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM MgCl 2 , 0.2% Tween 20), blocked with 5% goat serum and incubated with rat anti-BrU antibodies (IgG, SeraLab) followed by biotin-conjugated goat anti–rat IgG (1:50; Jackson ImmunoResearch Laboratories, Inc.) and Texas red–conjugated streptavidin (1:100; GIBCO BRL) to detect transcription sites. Replication sites were detected with FITC-conjugated sheep antidigoxigenin Fab fragments (1:10; Boehringer Mannheim). All incubations were performed at room temperature for 30 min. Alternatively, nascent RNA was labeled in vivo, by pulsing 3T3 cells for 2 min with 30 μM BrU (Sigma Chemical Co.). The biotin-strepavidin enhancement system resulted in greatly improved sensitivity for detected individual transcription sites compared with a standard secondary antibody approach and was absolutely essential for detecting transcription sites after 2-min in vivo pulses of BrU. Nuclear speckles were decorated with the Y12 mAb to Sm, which recognizes common core proteins of snRNPs involved in RNA processing , followed by goat anti–mouse IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.) conjugated to FITC or Texas red. Coiled bodies and the nuclear lamina were decorated with rabbit polyclonal anticoilin and antilamin B antibodies, respectively, and detected with FITC- or Texas red–conjugated goat anti–rabbit IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted on slides in Slow-Fade (Molecular Probes, Inc.) and stored at −20°C for microscopic examination. Optical sections (0.5 or 0.3 μm where indicated) were collected with a confocal microscope equipped with a Nikon Optiphot 2 microscope, a Nikon 60×, 1.4 NA objective, and a krypton argon laser to simultaneously excite FITC and Texas red at 488 and 568 nm, respectively. Emissions were collected with filters 522-DF32 for FITC and HQ598-40 for Texas red. The pixel intensity distribution was checked before image collection and adjusted so that the pixel intensities were all below saturation. The total number of transcription sites are calculated by a new and highly improved segmentation program, presented in detail elsewhere . In brief, by using two-dimensional image segmentation on the individual slices to obtain transcription site contours and combining this high level two-dimensional data using a modified three-dimensional connected component labeling algorithm with weak connectivity in the Z direction, we were able to reconstruct the network of three-dimensional transcription sites. Instead of using the equivalent of traditional two-dimensional 8-connectivity in three dimensions, we used a metric that evaluated the amount of overlap between successive sections to determine whether the two contours belong to the same site. After determining the three-dimensional boundary of transcription sites, the center of gravity of each site was calculated by averaging the (x, y, z) coordinates of all voxels that belong to the site . Segmentation of the nuclear speckles, nucleoli, the total nuclei, and measurement of the size and intensity of segregated areas was performed in IPLab (Signal Analytics Corp.). Distribution of transcription sites between the nuclear speckles and other nuclear regions was determined by direct measurement. Fluorescence signal intensities from unprocessed confocal microscopic images were measured in IPLab. In brief, the extranucleolar regions, nucleolar regions, and the internal region of the nuclear speckles were manually selected and the mean intensities were calculated after multiple scans through the areas under analysis. The nucleolar signal was selected as background and subtracted from the signal emitting from the speckle and extranucleolar regions. Previous studies indicated that incorporation of BrUTP into newly transcribed RNA in permeabilized cells is essentially a transcription readout system with little or no subsequent RNA processing , and that the labeled RNA is visualized predominantly if not exclusively at sites of transcription. Both nucleolar and extranucleolar incorporation sites are detected . When low levels of α-amanitin (1 μg/ml), which inhibits RP II- but not RP I- or RP III-mediated transcription , was included in the synthesis buffer, the entire extranucleolar signal is abolished . This demonstrates that all transcription sites visualized in the extranucleolar compartment are mediated by RP II. To confirm that nucleolar transcription is mediated by RP I, cells were treated with low concentration of actinomycin D (0.8 μg/ml), which preferentially inhibits RP I transcription of the ribosomal genes . When higher concentrations of actinomycin D (3.2 μg/ml) were used in the synthesis buffer, transcription activity is completely abolished in the nucleus . RNase A greatly reduced transcription , whereas DNase I and aphidicolin , a DNA synthesis inhibitor , had no effect on transcription sites. All transcription sites are confined within the boundary of the nuclear lamina detected with antilamin B antibody . Thus, transport of transcripts to the cytoplasm is undetectable, supporting the conclusion that the transcripts are still engaged on the templates in this transcription readout system . Using a laser scanning confocal microscope we find that extranucleolar RP II transcription is organized into granular-like sites with an average x-y diameter of 0.8 ± 0.05 μm. The nucleolar RP I transcription sites (average diameter of 0.7 ± 0.04 μm) are usually more intensely labeled than the extranucleolar RP II sites and are arranged into discrete clusters. The transcription sites are not distributed evenly in the extranucleolar compartment . Some regions have more sites and form clusters (active zones), whereas other regions have virtually no transcription sites (inactive zones). To study the three-dimensional organization of transcription sites in the cell nucleus, we reconstructed a series of optical images three dimensionally and displayed them as stereo pairs. This revealed that the individual sites in the extranucleolar compartment are arranged in a higher order networklike pattern . The individual sites that compose the overall network are readily observed at higher magnification . x, y, z coordinates (centers of gravity) for each transcription site were calculated by a computer program and displayed in three dimensions with an enlarged portion shown in Fig. 3 D. Consistent with the observation of the original confocal images , chainlike arrays and clusters of sites were observed that extended considerable distances in three dimensions. Since each site is assigned a unique x, y, z coordinate, individual transcription sites can be easily identified among the huge population present. This procedure is illustrated by the highlighting of two individual sites and has enormous potential as an approach for identifying active transcription of specific gene sequences by combining fluorescence in situ hybridization with transcription site labeling. After transcription synthesis, the cells were subjected to DNase I digestion and 0.2- or 0.6-M ammonium sulfate (AS) extraction to obtain the nuclear matrix . Transcription sites are maintained on the nuclear matrix with an organization similar to the intact nucleus . In particular, nucleolar sites maintained their high intensity and tight packing arrays, whereas the extranucleolar space was organized into clusters of sites (active zones) separated by relatively empty regions (inactive zones). We next determined if active transcription can occur after extraction for nuclear matrix. The cells were not treated with DNase I to maintain the DNA template in an intact state. After 0.2- or 0.6-M AS extraction, the cells were subjected to transcription incorporation followed by immunodetection of Br-RNA. We found that transcription activity is maintained on the extracted cells at levels comparable to permeabilized cells. Moreover, the transcription activity was organized into sites that were indistinguishable to those detected in permeabilized cells, including the characteristic intense nucleolar clusters and the arrangement of the extranucleolar compartment into clusters of sites and other areas devoid of sites . Examination of the nuclear matrix–associated transcription sites in three dimensions revealed an organization strikingly similar to that of the intact nucleus. Clusters of sites arranged into discontinuous networklike arrays were observed after extraction of cells for nuclear matrix and in transcription assays performed on the nuclear matrix . Chain-like arrays of transcription sites were also seen after visualization of the x, y, z coordinates (centers of gravity) of individual sites . It was recently reported that replication and transcription sites simultaneously labeled during early S phase are arranged predominantly into spatially separate higher order clusters or nuclear zones . As illustrated in Fig. 6 , this higher order arrangement is well maintained after nuclear matrix extraction. 75.2% ± 4.6 SEM ( n = 10) of the total extranucleolar area containing replication and transcription sites was occupied by separate zones of replication or transcription in cells extracted for nuclear matrix compared with 79.2% ± 5.2 SEM ( n = 12) for unextracted cells . A spot detection segmentation program was developed to quantify the transcription sites in the cell nucleus . Sites that can be detected are contoured in green. The segmentation program was applied to a series of optical sections and sites detected in each section were coordinated to accurately measure the total number of transcription sites. An example of this computer algorithm applied to a typical middle section image is shown in Fig. 2B and Fig. D . A large majority of all detectable transcription sites ranging from very intense to weakly stained sites are segmented with over 90% in the extranucleolar compartment. Of the small portion of sites not segmented by this program (<10% of the total), many represent relatively weak sites positioned between relatively intensely stained ones. The values obtained for the total number of transcription sites, therefore, represent minimal estimations. The transcription sites associated with the nuclear matrix had a similar range of intensity from strong to very weak and a similar efficiency of segmentation. From this analysis, we estimate a minimal average of 2,000 transcription sites per nucleus ( Table ). The great majority of transcription sites are maintained on the nuclear matrix ( Table ) after extraction of cells (1,500–1,900 sites) or transcription labeling on cells first extracted for the nuclear matrix (1,800–1,900). The widely held view that splicing components are highly enriched in nuclear speckles was recently challenged . Therefore, we measured the signal intensities of Y12 staining from unprocessed confocal microscopic images in the internal region of over 200 speckles and their surrounding nonspeckle regions in mouse 3T3 cells. An average enrichment of fivefold was found in the speckles compared with the diffuse staining in the extranucleolar regions . The Y12 staining associated with the speckles is about one third of the total signal measured in the extranucleolar region based on the speckles occupying <10% of the total extranucleolar volume ( Table ). The spatial relationships of RP II transcription sites and nuclear speckles were next examined. RP II transcription sites are associated with splicing factors sites at both speckles and diffusely stained regions . In contrast, RP I transcription sites are not associated with splicing factor sites . Speckle-associated RP II transcription sites are located not only along the periphery , but also in the interior regions of the speckles . Early S phase DNA replication sites, which are distributed throughout the cell nucleus and in numbers similar to transcription sites , are not associated with nuclear speckles or the more diffusely stained splicing factor sites . Overlap between replication and splicing factor sites was also not detected in cells of middle and late S phase (data not shown). Since nuclear coiled bodies also contain snRNPs , we determined whether coiled bodies were also associated with nascent transcripts. We found that transcription activity was rarely associated (<10%) with the 1–3 coiled bodies per cell . The resolution of the confocal microscope in the z-axis is two- to threefold less than in the x-y planes . Therefore, it is conceivable that peripherally localized transcription sites that happen to locate vertically in the center of the speckles could give the appearance of an internal location in a particular optical section . To investigate this possibility, we examined serial optical sections (0.3 μm) through individual speckles and found numerous examples of interior located transcription sites that occurred in the middle sections of the series, but were not present in the top or bottom sections . These serial sections also revealed many transcription sites located along the periphery of the speckles. Whereas the great majority of speckles had both peripheral and internally located transcription sites , a small percentage (<10%) displayed only peripheral ones . Quantitative image analysis was performed on the middle sections from 12 randomly selected optical image sets. An average of ∼400 extranucleolar transcription sites was counted per section. 45% of these sites were speckle-associated even though the speckles contained only 9.6% of the total extranucleolar area ( Table ). Whereas the nonspeckle extranucleolar regions of the nucleus contained the majority of transcription sites (55%), the speckle regions were significantly enriched in these sites (over 8-fold) compared with nonspeckle extranucleolar regions ( Table ). Of the 214 speckles analyzed, the majority of speckle-associated transcription sites (64%) were concentrated along the periphery of speckles either in close juxtaposition or partially overlapping with the speckles themselves ( Table ). All speckles examined had associated transcription sites and the great majority (92.2%) had a moderate to high level of associated sites (4–16 sites per speckle). Only a very small percentage of speckles (7.8%) were relatively weakly decorated with transcription sites (<4 sites per speckle). To determine whether transcription sites are also associated with speckles in vivo, 3T3 cells were pulsed with 30 μM BrU for 2 min to label nascent RNA transcripts, and then fixed immediately followed by dual color labeling for splicing factor and transcription sites. As seen in Fig. 10A–C , the double labeling pattern in the cell nucleus is virtually identical to that observed in the permeabilized cell system . Transcription sites are associated with both the peripheral and interior regions of the nuclear speckles as well as with the more diffuse Y12 staining regions . Nucleolar transcription sites are not associated with the Y12 staining regions as demonstrated in the permeabilized cell system . We have used BrUTP incorporation to label transcription sites (nascent RNA) in permeabilized cells (see introduction and Materials and Methods). By combining laser scanning confocal microscopy and a new computer segmentation algorithm , individual sites of relatively small size (x-y diameters of 0.2–1.0 μm) are detected that are closely associated and have a wide range of fluorescence intensity. Our estimation of 2,000 transcription sites per 3T3 cell nucleus is consistent with other recent reports . Since there are ∼30,000 genes expressed in a mammalian cell , these results can be explained in several ways. None of these explanations are mutually exclusive. Thus, more than one may be operative. First, the labeling system may only be sensitive enough to detect transcription sites for genes that transcribe at high copy number. The great majority of genes that are known to transcribe only a few copies in the cell may not be detected. Second, it is unlikely that all genes are being transcribed simultaneously and continuously during the cell cycle. The 2,000 sites that are detected may represent one window of time for the overall transcriptional expression pattern in the cell cycle. While there is little direct data supporting this model, Wei et al. 1998 has recently discovered that nuclear zones of early S replication sites do not contain significant levels of transcription sites and vice versa. Since early S replication sites contain predominantly actively transcribed genes , these results indicate that at least in S phase there is a higher order control of the qualitative pattern of gene transcription . Third, the large number of genes may be divided among the more limited number of transcription sites. In this multigene model, each site could be a transcription domain for a cluster of genes containing up to a dozen or more transcriptional units. This model has been favored by Cook and colleagues who envision a transcriptional factory where transcription of multiple genes can be regulated . If correct, it will be of great interest to determine what kinds of genes are organized at individual transcription sites. There are several possibilities that again are not mutually exclusive. Most simply, adjacent genes along the genomic DNA sequence could be clustered together at a transcription site. Since there are growing reports of gene clusters along the genomic DNA , this could be an effective way of regulating expression among members of the cluster. In contrast, active genes may randomly cluster at newly assembled transcription sites. Finally, genes that produce functionally related products or that are involved in a pathway of regulation may be assembled at the same transcription site and, in turn, may be subjected to a common regulation. Those genes could be far apart along the genomic sequence and even on different chromosomes. In this regard rDNA genes, which are clustered in tandem arrays on the short arms of several chromosome pairs , are packaged together in the nucleolar structure . It would be of interest to determine if genes regulated by a common activator are located on the same transcription domain. Future studies combining fluorescence in situ hybridization for specific gene localization with transcription site labeling should provide important insight into these issues. Using RP I antibodies, Scheer and Rose 1984 demonstrated that the presumptive sites of rDNA transcription show a punctate staining pattern over the fibrillar center regions of the nucleolus. While no measurements of these punctate sites were reported, we have estimated a diameter range of 0.5–1.0 μm with an average of ∼0.7 μm . This is in the same size range as the transcription sites we observed over the nucleolar regions. Similar to the number of RP I granular sites per nucleus , we report an average of 110 discrete and intensely stained nucleolar transcription sites per mouse fibroblast nucleus. This is considerably less than that of rRNA genes determined by saturation hybridization . It was proposed that each RP I site contains one active rRNA gene which would indicate that there are ∼115 and 106 active rRNA genes in human and mouse cells, respectively . The lower number of RP I transcription sites compared with the number of rRNA genes can also be explained by a limited clustering of rRNA genes at each transcription site (e.g., 2–3 genes per site). If this is the case, then gene clusters might represent a fundamental feature of transcription sites as units of gene expression. Jackson et al. 1993 have suggested that each RP I transcription site may contain approximately six transcriptional units, but this is based on a much lower estimation for the number of nucleolar transcription sites. We consistently observe that the transcription sites, rather than being scattered throughout the extranucleolar compartment as previously suggested , are arranged in nuclear zones containing clusters of sites and other regions devoid of sites. Three-dimensional reconstruction further reveals networklike arrays of the transcription sites. Contour mapping demonstrates that both transcription and replication sites in early S phase form separate nuclear zones and networklike arrays in three dimensions . Since the individual transcription sites are spatially separate, some elements of higher order nuclear architecture are likely involved in maintaining these networklike arrays. Previous reports have suggested that transcription sites are maintained on the nuclear matrix . Our study is the first detailed analysis of the association of transcription sites with the nuclear matrix. We also observe a remarkable maintenance of RP I and II transcription sites and their higher order arrangement into separate transcription and replication zones and networklike arrays after extraction for nuclear matrix. The question as to whether nuclear speckles, which are generally believed to be sites for storage of splicing factors, are also sites of active transcription and/or splicing has generated considerable interest in recent years . While studies of individual genes and poly(A) + RNA lend support to this view , the matter is complicated by the lack of correspondence between the overall pattern of RP II-mediated transcription sites, which are scattered throughout the extranucleolar compartment in hundreds to thousands of sites, and the 20–50 much larger nuclear speckles . Moreover, the lack of tritiated uridine labeling over the interchromatinic granule clusters , which are believed to represent the electron microscopic equivalent of nuclear speckles , has led to the view that at least most of the nuclear speckles are storage sites and transcriptionally inactive. Splicing factors are proposed to be recruited from nuclear speckles to other nonspeckled sites for coordinate transcription/splicing . Surprisingly few studies, however, have directly measured the degree to which nascent transcripts are associated with nuclear speckles. While Wansink et al. 1993 is often quoted as providing evidence that transcription sites are not associated with nuclear speckles, merged images or quantitation of the degree of overlap between transcription sites and nuclear speckles were not presented in that study. Direct overlay of the separate channel images shown in Fig. 7 of Wansink et al. 1993 , however, suggests significant levels of overlap of transcription sites with nuclear speckles (results not shown). In addition, Dundr and Raska 1993 observed labeling of nascent RNA over the interchromatin granular clusters after BrUTP incorporation in permeabilized cells. In our study, over 200 individual nuclear speckles were examined in detail by confocal microscopic optical sectioning and quantitative image analysis. We report that significant levels of RP II transcription sites (45% of total) are associated with nuclear speckles and concentrated over eightfold compared with the remaining extranucleolar compartment. The majority of speckle-associated transcription sites are found along the periphery or juxtaposed to the speckles (approximately two thirds) with the remainder located in interior regions of the speckles. Coupled with the concentration of transcription sites in the speckled regions is an average enrichment of approximately fivefold in snRNP splicing factors. Fay et al. 1997 previously measured the association of transcribed RNA with nuclear speckles. However, that study was limited by the relatively long in vivo pulse periods used to label the nascent RNA. Under those conditions, a significant portion of the labeled RNA would have completed transcription and might not be associated with the transcription sites. Consistent with this interpretation, the distribution of speckle-associated transcription sites in that study decreased from 21 to 10% as the in vivo labeling time increased from 9.5 to 19.5 min . The readout transcription approach employed in our study avoids this problem . Moreover, we observe a similar pattern of transcription sites associated with nuclear speckles after a brief in vivo pulse (2 min). Fay et al. 1997 also reported an enrichment of only twofold of the splicing factor, SC-35 in nuclear speckles and have questioned the view that speckles are indeed sites of enrichment of splicing factors . Our measurements using a similar approach, but on original confocal images without thresholding or image enhancement, revealed a fivefold enrichment for the snRNP splicing factors and are consistent with previous studies suggesting high levels of enrichment of SC-35 and other splicing factors in nuclear speckles . We propose that both nuclear speckles and the extraspeckle regions participate in coordinated transcription/RNA processing events. In this regard, Smith et al. 1999 have demonstrated that not all actively transcribed genes and their transcripts show an association with nuclear speckles. Further research is clearly needed to understand the relationship of nuclear speckles to the much smaller and diffuse sites of transcription and RNA processing in the cell nucleus. One simplistic view is that the speckles are sites of massive levels of transcription/RNA processing mediated by the limited number of genes that transcribe high copy numbers. Most genes that transcribe at low copy numbers would then be localized in the hundreds to thousands of additional sites arranged throughout the extranucleolar compartment. However, the situation is more complicated. Smith et al. 1999 recently showed that speckle association of RNA is not restricted to the limited number of highly abundant transcripts. Among four genes that are believed to have moderate levels of transcripts in the cell, two were speckle-associated and two were clearly not. These and previous studies suggest that a significant portion of the total population of different pre-mRNA transcripts are associated with nuclear speckles, whereas another portion is not. This agrees with our finding that ∼45% of total RP II transcription sites are speckle-associated. Several studies have stressed the dynamic nature of nuclear speckles and their role as recruitment sites for engaged transcription of activated genes . The dynamic recruitment of splicing factors from nuclear speckles to putative sites of transcription was recently observed in living cells using a GFP-expression system . Moreover, massive expression of an intron-containing RNA leads to recruitment of splicing factors at sites of transcription that assemble into nuclear speckle-like structures . These dynamic properties and studies demonstrating individual gene transcripts closely associated with nuclear speckles have led to the proposal that there are two populations of nuclear speckles in a cell: transcriptionally inactive speckles that serve as storage sites for splicing factors and transcriptionally engaged speckles, where splicing factors are massively recruited to active sites of transcription . Alternatively, it has been proposed that all nuclear speckles might be active in coordinate transcription/RNA splicing processes . Our results are most consistent with the latter model. All speckles examined (>4,000) had associated transcription sites. Of the 214 speckles examined in detail, the great majority (>90%) had moderate to high levels of associated transcription sites. The presence of a small population of speckles (<10%) with relatively low levels of transcription sites, however, also lends credence to the dynamic speckle model . Indeed, it is plausible that the population of speckles actively engaged in transcription and splicing may widely fluctuate depending on a variety of cellular conditions. Further research is needed to clarify these possibilities. While our studies demonstrate that nuclear speckles participate in transcription and are likely sites of coordinated transcription/RNA processing, only limited regions of the speckles likely participate in this activity at any given time. The majority of the RP IIo and splicing factors that are concentrated in the nuclear speckles are not colocalized with the transcription sites. What leads to recruitment of active RP IIo and splicing factors remains to be elucidated, but initial biochemical studies suggest an important role of the CTD of RP II and recently identified CTD-binding proteins of the SR class . One of these CTD-binding proteins, SCAF8, colocalizes with transcription sites and another, termed matrin CYP, is a novel cyclophilin-containing SR repeat that is highly concentrated in the nuclear speckles . Association of nuclear speckles , RP II transcription sites , RP IIo , transcription factors , SCAF8 , and matrin CYP with the nuclear matrix provide an in vitro approach for elucidating the molecular associations involved in the coordination of transcription and RNA processing and their architectural integration in the mammalian cell nucleus. | Study | biomedical | en | 0.999997 |
10444065 | Mouse Swiss 3T3 fibroblasts, HeLa cells, and human embryonic kidney 293 cells were grown in high glucose DME (Life Technologies, Inc.) supplemented with L -glutamine, 25 mM Hepes buffer, pyridoxine hydrochloride, 10% FBS, streptomycin (100 μg/ml), and penicillin (100 U/ml). Cells were incubated in 5% CO 2 at 37°C in 75-cm 2 flasks. For transient transfections, 1–5 × 10 5 cells grown in 60 × 15 mm style petri dishes containing glass coverslips were transfected with plasmid DNA (2–5 μg) using the SuperFect reagent (Qiagen) following the manufacturer's instructions. For stable transfection, 1–2 × 10 6 HEK 293 cells were transfected with 2 μg of pEGFP-Rpp38, split 24 h after transfection, and then grown in a selective medium supplemented with 0.5 mg/ml geneticin (G418; Life Technologies, Inc.). Mixtures of G418-resistant cell populations obtained were massively grown for RNase P purification. A PstI-NotI Rpp38 cDNA fragment subcloned in pBluescript was released by PstI and ApaI (located in the multiple cloning site) and subcloned in-frame in PstI-ApaI digested pEGFP-C1 (CLONTECH Laboratories) to generate pEGFP-Rpp38. pEGFP-Rpp38(246-283) was generated by cleaving pEGFP-Rpp38 with HindIII, deleting the first 245 amino acids of Rpp38, and then the plasmid was self-ligated in the presence of a short HindIII DNA adaptor to keep the carboxy terminal 37 amino acids of Rpp38 (positions 246–283) in-frame with GFP. pEGFP-Rpp38(1-245) was constructed by cleaving a PstI-HindIII Rpp38 cDNA subcloned in pBluescript with PstI and ApaI and subcloned in-frame in pEGFP-C1 first cleaved with PstI and ApaI. pEGFP-Rpp38(260-283) was generated by subcloning a PstI-ApaI deoxyoligonucleotide that codes for the last 24 amino acids of Rpp38 in pEGFP-C1 digested with PstI and ApaI. pNS38KN was constructed as pEGFP-Rpp38(260-283) with all the nine lysine residues in the carboxy terminal 24–amino acid sequence were substituted with asparagines. pNS38KN23, pNS38KN45, pNS38KN78, and pNS38KN59 were constructed as pEGFP-Rpp38(260-283) but with two lysine substitutions; numbers represent the substituted lysines . Constructs with a single substitution of arginine (pNS38R13A), serine (pNS38S18A), threonine (pNS38T22A), or proline (pNS38P23A) to alanine were also prepared as described for pEGFP-Rpp38(260-283). pNS38ATΔPP was obtained during the construction of pNS38R13A in which we found after sequencing that the arginine and lysine were substituted accidentally by alanine and threonine, respectively, whereas the consecutive proline residues were deleted, keeping the remaining amino acids in-frame with the upstream GFP. Two primers, one encompassing the translation initiation codon and the other one spanning the translation stop codon of Rpp29, were used to amplify the entire Rpp29 cDNA . The PCR product was digested with EcoRI, located in the designed primer sequences, and subcloned in-frame in pEGFP-C1 first cleaved with EcoRI. pEGFP-Rpp29(52-85) was constructed by digestion of a PCR DNA product, containing the sequence that codes for amino acids 52–85 of Rpp29 , with Hind III and BamHI located in the designed primers, and followed by subcloning in pEGFP-C1 cleaved first with the same two restriction enzymes. pEGFP-Rpp29(63-85) was generated by subcloning a BamHI-HindIII deoxyoligonucleotide that codes for the 23 amino acids encompassing positions 63–85 of Rpp29 in pEGFP-C1 digested with BamHI and HindIII. pNS29RN4 and pNS29KN4 were constructed as pEGFP-Rpp29(63-85), but the RQRR or KKKK residues were substituted with four asparagines, respectively. pEGFP-Rpp14 was generated by subcloning a XhoI-HindIII–digested PCR DNA product containing the entire Rpp14 open reading frame into pEGFP-C1 cleaved first with XhoI and HindIII. pPK-Rpp38 was generated by inserting a KpnI-digested PCR Rpp38 cDNA containing the entire open reading frame into the KpnI unique site of the myc-tagged chicken pyruvate kinase in pcDNA3-PK plasmid , provided to us by Dr. Gideon Dreyfuss (University of Pennsylvania, Philadelphia, PA). All DNA constructs described above were verified by sequencing to ensure in-frame subcloning of the desired inserts with the reporter gene. Cells (20% confluent) were grown overnight on coverslips (22 × 22 mm) before fixation with 2% paraformaldehyde (Electron Microscopy Sciences) diluted in 1× PBS for 30 min. Cells were treated with 0.5% Triton X-100 for 5–30 min, washed twice with 1× PBS (0.5 liter each), and then blocked with 3% BSA/PBS for 20 min. Rabbit polyclonal antibodies against Rpp subunits , p80-coilin , or Nopp140 peptide , diluted 1:50–400 in 3% BSA/PBS, were added to the fixed cells for 1 h, and then washed twice with PBS before incubation for 20 min with 1:50 diluted secondary antibody, Alexa™ 568 goat anti–rabbit IgG antibody conjugate (Molecular Probes Inc.). When the monoclonal antifibrillarin and anti-myc (9E10) mouse IgG antibodies or polyclonal anti-B23 goat IgG antibodies (Santa Cruz Biotechnology Inc., CA) were included, Alexa™ 488 goat anti–mouse IgG antibody or Alexa™ 594 donkey anti–goat IgG antibody conjugates (Molecular Probes Inc.) were used. Cells were washed twice with PBS and mounted on glass slides using boat sealer (Ernest Fullam). Confocal fluorescence microscopy of living or fixed cells was performed at 22°C (± 2°C) using a Bio-Rad MRC-1024 laser scanner mounted on a 2FL reflector slider on a Zeiss Axiovert equipped with differential interference contrast (DIC) optics (PlanApo 100× 1.4 NA oil immersion objective; Carl Zeiss). Fluorescent images were acquired by using Texas red and FITC filters, and then processed using LaserSharp software (Bio-Rad Laboratories). Bleedthrough was completely eliminated between fluorophore channels in colocalization studies. Nuclei of living cells were also visualized by DNA staining with 4′,6-diamidino-2-phenylindole. Digital processing and color adjustment of images were done using MetaMorph Image acquisition and processing software (Universal Imaging Corp.) and Adobe Photoshop (Adobe Systems, Inc.). RNase P from G418-resistant 293 HEK cells that constitutively express GFP-Rpp38 fusion protein was purified as previously described . In brief, 10 9 cells were pelleted, disrupted, and the cell homogenate was centrifuged at 7,000 rpm followed by another centrifugation at 42,000 rpm in a Beckman Ti50 rotor to obtain S100 crude extract. This S100 extract was loaded on a DEAE-Sepharose anion exchange chromatography column and RNase P was eluted from the column using a 100–500-mM KCl gradient. The flowthrough, wash, and the eluted fractions were assayed for RNase P activity, and then kept in 25% glycerol in −20°C for further analysis. Cleavage of the 5′ leader of the yeast suppressor precursor tRNA Ser (SupS1) by human RNase P was performed as described . For Western blot analysis, DEAE fractions were separated on 12% SDS–polyacrylamide gel, electrotransferred to a nitrocellulose filter, and immunoblotted with 1:3,000 diluted polyclonal anti-GFP antibodies (CLONTECH Laboratories) or with 1:100 dilution of affinity-purified polyclonal anti-Rpp38 rabbit antibodies . Peroxidase-labeled goat anti–rabbit IgG antibodies (Vector Labs, Inc.) were used at 1:5,000 dilution as secondary antibodies. Blots were washed and bands were visualized using the ECL plus kit (Amersham), following the manufacturer's instructions. Mouse Swiss 3T3 fibroblasts were transiently transfected with pEGFP-Rpp38, a derivative of the expression vector pEGFP-C1, which contains the Rpp38 open reading frame fused in-frame to the carboxy terminus of a GFP . Expression of the GFP-Rpp38 fusion protein in transfected cells was monitored by confocal fluorescence microscopy (see Materials and Methods). 48 h after transfection, the fluorescence signal of GFP-Rpp38 was seen in the nucleoplasm but was most visible in the nucleoli . Only background fluorescence was observed in the cytoplasm. By contrast, GFP alone was distributed diffusely throughout the cytoplasm and the nucleoplasm, but was completely excluded from nucleoli . Since GFP-Rpp38 was not seen in the cytoplasm, as was GFP alone, this fusion protein must have been retained in the nucleoplasm and nucleoli. Localization of GFP-Rpp38 in the nucleolus of transfected 3T3 fibroblasts was verified by the colocalization of this fusion protein with the nucleolar protein B23 using indirect immunofluorescence analysis . B23 is a nuclear localization sequence (NLS)–binding phosphoprotein that is found in the dense fibrillar component and the granular component of the nucleolus . GFP-Rpp38 is more uniformly distributed than B23 in the nucleolar compartments . That endogenous Rpp38 in 3T3 fibroblasts is also a nucleolar protein was confirmed by using affinity-purified, polyclonal anti-Rpp38 antibodies in indirect immunofluorescence analysis . As with GFP-Rpp38 , endogenous Rpp38 was uniformly distributed in the nucleolus. A weak signal around the nucleus that is typical of mitochondrial staining was also observed , but further work is required to confirm localization of Rpp38 in these cytoplasmic organelles. We also tested the ability of Rpp38 to target another reporter protein, the cytoplasmic chicken pyruvate kinase . A fusion protein of Rpp38 with a myc-tagged pyruvate kinase accumulated in the nucleoplasm of transfected 3T3 cells and a weak signal was seen in nucleoli (data not shown). This may suggest that this fusion protein is too large (∼100 kD) to be efficiently translocated and/or retained in the nucleolus, when compared with GFP-Rpp38. Therefore, GFP was used as the reporter protein throughout this study. In transfected HeLa cells, GFP-Rpp38 compartmentalized in nucleoli as well as in discrete, intranuclear organelles immunostained with an antibody against p80-coilin . These organelles represent coiled bodies as defined by the presence of p80-coilin . Diffuse immunostaining of p80-coilin was seen in the nucleoplasm and the nucleolus as well . The site of Rpp38 was further identified by the colocalization of GFP-Rpp38 with the nucleolar shuttling protein Nopp140 in nucleoli and coiled bodies . Nopp140 is confined to the dense fibrillar component of the nucleolus . Clearly, GFP-Rpp38 is more widely distributed in nucleoli than Nopp140. Similar results regarding coiled bodies were obtained with transfected 3T3 fibroblasts (data not shown). All these findings, taken together, demonstrate that the Rpp38 subunit of RNase P is localized in the nucleolus and in coiled bodies of cultured mammalian cells. Examination of the amino acid sequence of Rpp38 shows that it may possess three NLSs, located at positions 63–66, 241–244, and 262–281 . Two DNA constructs, pEGFP-Rpp38(1-245), which contains amino acids 1–245 of Rpp38 and pEGFP-Rpp38(246-283), which possesses the remaining 37 amino acids of the polypeptide , were separately transfected into 3T3 fibroblasts and the localization of these truncated fusion proteins was determined. GFP-Rpp38(1-245) was concentrated in the nucleoplasm but not in the nucleoli , an observation that was also confirmed in a double label experiment in transfected cells immunostained for the nucleolar B23 protein . By contrast, GFP-Rpp38(246-283) was targeted to the nucleoli and nucleoplasm of cells with no significant signal seen in the cytoplasm . Similar nuclear and nucleolar localization patterns were obtained in 293 HEK cells transfected with the two constructs described above (not shown). Thus, the sequence required for nucleolar localization of Rpp38 exists between positions 246–283. The carboxy terminal 24 amino acids of Rpp38 fused to GFP in pEGFP-Rpp38(260-283) are capable of introducing the reporter protein into the nucleoli of 3T3 fibroblasts ( Table and data not shown). Nucleolar staining with GFP-Rpp38(260-283) was as intense as with GFP-Rpp38 and GFP-Rpp38(246-283) (compare relative intensities in Table ), an indication that the last 24–amino acid sequence of Rpp38, designated NS38, are sufficient for nucleolar localization of the reporter protein. We investigated whether the lysine residues in NS38 were important for its function by amino acid substitution analysis. The intracellular distributions (cytoplasm, nucleoplasm, and nucleoli) as reflected by the fluorescence signals of several mutants shown in Fig. 1 A are also summarized in Table . Thus, pNS38KN, in which all the nine lysines in the NS38 sequence were substituted by similar, positively charged asparagine (N) residues, was introduced into 3T3 fibroblasts. The resultant fusion protein failed to enter the nucleoli and the fluorescent signal was detected in the cytoplasm as well as the nucleoplasm ( Table ). When cells were transfected with pNS38KN23, in which lysines 2 and 3 in NS38 sequence were replaced by asparagines, or with pNS38KN45, in which lysines 4 and 5 were substituted, a marked decrease in the nucleolar fluorescence was measured ( Table ). These latter two fusion proteins were also distributed evenly throughout the nucleus, when compared with the prominent concentration of GFP-Rpp38(260-283) in the nucleoli ( Table ). Moreover, as in the case of GFP-Rpp38(260-283), NS38KN23 and NS38KN45 accumulated in the nucleoplasm ( Table ), an indication that their nuclear retention was not completely abolished. Similar results were obtained with NS38KN59 in which lysines 5 and 9 were substituted ( Table ). However, the nucleolar localization capability of NS38 was completely abolished when the double mutant NS38KN78, in which lysines at position 7 and 8 in NS38 were replaced by asparagines , was introduced into cells ( Table ). NS38KN78 was concentrated in the nucleoplasm rather than the cytoplasm when compared with NS38KN ( Table ). Next, we substituted alanine separately for each of the arginine (R13A), serine (S18A), threonine (T22A), or proline (P23A) residues in the NS38 sequence and tested the ability of these mutants to localize GFP to the nucleolus. The single mutants, R13A, S18A, and T22A, were found to have no profound effect on the nucleolar localization capability of NS38 ( Table ). Therefore, phosphorylation of serine or threonine appears not to be an obligatory modification for NS38 function. The proline substitution to alanine (P23A), however, seemed rather to increase the ratio of the nucleolar to the nucleoplasmic staining when compared with the ratio obtained with the NS38 parental construct ( Table ). The two prolines in the RKPP sequence of NS38, by contrast, had no critical role in nucleolar localization as corroborated by the ATΔPP construct in which the arginine and lysine (at position 5) were replaced with alanine and threonine, respectively, and the two consecutive proline residues were deleted from NS38 ( Table ). The findings described above show that lysine residues throughout the NS38 sequence are required for its nucleoplasmic retention and nucleolar localization, with the lysines at position 7 and 8 being most critical for its entry to the nucleolus. We obtained evidence that the GFP-Rpp38 fusion protein actually resides in a catalytically active RNase P complex. pEGFP-Rpp38, which expresses the neomycin resistance gene (G418 resistance), was used to establish stably transfected human embryonic kidney (HEK) 293 cells in culture. G418-resistant cell populations obtained in this manner exhibited fluorescent signals in the nucleoli and nucleoplasm, as judged by confocal microscopy (data not shown). To determine if GFP-Rpp38 expressed in these cells can be found in RNase P, S100 crude extracts from these stably transfected cells were fractionated on a DEAE-Sepharose anion exchange column. As determined by processing of a yeast tRNA Ser precursor, RNase P activity was eluted at 280–340 mM KCl , a salt concentration shown previously to elute active RNase P from untransfected human cells from DEAE columns . When fractions across the peak of enzymatic activity were subjected to Western blot analysis using anti-GFP antibodies (see Materials and Methods), a protein of ∼75 kD that copurified with enzymatic activity was detected . This protein corresponds to the GFP-Rpp38 fusion protein and apparently has an anomalous migration in SDS-PAGE, a property that is shared by several Rpp proteins including Rpp38 . When polyclonal anti-Rpp38 rabbit antibodies were used in Western blotting, the same 75-kD protein was detected . A protein of ∼65 kD visible in the blot may be a truncated fragment of GFP-Rpp38. Neither flowthrough nor wash fractions from the column contained GFP-Rpp38 or endogenous Rpp38 protein , an indication that both polypeptides were tightly bound to the column and eluted only with RNase P. At least as demonstrated in vitro, the expression of GFP-Rpp38 in human cells does not abolish RNase P function in tRNA processing, although constitutive expression of GFP-Rpp38 resulted in cell death after 10–15 passages in culture (data not shown). As with Rpp38, sequences of contiguous basic residues that may function in nuclear localization are found in Rpp29, another protein subunit of RNase P . 3T3 mouse fibroblasts were transfected with pEGFP-Rpp29, in which the open reading frame of the Rpp29 cDNA was fused in-frame with GFP . GFP-Rpp29 was localized in the nucleoplasm and exhibited very intense staining of nucleoli . However, GFP-Rpp29 was not evenly distributed in the nucleoli but was concentrated in subregions inside these structures . The smaller punctate stainings seen in the nucleoplasm may represent discrete structures other than nucleoli such as coiled bodies (see below). Indirect immunofluorescence analyses revealed that GFP-Rpp29 localized in nucleoli with B23 . Moreover, endogenous Rpp29 colocalized with fibrillarin in untransfected fibroblasts . The immunostains of Rpp29 and fibrillarin seen in nucleoli are strikingly similar , suggesting that Rpp29 resides in the dense fibrillar component as fibrillarin. As shown in Fig. 6D–F , the sequence responsible for the nucleolar localization of Rpp29 is located between positions 52 and 85 of this protein, as demonstrated by the use of pEGFP-Rpp29(52-85) in transfected fibroblasts. This domain was able to localize GFP to subnucleolar regions, but in a less distinct manner than the full-length Rpp29 protein. GFP-Rpp29(52-85) was almost exclusively retained in the nucleus and only background fluorescence was seen in the cytoplasm. Amino acids 63–85 of Rpp29 were still sufficient for nucleolar localization ( Table ; construct pEGFP-Rpp29(63-85)). This sequence of 23–amino acid of Rpp29 is now designated NS29 . Mutational analysis of NS29, summarized in Fig. 1 B and Table , showed that the KKKK residues in NS29 were required for efficient nucleolar localization but were not crucial for function, as determined through the use of NS29KN4 mutant. All the other multiple and single mutants tested, including the substitution of the RQRR residues to asparagines, had no dramatic effect on NS29 function ( Table ). In fact, the RHKRK motif is sufficient for nucleolar entry ( Table ). We concluded that NS29 and NS38 represent distinct domains required for nucleolar localization. In HeLa cells, coiled bodies immunostained for p80-coilin contained GFP-Rpp29 . In some transfected cells, however, coilin-immunostained structures that were on the periphery of nucleoli exhibited no intense signal of GFP-Rpp29 . In contrast to GFP-Rpp29 and GFP-Rpp38, no prominent signal of GFP-Rpp14 fusion protein was seen in the coiled bodies of HeLa cells transiently transfected for 48 h with pEGFP-Rpp14 construct . Whether Rpp14 requires a longer time to localize in coiled bodies, as was the case with its inefficient localization in the nucleolus (see below), remains unknown. A detailed kinetic study, however, is required to determine whether the several GFP-Rpp fusion proteins presented in this study enter the nucleoli first on their way to coiled bodies, as has been shown with Nopp140 . Indirect immunofluorescent analysis using affinity-purified, polyclonal antibodies against Rpp14 showed localization of this RNase P subunit in the nucleolus of 3T3 fibroblasts . Yet, Rpp14 has no sequences of basic residues typical of NLSs, in contrast to Rpp29 and Rpp38 . Nevertheless, Rpp14 fused to GFP was directed to subnucleolar regions in 3T3 fibroblasts transfected for 48 h with pEGFP-Rpp14 . No prominent punctate staining was seen in the nucleoplasm, as was the case with Rpp29 and Rpp38. However, we found that GFP-Rpp14 was seen exclusively in the nucleolus only in cells producing low levels of this fusion protein, as reflected in the relatively weak fluorescent signals observed in transfected cells . In transfected cells that showed more intense signals, comparable in their intensity to those reported here for Rpp38 and Rpp29, most of the GFP-Rpp14 synthesized was not transported to the nucleoli, but rather remained in the cytoplasm . Moreover, when cells were tested at earlier times (<24 h) after transfection, most of the GFP-Rpp14 stain was visible in the cytoplasm (data not shown). These observations suggest that, when compared with the rapid and efficient entry of Rpp38 and Rpp29 into the nucleolus, the localization process of Rpp14 under the same conditions is inefficient, and thus may require a limiting, endogenous factor. As demonstrated by co-localization analysis, GFP-Rpp14 was compartmentalized into subnucleolar regions occupied also by endogenous Rpp29 in transfected cells , an indication that these two subunits colocalized in the dense fibrillar component. This study shows that the nucleolus of cultured mammalian cells serves as the major site of localization of several protein subunits of human RNase P. In contrast to the dispersed distribution of Rpp38 in nucleoli, the Rpp14 and Rpp29 subunits are localized in the dense fibrillar component. The differential pattern of nucleolar localization was also observed for these subunits when fused to GFP and expressed in living cells. These RNase P subunits, thus, define different sites that, in turn, may reflect distinct biological functions. This conclusion is supported further by our findings that at least two subunits, Rpp29 and Rpp38, are also found in functionally distinct organelles, the coiled bodies. The nucleolus and the coiled bodies appear to be involved in the biogenesis of the ribonucleoprotein RNase P and, therefore, in the process of maturation of tRNA precursors. Nucleolar localization of proteins usually involves multiple domains in targeting sequences that can interact with ribonucleic acids or with other proteins . Nucleolar localization domains of some proteins, such as nucleolin, p120 nucleolar protein, and ribosomal proteins L5 and L7a are not functional by themselves when transferred to a reporter protein; they require additional, noncontiguous domains for function . On the other hand, NS38 and NS29 are functional and transferable. However, these two domains seem not to be sufficient for targeting a reporter protein to the coiled bodies as well. NS38 has no arginine- or arginine/glycine–rich motifs , as has been found in domains in nucleolin (C23) and in the human immunodeficiency virus Tat protein that may facilitate RNA-binding and/or protein–protein interactions . The single arginine residue found in NS38 has no essential role either in the nucleoplasmic retention or in the nucleolar localization capability of this domain. Lysine residues at different positions throughout the NS38 sequence, instead, are required for efficient nucleolar localization. Adjacent lysines at positions 7 and 8, but not at positions 2 and 3, have a critical role in NS38 function. Numerous KKX repeats are found in several protein subunits of yeast nuclear RNase P , but as in many other cases of nucleolar proteins such repeats were proved nonessential for nucleolar targeting . NS38 shows no identity at the primary amino acid sequence to NS29. It is thus likely that structural features and the placement in space of side chains of both hydrophobic and charged amino acids (lysines) determine the function of these sequences. Both NS29 and NS38 act early and efficiently to introduce a reporter protein to the nucleoli of mouse and human cultured cells. Similar conclusions were made for the full-length proteins, Rpp29 and Rpp38. In contrast, Rpp14 entry to the nucleolus seems slow and limited. Rpp14, which lacks any basic residues typical of nuclear or nucleolar targeting domains, may require other proteins that occur in limited amounts in the cell for its nucleolar transport. Furthermore, we were able to show that the nucleolar localization processes of Rpp subunits are dependent on ongoing transcription in functional, intact nucleoli. Thus, selective inhibition of rRNA transcription by a low concentration of actinomycin D leads to disintegration of the nucleoli and to dispersed nucleoplasmic staining by Rpp29, Rpp38, or their nucleolar localization domains fused to GFP (data not shown). Inhibition of protein synthesis by cycloheximide, by contrast, seems to have no effect on the nucleolar localization properties of these subunits. As judged by RNA hybridization analysis in situ, most of the RNA subunit of the yeast nuclear RNase P is localized in the nucleolus with some unprocessed tRNA precursors that contain 5′ leader sequences . In contrast, the majority of the human RNase P RNA is concentrated in the nucleoplasm rather than the nucleolus . Moreover, H1 RNA that was microinjected to the nucleoplasm only transiently enters the dense fibrillar component of the nucleolus before it is redistributed in the nucleoplasm . Our study now shows, using both indirect immunofluorescence and cell transfection analyses, that several protein subunits of human RNase P reside in the nucleolus. Since the estimated copy numbers of RNase P RNA and RNase MRP RNA in a metazoan cell are ∼2 × 10 5 and ∼10 5 , respectively , there are at least two explanations of these differences in the location of the RNA and the protein subunits: newly synthesized H1 RNA enters the nucleolus for assembly with the Rpp subunits before it exits to the nucleoplasm, or that the nucleolus acts as a sequestration compartment for several Rpp subunits that can be recruited to other nucleoplasmic sites, where H1 RNA exists, to form an active RNase P complex under certain physiological conditions. The localization of these subunits in the coiled bodies supports the idea that these organelles may be involved in sorting and transport of RNase P and RNase MRP components from the nucleolus to other destinations and vice versa. Finally, RNA and protein subunits of RNase P and RNase MRP colocalize in the dense fibrillar component of the nucleoli and both utilize a common conserved RNA structural element, the P3 domain , for their nucleolar entry . The localization pattern of Rpp29 in the nucleolus itself suggests that this subunit localizes RNase P and RNase MRP to the dense fibrillar component in which transcription and early rRNA processing events take place . Colocalization studies with Nopp140 and B23, the latter is found in the granular component in addition to the dense fibrillar component , indicate that Rpp38 may also reside in other compartments known as sites of preribosome assembly . Whether these compartments are involved in the processing of some precursor tRNAs remains unknown. However, a common molecular process may govern the localization and assembly of RNase P and RNase MRP to ensure the coordinated processing of stable RNA in mammalian cells. | Study | biomedical | en | 0.999997 |
10444066 | HeLa cells were grown in DME supplemented with 5% newborn calf serum, 5% FCS, and antibiotics. HeLa cells were synchronized using a thymidine-aphidicolin protocol . Cells were cultured on the microscope stage in a CO 2 -independent medium without phenol red (GIBCO BRL). The medium was overlaid with mineral oil (Sigma Chemical Co.) to prevent evaporation. Hydroxyurea was used at a final concentration of 2.5 mM and was added immediately after release from the aphidicolin block. The cyclin B1-MmGFP, cdc25C(S216G), and Wee1 constructs have been previously described . Cyclin B1 R42A -MmGFP was the gift of Paul Clute (Wellcome/CRC Institute). Cdc25B3 and Cdc25C were tagged at the amino terminus with MmGFP by PCR using Taq polymerase and cloned into the pCMX vector . The stop codon of GFP was mutated to a Hind III site to link it with the first amino acid of Cdc25B or Cdc25C creating a 3–amino acid linker (Gly-Ile-Pro). Myc-tagged cdc25B2 was cloned into the pCDNA3 vector for expression in HeLa cells. All constructs were sequenced using an Applied Biosystems DNA sequencer. Cyclin B1–cdc2 K33R complexes were expressed in and purified from baculovirus-infected cells as described . Cyclin B1 F146A was expressed in Escherichia coli and purified as described . Constructs expressing cDNAs under the control of the cytomegalovirus promoter were microinjected into cell nuclei using an Eppendorf semi-automatic microinjection apparatus. To assay for condensed chromatin, Hoechst 33342 was added to cells at a concentration of 1 μg/ml at the end of the experiment. Injected cells were identified by green fluorescent protein (GFP) fluorescence and those that had rounded up with abnormally condensed chromatin were scored. At least 50 cells were scored for each injected construct and experiment. Apoptotic cells were assayed using the apoptosis detection kit (R&D Systems, Inc.) and HeLa cells treated with cycloheximide plus tumor necrosis factor α were used as positive controls. To visualize GFP-chimeras in living cells, cells were cultured on an inverted Leica DMIRB/E microscope using the ΔTC3 system (Bioptechs) to maintain cells at 37°C. Images were captured with a PentaMax CCD camera (Princeton Instruments) fitted to the lateral photo port. GFP- and yellow fluorescent protein (YFP)–chimeras were detected with custom filter sets JP1 and JP2 (Chroma Technology Corp.) and two Lambda 10-2 filter wheels (Sutter Instrument) controlled by a PowerWave computer (PowerComputing). One filter wheel was used to control the wavelength of the excitation light. The other filter wheel controlled the wavelength of the emission light and also the polarizer for DIC images. To distinguish between GFP and YFP we used the JP3 filter set as described . Images were collected and processed using IP Lab Spectrum software (Scanalytics Inc.) and exported to Adobe Photoshop for printing. For β-tubulin and MPM2 staining, cells were fixed with 3% PFA/Triton X-100 and stained as described 3–4 h after microinjection. Tubulin was detected using an anti–β-tubulin mAb (Nycomed Amersham) and mitotic epitopes were detected using the MPM2 mAb (Upstate Biotechnology, Inc.). To detect myc-cdc25B2, pCDNA3/myc-cdc25B2 was microinjected (0.1 μg/μl) and cells were fixed with methanol/acetone (1:1) 3 h after injection and stained with the mAb 9E10 (gift of Erich Nigg, University of Geneva, Geneva). A Cy5 conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) was used as the secondary antibody. Cells were analyzed by confocal laser scanning microscopy using a Bio-Rad 1024 confocal microscope set on 10% laser power and Kalman averaging. Stacks of images were projected using Lasersharp software (Bio-Rad Laboratories) and exported to Adobe Photoshop for processing and printing. Human cdc25B2, cdc25B3, cdc25C, and cyclin B1 were in vitro translated from pBSK/cdc25B cDNA using the TNT-coupled reticulocyte system (Promega Corp.). GST-cyclin B1, GST-Cdc25B2, GST-Cdc25B3, and GST were expressed in BL21(DE3) cells using the pGEX-4T expression vector and purified on GSH-Sepharose. To assay the role of the two types of mitotic Cdc25 phosphatases in the initiation of mitosis, expression constructs encoding Cdc25B or Cdc25C, or the equivalent GFP-chimera were introduced into synchronized HeLa cells by microinjection. The GFP-chimeras became visible about 3 h after microinjection, and comparing the fluorescence between GFP-Cdc25B– and GFP-Cdc25C–expressing cells showed that these proteins are expressed to similar levels (data not shown). The behavior of the cells was followed by DIC microscopy and the cell cycle phase was determined by flow cytometry and by incorporation of BrdU. In these experiments, we primarily used the full-length Cdc25B3 clone that is found in all cells , but we detected only minor differences between the behavior of this and the other two forms of Cdc25B (data not shown). Using the GFP-chimeras we were able to estimate the half-life of the proteins in cycloheximide–chase experiments and found that there was little or no difference in their turnover rate compared with the endogenous proteins (data not shown). We found that overexpressing GFP-Cdc25B in early S phase rapidly caused cells to round up and break down their nuclear envelope . Staining the cells with Hoechst 33342 showed that the chromatin had condensed abnormally and anti–β-tubulin immunofluorescence staining revealed the presence of mini-spindles as previously described . This suggested that the cells had entered mitosis prematurely rather than undergone apoptosis. To confirm this, we stained cells with the MPM2 mAb that recognizes mitosis-specific phospho-epitopes and for the apoptosis-specific surface marker annexin V. Cells expressing Cdc25B with abnormally condensed chromatin stained very strongly with the MPM2 antibody but not for annexin V (data not shown). Thus, we concluded that Cdc25B caused cells to enter mitosis prematurely (premature chromosome condensation, PCC) rather than undergo apoptosis. The GFP tag was not responsible for this effect because the untagged Cdc25B cDNA induced premature mitosis with a similar frequency to GFP-Cdc25B (not shown) and an inactive mutant of Cdc25B that lacked the catalytic cysteine residue (C488S) could not induce premature mitosis (not shown). Coinjecting a cyclin B1 expression vector with Cdc25B had only a minor effect on the frequency of premature mitosis . In these experiments, cells were released from an aphidicolin block and, therefore, progressed through S phase in the course of the experiment. We also wished to assay the effect of overexpressing cdc25B in cells in which the unreplicated DNA checkpoint was strongly activated. Therefore, we microinjected cells with the Cdc25B expression construct and released them from the aphidicolin block in the presence of hydroxyurea. Under these conditions cells progressed only very slowly through S phase as determined by flow cytometry (not shown). We found that Cdc25B efficiently induced PCC in these cells, and, in this case, coexpressing cyclin B1 greatly increased the frequency of PCC . The effect of overexpressing Cdc25C in HeLa cells was markedly different from that of overexpressing Cdc25B; Cdc25C could not induce PCC when overexpressed alone. Cdc25C was only able to cause a significant fraction of cells to enter mitosis prematurely when cyclin B1 was also overexpressed. Furthermore, overexpressing Cdc25C and cyclin B1 could only cause PCC after cells had entered the G2 phase , not in the S phase cells. The unreplicated DNA and G2 phase DNA damage checkpoints prevent mitosis, at least in part, by acting on Cdc25C. Unreplicated DNA activates the Cds1 and Chk1 kinases; Chk1 is also activated by DNA damage. Both kinases phosphorylate Cdc25C on serine 216 to create a binding site for a 14-3-3 protein that inactivates Cdc25C. Therefore, to test whether Cdc25C was less potent than Cdc25B because it was downregulated by Cds1 and/or Chk1, we used a Cdc25C mutant that cannot be phosphorylated on serine 216 (S216G). Cdc25C S216G was more efficient than wild-type Cdc25C in causing PCC, but again this was only when cyclin B1 was coexpressed . Therefore, downregulation by Cds1 and/or Chk1 is not sufficient to explain the difference in potency between Cdc25B and Cdc25C. The potent ability of cdc25B to activate cyclin B1 and induce premature mitosis raised the possibility that cdc25B might be able to force cells into mitosis before they had begun to replicate their DNA. At this stage in the cell cycle, the proteolysis machinery that degrades the mitotic cyclins is still active , therefore, we microinjected Cdc25B expression constructs with and without a cyclin B1 mutant (R42A) that was resistant to degradation. We injected the constructs into early G1 cells that had just flattened out after cytokinesis. We found that overexpressing GFP-cdc25B alone could not force cells into mitosis, but coexpressing the nondegradable form of cyclin B1 caused PCC in ∼50% of the injected cells 6 h after injection. This would normally correspond to the middle of G1 phase . Furthermore, overexpressing Cdc25C, with or without cyclin B1, or cyclin B1 alone did not force G1 phase cells into premature mitosis . The balance between the inhibitory kinases of cdc2, Wee1, and Myt1, and Cdc25C phosphatase is crucial to the timing of mitosis . Therefore, we coinjected varying amounts of expression constructs for Cdc25B and for Wee1 kinase to see whether Wee1 could counteract the ability of Cdc25B to cause PCC. We found that Cdc25B induced PCC in a dose-dependent manner and that Wee1 could rescue Cdc25B-expressing cells, again in a dose-dependent fashion . We considered the possibility that the differences in the abilities of Cdc25B and Cdc25C to force cells into mitosis might be related to the differences in their localization. Human Cdc25C is usually found to be a nuclear protein and our Cdc25C chimera is also nuclear throughout interphase . Cdc25B has been previously described as a cytoplasmic protein that colocalizes with centrosomes and becomes nuclear at the end of the G2 phase . To verify that the GFP-Cdc25B chimeras localized correctly, we compared their localization with a myc epitope-tagged Cdc25B, reasoning that the nine amino acids of the myc epitope would be unlikely to perturb the localization of Cdc25B. HeLa cells were synchronized in the G1, S, and G2 phases and the myc-tagged Cdc25B protein localization was determined by immunofluorescence after microinjection of a myc-cdc25B expression construct . Cells expressing the GFP-cdc25B chimera were followed by time-lapse fluorescence microscopy at different stages of the cell cycle. We found that Cdc25B was nuclear in the G1 phase and gradually moved from the nucleus to the cytoplasm as cells progressed through S phase. Unfortunately, we were unable to visualize the endogenous Cdc25B by immunofluorescence, but we were able to confirm that Cdc25B was substantially nuclear in G1/S phase cells and accumulated in the cytoplasm by immunoblotting nuclear and cytoplasmic fractions through the cell cycle (not shown). These results suggested that Cdc25B might be exported from the nuclei of S and G2 phase cells. Most nuclear export pathways reported so far depended on exportin 1/Crm1, which can be inactivated by leptomycin B (LMB) . Therefore, we added LMB to cells expressing GFP-cdc25B in G2 phase and found that LMB caused GFP-cdc25B to accumulate in the nucleus within 30 min , indicating that the localization of Cdc25B is regulated by nuclear export. Although the cytoplasmic localization of Cdc25B in S and G2 phases depended on Crm1-mediated nuclear export, Cdc25B was nuclear in the G1 phase. The cytoplasmic localization of Cdc25B correlated with the point in the cell cycle when cyclin B1 started to accumulate and cyclin B1 has been shown to be exported from nuclei in an LMB-sensitive manner . Taken together these observations suggested that cyclin B1 might be involved in mediating the cytoplasmic localization of Cdc25B. To test this hypothesis, we expressed GFP-cdc25B in early G1 phase or early S phase cells (i.e., cells that lack any endogenous cyclin B1), and then microinjected purified cyclin B1 or cyclin B1/CDK1 K33R protein into the nucleus. GFP-cdc25B was immediately and rapidly exported from G1 (not shown) or S phase nuclei after injecting cyclin B1/CDK1 K33R and reentered the nuclei when LMB was added to inhibit export . This effect was observed using cyclin B1 in a complex with a kinase-dead mutant of CDK1, which strongly suggested that cyclin B1 itself and not CDK1 kinase activity was required for cdc25B export. In support of this, we found that GFP-cdc25B still accumulated in the cytoplasm of G2 phase cells in the presence of the CDK1 inhibitor, roscovitin (data not shown). Furthermore, a cyclin B1 mutant with a defective nuclear export signal, cyclin B1 F146A , did not cause nuclear Cdc25B to be exported when microinjected into G1 or S phase nuclei (not shown). These observations suggested that Cdc25B export could be due to a direct interaction between cyclin B1/CDK1 and Cdc25B. In support of this, we were able to detect an in vitro interaction between Cdc25B and cyclin B1 that appeared to be slightly stronger than an association between cyclin B1 and Cdc25C . In this paper, we have used time-lapse microscopy to show that Cdc25B and Cdc25C differ markedly in their abilities to induce mitosis. Our assay allowed us to determine the effects on progress through the cell cycle of overexpressing specific proteins at defined points in the cell cycle. In this way, we were able to avoid the problems of trying to analyze the effects of overexpressing proteins by transfection in a heterogeneous population of cells that must be fixed for immunofluorescence at arbitrary time points. We have shown that simply overexpressing Cdc25B is sufficient to induce premature mitosis when there is cyclin B1 in the cell regardless of the replication state of the DNA. In contrast, Cdc25C will only cause premature mitosis when it is overexpressed with cyclin B1 and this does not override the negative signal generated by unreplicated DNA. We have also shown that the subcellular localization of Cdc25B changes through the cell cycle and is regulated by nuclear export, most likely by binding to cyclin B1. Our demonstration that overexpressing Cdc25C alone cannot overcome the unreplicated DNA checkpoint, but that Cdc25B can, suggests that Cdc25C and Cdc25B are regulated in different ways. Cdc25C appears to require a further activation step that can be partially supplied by increasing the level of cyclin B . Indeed, an extra copy of cyclin B (NIME) can partially suppress a mutation in cdc25 (NIMT) in Aspergillus nidulans . This activation step is most likely to be phosphorylation of the amino terminus of Cdc25C, which activates its phosphatase activity ∼10-fold in vitro, and cyclin B/Cdk1 is able to phosphorylate and activate Cdc25C . More recently, members of the polo-like family of kinases have been shown to phosphorylate and activate Cdc25C , but it is unclear whether they initiate Cdc25C activation at the end of G2 phase. In this regard, we were unable to activate prematurely Cdc25C by coexpressing human plk1 (data not shown), although this may be because plk1 itself needs to be activated by phosphorylation . Our data show that Cdc25C activation is either prevented, or rapidly reversed, in the presence of unreplicated DNA. The inhibition of Cdc25C may be partially effected through phosphorylation on S216 and the consequent binding of a 14-3-3 protein . However, this cannot provide a full explanation, because we found that although an S216G mutant form of Cdc25C is able to promote premature mitosis more rapidly than the wild-type Cdc25C, it is unable to overcome the DNA replication checkpoint. In contrast, it appears that Cdc25B either does not need an activation step or that the activator is present whenever cyclin B1 is present. Furthermore, cyclin B/Cdk1 phosphorylates Cdc25B in vitro and this correlates with a fourfold increase in Cdc25B activity . One explanation could be that cyclin B/CDK1 activates Cdc25B and vice versa in a positive feedback loop, and that this is more potent than the positive feedback loop between Cdc25C and cyclin B1/CDK1 because Cdc25B and cyclin B1/CDK1 are both cytoplasmic. If Cdc25B and cyclin B do activate one another, then the resulting positive feedback loop must be carefully regulated because overexpressing Cdc25B is sufficient to cause premature mitosis, regardless of whether the DNA has been replicated. One way in which Cdc25B might be regulated is by protein turnover. Cdc25B is an unstable protein, with a half-life of <30 min in hamster and HeLa cells , which can be targeted for degradation in vitro by cyclin A-CDK2 . Thus, Cdc25B may be primarily regulated by ubiquitin-mediated proteolysis in an analogous fashion to cdc25 in fission yeast . Remarkably, we found that when Cdc25B and cyclin B1 were coexpressed in G1 phase cells the cells attempted to enter mitosis, showing that cell division and DNA replication can be uncoupled in human cells. This may also be relevant to how the cell causes two sequential M phases in meiosis and it will be interesting to determine whether Cdc25B is required for gametogenesis. Despite the differences in their regulation, both Cdc25B and Cdc25C are antagonized by Wee1. Wee1 and Cdc25C are both nuclear proteins and, therefore, their relative activities will determine whether the nucleus will activate or inhibit cyclin B/CDK1 when it is imported into the nucleus. However, Cdc25B is primarily a cytoplasmic protein in the G2 phase and so might act on the predominantly cytoplasmic pool of cyclin B/CDK1. Therefore, Wee1 may only counteract the effects of Cdc25B when active cyclin B1/Cdk1 shuttles into the nucleus . Human Cdc25B was originally described as being a cytoplasmic protein that translocated to the nucleus at mitosis in concert with cyclin B . However, these studies were performed by immunofluorescence in which the exact cell cycle stage of an individual cell was often difficult to judge, except by the appearance of condensed chromosomes. We have shown here that Cdc25B is nuclear in G1 cells, but gradually accumulates in the cytoplasm as cells progress through the S and G2 phases. Furthermore, Cdc25B will accumulate in the nucleus when S or G2 phase cells are treated with LMB, a specific inhibitor of crm1/exportin1–mediated nuclear export . This suggests that the localization of Cdc25B, like that of cyclin B1 , is primarily determined by nuclear export. However, Cdc25B does not have a recognizable nuclear export signal and is nuclear in the G1 phase. When we microinjected wild-type cyclin B1 into the nuclei of G1 cells, we observed that cdc25B was immediately exported, but not when we microinjected an export-defective cyclin B1. Furthermore, we found that cyclin B1 binds to Cdc25B in an in vitro binding assay. These results suggest that the cytoplasmic localization of cdc25B depends upon cyclin B1, and that the two proteins are likely to be exported together from the nucleus. This is reminiscent of recent results showing that fission yeast cdc25C is exported from the nucleus after DNA damage by binding to the rad24 14-3-3 protein . In both cases, the Cdc25 protein does not have a nuclear export signal itself, but is exported by virtue of its association with another export-capable protein. In conclusion, we have demonstrated marked differences in the abilities of Cdc25B and Cdc25C to promote mitosis and in their cell cycle behavior. Our results suggest that controlling the level of Cdc25B is crucial to prevent premature mitosis, whereas Cdc25C can be regulated at a subsequent step, most likely by phosphorylation. The activity of Wee1 in the nucleus is very important in counteracting the effects of both the nuclear Cdc25C and the primarily cytoplasmic Cdc25B, emphasizing the dynamic nature of the interactions between cell cycle components that shuttle between the nucleus and the cytoplasm. | Study | biomedical | en | 0.999995 |
10444067 | To construct a γ-tubulin/green fluorescence protein (γTGFP) expressing plasmid we started with a full-length human γ-tubulin sequence . The original stop codon was destroyed and a 5′-terminal BamHI and a 3′-terminal HindIII site were introduced by PCR. The resultant fragment was then cloned into pcDNA3 vector (CLONTECH Laboratories, Inc.). The S65T variant of GFP (CLONTECH Laboratories, Inc.) was fused to the γ-tubulin COOH terminus. This resulting plasmid was designated as pcDNA3–γTGFP. PtK 1 (rat kangaroo kidney epithelial) cells were purchased from American Type Culture Collection at passage 69 and grown in antibiotic-free Ham's F12 media supplemented with 10% FCS. At passage 80, cells were transformed with the pcDNA3–γTGFP plasmid by electroporation. γTGFP-expressing clones were initially isolated by G418-resistance selection (1 mg/ml). Of these clones, several were then selected for the lowest expression level that still yielded a sufficient GFP-fluorescence signal for time-lapse microscopy with a Photometrics PXL cooled CCD camera. This strategy enabled us to avoid potential abnormal phenotypes due to γ-tubulin overexpression . Three selected clones (PtKG-22, PtKG-23, and PtKG-36) were finally purified by limited-dilution cloning on a feeder layer of wild-type PtK 1 cells. All three of these clones appear to be stable and continue to express γTGFP (as judged by centrosome-associated GFP signal) after >20 passages in the absence of G418. All three clones behaved identically in the experiments described in this paper. A similar strategy was used to isolate clones constitutively expressing γTGFP from PK (pig kidney epithelial) and CV-1 (green monkey kidney fibroblasts). All experiments were conducted on cells grown on #1 1/2 coverslips mounted in Rose chambers in L-15 media, as previously described . Cells were kept at 37°C using a custom built microscope stage heater . For experiments involving Mt disassembly, Rose chamber cultures were treated with 4 μM nocodazole 1 h before observations. Near simultaneous GFP fluorescence/DIC time-lapse sequences were collected using a custom-modified Nikon Optiphot microscope equipped with De Senarmont compensation long-working-distance DIC optics (60× 1.4 NA PlanApo lens), a Quad-Fluor epifluorescence attachment (Nikon, Inc.), a stepping motor for Z-positioning (Ludl Electronics), and a Photometrics PXL cooled CCD camera (Photometric). The microscope system was driven by Isee software (Inovision Corp.), and images were recorded as 12-bit computer files . The intensity of brightest pixels in the fluorescence images were kept at <600, which guaranteed that none of the images were saturated. The CCD chip was read out at 800 kHz with an electronic gain of four, which assured a linear correspondence between the well-charge and light intensity for the PXL camera. To capture the full in-focus intensity for centrosomes that move in all three axes (X, Y, and Z) within a living cell, the GFP image for any one time point was collected as Z-series of 16 images at 0.5-μm steps. From these Z-series, a single maximal intensity projection was computed for each individual time point. These computations were done concurrent with image collection and only the resultant maximal intensity projections were saved to the disk and subsequently used for image analysis. The DIC images were acquired at the focal plane corresponding to the middle of the GFP Z-sequence. All images were corrected using standard algorithm , 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*}{\mathit{Ic}}=\frac{ \left \left({\mathit{Ir}}-{\mathit{Ib}}\right) \right {\cdot}{\mathit{M}}}{{\mathit{If}}-{\mathit{Ib}}}\end{equation*}\end{document} where Ic is the corrected image, Ir is the noncorrected or raw object exposure, Ib is an electronic or dark background frame obtained with the shutter closed, M is the mean pixel value of the object exposure, and If is flat field image obtained with no specimen, but a homogeneous fluorescent field. To measure the amount of γTGFP associated with the centrosome, a circle of 20 pixels in diameter (Ø1.75 μm) was centered on the centrosome and the sum of pixel intensities was calculated. The results of these intensity measurements were normalized so that the highest value for the centrosome was equal to ten while the background intensity outside of the cell was zero. For fluorescence imaging, cells were illuminated with light from a 75 W xenon burner that was filtered with a GG400 (to eliminate UV), a KG5 (to eliminate IR), and a 4× or 8× ND filter to decrease light intensity to a level safe for the cells. The DM505 filter cube (450–490 nm excitation − 520–560 nm emission; Nikon, Inc.) was used for GFP detection. For DIC imaging, cells were illuminated with light from a 50 W tungsten filament, filtered with GG400, KG5, and GIF546 (green) filters. Both the fluorescence and DIC light sources were shuttered by UniBlitz shutters (Uniblitz Electonics) so that cells were illuminated only during image acquisition (200 ms/frame for GFP and 600 ms/frame for DIC mode). Under these conditions, we were able to follow centrosomes in interphase cells for more than six hours at a framing rate of one fluorescence sequence (i.e., 16 frames × 200 ms = 3.2 s total illumination) every 2.5 min without detectable photobleaching. We find, as reported by others , that vertebrate somatic cells are extremely sensitive to illumination, even with monochromatic light, and that overillumination can forestall progression through the cell cycle and even send cells in prophase back to interphase (see Results). For photobleaching experiments, we used a continuous-wave argon ion laser . The output light was filtered by a laser-quality 488-nm interference filter and extended using a 10× beam-extender. The 12-mm-diam beam was directed to the back aperture of the lens through a custom-made additional epiport. The objective lens then focused the beam into a small spot (∼1.5 μm) within the specimen plane. This approach allows one to photobleach individual objects 1–2 μm in diam with minimal light exposure to the surroundings. In our experiments, we were able to photobleach one of two replicated centrosomes that were separated by ∼5 μm with no detectable decrease in the signal intensity associated with the other centrosome. For photobleaching, the light intensity was empirically adjusted so that an ∼5–10 s exposure completely abolished the GFP signal associated with the centrosome. Under this condition, the centrosomes always recovered after photobleaching and the cells eventually entered mitosis and formed a normal spindle (see Results). For immunostaining, cells were briefly rinsed in warm (∼37°C) PEM buffer (100 mM Pipes, pH 6.9, 5 mM EGTA, 1 mM MgCl 2 ), permeabilized for 30 s in PEM with 0.1% Triton X-100, and fixed in 1% glutaraldehyde in PEM. After fixation, free aldehyde groups were reduced by a 5-min incubation in NaBH 4 (1 mg/ml). γ-Tubulin was stained using a mouse mAb (clone GTU-88, Sigma Chemical Co.) and a TRITC-conjugated goat anti–mouse IgG secondary antibody (Sigma Chemical Co.). α-Tubulin was stained using a rat mAb (clone YL1/2; kind gift of Dr. J.V. Kilmartin, MRC, Cambridge, UK) and an FITC-conjugated donkey anti–rat IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Since two separated centrosomes are often located in different focal planes, all fluorescence images were collected as Z-series (200-nm steps) on the same microscope workstation used for GFP-imaging. These datasets were then deconvolved using Delta Vision deconvolution software (Applied Precision Inc.) and presented as maximal intensity projection. We have established several stable cell lines that constitutively express the γTGFP. Among them are clones isolated from two different epithelial cell lines, including PtKG (from the parental PtK 1 , rat kangaroo kidney) and PKG (from the parental PK, pig kidney), as well as a CVG fibroblastic cell line (from parental CV-1, green monkey kidney). In this report, we illustrate our findings primarily with video sequences obtained from PtKG cells. However, without exception, the same results were obtained from CVG and PKG cells. The constitutive expression of low γTGFP levels is not toxic to the cells. All of our γTGFP expressing lines exhibited growth rates similar to the parental cell lines. The mitotic index and percent of multinucleated cells and abnormal (multipolar) spindles all appeared similar to those of the parental cell lines. When expressed in mammalian cells, γTGFP associates with the centrosome and fluorescently labels this organelle throughout the cell cycle. In interphase cells, the γTGFP-labeled centrosome appeared as two fluorescent dots that varied widely in their separation . Interphase cells with widely separated centrosomes were common in PKG and CVG cells, but less abundant in PtKG cells. Previous correlative LM/EM observations have shown that each of these dots contains either a single centriole (G 1 ) or a pair of centrioles (G 2 ). For any one interphase cell, the γTGFP fluorescence intensity of each of the two dots was usually fairly similar. However, in some cells, they differed substantially and one of the dots could contain up to twice as much γTGFP as the other. In time-lapse sequences, these dots were motile, often seen to separate and then reform a common complex, only to separate again several times during the period of observation. The γTGFP fusion protein was excluded from the nucleus, but was present in significant quantities in the cytoplasm of all cells . This observation is consistent with the biochemical analyses of Moudjou et al. 1996 , which revealed that only 20% of the γ-tubulin in cells is associated with the centrosome, whereas ∼80% remains in the cytoplasm. We followed interphase cells by quantitative time-lapse imaging for up to 20 h ( n = 20). In all cases, the intensity of the γTGFP signal associated with the centrosome remained roughly constant throughout the observation period (data not shown). As cells entered mitosis, as defined by the initiation of chromosome condensation, the intensity of the γTGFP signal did not change significantly . However, 20–30 min before nuclear envelope breakdown the γTGFP signal associated with the centrosome suddenly began to increase . It then reached its maximum level, greater than three times that seen during early prophase, shortly after nuclear envelope breakdown, which can be clearly defined in these cells as the point when γTGFP entered the previously nonfluorescent nuclear volume . In vertebrates, the duration of spindle formation, as defined by the interval between nuclear envelope breakdown and anaphase onset, is highly variable and depends on how long the cell contains monooriented chromosomes . In cells in which spindle formation was prolonged, the γTGFP signal remained at its peak level until anaphase onset, which took one or more hours . As soon as the cell initiated anaphase, the γTGFP content of both centrosomes progressively decreased until it reached a minimal level after cytokinesis . At this point in G 1 , the γTGFP content of the centrosome was ∼50% of what it was during the previous G 2 , before its mitotic activation . Thus, from early G 1 until late G 2 , the γTGFP content of the centrosome increased only about two times, in contrast to the sudden greater than three times increase during prophase. In addition to an increased intensity of γTGFP, the apparent diameter of the centrosome also increased during spindle formation . Shortly after nuclear envelope breakdown, and after the central part of the centrosome reached its maximal intensity, the γTGFP signal continued to progressively accumulate around the centrosomal periphery. As a result, the area occupied by the centrosome in metaphase, as defined by a γTGFP intensity similar to that of an interphase centrosome, was much larger than that seen at nuclear envelope breakdown. Here it is noteworthy that our measurements of the γTGFP amount associated with the centrosome, as presented in Fig. 3 , only account for the γTGFP signal contained within the central part of the centrosome (defined by a 1.75-μm-diam circle) and, therefore, underestimates the total amount of γTGFP recruited during mitosis. We chose to use the same-size cursor for both interphase and mitotic centrosomes because it was impossible to define the exact boundary of the centrosome during the later stages of spindle formation/maturation. As the spindle matured, the γTGFP signal extended from each centrosome into its associated half-spindle so that at anaphase onset both half-spindles also contained a γTGFP intensity equivalent to that of an interphase centrosome . After the chromatids disjoined the γTGFP, signal in the spindle rapidly decreased to background levels by late anaphase . In some cells, we observed an accumulation of the γTGFP signal at the ends of midbody Mts during cytokinesis . This phenomenon was regularly seen in PKG cells, but only rarely in PtKG and CVG cells. Is the sudden increase of centrosome-associated γTGFP during prophase mediated by Mts? To answer this question, we followed cells by time-lapse microscopy ( n = 10) as they entered mitosis under conditions in which they lacked Mts (4 μM nocodazole for 1 h). As expected, this treatment inhibited centrosome movement and spindle formation . However, it did not inhibit the sudden accumulation of γTGFP at the centrosome as the cell progressed through prophase . This accumulation occurred with kinetics similar to those seen in untreated cells , and the γTGFP level remained maximal for as long as the cell was blocked in mitosis . Thus, the recruitment of γTGFP to the centrosome during mitosis does not depend on the presence of Mts. Unlike mitosis in control cells, the γTGFP signal in nocodazole treated cells remained closely associated with the centrosome, and, after reaching a maximal level, it did not continue to accumulate around the centrosomal periphery . When vertebrate cells in prophase are excessively irradiated, they decondense their chromosomes and return to G 2 . In PtK 1 cells, this reversal of the cell cycle is correlated with the dephosphorylation of those epitopes phosphorylated during the nuclear events of prophase . In some instances, the prophase cells that we were following by time-lapse LM decondensed their chromosomes and returned to interphase ( n = 4), which we assume was due to excessive illumination . Under this condition, we found that this reversal of the cell cycle can occur even after the centrosomes have recruited near maximal levels of γTGFP . During the reversion process, the chromosomes decondensed and the amount of γTGFP associated with the centrosomes progressively decreased to typical G 2 levels . It then remained at this level for as long as the cell was blocked in G 2 . At this point, our data clearly demonstrated that centrosomes suddenly recruit additional γTGFP at the G 2 /M transition, and that this γTGFP is then lost as the cell exits mitosis. This finding raises the question as to whether centrosome-associated γTGFP is in continuous exchange with a cytoplasmic pool during interphase and mitosis. To examine this issue, we performed FRAP studies on γTGFP-labeled centrosomes. For these studies, we chose cells with widely separated centrosomes so that we could follow fluorescence recovery of the experimental centrosome in the presence of an internal control. In all cases, when one of the centrosomes was photobleached the fluorescence intensity of the remaining centrosome remained unaffected. To determine if our photobleaching protocol causes functional damage to the irradiated centrosome, we photobleached one of two separated centrosomes in cells treated with 4 μM nocodazole for 2 h before the experiment. This nocodazole concentration completely depolymerizes Mts. Immediately (less than one minute) after photobleaching, the cells were washed in a large volume of warm culture media for about three minutes, and then fixed and immunostained for γ-tubulin and α-tubulin (to visualize Mts). In all cases, the photobleached centrosome was found to contain a normal amount of γ-tubulin and was capable of nucleating the same number of Mt as the nonirradiated centrosome in the same cell . Thus, based on functional criteria, our photobleaching protocol does not damage the centrosome. We found that when a centrosome was photobleached during interphase, it rapidly recovered ∼50% of its original signal intensity over a 60 min period . It then remained at this level for several hours. In some cases ( n = 7), the photobleached centrosome eventually recovered to its original intensity 5–6 h after photobleaching. However, some cells ( n = 4) entered mitosis before the slow phase of recovery was completed, and when this occurred, the γTGFP content of both centrosomes increased with normal kinetics (data not shown). The same recovery curves were observed when photobleaching was performed on interphase cells treated with 4 μM nocodazole (data not shown). Together, these data reveal that two populations of γTGFP are associated with an interphase centrosome: one that turns over relatively rapidly and another that exchanges very slowly. Importantly, this dynamic exchange does not require the presence of Mts. We could not determine the FRAP characteristics of centrosomes that were photobleached during mitosis. This was because the recovery process was superimposed on natural intensity changes that occurred in the centrosome as the cell progressed through mitosis. Therefore, we conducted this experiment on centrosomes in nocodazole-arrested mitotic cells ( n = 7). Under this condition we found that, as during interphase, the centrosome recovered ∼50% of its intensity 30–40 min after photobleaching . However, in contrast to interphase centrosomes, mitotic centrosomes fully recovered to their original intensity within 60–90 min of photobleaching and then remained at that level as long as the cell was blocked in mitosis . Although the centrosome can be discerned by video-enhanced DIC microscopy in living cells during mitosis, it cannot be distinguished in interphase with certainty from small granules and vacuoles that appear similar in size and contrast. As a result, it is seldom possible to follow the dynamic behavior of this organelle in living cells. With the introduction of GFP-labeling, the position and boundary of the centrosome can now be clearly defined in vivo which, in turn, greatly facilitates studies on centrosome function and behavior (including the isolation of glowing centrosomes). For example, using GFP-labeled γ-tubulin, Ueda et al. 1997 have shown that the centrosome in Dictyostelium does not direct cell migration. Similar methods were also used to demonstrate unique structural changes within the Dictyostelium centrosome as it duplicated and separated during mitosis . To investigate how centrosomes behave during the cell cycle in vertebrates, we have established several cell lines in which this organelle is clearly delineated by γTGFP. As a rule, overexpression of γ-tubulin in vertebrates leads to gross defects and a loss of cell viability . However, modern low-light-level CCD cameras are sensitive enough to detect very few GFP molecules . By selecting cells expressing only a low-level of the fusion protein, we were able to establish clones that appear normal in every aspect. Thus, substituting at least part of the centrosome's endogenous γ-tubulin with our γTGFP does not deleteriously affect the ability of the centrosome to function normally throughout the cell cycle. The cell cycle-specific redistribution of γTGFP, including its enhanced association with mitotic centrosomes, as well as its transient association with the spindle and midbody, are all consistent with previous immunofluorescence studies of fixed cells . In vertebrates, it has been shown that Mts generated by the centrosome can detach and move away from their site of nucleation . This shedding of Mt minus ends has also been observed in some neuronal cells and grasshopper spermatocytes . Since free Mt minus ends are not stable, it is likely that they are stabilized transiently, perhaps by a γ-TuRC cap. If this is true, then centrosome-associated γ-tubulin must exist in dynamic exchange with a cytoplasmic pool. In this context, only ∼20% of the γ-tubulin within a cell is associated at any one time with the centrosome while the remainder resides in the cytoplasm . In addition, the biochemical properties of centrosomal and cytoplasmic γ-tubulin appear to be similar, and, in both cases, the γ-tubulin exists in large complexes, whose exact composition remains to be determined . Using FRAP methodology, we have directly tested the idea that centrosome-associated γ-tubulin is in dynamic exchange. We found that when the γTGFP associated with an interphase centrosome is photobleached, the centrosome recovers ∼50% of it original intensity relatively rapidly (within 60 min), but that the remainder of the photobleached γ-tubulin takes much longer to turn over (greater than five hours). Thus, the centrosome contains two distinct populations of γ-tubulin: one that rapidly exchanges with the cytoplasmic pool and one that is more stable. It is tempting to speculate that the stable population represents γ-tubulin that is allied with the centrioles, while the rapidly exchanging γ-tubulin resides in the pericentriolar material. This is consistent with immunoelectron microscopy data demonstrating γ-tubulin association with the core of centrioles and biochemical studies showing that half of the γ-tubulin is tightly associated with isolated centrosomes, while the other half can be easily extracted . Our FRAP observations on nocodazole-treated cells reveal that the dynamic exchange of centrosome-associated γ-tubulin occurs even when Mts are not present. This is a surprising finding since, based on prior studies , one would have predicted that the rate at which centrosomal γ-tubulin exchanges should depend on Mt dynamics. However, our data clearly reveal that the dynamic exchange of γ-tubulin is not caused by the constant loss of γ-tubulin leaving the centrosome on the tips of released Mts. Instead, centrosomes intrinsically shed γ-tubulin regardless of whether it is associated with the end of a Mt. We also found that the kinetics of exchange do not differ significantly between interphase and mitotic centrosomes, i.e., that the exchangeable population turns over completely within one hour. However, within this time, mitotic centrosomes fully recover their fluorescent intensity, whereas the intensity of interphase centrosomes only recovers to ∼50%. This could mean that mitotic centrosomes no longer possess a nonexchangeable fraction of γ-tubulin. However, an equally plausible explanation is that the nonexchangeable population represents a minor fraction of centrosome-associated γ-tubulin during mitosis. Considering that the γ-tubulin content of the centrosome increases at least threefold at the onset of mitosis, assuming that all of this excess is exchangeable, then the nonexchangeable signal would become diluted to the point that it is no longer detectable by our methods. Early immunofluorescence studies on the distribution of γ-tubulin noted that more of this protein is associated with mitotic than interphase centrosomes . This difference in γ-tubulin content correlates with the fact that mitotic centrosomes generate about five to ten times more Mts than interphase centrosomes . When does the centrosome acquire its additional γ-tubulin so that it can generate enhanced numbers of Mts during mitosis? One possibility is that γ-tubulin gradually accumulates in the centrosome during the cell cycle, but it is maintained in an inactive form until spindle formation. The other possibility is that it is suddenly recruited to the centrosome near the onset of mitosis. The former hypothesis has recently been supported by Dictenberg et al. 1998 who concluded, from an immunofluorescence analysis of fixed synchronized CHO cells, that the amount of pericentrin and γ-tubulin associated with the centrosome gradually increases from G 1 until mitosis. Our results on living cells are not consistent with this conclusion. Instead, we find that the γ-tubulin content of each centrosome, at best, doubles during interphase, and then suddenly increases more than three times as cells progress through prophase. We also demonstrate that this sudden increase occurs even in the absence of Mts. This means either that the centrosome suddenly acquires the ability to bind more γ-tubulin, or that a sudden global biochemical change within the cell modifies cytoplasmic γ-tubulin so that it binds more efficiently to the centrosomal lattice. Since our FRAP data reveal that recovery occurs with similar kinetics during interphase and mitosis, the affinity of γ-tubulin for the centrosome does not appear to change significantly between these two phases of the cell cycle. Thus, the sudden recruitment of γ-tubulin to the centrosome during prophase is due to changes that occur within the centrosome that allow it to bind more γ-tubulin. Our results are not inconsistent with the idea that the centrosome, as a structural entity, grows throughout the cell cycle by the gradual accumulation of constituents (e.g., pericentrin). However, our data demonstrate clearly that a key functional component required for enhancing the Mt-nucleating potential of the centrosome during mitosis appears suddenly as the centrosome becomes activated at the G 2 /M transition. The fact that this process occurs normally in nocodazole-treated cells reveals that the Mt-nucleating potential activity of the centrosome is linked directly to the stage of the cell cycle and not to the functional state of its associated Mt array. This is consistent with accumulating data linking centrosome activation at the G 2 /M boundary with the phosphorylation of various centrosomal components by CDK1, Polo, and other kinases that regulate progression through the cell cycle . In this context, we also note that cells can be induced to return to G 2 , even after their centrosomes have been activated, as defined by an enhanced accumulation of γ-tubulin. This means that the sudden accumulation of γ-tubulin at the centrosome is not an event that commits the cell to mitosis, i.e., that the cell cycle checkpoint leading to the reversal of prophase can still operate, even after the centrosomes have been activated . While the initial recruitment of γ-tubulin to the centrosome at the G 2 /M transition is independent of Mts, the presence of Mts leads to subsequent changes in the distribution of γ-tubulin in mitotic cells. We found that, as the spindle formation proceeds, γ-tubulin continues to accumulate around the centrosomes and subsequently spreads into the spindle. This increased γ-tubulin content of the spindle, which has been noted by others on fixed cells , appears in time-lapse recordings to be derived from the centrosome. The accumulation of γ-TGFP in the spindle occurs after the centrosome has reached its maximum fluorescence intensity and is restricted during the initial stages of spindle formation to those parts of half-spindle immediately adjacent to the centrosome . Only later, as the spindle becomes compacted during metaphase , does it extend to permeate each half-spindle. This changing pattern of γ-tubulin distribution may arise as each centrosome sheds Mts, capped by γ-tubulin, into its associated half-spindle. Alternatively, the recruitment of γ-tubulin to the spindle may be independent of the centrosome and/or Mt minus ends . Regardless of why the spindle accumulates γ-tubulin, our observations clearly demonstrate that this phenomenon occurs progressively as the spindle matures, and its progress can even be used to distinguish old from young metaphase spindles . Our data on living cells also confirm previous reports that γ-tubulin becomes transiently associated with the ends of midbody Mts after cytokinesis. For example, Julian et al. 1993 found that some, but not all, of the midbodies in fixed cell populations labeled with anti–γ-tubulin antibody. They interpreted this to mean that γ-tubulin associates with all midbodies, but only transiently. Our observations, however, suggest that γ-tubulin may become associated with the midbodies in some, but not all, cells and even that this phenomenon may be cell-type specific. Whereas most of the midbodies in our PKG cells contain elevated levels of γ-TGFP, the γ-TGFP content of midbodies in the majority of our CVG and PtKG cells is seldom above background. Importantly, these cells complete cytokinesis normally. Midbody Mts are thought to be derived during anaphase from the centrosomes . As a result, the accumulation of γ-tubulin at the midbody may be due to the relocation of γ-tubulin, originally associated with spindle Mts, as these Mts become concentrated in the midzone during cytokinesis. Under this scenario, the presence or absence of γ-tubulin in the midbody may simply manifest how rapidly this molecule dissociates from the midzone Mts. | Study | biomedical | en | 0.999996 |
10444068 | The isolation of Drosophila Dhc64C alleles used in this study has been described . To analyze cytoplasmic dynein mutant phenotypes in syncytial blastoderm embryos, reciprocal crosses were made using the balanced Dhc64C mutant stocks mwh Dhc64C 6-6 h st p p ss e s /TM6B, D h Hu e ca and mwh jv Dhc64C 6-8 ca/TM6B, D h Hu e ca . Transheterozygous Dhc64C 6-6 /Dhc64C 6-8 progeny contained wild-type non- Dichaete wings and non- Humeral phenotypes. Dhc64C 6-6 /Dhc64C 6-8 progeny were further identified by the characteristic recessive Dhc64C short thin bristle mutant phenotype previously reported for this and other viable transheterozygous dynein mutant combinations . Analysis of maternal effect lethality was performed on embryos derived from mothers that contained Dhc64C 6-6 /Dhc64C 6-8 that had been mated with wild-type Oregon R males for 3–4 d. Similarly, females mated with sibling Dhc64C 6-6 /Dhc64C 6-8 males results in the maternal effect lethal phenotype. A wild-type dynein transgene, P{Dhc64C T } , was used to rescue all the dynein alleles used in this study. Hemizygous larvae derived from both hypomorphic dynein alleles Dhc64C 6-10 and Dhc64C 6-6 were used to examine mitosis within neuroblasts in situ (below). Df(3L)10H is a chromosomal deficiency which removes the entire Dhc64C gene . Embryos from wild-type Oregon R or Dhc64C 6-6 /Dhc64C 6-8 females were collected on agar culture media containing grape juice at 20–45-min intervals. In preparation for microinjection, embryos were dechorionated by hand and mounted onto glass 24 × 50-mm coverslips (#1 thickness) coated with a thin film of glue that was prepared by dissolving double-sided tape adhesive in heptane . Depending upon the relative humidity, embryos were briefly desiccated for 4–8 min using a covered dish containing anhydrous CaSO 4 . Prepared embryos were covered with oxygenated halocarbon oil (series 700; Halocarbon Products) and injected using a Narishige MN-151 apparatus attached to a Zeiss Axiovert microscope. The embryos were injected with mammalian brain tubulin conjugated with TRITC . To address whether the observed phenotypes are specific to dynein dysfunction and not artifacts resulting from the microinjection of exogenous tubulin, we genetically introduced a tau-GFP chimeric transgene (kindly provided by Prof. Daniel St. Johnston, Cambridge, United Kingdom) into the background of Dhc64C 6-6 /Dhc64C 6-8 animals. The expression of this chimeric transgene, driven by the maternally expressed Drosophila α-tubulin 67C promoter, provides an excellent marker for microtubules during early embryogenesis . Virgin Dhc64C 6-6 /Dhc64C 6-8 females were mated with wild-type Oregon R males for 3 d at 25°C on standard cornmeal media. To avoid embryo crowding and lethality due to anoxia, embryos were collected for up to 6 h at 3-h intervals on grape juice–agar media with agar plates at 25°C. After the collection of embryos, the total number of embryos was determined. At 30–36 h after egg lay, the number of hatched first instar larvae and empty chorions was determined. Subsequently, viable larvae were counted, transferred to glass vials containing cornmeal agar media, and incubated at 25°C. At 3–6 d of development at 25°C, the number of second and third instar larvae was determined. Similarly, the numbers of surviving third instar larvae, pupae, and adults were counted on days 8–13 of development. Standard light microscopy was performed on a Zeiss Axioskop microscope equipped with both phase-contrast and DIC lenses. Images from embryos and larval brains were collected using a Bio-Rad MRC 600 or 1024 scanning confocal system mounted on a Nikon Diaphot 300 microscope equipped with a 15 mW krypton/argon laser. A 60×/1.4 NA Planapochromatic and objective lens was used for all analyses. Injections were made using a Narishige MN-151 injection apparatus attached to the microscope. After injection, embryos were previewed using epifluorescence to assess the success of the injections and to determine the developmental stages of injected embryos. Images were collected and saved either digitally to disk or directly to optical memory disk using a Panasonic model 2028 OMDR. Individual 640 × 480 pixel frames were collected at 3–6-s intervals using Bio-Rad COMOS or Lasersharp software time-lapse features at 2 Kalman filtering at normal speed settings, or slow scan mode. Digital files were processed in NIH Image. Individual still frames were saved as PICT or TIFF files and Adobe Photoshop was used to adjust image size and contrast and to crop and pseudocolor images. Prints were made using the Fujix Pictography 3000 and Tektronix Phaser 340 color printers. Series of time-lapse images were opened using custom macros and individual nuclei within an injected embryo were analyzed at the point nearest the time of nuclear envelope breakdown (NEBD). NEBD of all nuclei was not entirely synchronous in a single frame; thus, data were obtained by moving up or down frames within the series to determine the point of NEBD for an individual nucleus. Measurements were recorded for a given nucleus at NEBD. An X-Y center was determined for an individual nucleus using the select line tool and the angle tool was used to measure the angle between the separating centrosomes at the point of NEBD. The results of these measurements were analyzed and plotted using Microsoft Excel. The statistical significance of these measurements was determined using a t test module in Microsoft Excel. The attachment of centrosomes to spindle poles was analyzed in fixed preparations by measuring the distance between a centrosome and the spindle pole to which it was attached . Centrosome position was established by determining the centroid of the γ-tubulin–stained foci in fixed preparations using NIH Image. The end of the spindle pole was defined by the position at which the α-tubulin fluorescence intensity in the half spindle narrowed to a minimum width at the pole. The distance between the centrosome and the spindle pole positions was determined using the NIH Image line tool and the data were analyzed using Microsoft Excel. Nuclear diameter measurements were accomplished in Image Pro Plus (Media Cybernetics) or NIH Image. Stacks of optical sections through the cortex region of four embryos for each genotype were collected. At the point in time nearest NEBD, maximum projections were made to determine the diameter of the nuclei from multiple focal planes. The dark areas corresponding to the nuclei were selected by density slicing and the nuclei were counted and measured. The resulting data were analyzed and plotted using Microsoft Excel. The statistical significance of these measurements was determined using a t test module in Microsoft Excel. Embryos from dynein mutant Dhc64C 6-6 /Dhc64C 6-8 , Oregon R wild-type, or P{Dhc64C T }; Dhc64C 6-6 /Dhc64C 6-8 (containing the wild-type Dhc64C transgene) were collected for up to 3 h and dechorionated using a 50% bleach solution. After dechorionation, embryos were rinsed in 0.1% Triton X-100 and fixed in heptane/methanol/EGTA . Fixed embryos were rehydrated for 5-min periods in a 70:30, 50:50, 30:70 PBS/methanol series followed by 5-min incubations in PBS and PBS containing 0.1% Triton X-100 (PBT). Before antibody labeling, embryos were blocked for 1 h at room temperature in PBS containing 0.1% Triton X-100, 1% BSA, and 0.02% sodium azide (PBT-BSA). All antibodies were diluted into PBT-BSA and incubations were performed at room temperature for up to 3 h or at 4°C for up to 18 h. After each antibody incubation, embryos were rinsed at 15–20-min intervals for 2–3 h in PBT-BSA at room temperature. Microtubules were labeled using a rat monoclonal anti–β-tubulin antibody (clone YOL1/34; Accurate Chemical Co.) diluted 1:10 and a Texas red–conjugated goat anti–rat secondary antibody (Jackson ImmunoResearch Labs). Centrosomes were labeled using a rabbit anti–γ-tubulin polyclonal antibody diluted 1:200 (kindly provided by Dr. Yixian Zheng, Carnegie Institute, Baltimore, MD) and a Cy-5 goat anti–rabbit (Amersham) or FITC-conjugated goat anti–rabbit secondary antibody (Boehringer Mannheim) diluted 1:100. For DNA labeling, embryos were treated with RNase (1 μg/ml) in PBS for 1 h at 37°C, then labeled with the nucleic acid probe Oligreen (1:200 in PBS) (Molecular Probes, Inc.) for 30 min at room temperature. After labeling, embryos were mounted in glycerol containing PBS and 1 mg/ml p -phenylenediamine and stored at −20°C. Third instar larval brains of the genotype Dhc64C 6-10 /Df(3L)10H and wild-type Oregon R were dissected in 0.7% saline and prepared for immunofluorescence according to published procedures . Microtubules were visualized with mouse anti–α-tubulin diluted 1:200 and Texas red–conjugated goat anti–mouse secondary antibody (Jackson ImmunoResearch Labs) diluted 1:200. Centrosomes were visualized with rabbit anti-CP190 (kindly provided by Dr. Will Whitfield, University of Dundee, Dundee, United Kingdom) diluted 1:250, and Cy-5 goat anti–rabbit (Amersham) diluted 1:200. DNA was stained with Sytox green (Molecular Probes, Inc.) diluted 1:1,500. To investigate the function of Dhc in mitosis we obtained animals that lack a functional Dhc and examined them in vivo. Using a collection of hypomorphic Dhc alleles in Drosophila , we obtained mutant animals that are compromised for dynein function at specific developmental stages or in specific tissues . We have identified heteroallelic combinations of Dhc64C that enable us to examine the role of dynein in mitosis during early embryogenesis. A combination of lethal hypomorphic alleles, Dhc64C 6-6 and Dhc64C 6-8 , exhibits complementation for the zygotic requirement of dynein function, but results in maternal effect lethality. Adults of the Dhc64C 6-6 /Dhc64C 6-8 genotype are recovered in equal proportion to nonmutant sibling classes, indicating that these heteroallelic adults are not less viable than wild-type animals. Dhc64C 6-6 /Dhc64C 6-8 adult females lay eggs that lack wild-type dynein heavy chain before the onset of zygotic transcription. When Dhc64C 6-6 /Dhc64C 6-8 adult females are mated to wild-type males (+/+), 94% of the resultant embryos fail to survive beyond the embryogenesis ( Table ). Most of these embryos fail to properly cellularize and cannot complete gastrulation. Approximately 5.7% of the remaining embryos survive through hatching, with 1.4% of these embryos failing to complete larval development. The observed maternal effect lethality can be rescued by addition of one copy of a wild-type dynein transgene , which confirms that the maternal effect lethality results from loss of dynein function. The ∼3% of embryos that escape and complete larval development survive to adulthood. These escapers may be rescued by the initiation of zygotic expression of the paternally provided wild-type dynein heavy chain gene. While most transheterozygous Dhc64C 6-6 /Dhc64C 6-8 embryos display early embryonic lethality, neither embryos containing a wild-type dynein transgene nor embryos derived from heterozygous adult females that carry a single mutant allele show dramatic reduction in viability with 96% or greater of these embryos surviving and completing development. The failure to complete cellularization of the early embryo suggests that dynein plays a critical role in the early syncytial mitotic divisions and is consistent with clonal analysis demonstrating that dynein function is required for cell division and/or viability . In the studies that follow, we have further characterized the maternal effect lethal phenotype to directly establish a requirement for cytoplasmic dynein in several aspects of syncytial mitotic divisions. The majority of Dhc64C 6-6 /Dhc64C 6-8 syncytial blastoderm embryos arrest in early embryogenesis before cellularization. We examined the fidelity of the syncytial mitotic divisions in situ within fixed whole-mount wild-type and mutant specimens using confocal microscopy. Indirect immunofluorescence using antibodies that recognize γ-tubulin and β-tubulin, as well as the DNA dye Oligreen, was used to visualize the centrosome, mitotic apparatus, and chromosomes, respectively . A range of abnormalities in the structural configurations of individual mitotic spindles within the syncytium was apparent in mutant embryos . In all studies, the defects were shown to be specific to mutant embryos by comparison to wild-type or sibling embryos that were processed for immunofluorescence in parallel. Two predominant mitotic defects, free centrosomes and multipolar spindle arrays, were commonly found in fixed preparations of embryos that lack wild-type dynein function. First we will address our data concerning defective centrosome attachment. Free centrosomes can be found singly or in numbers . The origin of some of these centrosomes may be deduced from the presence of spindles lacking one or both centrosomes at their poles, and is suggestive of poor affinity between centrosomes and spindle microtubules in dynein mutant embryos. Corroborating evidence of a disrupted association between centrosomes and spindle poles was obtained from an analysis of γ-tubulin and β-tubulin distribution in the spindles of mutant and wild-type embryos . The γ-tubulin antigen (red) is generally restricted to the centrosome, while β-tubulin (green) is present in microtubules throughout the spindle. In wild-type embryos, the overlap in the immunolocalization of the two antigens reveals a tight association between the centrosomes and spindle poles . Measurement of the distance between the center of γ-tubulin staining and the end of the spindle pole gave a mean value of 0.8 μm (number of poles measured = 164; SD = 0.05). In contrast, the centrosomes are not tightly associated with the ends of the fusiform spindle in the mutant embryos, but are visible as distinct foci displaced from the spindle pole . The mean distance between centrosomes and the associated spindle poles in the mutant embryo was 1.8 μm (SD = 0.15; number of poles measured = 132) and is significantly different from wild-type ( t stat = 2.83; 97% significance). To test the relationship between dynein dysfunction and centrosome detachment from spindle poles, we examined mitosis in Drosophila larval neuroblasts from both wild-type and dynein mutants. The rapid Drosophila syncytial divisions, described above, undergo a cell cycle lacking in gap phases and take place within a unicellular environment. In contrast, the Drosophila central nervous system is a cellularized tissue that, unlike the abbreviated syncytial cell cycle, undergoes complex and patterned cell divisions . Mitotic neuroblasts from flies hemizygous for either of two independent lethal dynein alleles ( Dhc64C 6-10 , Dhc64C 6-6 ) were immunohistochemically examined in whole-mount fixed larval brains . Significantly, a reduced affinity of centrosomes (blue) for spindle poles was frequently observed in the Dhc64C 6-10 and Dhc64C 6-6 mutant lines . In addition, spindle microtubule bundles were often disrupted and curved in the dynein mutant neuroblasts . This phenotype was never observed in wild-type neuroblasts . These defects are rescued by addition of wild-type dynein transgene. The above observations suggest that dynein function is required for the proper association between centrosomes and spindle poles during mitosis throughout Drosophila development. A second class of mitotic defects that we noted in the fixed preparations of mutant embryos included multipolar spindle arrays. While rare in wild-type embryos, aberrant spindle configurations occurred at high frequency in the dynein mutants. Multipolar spindle arrays and bipolar spindles with aberrant numbers of centrosomes associated with each pole were abundant during nuclear cycles 10–13 in mutant embryos. In addition, spindle configurations frequently were excessively curved and the normally uniform spacing between spindles within the syncytium was disrupted . Multipolar microtubule arrays were judged to result from fusion of a number of neighboring spindles and associated chromatin . In addition to multipolar spindles, we also observed abnormal spindles in which an apparently normal half-spindle containing a single centrosome, spindle pole and chromatin, was flanked by an abnormally blunt-ended pole lacking a detectable centrosome . Significantly, these defects in spindle bipolarity and centrosome associations are also detected during very early nuclear cycles, well before cycle 10 and the migration of nuclei to form a closely packed monolayer within the cortical cytoplasm. To extend our understanding of how the mitotic phenotypes in fixed preparations arise, we analyzed syncytial mitotic divisions in living embryos. We visualized mitotic spindles after microinjecting rhodamine-labeled tubulin into living wild-type embryos and mutant embryos from Dhc64C 6-6 /Dhc64C 6-8 mothers, and then recorded time-lapse movies of syncytial mitosis. After injection, wild-type embryos progressed normally through several rounds of mitosis . Nuclear divisions were highly synchronous and proceeded in well-organized waves across the embryo. The orderly progression of nuclear cycles results in an evenly spaced monolayer of nuclei at the surface of the syncytial blastoderm. As noted by others , we occasionally did observe nuclei that failed to complete mitosis. Such nuclei lose their association with the cell cortex and rapidly depart into the interior of the embryo; this event is termed “nuclear fallout.” Unlike wild-type syncytial nuclear divisions, dynein mutant divisions progressed with poor synchrony and displayed profound defects in the behavior of the mitotic apparatus . These defects occurred during any syncytial nuclear cycle with no discernible temporal or spatial pattern. In this regard, our in vivo study is entirely consistent with the analysis of dynein mutant phenotypes in fixed preparations of embryos. Moreover, the mutant phenotypes that we characterized in vivo using microinjection of rhodamine-labeled tubulin were also apparent when using a tau-GFP transgene to visualize microtubule arrays and dynamics . This strategy circumvents the potential artifacts that may ensue after embryos undergo microinjection. The parallel observations made in fixed and living embryos substantiate that the observed defects are the consequence of dynein dysfunction. In presenting the results of our time-lapse analyses, we have divided the defective mitotic behaviors into four main categories: (1) abnormal centrosome migration; (2) pathways to the formation of free centrosomes; (3) pathways to multipolar spindle arrays; and (4) defects in karyokinesis. In fixed mutant specimens, we frequently observed the improper positioning of centrosomes off the spindle pole . To determine a possible pathway to this condition, we examined centrosome separation and positioning in mutant embryos. A large fraction of the syncytial nuclei in mutant embryos failed to separate their centrosomes to a position fully 180° apart before the onset of NEBD. To quantify this defect we analyzed time-lapse records of mitosis in both mutant and wild-type embryos. In wild-type embryos, ∼98% of the centrosomes migrated around the nuclear envelope to final positions at NEBD to a mean displacement angle of between 170° and 180° . Only 2% of nuclei examined underwent less than 170° of separation and these typically were culled from the syncytial monolayer by nuclear fallout ( Table ). In contrast, we determined that the mean centrosome migration and separation angle in mutant embryos at the time of NEBD is 136.47° . The aberrant migration of centrosomes in mutant embryos correlates with subsequent defects in the affected mitotic apparatus. For instance, nearly all nuclei that later gave rise to multipolar spindles were found to produce a mean centrosome migration angle of 119.68° at the preceding NEBD . The most severe defects in centrosome separation concluded with a centrosome separation angle of 102.28° and were associated with the most aberrant spindle arrays, including bipolar and multipolar configurations that failed to progress through the mitotic cycle. However, nuclei that exhibited less extreme defects in centrosome separation and subsequent spindle assembly continued through the cycle. Thus, it is likely that a primary defect resulting from dynein dysfunction is the compromised migration of centrosomes. Analyses of time-lapse sequences were conducted to establish the origin of free centrosomes present in mutant embryos. We find that free centrosomes can arise during both early and late nuclear cycles by different pathways that are independent of cell cycle stage. First, we observed centrosomes departing the nuclear envelope during prophase in mutant embryos . The affected nucleus, bearing only a single centrosome, would often attempt to complete the current mitotic cycle. Free centrosomes observed in the cortical layer persisted and replicated during the final nuclear cycles in synchrony with surrounding nuclei. We never observed this pathway in wild-type embryos. The detachment of centrosomes from bipolar and multipolar spindles was also observed. Relative to the loss of centrosomes from the nuclear envelope, the detachment of centrosomes from mitotic spindle poles was more frequently captured in a single focal plane during time-lapse imaging. Centrosome loss from bipolar spindles often resulted in the partial collapse of the affected spindle pole . Such nuclei invariably failed to complete the current cycle successfully and dropped into the interior of the embryo. This result is supported by observations in fixed mutant embryos of normal bipolar spindles that lack a centrosome at one pole . However, in the case of multipolar spindle configurations, the loss of a centrosome was always accompanied by the complete collapse of the microtubule array associated with the centrosome. Fig. 7 b shows a time-lapse sequence of a spindle that has four poles. The upper centrosome completely detaches from its spindle pole while the remaining three centrosomes appear to be tenuously associated with the spindle poles. In this example, the upper centrosome loses its association with the spindle pole and, subsequently, the associated spindle pole collapses . A final pathway that contributes to the accumulation of free centrosomes in the dynein mutant embryos involves the removal of defective nuclei. Aberrant spindle configurations produce aberrant mitotic products that are eliminated during cycles 10–13 by nuclear fallout. As the defective nuclei drop from the cortex into the interior cytoplasm, the centrosomes associated with such nuclei remain in the cortical layer. This is likely to be the predominant mechanism that contributes to the patches of free centrosomes observed at the surface of embryos. The formation of multipolar arrays in mutant embryos produced by Dhc64C 6-6 /Dhc64C 6-8 females most commonly occurred by the aberrant fusion of adjacent mitotic spindles in the syncytium. This event frequently correlated with the improper migration and separation of duplicated centrosomes (described above) before the assembly of the spindle. The fusion of neighboring spindles would often result in the formation of a bipolar or quadripolar structure which would fail to undergo anaphase . Instead, such an array would often progress directly into interphase. The abnormal metaphase array would disassemble and reform a nuclear envelope with an aberrant number of associated centrosomes. At the next nuclear cycle, the presence of multiple centrosomes on a single nucleus would provide another pathway toward the formation of multipolar spindle arrays . In addition to nuclei retaining aberrant numbers of centrosomes following an aborted mitosis, nuclei were also occasionally observed to capture a free centrosome in close proximity. Multipolar configurations were also generated by the dominant influence of centrosomes on neighboring spindles in the syncytial cytoplasm. In the mutant embryos, spindle-associated or single free centrosomes were capable of inducing ectopic spindle poles on adjacent mitotic arrays. Fig. 10 shows such an example where the resident centrosome of one spindle interacts with a bipolar spindle array in close proximity. In this case, an ectopic spindle pole is formed when a bundle of microtubules is split off from the bipolar spindle and becomes focused towards the neighboring centrosome. We never observed such activity by centrosomes of closely opposed spindles in wild-type embryos. These results suggest that the reduced affinity of centrosomes for spindle poles in mutant embryos can promote inappropriate interactions between centrosomes and microtubule arrays. An additional defect in chromosome segregation is suggested by the variable size of interphase nuclei in mutant embryos. Z-series confocal optical sections were collected through the cortical layer of nuclei in late stage syncytial embryos and maximum projections were made to determine nuclear diameters. As shown in Fig. 11 , nuclei in mutant embryos showed a nearly twofold greater range in nuclear diameters relative to nuclei in wild-type embryos. Because the relative size of syncytial nuclei is held to be indicative of DNA content, such variation indicates that karyokinesis in dynein mutants is defective. The analysis of optical sections that extended through the cortical layer and into the interior of mutant embryos also revealed a nonuniform spacing of nuclei, as well as frequent patches at the surface that were devoid of nuclei. Such defects in the cortical monolayer of nuclei were never observed in wild-type embryos. As previously proposed, a mechanism of nuclear fallout may serve to remove aberrant nuclei that result from defective mitosis in dynein mutant embryos. In this study, we identified recessive lethal alleles of the dynein heavy chain gene, Dhc64C , that exhibit intragenic complementation and reveal mitotic phenotypes during the rapid divisions of the syncytial embryo. The nonlethal Dhc64C 6-8 /Dhc64C 6-6 combination of mutations results in fully viable transheterozygous adult females that produce eggs that are endowed with strictly mutant dynein heavy chain. The rapid rounds of nuclear divisions that follow fertilization of these mutant embryos are compromised by the defective dynein and result in maternal effect lethality with >94% of the embryos dying. The nature of the lesions within the dynein heavy chain mutations, Dhc64C 6-8 and Dhc64C 6-6 , are not known. However, the presence of a wild-type dynein heavy chain transgene rescues the mitotic defects, as well as maternal effect lethality, demonstrating that the phenotype is specific to a loss in dynein function. Importantly, the mitotic defects we uncovered are not unique to the syncytial nature of early nuclear divisions. We discovered similar defects occurring in the larval neuroblasts of late-lethal alleles of the dynein heavy chain. Within the mutant syncytium, nuclear cycles proceed and repeatedly show defects in specific centrosome behaviors and spindle morphogenesis at each nuclear cycle. This progression of the nuclear cycles and the repetitive occurrence of centrosome misbehavior and aberrant multipolar spindle formation is consistent with the defects being a primary consequence of dynein dysfunction. In this regard, we suggest that the combination of hypomorphic heavy chain alleles provides a means to specifically attenuate dynein function in order to investigate its mitotic function in early syncytial embryos. Strong loss of function alleles or null mutations in the dynein heavy chain are cell lethal and prohibit such analysis. This result contrasts with findings in other genetically tractable systems, such as yeast, in which it has been shown that dynein is not an essential gene . Our results demonstrate that the mitotic function(s) of cytoplasmic dynein are essential in Drosophila . Analysis of centrosome behavior in vivo within the dynein mutant embryos occasionally revealed centrosomes detaching from the nuclear envelope. In time-lapse movies some centrosomes left the envelope never to return, while other centrosomes detached briefly and then moved back to the nucleus and reattached. These events are never seen in wild-type embryos and provide evidence for a novel function for dynein in maintaining the association of centrosomes with the nuclear envelope. The reattachment of centrosomes, as well as the low penetrance of the detached centrosome phenotype, is consistent with the prediction that the hypomorphic dynein gene products are only partially compromised for nuclear attachment. One interpretation of these phenotypes is that dynein is associated with the nuclear envelope, where it acts as a minus-end motor to draw in centrosomal microtubules and secure the centrosomes to the nuclear membrane. Alternatively, or in addition, dynein may reside in the centrosome and act to stabilize the attachment of nucleated microtubules that are themselves required for nuclear attachment. In this case, loss of dynein function may increase the frequency of microtubule release from centrosomes and thus weaken nuclear attachment. Evidence for active dynein complex associated with the nuclear envelope has been reported in vitro in Xenopus extracts . In Drosophila we have previously noted that cytoplasmic dynein is localized to the oocyte nucleus, where it might power nuclear migration . However, in embryos, dynein is present on the mitotic spindle and appears concentrated at spindle poles, but no accumulation on nuclear envelopes has yet been detected . In mammalian cell lines, dynein is localized to kinetochores, centrosomes, and at the nuclear periphery . Most centrosomes observed in mutant embryos retained their nuclear attachments, but exhibited defects in migration along the nuclear membrane during prophase. Our time-lapse analysis in living embryos demonstrates that dynein is required for the initial separation of centrosomes along the nuclear envelope and is distinct from the function of antagonistic motors that maintain the separation of centrosomes once initial separation is complete . We frequently observe a failure of the duplicated centrosomes to fully migrate along the nuclear envelope to a position 180° apart before NEBD. The centrosome migration defect is consistent with results from antibody knockout experiments performed in a vertebrate cell culture system . The predominant defect in centrosome migration can be viewed as an intermediate phenotype, the consequence of only partial loss of dynein function. The “detached-centrosome” phenotype might occur when the same dynein-based mechanism is further compromised. However, whether dynein function in centrosome attachment and centrosome migration are mechanistically related remains to be determined. Indeed, recent studies show that centrosome migration in Xenopus extracts depends upon the activity of the plus-end directed kinesin-like protein Xklp2 . Xklp2 is a member of the BimC class of conserved kinesin-like proteins which likely play similar roles in several different eukaryotes . Furthermore, the localization of a COOH-terminal fragment of Xklp2 to the minus-ends of spindle and astral microtubules requires the activity of cytoplasmic dynein . Mutations in dynein may affect centrosome separation by reducing or preventing the normal accumulation of BimC class motor proteins to astral and spindle microtubules. An opposing category of models for dynein involvement in centrosome separation predicts that force production occurs within the cortical cytoplasm and acts to pull on centrosomal microtubules. While such a model readily explains the centrosome migration defect, this mechanism does not account for the observed detachment of centrosomes. In dynein mutant embryos, we observed the release of centrosomes from spindle poles, as well as “loosely attached” centrosomes, where the distance between a centrosome and the associated metaphase spindle pole is significantly greater than in wild-type. Furthermore, the morphology of the spindle poles which lose a centrosome is affected in a manner consistent with current models of dynein function in spindle morphogenesis . In mutant embryos the detachment of a centrosome from bipolar spindles results in the partial collapse of the affected pole. In some cases, the blunt-ended pole becomes refocused, suggesting that either a residual function of the mutant dynein or an additional minus-end motor can rescue the spindle pole. Loss of a centrosome from multipolar spindles also results in collapse of the affected pole. Our observations demonstrate in living embryos that the maintenance of a focused spindle pole requires dynein and the stabilizing influence of a centrosome. This result is not contingent upon the syncytial environment of the embryo since a similar requirement for centrosomes in the organization of spindle poles is apparent in Drosophila larval neuroblasts. Our in vivo time-lapsed analysis provides a temporal understanding of the relationship between centrosome behavior and spindle morphogenesis, and reveals another novel aspect to the dynein mutant phenotype. We find that nuclei which undergo incomplete centrosome migration are predisposed to suffer further defects in spindle assembly that frequently lead to multipolar spindle configurations. As a further consequence, the size of interphase nuclei is variable and suggests a significant defect in karyokinesis. Although dynein is likely to be present on Drosophila kinetochores , as it is in other organisms , evidence for a direct role for dynein in chromatid congression or segregation is lacking. The alignment of chromosomes at metaphase is apparently normal in mutant embryos and we favor the interpretation that aberrant nuclear size results predominantly from abnormal chromatid segregation on multipolar spindles, rather than a direct effect on kinetochore-mediated chromatid movement. How does a loss in dynein function and defective centrosome behavior lead to multipolar spindle assembly? It previously has been shown that the regular spacing between metaphase spindles in the Drosophila syncytium is dependent upon centrosome positions on the nuclear envelope before M phase . One interesting possibility is that organization of the cortical cytoskeleton and the pseudocleavage furrow acts to help isolate nuclei from one another during late nuclear division cycles and is disrupted by mispositioned centrosomes. In this case, loss of spindle autonomy and the formation of multipolar spindle configurations is an indirect effect of the role of dynein in centrosome positioning. However, mutations known to disrupt the cortical cytoskeleton and pseudocleavage furrows are reported to promote spindle fusions during late nuclear cycles when nuclear density is high . For dynein mutant embryos, spindle fusions are detected during the earliest rounds of division before cortical migration and when nuclear density is quite low. Alternatively, a reduction in dynein function in mitotic spindles and/or the syncytial cytoplasm may allow spindle or centrosomal microtubule bundles to interact inappropriately with neighboring arrays. Measurements of an increase in spindle girth in the mutant embryos is consistent with a reduced organization of microtubules within mutant spindles (Sanders, M.A., unpublished data). In spite of the reduced affinity of centrosomes for both nuclear envelopes and spindle poles in the dynein mutants, centrosomes retain a strong capacity to organize spindle poles. We have observed ectopic spindle pole formation on neighboring mitotic arrays by both free centrosomes and spindle-associated centrosomes. The ectopic poles form by splitting off bundles of microtubules from the adjacent spindle, rather than by nucleation of microtubule bundles from the errant centrosome toward the adjacent spindle. Subsequently, the formation of interspindle microtubule bundles and the fusion of neighboring spindles may result from the action of other motor activities. For example, it was recently shown that the separation of spindles during late nuclear cycles in Drosophila embryos requires the function of KLP61F . In summary, we have provided the first evidence that cytoplasmic dynein is required for the attachment of centrosomes to prophase nuclei. Our time-lapsed analysis has further demonstrated in living embryos the role of dynein in the initial migration of centrosomes along the nuclear envelope before spindle assembly, as well as in the attachment of centrosomes to spindle poles. The inappropriate behaviors of centrosomes that result from the reduction in dynein function disrupt spindle morphogenesis. Our results show that dynein function in centrosome attachments is essential for autonomous spindle function, and the global spatial organization of early development in the Drosophila embryo. | Study | biomedical | en | 0.999997 |
10444069 | A rabbit polyclonal antibody raised against the carboxy terminus of GLUT4 (MC2A) was a gift of Dr. Maureen Charron (Albert Einstein College of Medicine, Bronx, NY). A goat polyclonal carboxy-terminal GLUT4 antibody was purchased from Santa Cruz Biotechnology. Rabbit ACRP30 and α3 (VI) collagen antisera were a gift of Dr. Philipp Scherer (Albert Einstein College of Medicine). A rat TfnR mAb was purchased from PharMingen. Rabbit antiserum to β-COP and rat anti-GRP94 mAb antibody (clone 9G10) were from Affinity Bioreagents. A mouse mAb to adaptin-γ was purchased from Transduction Laboratories. Normal goat and donkey sera and fluorophore-conjugated secondary antibodies cross-adsorbed against the relevant species-specific IgGs were purchased from Jackson ImmunoResearch, Inc. Paraformaldehyde (16% solution) was purchased from Electron Microscopy Sciences. Murine 3T3-L1 fibroblasts were kindly provided by E. Santos (National Institutes of Health, Bethesda, MD) and were cultured in DME containing 10% fetal calf serum . Differentiation was induced as described . Appropriate differentiation was confirmed by noting accumulation of lipid droplets, and cells were used 8–12 d after induction of differentiation. The protocol used was modified from Scherer et al. 1995 . 3T3-L1 adipocytes grown and differentiated in 10-cm tissue culture dishes were starved in DME for a total of at least 3 h. During the final hour of serum starvation, the medium was changed to DME lacking cysteine and methionine (ICN Radiochemicals). For some experiments, 100 nM wortmannin (Sigma Chemical Co.), 10 μM LY294002 (Calbiochem), or 10 ng/ml rapamycin (Calbiochem) was added during the last 45 min of starvation. Cells were then pulse-labeled for 15 min in the same medium supplemented with 0.5–0.7 mCi/ml Express Protein Labeling reagent, a mixture of 35 S-labeled cysteine and methionine (1,000 Ci/mmol; DuPont/New England Nuclear). Cells were then washed twice with DME containing unlabeled cysteine and methionine, and then incubated during a 2-h chase period at 37°C in DME containing 300 μM cycloheximide to prevent further protein synthesis, with or without 160 nM insulin or 200 μM A23187 (Calbiochem). Cells that were pretreated with wortmannin, LY294002, or rapamycin were maintained in the presence of these drugs during the chase period. At 30-min intervals, the medium was collected from each plate and was replaced with identical fresh medium. Samples were kept on ice until all were collected, then insoluble debris was removed by centrifugation (15,000 g , 10 min, 4°C). Samples were precleared by incubation with 60 μl protein A–Sepharose for 30–60 min at 4°C. Immunoprecipitations each used 6 μl of antisera directed against ACRP30 or α3 (VI) collagen, and were allowed to proceed for a minimum of 4 h at 4°C, after which 100 μl protein A–Sepharose was added and the incubations were continued for an additional 45 min. ACRP30 and α3 (VI) collagen were immunoprecipitated sequentially from the same samples. Immunoprecipitates were washed five times in TNET (1% Triton X-100, 150 mM NaCl, 20 mM Tris, pH 8.0, 2 mM EDTA), electrophoresed on SDS-PAGE, and the dried gels were fixed, treated with sodium salicylate or Enhance (Amersham), exposed, and quantified using a Fuji PhosphorImager. Experiments were performed at least twice, with similar results each time. 3T3-L1 adipocytes were reseeded 1 d before fixation to either coverslips or to the wells of teflon-coated microscope slides (Cel-Line Associates, Inc.). Cells were serum starved in DME for a minimum of 3 h, then stimulated or not with 160 nM insulin for 12 min. Cells were washed with cold PBS containing 0.9 mM Ca ++ and 0.5 mM Mg ++ (PBS++). Fixation was with 3–4% paraformaldehyde in PBS for 45 min at room temperature, followed by permeabilization with 0.2% Triton X-100 in PBS++ at 4°C. In some instances, cells were washed with PBS++ and stored before staining at 4°C in PBS++, 2% BSA, and sodium azide. After washing the cells again with PBS++, nonspecific antibody binding was blocked with PBS++ containing 2% BSA and 4% goat or donkey serum (blocking buffer), as appropriate to the source of the secondary antibody, for 30 min. In experiments using the goat GLUT4 antisera, only donkey secondary antibodies were used. Primary antibodies were used at dilutions ranging from 1:200 to 1:500 in blocking buffer. Incubation with primary antibodies was for 60–120 min at 25°C or 37°C in a humidified chamber. The cells were again washed with PBS++, and then incubated with FITC- or Cy3-conjugated goat or donkey secondary antibodies at dilutions of 1:200 to 1:400 in blocking buffer. Incubation with secondary antibodies was for 30–45 min at 25°C or 37°C in a humidified chamber. The cells were rinsed twice with PBS++ and then washed at least three times, for 10 min each, with PBS++. Coverslips were mounted using Vectashield (Vector Laboratories). For all microscopy experiments, controls were done to demonstrate that the binding of secondary antibodies was specific for the intended primary antibody. Additionally, the specificity of the rabbit ACRP30 and rabbit and goat GLUT4 antisera was shown in peptide competition experiments (data not shown). Deconvolution microscopy was performed using a CELLscan system by Scanalytics, Inc., attached to either an Olympus or a Nikon Eclipse E-800 microscope. Both microscopes were equipped with an excitation filter wheel and a triple bandpass cube containing a dichroic mirror and emission filter, ensuring registration of two or three color images in both the X–Y plane and along the Z-axis. Empirical point spread functions were obtained immediately before image acquisition by using 0.1-μm diam latex beads fluorescent at the appropriate wavelengths (Molecular Probes). Beads were diluted in PBS and briefly sonicated to disrupt aggregates, and were then by mounted on microscope slides using Vectashield under conditions identical to those for 3T3-L1 cells. For both point spread function and image acquisition, through-focusing was done using the 100×/1.4 NA objective and acquiring data at 250-nm intervals. For cross-sectional images of cells, at least nine planes of raw image data were obtained so as to optimize reconstruction of the center plane image. Deconvolution was done on a CSPI high speed processor, and both raw and reconstructed images were acquired with 12-bit/pixel resolution. Under these conditions, we estimate lateral resolution at 180 nm. Images were pseudocolored and merged using Photoshop (Adobe Systems, Inc.); final pixel depth is 8 bits/channel. To determine if the insulin-stimulated enhancement of ACRP30 secretion is mediated by a regulated secretory compartment, or if it represents a nonspecific acceleration of the entire secretory pathway, we compared secretion of ACRP30 and α3 (VI) collagen in a pulse-chase experiment. Previous work done in our laboratory using a complex antisera raised against many proteins secreted from 3T3-L1 adipocytes demonstrated that insulin enhances secretion of some, but not all, of these proteins . We hypothesized that one specific protein that does not participate in an insulin-regulated secretory compartment might be α3 (VI) collagen . Accordingly, we followed secretion into the media of a discrete population of newly synthesized protein, synthesized during a short pulse of labeled amino acids and chased in the presence of cycloheximide and in the presence or absence of insulin or a calcium ionophore, A23187. The media were collected at intervals throughout the chase period, and α3 (VI) collagen and ACRP30 were immunoprecipitated sequentially from the same samples. Fig. 1 A shows a representative experiment; in Fig. 1 B these data are used to plot the cumulative amount of each protein secreted. Whereas insulin clearly enhances ACRP30 secretion, there is no effect on α3 (VI) collagen secretion. The insulin-stimulated increase in ACRP30 secretion is most marked during the early part of the chase period, consistent with an effect of insulin to accelerate the early part of the secretory pathway. Yet, insulin's effect may not be limited to this part of the secretory pathway, since ACRP30 secretion is increased even at later time points. A23187 has a minimal effect early in the chase period, but enhances ACRP30 secretion, and not α3 (VI) collagen secretion, after 90 min. This result is consistent with an effect late in the secretory pathway, involving a compartment to which ACRP30, but not α3 (VI) collagen, is targeted. Thus, the data indicate that the insulin-stimulated enhancement of ACRP30 secretion is not due to nonspecific acceleration of the entire secretory pathway. Rather, ACRP30 is targeted to a regulated secretory compartment in 3T3-L1 adipocytes, and α3 (VI) collagen is excluded from this compartment. PI-3 kinase activity is stimulated by insulin, and has been implicated in the insulin signal transduction pathway leading to GLUT4 exocytosis. To determine if PI-3 kinase activation is also required for insulin-stimulated ACRP30 secretion, we performed pulse-chase experiments similar to those described in Fig. 1 , but in the presence or absence of two pharmacologic inhibitors of PI-3 kinase, wortmannin and LY294002. Fig. 2 plots cumulative ACRP30 secretion, and demonstrates that ACRP30 secretion is enhanced by insulin in cells left untreated or in the presence of rapamycin, used here as a negative control, and that this effect is absent in the presence of wortmannin or LY294002. Like the results shown in Fig. 1 , the increased secretion is especially marked during the first 30 min of the chase period in the untreated and rapamycin-treated cells. In the presence of wortmannin, ACRP30 secretion at the 30-min time point is not significantly enhanced by insulin, and at subsequent time points the cumulative secretion in the presence of insulin is even less than in the absence of insulin. Similarly, LY294002 completely abolishes the effect of insulin, so that cumulative ACRP30 secretion is essentially identical in the presence or absence of insulin in cells treated with this drug. No definite effect is seen on basal ACRP30 secretion under any of the conditions tested. Thus, PI-3 kinase inhibitors specifically block insulin-stimulated exocytosis of ACRP30. Together with the data presented in Fig. 1 , we conclude that ACRP30 is targeted to a regulated secretory compartment, the exocytosis of which is stimulated by insulin through a PI-3 kinase activity. It has been reported that leptin largely colocalizes with calnexin, a marker for the ER, in isolated rat adipocytes . To determine whether this is also true of ACRP30 in 3T3-L1 adipocytes, we used deconvolution immunofluorescence microscopy to obtain cross-sectional images of cells stained for ACRP30 and GRP94. GRP94 (also called endoplasmin) is a well-characterized lumenal protein of the ER that contains a carboxy-terminal KDEL motif, and that functions in concert with GRP78/BiP to assist in protein folding . As shown in Fig. 3 , some but not all ACRP30 staining overlaps with that of GRP94. GRP94 staining (shown in red) is reticular with some pronounced punctae, and is present throughout the cytoplasm in both unstimulated and insulin-stimulated cells. ACRP30 staining is mostly punctate, but has a reticular component as well. In unstimulated cells, there is moderate overlap of these two proteins , though it is also clear that some ACRP30 staining does not overlap with GRP94 staining . ACRP30 expression was heterogeneous, in that it was commonly observed that not all cells that had differentiated by morphological criteria (as assessed by the presence of lipid droplets) stained for ACRP30 on immunofluorescence microscopy. The staining for GRP94 (red), but not ACRP30, present in the lower left quadrant of Fig. 3 c represents an adjacent cell not expressing ACRP30; there is very little staining for GRP94 by itself (i.e., not overlapping ACRP30) in the remainder of the image. In comparison to unstimulated cells, insulin-stimulated cells have more prominent staining for GRP94 that does not overlap with ACRP30 , though some overlap also remains (yellow). These findings are subtle, but are observed consistently in the highest quality images. Thus, the data suggest that insulin stimulates movement of ACRP30 out of the ER. More significantly, a proportion of ACRP30 does not overlap with GRP94, and is presumably in a more distal compartment of a regulated secretory pathway. To determine if GLUT4 recycles to the insulin-regulated secretory pathway containing ACRP30, we employed deconvolution immunofluorescence microscopy to obtain cross-sectional images of GLUT4 and ACRP30 in 3T3-L1 adipocytes. Fig. 4 presents representative images of adipocytes stained for GLUT4 (shown in red) and ACRP30 (shown in green). The staining for GLUT4 is punctate, consistent with a vesicular compartment, with prominent perinuclear staining that is in a more tubulovesicular pattern . This distribution has been noted previously by others, and is characteristic of GLUT4 . ACRP30 staining, too, is mainly punctate, but also has a reticular component and is present more equally throughout the cytoplasm, with less perinuclear accumulation . The merged images demonstrate that there is essentially no overlap (yellow) in the subcellular distribution of the two proteins. This is the case for both unstimulated and insulin-stimulated cells . Indeed, because the staining for these two proteins is so distinct, we have presented views through several cross-sections in Fig. 4 and still see no significant overlap. Insulin stimulation (160 nM, 12 min) of cells before fixation clearly results in accumulation of GLUT4 at the plasma membrane , but did not cause any obvious change in the pattern of ACRP30 staining. Thus, GLUT4 does not appear to participate in the regulated secretory compartment that contains ACRP30. Fig. 5 shows immunofluorescence images of cells stained with GLUT4 (in red) and TfnR (in green). Both GLUT4 and TfnR are present in a punctate pattern at the periphery of the cell and in a more tubulovesicular pattern adjacent to the nucleus. The merged images demonstrate that there is significant colocalization (yellow) of the two proteins, with perhaps half of each colocalizing with the other. This colocalization is present in the periphery of the cell as well as in the perinuclear region. In cells stimulated with insulin , there is more prominent staining of both GLUT4 and TfnR at the plasma membrane . Of note, neither the perinuclear GLUT4 nor the peripheral GLUT4 overlaps completely with TfnR. After insulin stimulation, prominent GLUT4 staining remains in the perinuclear region, whereas TfnR staining is somewhat less marked . Though this effect of insulin was not seen in all images, it may represent participation of TfnR, but not GLUT4, in a perinuclear recycling endosome. As an additional control experiment, we stained cells for TfnR and ACRP30. As shown in Fig. 6 , there is essentially no overlap in the distribution of these two proteins. ACRP30 (in red) is once again seen to have punctate and reticular components in both unstimulated and insulin-stimulated cells. The apparent perinuclear staining in Fig. 6 d is due, at least in part, to the presence of several lipid droplets throughout the periphery of the cell. TfnR staining is present in a more tubulovesicular pattern. There is some TfnR present at the plasma membrane after insulin stimulation , though this is less marked than in Fig. 5 . The merged images demonstrate very little overlap (yellow) in the subcellular distributions of these proteins, in both unstimulated and insulin-stimulated cells , corroborating the data shown in Fig. 4 and Fig. 5 . We conclude that GLUT4 and TfnR are targeted to compartments that are distinct from those containing ACRP30. If GLUT4 does not recycle to a regulated secretory compartment, then GLUT4 in the perinuclear region likely does not represent recycling to the Golgi complex or TGN. We sought to confirm this in 3T3-L1 adipocytes by microscopy of cells costained for GLUT4 and Golgi markers. We first used an antibody to detect endogenous β-COP, a component of the nonclathrin COPI vesicle coat that has been localized previously to the Golgi complex . Fig. 7 demonstrates that the distributions of GLUT4 and β-COP are closely apposed, but do not overlap either in unstimulated or in insulin-stimulated cells. GLUT4 (in red) is present in unstimulated cells and in insulin-stimulated cells in its characteristic perinuclear location. Compared with Fig. 4 and Fig. 5 , less plasma membrane GLUT4 staining is seen in the insulin-treated cells. This is in part due to cell-to-cell variability; also, data from only one optical cross-section are shown because views through several cross-sections resulted in the artifactual appearance of overlap due to staining of GLUT4 and β-COP in different planes. β-COP staining (in green) is also perinuclear, and is not markedly different in basal and in insulin-treated cells . The merged images clearly show that there is essentially no overlap (yellow) in the distributions of GLUT4 and β-COP staining in either unstimulated or in insulin-stimulated cells. We next stained cells for GLUT4 and γ-adaptin, a component of the AP-1 adaptor complex involved in ADP-ribosylation factor–dependent budding of clathrin-coated vesicles from the TGN . Single optical cross-sections of unstimulated and insulin-stimulated 3T3-L1 adipocytes are shown in Fig. 8 ; whereas there is partial overlap of GLUT4 and γ-adaptin staining, the overall impression is that these compartments are distinct. GLUT4 is shown in red and can more readily be detected on the plasma membrane of insulin-stimulated cells than in unstimulated cells . As noted for the other images of GLUT4 presented above, no qualitative differences are observed in the perinuclear GLUT4 distribution before and after insulin stimulation. Similarly, γ-adaptin staining (in green) is perinuclear and tubulovesicular in nature, and is not noticeably different in unstimulated and in insulin-stimulated cells. The merged images show very little overlap (yellow); most staining is exclusively for either GLUT4 or for γ-adaptin. Review of several images revealed no difference between unstimulated and insulin-stimulated cells in the degree of overlap . Thus, whereas the possibility of GLUT4 recycling to the TGN cannot be excluded, the perinuclear GLUT4 and γ-adaptin staining patterns are clearly not identical, and the data support the notion that GLUT4 does not rejoin the secretory pathway at the level of the Golgi complex or TGN. We have shown that insulin stimulates exocytosis of a regulated secretory compartment, containing ACRP30, in 3T3-L1 adipocytes. This compartment is distinct from that containing α3 (VI) collagen, because insulin has no effect on secretion of this protein. Therefore, it is not the case that insulin enhances ACRP30 secretion merely by accelerating the entire constitutive secretory pathway. Our observation that a calcium ionophore also stimulates ACRP30 secretion, and not α3 (VI) collagen secretion, supports this inference. Thus, we propose that a portion of ACRP30 is sorted into regulated secretory vesicles whose exocytosis is stimulated by insulin, and that the remainder is sorted into vesicles that undergo constitutive exocytosis. We propose that the latter population accounts for secretion of ACRP30 in the absence of insulin, and also contains α3 (VI) collagen and other proteins whose secretion is not enhanced by insulin. Partial sorting of protein hormones into regulated secretory vesicles has been observed in other types of cultured cells . We also present immunofluorescence microscopy data demonstrating that whereas some ACRP30 staining overlaps with that of GRP94, and is presumably in the ER, additional ACRP30 staining does not overlap this marker and likely represents protein in peripheral storage vesicles. Insulin apparently accelerates movement of ACRP30 out of the ER, because staining for GRP94 that did not overlap with ACRP30 was only observed in cells that had been stimulated with insulin before fixation. Stimulation of ACRP30 export from the ER may account for observation that the insulin-stimulated enhancement of ACRP30 secretion is most marked in the early part of the chase period. Such export would have to be selective, since insulin did not increase α3 (VI) collagen secretion. Yet, this may not be the only site of insulin action: our observation that insulin also increases ACRP30 secretion late in the chase period, combined with our immunofluorescence data showing that some ACRP30 is in peripheral vesicles, suggests that it mobilizes a pool of regulated secretory vesicles in the periphery. Our proposal that insulin stimulates exocytosis of regulated secretory vesicles is in contrast to that of Barr et al. 1997 , who found no light microscopic evidence that leptin is targeted to such vesicles in rat adipose cells. These investigators found that the vast majority of leptin staining coincided with a marker for the ER, and that insulin stimulated export from this compartment. In this respect, our data that insulin stimulates ACRP30 export from the ER result is similar. It is possible that the presence of a small proportion of leptin in regulated vesicles in the periphery could have gone undetected by Barr et al. 1997 , especially given the morphological challenge presented by primary adipocytes. Moreover, whereas insulin accelerates secretion of leptin and adipsin as well as ACRP30, it is not known if all of these proteins share a common exocytic pathway . Finally, these different interpretations may reflect actual differences in the cells used for experiments. Our work demonstrates that insulin-stimulated enhancement of ACRP30 secretion is blocked by pharmacologic PI-3 kinase inhibitors, suggesting that PI-3 kinase activation is necessary for insulin stimulation of ACRP30 secretion. PI-3 kinase has been previously implicated in the insulin signal transduction pathway leading to GLUT4 exocytosis, and treatment of intact fat or muscle cells with LY294002 or wortmannin blocks insulin-stimulated translocation of GLUT4 to the plasma membrane . Rapamycin, which we used as a negative control, acts downstream of PI-3 kinase to block insulin-stimulated p70 S6 kinase activation, but has no effect on glucose transport in 3T3-L1 adipocytes . Though the specificity of wortmannin and LY294002 has been questioned , recent work demonstrates that the effect of wortmannin on insulin-stimulated hexose uptake is largely reversed by membrane-permeant esters of phosphatidylinositol-3,4,5-trisphosphate, a product of PI-3 kinase activity . Therefore, the notion that PI-3 kinase activation is necessary for insulin-triggered GLUT4 exocytosis is supported, and it is through inhibition of this activity that wortmannin and LY294002 block insulin-stimulated GLUT4 trafficking. By extension, a strong argument is made that it is through inhibition of PI-3 kinase activity that these drugs block the effect of insulin to augment ACRP30 secretion. Similar to our results with ACRP30, it has been shown very recently that the PI-3 kinase inhibitor, LY294002, blocks insulin-stimulated leptin secretion from rat adipocytes . These investigators also found that rapamycin decreased insulin-stimulated leptin secretion, though not as completely as LY294002; in contrast, we observed no effect of rapamycin on insulin-stimulated ACRP30 secretion. Aside from the observation that ACRP30 and leptin may not share a common regulated secretory pathway, this apparent contradiction can be resolved because our experiments using rapamycin were done in the presence of cycloheximide, whereas those of Bradley and Cheatham 1999 were not. Thus, as pointed out by these authors, it may be the case that the major effect of rapamycin was to prevent insulin-stimulated mRNA translation. We have shown that there is no overlap in the subcellular distributions of GLUT4 and ACRP30 in unstimulated and in insulin-stimulated 3T3-L1 adipocytes. Together with our result that ACRP30 participates in a regulated secretory compartment, we conclude that GLUT4 does not recycle to a regulated secretory compartment, as defined by ACRP30. Important control experiments demonstrate that the subcellular distributions of GLUT4 and TfnR overlap substantially, whereas those of TfnR and ACRP30 are distinct and nonoverlapping. Further support that GLUT4 does not recycle to a regulated secretory pathway are our findings that perinuclear GLUT4 does not colocalize with a Golgi marker (β-COP) and is mostly distinct from that of a TGN marker (γ-adaptin). Thus, these parts of the secretory pathway are not major sites to which GLUT4 is distributed, either in the absence of insulin or after insulin stimulation. The question of whether GLUT4 recycles to a regulated secretory pathway, or is instead targeted to synaptic-like vesicles, has been controversial. Conflicting results may have been obtained because previous studies relied on exogenously expressed GLUT4 in cells that do not normally take up glucose when stimulated by insulin, or were conducted using tissue that does not have a secretory pathway that is regulated by insulin . We have addressed these concerns by studying endogenous GLUT4 in a well established adipose cell culture system, by showing that ACRP30 participates in a regulated secretory compartment in these cells, and by comparing the subcellular distributions of ACRP30 and GLUT4. Our conclusion that GLUT4 does not recycle to a secretory compartment is similar to that of Malide and Cushman 1997 , who showed that in primary adipocytes wortmannin disrupts endocytic trafficking of GLUT4, but has no effect on trafficking of late endosomal or TGN markers. Their data suggest that GLUT4 does not recycle through the late endosome to the Golgi complex. Yet, this conclusion rests on the assumption that if this were the case, GLUT4 would traffic together with these markers. Additionally, proteins such as TGN38 recycle from the TGN to the plasma membrane quite slowly, and relatively short term wortmannin treatment might not appreciably alter their distribution . By examining the distribution of GLUT4 relative to a protein that participates in regulated secretion, we have avoided these potential pitfalls. We observed significant overlap in the subcellular distributions of GLUT4 and TfnR. Other investigators have also noted overlap of GLUT4 and endosomal markers, including TfnR, yet contradictory data have been reported . The variability in the literature may be explained, at least in skeletal muscle, by the recent observation that there are two distinct intracellular GLUT4 compartments: one that cofractionates with the early endosomal markers TfnR and annexin II, and a second compartment from which GLUT4 is depleted after insulin stimulation . Likewise in 3T3-L1 adipocytes, experiments involving either immunoisolation of GLUT4 vesicles or ablation of transferrin (Tfn)–containing compartments determined the presence of distinct GLUT4 populations . In both of these reports, ∼40% of GLUT4 colocalized with the TfnR, and ∼40% of the TfnR colocalized with GLUT4, consistent with our data as well. Moreover, both reports suggest that insulin stimulates movement of GLUT4 from the TfnR-negative pool to the plasma membrane. Although it seems likely that the TfnR-positive GLUT4 compartment is the precursor of the TfnR-negative, insulin-regulated GLUT4 compartment, this has not been definitively established. Our data add that the TfnR-negative, insulin-regulated GLUT4 compartment is not identical to the insulin-regulated compartment for ACRP30 secretion. We also found that perinuclear GLUT4 does not appear to rejoin the secretory pathway at the level of the Golgi complex. GLUT4 immunostaining does not overlap with that of β-COP, a protein found on the cis side of the Golgi complex in pancreatic acinar cells and on the lateral rims and trans face of the Golgi complex in spermatids . An additional pool of β-COP has been described on membranes of the TGN, though this pool appears not to participate in budding of transport vesicles . We also found that GLUT4 immunostaining is mostly separate from that of γ-adaptin; overall, the data show that the perinuclear GLUT4 compartment does not correspond to the TGN. Our interpretation is consistent with previous light microscopy data that in insulinoma cells, transfected GLUT4 did not significantly overlap with the TGN marker protein, TGN38 . Other reports have described minimal overlap of GLUT4 and TGN38 in rat adipose cells, and of GLUT4 and giantin in cultured myotubes . Biochemical studies of 3T3-L1 adipocytes found that only 5–10% of low density microsomal GLUT4 was copurified by immunoadsorption of vesicles using an antiserum to TGN38; the copurified GLUT4 did not correspond to the insulin-regulated GLUT4 compartment within the low density microsomal fraction . Thus, our data concerning the perinuclear GLUT4 compartment are in agreement with other studies noting close association, but not identity, with the TGN. In summary, we have shown that ACRP30 participates in an insulin-regulated secretory compartment in 3T3-L1 adipocytes, and that α3 (VI) collagen does not. Insulin appears to accelerate ACRP30 secretion at both early and late steps in its secretory pathway, possibly corresponding to export of ACRP30 from the ER and to exocytosis of regulated secretory vesicles containing this protein. Like GLUT4 exocytosis, insulin-stimulated ACRP30 secretion is blocked by inhibitors of PI-3 kinase. Finally, we show that GLUT4 does not recycle to the regulated secretory pathway containing ACRP30. Insulin-stimulated PI-3 kinase activity must therefore act through effectors present at multiple locations within the cell. | Other | biomedical | en | 0.999997 |
10444070 | Generation of RyR1−/− and RyR3−/− mice has been described elsewhere . For tissue preparation, normal, homozygous RyR1−/−, and homozygous RyR3−/− mice were anesthetized with carbon dioxide, decapitated, and tissue samples were removed. Small muscle pieces were infiltrated in 5% DMSO in PBS for 5 to 15 min and then rapidly frozen in precooled 2-methylbutane and stored at −70°C. Frozen samples were mounted in OCT compound, sectioned on a cryostat, and the sections were mounted on glass microscope slides. Tissue sections were rinsed in PBS containing 0.2% BSA and 0.2% Triton X-100 (PBS/BSA/Triton) and then incubated in 5% normal goat serum (NGS) in PBS/BSA/Triton for 30 min at room temperature. Then, the sections were incubated in primary antibody for 4 h at room temperature or overnight at 4°C and subsequently washed with five changes of PBS/BSA/Triton for a total of 30 min at room temperature. Sections were then incubated in fluorochrome-conjugated secondary antibody for 1 h at room temperature and washed as before. The sections were then incubated in 90% glycerol, 0.1 M Tris, pH 8, with 5 mg/ml p-phenylenediamine to retard photobleaching and covered with a coverglass. The following primary antibodies were used at the indicated concentrations: affinity-purified rabbit antibody against RyR1 at 1:5,000 ; rabbit antiserum against RyR3 at 1:1,000; monoclonal mouse antibody against the skeletal muscle dihydropyridine receptor α 1 subunit at 0.1 mM ; monoclonal mouse antibody against fast isoform of the skeletal muscle Ca 2+ ATPase, SERCA1 at 1:500 ; and monoclonal mouse antibodies against the following isoforms of myosin heavy chain, which were generously supplied by Dr. S. Schiaffino (University of Padova, Italy): antibody BF-G6 against embryonic MHC used at 1:4,000; BA-D5 against slow MHC used at 1:500; and SC-71 against fast red, 2A MHC used at 1:500. Secondary antibodies were Cy3- and fluorescein-conjugated goat anti–rabbit and goat anti–mouse IgG (Jackson ImmunoResearch Laboratories) used at a dilution of 1:5,000. All antibodies have been characterized elsewhere and, except anti-RyR3, extensively used in immunocytochemistry . As controls, primary antibodies were omitted or inappropriate secondary antibodies were applied. Isoform-specificity of the RyR antibodies was evaluated in situ using tissues from the homozygous RyR1−/−, RyR3−/− (see Results), and from a double knockout strain of mice for both RyR1 and RyR3 genes . Immunostained sections were evaluated and analyzed on a Zeiss fluorescence microscope and images were captured with a Zeiss laser scanning confocal microscope using the 63×, 1.4 NA, Plan-Apochromat objective lens. Skeletal muscles isolated from mice at indicated ages were used to prepare the microsomal fractions as previously described . Muscles were homogenized in ice-cold buffer A (320 mM sucrose, 5 mM Na-Hepes, pH 7.4, and 0.1 mM PMSF) using a Dounce homogenizer. Homogenates were centrifuged at 7,000 g for 5 s at 4°C. The supernatant obtained was centrifuged at 100,000 g for 1 h at 4°C. The microsomes were resuspended in buffer A and stored at −80°C. Protein concentration of the microsomal fraction was quantified using the Bradford protein assay kit (BioRad). Microsomal proteins were separated by SDS/PAGE and then transferred to a nitrocellulose membrane (Schleicher & Schuell). Membranes were incubated for 3 h in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.2% Tween 20, plus 5% nonfat milk. Primary antibodies used were polyclonal rabbit antisera (diluted 1:3,000) against the RyR isoform . Antigen detection was performed using the amplified alkaline phosphatase detection method. To determine to what extent RyR3 is expressed in developing skeletal muscle and whether it is coexpressed with RyR1 in the same fibers, unfixed cryosections of muscles from normal, homozygous RyR1−/−, and homozygous RyR3−/− mice at embryonic day 18 (E18) were immunolabeled with specific antibodies against RyR1 and RyR3 . In wild-type muscles, both RyR antibodies stained all myofibers with similar intensity, indicating that, at this developmental stage, RyR1 and RyR3 are coexpressed in mouse skeletal muscles. The labeling patterns for both RyR isoforms were punctate and irregularly distributed throughout the myoplasm , resembling the distribution pattern of triad proteins that is typically found in E18 muscle fibers. Since the nuclei were still centrally located in the myofibers, the labeling pattern appeared ring-shaped in cross-sections. RyR1−/− muscles were labeled with anti-RyR3, but not with anti-RyR1 . Conversely, RyR3−/− muscles were labeled with anti-RyR1, but not with anti-RyR3 . This is consistent with previous immunoblot experiments and shows that there are no cross-reactions of anti-RyR1 with RyR3 and of anti-RyR3 with RyR1. Thus, the immunofluorescence assay is highly specific for the respective RyR isoforms. Furthermore, the absence of immunostain with anti-RyR3 and anti-RyR1 in muscles of RyR1−/− and RyR3−/− mice, respectively, provides additional evidence that the targeted mutations of the genes encoding the RyR isoforms resulted in the complete and specific loss of the respective proteins. Expression of RyR3 in skeletal muscles of RyR1−/− mice was also observed in a second independent RyR1 knockout mouse strain (data not shown) generated by Dr. P.D. Allen (Brigham and Women's Hospital, Boston, MA). Normal expression of RyR1 in RyR3−/− mice was also detected in skeletal muscles from mice 15-, 25-, and 60-d-old (D15, D25, and D60; not shown). If RyR3 is involved in the initial aspects of excitation–contraction coupling, then its localization in the triad would be expected. With fluorescent microscopy, triad proteins exhibit characteristic labeling patterns . In longitudinal sections of differentiated skeletal muscles, a cross-striated banding pattern with a center-to-center distance between the bands of ∼2 μm can be seen. At higher resolution, the cross-striated bands can be identified as double rows of fluorescent dots, representing the pairs of triads on either sides of the Z-line. Pairs of neighboring fluorescent dots are aligned along the myofibril bundles. In cross-sections, triad proteins show a network of membranes encircling the myofibril bundles. Fig. 2 compares the distribution pattern of a known triad protein, the α 1 subunit of the dihydropyridine receptor , with that of RyR3 in D15 normal diaphragm muscle. The distribution of the RyR3 immunolabel shows the characteristics of a triad protein, most importantly the double rows of fluorescent dots . The fact that a T-tubule protein, the dihydropyridine receptor, and a SR protein, RyR3 (or RyR1) stain the same cytoplasmic structure is further evidence that this structure represents the T-tubule/SR junction, i.e., the triad. No other cytoplasmic structure showed significant RyR3 label. Thus, RyR3 is localized in the triad junctions of skeletal muscle and not to any significant amount in other regions of the ER/SR system. The coextensive labeling patterns of RyR1 and RyR3 observed in E18 hind limb muscles combined with the localization of RyR3 immunolabel in the triads of differentiated muscle suggests that the two RyR isoforms coexist in the same triads. Fig. 3 allows the direct comparison of the subcellular distribution of RyR3 with that of RyR1 in D15 normal mouse diaphragm. Specific antibodies against RyR1 and RyR3 labeled all muscle fibers to the same extent and the two RyR isoforms showed identical triad labeling patterns in longitudinal and cross-sections. Thus, RyR3 and RyR1 coexist in, or in close proximity to, young skeletal muscle triads. Triad staining of RyR3, as shown for D15 diaphragm in Fig. 3 was also observed in RyR3-expressing fibers from hind limb muscles (not shown) and from muscles of later developmental stages. However, in older muscles, RyR3 was not expressed uniformly in all muscle fibers, but only in a subset of fibers. Fig. 4 shows an example of normal D25 diaphragm labeled with antibodies against RyR1 and RyR3. Whereas anti-RyR1 uniformly stained all muscle fibers in the fields, only one fiber in each field is stained with anti-RyR3. These RyR3-positive muscle fibers were labeled as intensively as those of D15 and also expressed RyR1 . This shows that, during postnatal development, the majority of muscle fibers lose RyR3 from their triads. However, those fibers that continue to express RyR3 also express RyR1. The finding described above indicates that the RyR3 content does not decrease gradually and simultaneously in all muscle fibers, but that it decreases rapidly in some fibers and not in others. Fig. 5 shows that during postnatal development the number of RyR3-containing fibers dramatically decreases with age and that this occurs at different rates in different muscles. Whereas RyR3-positive fibers could be found even in adult (D60) diaphragm muscle, extensor digitorum longus (EDL) muscle was devoid of RyR3-positive fibers as early as D25. Semiquantitative analysis showed that in diaphragm, the decline in numbers of RyR3-containing fibers did not begin until after D15. By D60, only 13% of the fibers were positive for RyR3. In contrast, in EDL the fraction of RyR3-containing fibers declined to 17% by D15 and disappeared before D25. The overall morphology of the RyR3-containing fibers gave no indication that these fibers were different or at a different stage of differentiation than the neighboring RyR3-lacking fibers in the section. Comparison of the tissue distribution of RyR3 with the fiber type composition of the studied muscles did not suggest a correlation of the RyR3-containing fibers with a distinct fiber type . Comparison of the fractions of RyR3-containing fibers with fibers labeled with specific antibodies against myosin heavy chain isoforms of embryonic, slow, and fast (2A) fibers, or with an antibody against the fast calcium ATPase, also failed to show any correlation of RyR3 expression with a specific fiber type ( Table ). For instance, in D15 diaphragm, 100% of the fibers were positive for RyR3, but only 10%–80% of the fibers reacted with any one of the fiber type markers. Conversely, in D25 EDL muscle, none of the fibers contained RyR3, whereas between 5 and 30% of the fibers reacted with one of the tested markers. As to the onset of RyR expression during embryonic development, we observed both RyR1 and RyR3 in leg muscles fibers at E14 (not shown). To directly correlate the immunocytochemical data with the relative content of RyR1 and RyR3 in crude membrane preparations of developing hind limb muscles and diaphragm, muscle tissue was isolated from mice at D2 and D15, and from adult mice (i.e., D60) and subsequently analyzed in Western blots. As shown in Fig. 6 , the expression patterns of RyR1 and RyR3 differ between hind limb and diaphragm muscles. In neonatal mice, levels of RyR1 and RyR3 are higher in diaphragm than in hind limb muscles. In D2 and D15 diaphragm, RyR3 was found at similar levels, while in hind limb muscles, RyR3 levels increased markedly during the same period. A similar expression pattern (steady expression within the first two weeks after birth in diaphragm and an increase in expression in hind limb muscles) was observed for the RyR1 isoform. In line with previous results, RyR3 expression was reduced below the level of detection in hind limb muscles of adult animals. In contrast, in diaphragm, RyR3 was still detected in adult animals, although at lower levels. These experiments were repeated with five different microsome preparations, always confirming the expression pattern reported in Fig. 6 . Densitometric analysis of seven representative Western blots from these experiments indicate a 5–10-fold difference in RyR3 content of diaphragm of newborn and adult mice, which is consistent with the decreased number of muscle fibers expressing RyR3, as observed with immunocytochemistry. Most of our current knowledge of the physiological role of RyRs is derived from studies of RyR1 and RyR2 in striated muscles, where these two RyR isoforms function in depolarization-induced calcium release from the SR. The skeletal muscle RyR1 is controlled directly by the voltage sensor, the dihydropyridine receptor, whereas the cardiac RyR2 opens in response to calcium entering through the dihydropyridine receptor and calcium being released elsewhere from the SR. In contrast, we know little about the biological functions of the ubiquitously expressed RyR3 isoform. Studying RyR3 in skeletal muscle, where we understand the mechanisms of calcium regulation best, may give us important insights into its physiological role. RyR3 is not essential for muscle function and it cannot substitute for the function of RyR1 in skeletal muscle. Expression of RyR3 is downregulated during early development and some adult mammalian muscles, like EDL, do not express RyR3. Furthermore, RyR3−/− mice develop and move normally. However, depolarization-induced calcium release is weaker in young RyR3−/− muscle fibers than in normal fibers . Thus, it has been suggested that RyR3 may play an accessory function in skeletal muscle excitation–contraction coupling. This hypothesis is strongly supported by our present finding that RyR3 is localized in skeletal muscle triads. The immunolocalization pattern of RyR3 resembled exactly that of RyR1 in young mammalian muscle fibers and the colocalization of RyR3 with the skeletal muscle dihydropyridine receptor indicates that the RyR3-containing structure corresponds to T-tubule/SR junctions. This finding is in agreement with the preferential distribution of RyR3 in the heavy SR fraction . Within the triad, the observed immunofluorescence labeling pattern is consistent with one of two possible subcellular localizations. Either RyR3 is interspersed between RyR1 in the junctional face of the terminal SR cisternae, or RyR3 and RyR1 are localized adjacent to each other in separate membrane domains. The alternating organization of the RyRs, with respect to the dihydropyridine receptors , would allow RyR3 to occupy the positions of the uncoupled RyRs within the junction. The preferential association of dihydropyridine receptors with every other RyR foot structure during early formation of junction is suggestive of an alternating organization of two different RyR isoforms within these junctions: one that associates with dihydropyridine receptors and one that does not. However, even if an alternating organization of RyR1 and RyR3 would exist in developing muscle fibers, this can hardly be the mechanism by which this molecular arrangement of the dihydropyridine receptor tetrads is formed, because muscles that do not express RyR3 have been shown to contain alternating coupled and uncoupled RyRs . Alternatively, the RyR3 could occupy regions adjacent to the RyR1-containing junctional domain. This arrangement would be more consistent with the observation that junctions of the RyR1−/− mouse, in which we detect RyR3 with immunofluorescence labeling, apparently lack feet and are significantly narrower than normal T-tubule/SR junctions . However, in that and another report , junctions with few feet have occasionally been found. Whether RyR3 is located in one or the other of these two domains of the terminal SR cisternae has implications on current models of excitation–contraction coupling . But either location, within or adjacent to the junctional face, would be consistent with a role of RyR3 in the amplification of calcium release immediately after initiation of depolarization-induced calcium release. The fact that RyR3 was not localized in any compartments other than the triad is also important. Even though immunolabeling does not exclude the possibility that RyR3 is expressed at low concentrations in other regions of the SR, its distribution pattern was clearly that of a triad protein and distinct from that of the calcium ATPase, which is concentrated in the longitudinal SR . Thus, it is rather unlikely that RyR3 would primarily serve general housekeeping functions in developing muscles and that its contribution to excitation–contraction coupling would be only secondary. In cardiac ventricular muscle, SR calcium stores have been observed (extended or corbular SR) that contain RyRs, but are not coupled to dihydropyridine receptors . Presumably, these calcium release sites serve in the wave-like propagation of the calcium signal into the interior of the cardiac myocytes. The properties of RyR3 would be consistent with a similar function in skeletal muscle, but such structures have not been described in skeletal muscle, and our immunolocalization does not indicate the existence of calcium release sites outside the triads either. Thus, the lack of RyR3 immunolocalization outside the triad junctions further supports the notion that the primary biological role of RyR3 in skeletal muscle is in excitation–contraction coupling. This is in agreement with the results of several recent functional studies of the RyR3 , which indicate that RyR3 channels are less sensitive to inactivation at high calcium concentrations than RyR1. Thus, the function of RyR3 in the skeletal muscle triad could be to amplify the calcium signal coming from the directly voltage-activated RyR1 channels . The developmental expression of RyR3 differs from that of RyR1. The earliest stage in which we looked for RyR3 expression in skeletal muscle was E14. Even at this time in development, RyR1 and RyR3 were both coexpressed in mouse muscle fibers. This differs from the onset of expression of the corresponding RyR isoform in chicken, where the α RyR precedes the β RyR by as much as five days . At E18, all hind limb and diaphragm muscle fibers expressed both RyR isoforms. At this developmental stage, hind limb and diaphragm muscles of RyR1−/− mice also contained normal concentrations of RyR3 . This finding is consistent with data showing the expression of RyR3 message and the ryanodine and caffeine sensitivity in RyR1−/− myotubes , but it contradicts the results of Buck et al. 1997 , who did not detect ryanodine binding or RyR3 immunoreactivity in muscles of a different RyR1 knockout mouse. To rule out that our positive RyR3 immunoreaction resulted from a peculiarity of one particular RyR1 knockout mouse, we performed the immunofluorescence experiments on muscles from RyR1−/− mice generated in two different laboratories . In both cases we found RyR3 label in triads of embryonic muscle. Soon after birth, expression levels of RyR3 began to decline, first in EDL muscle and later in diaphragm. This time course was expected from our earlier results of a Western blot analysis . However, to our surprise, the loss of the RyR3 isoform in developing muscles occurred not by a continuous decline of RyR3 throughout the muscle, but occurred rapidly in some fibers and delayed or not at all in others. Available estimates of RyR3 content in adult diaphragm indicate that the content of this isoform varies between 0.7 and 5% of total RyRs in whole muscle homogenates . Our Western blot analysis revealed a 5–10-fold reduction in the RyR3 levels of adult diaphragm compared with newborn diaphragm. Immunostaining results presented in Fig. 4 and Fig. 5 show that all fibers of skeletal muscles of fetal and newborn mice contain RyR3, while in adult diaphragm, only a subset of ∼10–15% of the fibers contain RyR3. Together, these findings suggest that the developmental decline in RyR3 protein in adult diaphragm results from the decreased number of RyR3 positive fibers. At the same time, it can be inferred that the content of RyR3 in the subset of fibers expressing this isoform in adult mouse diaphragm is roughly the same as that of newborn fibers. This is of particular importance since it indicates that the relative amount of RyR3 in skeletal muscle fibers that express this isoform is higher than previously appreciated from biochemical estimates based solely on total muscle homogenates. With the aim of correlating the RyR3-expressing fibers with a functional muscle fiber type, we compared the expression pattern of RyR3 in D15 and D25 diaphragm and EDL muscles with those of different isoforms of the myosin heavy chain and with that of the fast calcium ATPase. However, the expression pattern of none of these marker proteins mirrored that of RyR3, indicating that the regulation of RyR3 expression in developing mammalian muscles is independent of regulation of the fiber-type specific set of proteins. This result is supported by data from an earlier denervation experiment in which no changes of RyR3 expression levels were observed after denervation , even though the fiber type composition is known to change after denervation . Thus, neither the physiological function of RyR3 nor the regulation of its expression are directly correlated with a single specific fiber type. The differential regulation of RyR3 expression in different muscle fibers suggests a functional difference between the fibers that contain RyR3 in addition to RyR1 and those that do not. The physiological significance of a second RyR isoform in a skeletal muscle triad is not clear, although expression of two isoforms of RyRs is common in nonmammalian vertebrate skeletal muscles . Two components of Ca 2+ release can be identified in voltage clamp experiments: one that is strictly related to membrane depolarization and a second with fast activation and inactivation properties, presumably representing Ca 2+ -induced Ca 2+ release . While RyR1 alone seems to be able to support both components of Ca 2+ release, as they have also been observed in muscle fibers containing only a single RyR isoform, a second RyR isoform with different properties of Ca 2+ -dependent activation and inactivation may affect the Ca 2+ release component that is not under the direct control of membrane depolarization . In summary, the results of this study demonstrate that considerable amounts of RyR3 are expressed in the triads of skeletal muscle where it can perform an accessory function to that of RyR1 in excitation–contraction coupling. Based on the expression pattern in developing muscles, the function of RyR3 appears to be important for all muscles during early development, but only to a subset of muscle fibers of certain adult muscles. | Study | biomedical | en | 0.999998 |
10444071 | For titin antibody epitope positions, see Fig. 1 . The following monoclonal antibodies were used: T12 , I18, S54/56 , and I17. I17 was prepared according to the nomenclature and domain boundaries of the Ig domain I17 as in Labeit and Kolmerer 1995 . The cDNA fragment coding for I17 was subcloned into a modified pET vector , and His 6 -tagged protein was expressed and purified as described previously . BALB/c mice were immunized in a standardized scheme with the purified I17 titin using titerMax adjuvans (Vaxcel), and antibodies were generated as described . The antibody was found to be specific for cardiac titin, recognizing a single defined epitope in the assay with recombinant titin domains (data not shown). The following affinity-purified polyclonal antibodies were used: I20/22 , MIR , and N2B. For N2B, base pairs 11,551–11,928 of the human cardiac titin cDNA entry were isolated by PCR . The fragments were subcloned into modified pET9D vectors that expressed their insert sequences as fusions with NH 2 -terminal His 6 tags. The recombinant peptides were purified from the soluble fractions by nickel chelate affinity chromatography on NTA (Ni-NTA) resins as specified by the manufacturer (Qiagen). Antibodies were raised in rabbits by Eurogentec and the specific IgG fraction was isolated by affinity chromatography. Western blot analysis of extracts of cardiac muscle was used to verify the specificity of the antibodies (data not shown). The antibody is directed against an epitope within a 162-residue-long sequence near the NH 2 terminus of the middle N2-B region . Myofibrils were isolated from freshly excised rabbit left ventricle as described . In brief, trabeculae were dissected, tied at both ends to a thin glass rod, and skinned in ice-cold rigor solution containing 0.5% Triton X-100 for a minimum of 4 h. The skinned strips were minced and homogenized in rigor buffer. A drop of the suspension was placed on a coverslip, and a single myofibril (sometimes a doublet) adhering lightly to the glass surface was picked up under a Zeiss Axiovert 135 inverted microscope with the help of water-hydraulic micromanipulators (Narishige) by two glass needle tips coated with water-curing silicone adhesive. Experiments were performed at room temperature in relaxing solution of 200 mM ionic strength, pH 7.1. All solutions were supplemented with the protease inhibitor leupeptin to minimize titin degradation . For immunofluorescence microscopy, a myofibril was stretched in relaxing buffer to a desired SL and was labeled with primary titin antibody and Cy3-conjugated secondary IgG (anti–rabbit or anti–mouse; Rockland). Sometimes, a stretched myofibril was fixed in 3.5% paraformaldehyde solution for 20 min before the staining procedure. Using both approaches, identical results were obtained, although fixation appeared to decrease fluorescence intensity. Control measurements with secondary antibody only showed no fluorescence. Antisera were used at the following concentrations (antibody/relaxing buffer): T12, 1:50; I17, 1:5 or undiluted; N2B, 1:5 or 1:10; I18, 1:5 or 1:10; S54/56, 1:20; I20/22, 1:50; MIR, 1:100; secondary antibodies, 1:50. For most experiments the incubation time was 20 min for both primary and secondary antibodies. Images were recorded in the epifluorescence mode of the microscope (100×, 1.4 NA oil immersion objective). Three images were recorded at a given SL and automatically superimposed by using a feature of the software (Global Lab Image, Data Translation). For measurements at another SL, a new preparation was used. The intensity profile along the myofibril axis was plotted, and the distances between intensity peaks were determined by two independent methods. First, a commercial peak detection program (AIDA; Raytest) was used to measure distances between the highest points on the plots. Second, to exclude the possibility that a distorted shape of some maxima influences the results, custom-written software (Borland Turbo Pascal) was used to determine the peak distances by a center-of-mass analysis . The program calculates the area under an intensity maximum, above a baseline drawn parallel to the abscissa . This baseline was either set to pass through the relative minimum between peaks reflecting the unlabeled Z-line region or was positioned by eye to above that minimum. The latter case was applicable when peak spacings were increased and peak amplitudes were relatively large in highly stretched sarcomeres. Next, the program calculates those points on the abscissa at which a vertical straight line can be constructed that divides the area under each maximum into two equal parts . These points were then used to measure the epitope spacings across the Z-line. Results obtained with the two analysis methods were similar within experimental error; in this study, we exclusively show data from the center-of-mass analysis. Because the method could be applied successfully only when peaks did not overlap, reliable detection required epitope spacings of at least ∼200 nm. Finally, the calculated peak distances were divided by two to obtain the Z-disk center to epitope distance in a half-sarcomere. Immuno-EM was essentially carried out as follows: rapidly excised rabbit left ventricular tissue (mostly trabeculae, sometimes papillary muscles) was stretched to different degrees in relaxing buffer. Then, a solution containing 3.5% paraformaldehyde was added as fixative for no more than 2 h. As described elsewhere, specimens were processed for cryosectioning and immunolabeling with the titin antibodies and secondary antibody conjugated to 10-nm gold particles . Titin antiserum dilution was as follows: T12, 1:50; N2B, 1:5; I18, undiluted; I20/22, 1:40. For double staining experiments with the titin antibodies, N2B and I18, differently sized gold particles were used: N2B was visualized with 10-nm particles, I18 with 15-nm particles. Micrographs were taken with a Philips EM 301 at 80 kV. The center of the nanogold particles was used to measure the distance of a given antibody epitope from the center of the Z-disk. Myofibrils were suspended between a micromotor and a sensitive force transducer, and passive length-tension curves were measured as described previously . Solutions contained 20 mM 2,3-butanedione monoxime to suppress force-generating actin-myosin interactions possibly remaining even under relaxing conditions. In a typical protocol, preparations were stretched in stages from slack length to a series of desired SLs. Stretch duration was ∼20 s; the hold period (to wait for stress relaxation) was 2–3 min. After stretching to a maximum SL, the specimen was released in stages to slack length. Then, the cycle was repeated two more times, extending the myofibril to a higher maximum SL in each cycle. To obtain passive tension, the cross-sectional area of a preparation was inferred from the diameter of the specimens as described . GFP (green fluorescence protein)–N2-B fusion constructs: For the expression of N2-B sequences in cardiac myocytes, PCR fragments were amplified from a human cardiac cDNA library that contained either the complete cardiac-specific N2-B sequence in the center of I-band titin , or fragments of it. For the amplification, the following primers were used : X218, tttagatct-GAA GGC ACT GGC CCA ATT TTC ATC AAA GAA; X219, tttgtcgac-ta-GTC TGT GTC TTC CAG AAG CAC AAG CAG CTC; X220, tttagatct-GAG GAT GGC CCC ATG ATA CAT ACA CCT TTA; X221, tttgtcgac-ta-CAC TGT CAC AGT TAG TGT GGC TGT ACA GCT; X222, tttagatct-ATG ACT GAT ACC CCC TGC AAA GCA AAG TCC; X223, tttgtcgac-ta-GCC ATC CTC TTT GAT TAA GCC ACC CTC AGC. The NH 2 -terminal fragment X218-X219 encompasses the Ig repeats N2-B I16/17 and the COOH-terminal fragment X220-X221 contains the Ig repeats N2-B I18/I19 . The intervening, cardiac-specific sequence (middle N2-B region) is contained in the construct X222-X223. The N2-B fragments amplified by the X218-X223 primers were inserted into the pEGFP-C1 vector (Clontech) that was linearized by digestion with BglII and Sal I restriction endonucleases (MBI Fermentas). The recombinant plasmid inserts were transformed into competent DH5-alpha E . coli cells, and recombinant plasmid DNAs were purified using Qiagen columns (Qiagen). Plasmids were verified by sequencing. Cardiac myocytes were prepared from day 6 embryonic chick hearts and cultured as described previously . Isolated cells were plated in 35-mm tissue culture dishes containing 12-mm round coverslips (1 × 10 6 cells/dish). Approximately 15% of the cells in most of our primary cultures are fibroblasts. 24 h after plating, cultured myocytes were washed two times in OptiMEM, placed in 800 μl fresh OptiMEM, and returned to the incubator while DNA liposome complexes were prepared. Such complexes were prepared by combining 1 μg plasmid with 4 μl Lipofectamine and 6 μl PLUS Reagent in 200 μl serum-free OptiMEM. After 15 min, the complexes were added dropwise to the culture dish. 3 h later, 1 ml of MEM 10% FBS (Hyclone Laboratories, Inc.) was added to the dish. One to six days later, cells were gently washed with PBS and fixed with 2% formaldehyde in PBS for 10 min. Coverslips were washed and stored in PBS at 4°C until staining. Over 300 transfected cells per construct were analyzed. All tissue culture reagents (except where noted) were purchased from Life Technologies. Cardiac myocytes were essentially stained as described . In brief, coverslips were permeabilized in 0.1% Triton X-100 and PBS for 15 min. To minimize nonspecific binding of antibodies, the coverslips were preincubated in 2% donkey serum, 2% BSA, and PBS for 30 min. Antibodies specific to various sarcomeric components and phalloidin (to stain for actin filaments) were used to analyze the transfected cells. Details on the staining protocols used in the micrographs presented are described. To analyze the distribution of α-actinin, rabbit anti–α-actinin antibodies (1:1,000) (generously provided by Dr. S. Craig, Johns Hopkins University, Baltimore, MD) were used, followed by Texas red–conjugated F(ab) fragments of donkey anti–rabbit antibodies (1:200). To analyze the distribution of MyBP-C, polyclonal anti–MyBP-C antibodies (1:50) (see below) were used followed by Texas red–conjugated F(ab) fragments of donkey anti–rabbit antibodies (1:200). For triple labeling, rabbit anti-GFP antibodies (1:100) (Clontech) were used, to visualize transfected cells before the appearance of GFP fluorescence, followed by Cascade blue–conjugated goat anti–rabbit IgG antibodies (1:100; Molecular Probes Inc.). Monoclonal anti-striated muscle myosin antibodies (1:10) (F59, generously provided by Dr. F. Stockdale, Stanford University, Stanford, CA) were used to visualize thick filaments, followed by Texas red–conjugated F(ab′) 2 fragments of donkey anti–mouse antibodies (1:600; when costained with titin antibodies) or FITC-conjugated donkey anti–mouse antibodies (1:200; when costained with phalloidin). Chicken anti-titin N2A antibodies were used to visualize titin , followed by FITC-conjugated donkey anti–chicken antibodies. Texas red–conjugated phalloidin (Molecular Probes Inc.) was used to visualize actin. Stained cells were analyzed on a Zeiss Axiovert microscope using 63× (NA 1.4) and 100× (NA 1.3) objectives and micrographs were recorded as digital images on a SenSys cooled HCCD camera (Photometrics). Images were processed for presentation using Adobe Photoshop and printed using a Codonics NP1600 dye sublimation printer. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc., unless otherwise noted. For the cardiac MyBP-C antibodies, residues 268–375 of mouse cardiac cDNA were amplified from total mouse heart cDNAs by PCR , and fragments were expressed as described above (see Antibodies to Cardiac Titin). The antibodies were raised in rabbits by Eurogentec and their specific IgG fraction was isolated by affinity chromatography. Antibody specificity was confirmed by Western blot analysis of extracts of rat cardiac muscle (data not shown). By using a panel of sequence-assigned antibodies we aimed to establish the extension behavior of structurally distinct segments in both N2-A and N2-B cardiac titins. The epitope locations of the titin antisera are indicated in Fig. 1 . The mobility of titin antibody epitopes in each half-sarcomere was investigated in stretched single myofibrils isolated from rabbit left ventricle by immunofluorescence microscopy. Fig. 2 a shows typical images of myofibrils extended to two different SLs, 2.3 and 2.7 μm, and stained with the respective primary antibody and Cy-3–conjugated secondary antibody. Of the antibodies used, I17, N2B, and I18 are specific to the cardiac N2-B–titin region and did not stain rabbit soleus myofibrils (data not shown), whereas S54/56 is directed to Ig-repeats from the N2-A region. T12 , I20/22 and MIR recognize epitopes outside the differentially expressed I-band titin and should stain all titin isoforms. Both N2-A and N2-B–titin were found to be highly expressed in rabbit cardiac myofibrils, as judged from the strong staining intensity obtained with all antibodies except I17 (see below). At least at high stretch it was obvious that I17/N2B, I18, I20/22, and MIR stained different positions on N2-B–titin. I18 colocalized with the N2-A–specific S54/56 epitope. To more precisely map the epitope positions in a half-sarcomere, we plotted the intensity profiles along the myofibril axis and calculated the center-of-mass pixel position for each intensity peak with custom-written software . The epitope spacing across the Z-disc could now be measured with satisfactory resolution at modest to long SLs. At short SLs, it was more difficult to obtain reasonable results with this analysis method, since the two epitopes around the Z-disk frequently (but not always) were seen to merge into one broader strip. Whereas the antibodies, T12, N2B, I18, and I20/22 were subsequently used for immunoelectron microscopy (immuno-EM), I17 appeared to be unsuitable for this technique. Possibly, the immunoreactive sequence in the Ig domain I17 becomes accessible only at very high sarcomere stretch. Although a tendency for stronger labeling at long SLs was observed sometimes by immunofluorescence microscopy, we regularly found both stained and unstained sarcomeres of comparable length side by side in a myofibril , a finding never observed with any other antibody. Hence, we speculate that the native I17 domain of N2-B–titin may be partially inaccessible for the antibody, because its immunoreactive site is covered by some ligand (see Effects of Titin N2B Overexpression in Cardiac Myocytes). The distance of a given epitope from the center of the Z-disk was determined at shorter SLs by immuno-EM. Fig. 3 a shows images of rabbit cardiac sarcomeres stretched to 1.9 and 2.3 μm, respectively, and labeled with one of four different antibodies. Using these antibodies, which flank the structurally distinct regions in N2-B–titin (proximal Ig-segment, middle N2-B region, PEVK domain), we confirmed that each region contributes to titin extensibility already at short to modest SLs. Whereas extension of the Ig-chains and the PEVK segment has been recognized previously , the observation of considerable extensibility of the middle N2-B region is novel. To determine the SL at which this region begins to extend, we double-stained cardiac sarcomeres with both N2B and I18 and used secondary antibodies conjugated to different sized nanogold particles. As shown in Fig. 3 b, the epitopes could not yet be separated at 2.1 μm SL. However, separation was apparent in sarcomeres stretched to 2.35 μm. At 2.6 μm SL, the N2-B segment flanked by the antibody epitopes contributed substantially to I-band titin extensibility. The results of the immunolabeling experiments are summarized in Fig. 4 ; separate graphs are shown for immunofluorescence and immuno-EM data . For each antibody type, individual data points were pooled in 50-nm-wide SL bins, plotted as mean ± SD, and fitted by a third-order regression. When we compared results obtained with the two different techniques, epitope mobility was found to be similar within experimental error. The I17 and N2B antibodies stained almost the same position on N2-B–titin, as expected from their epitope location. I18, which comigrated with the N2-A–specific S54/56, labeled a position clearly different from that stained by I17/N2B or I20/22. The unique N2-B sequence flanked by I18 and N2B was confirmed by statistical analysis (unpaired Student's t test) of immuno-EM data to begin to lengthen significantly at ∼2.15 μm SL ( P < 0.001; n = 17 for the I18 data set and n = 19 for the N2B data set at SL bin 2.15–2.19 μm). By calculating the distances between fit curves at a given SL, we plotted the extensions of individual cardiac titin segments versus SL . Data from both immuno-EM and immunofluorescence measurements were compiled because of the similarity of results obtained with the two techniques. Curves in the main Fig. 5 a indicate extensibility of the four structurally distinct regions that make up the entire elastic N2-B–titin. The proximal poly-Ig region elongated continuously up to the longest SL investigated, whereas the other three elements approached a plateau in their extension curves: the N2-B–PEVK domain at a SL of 2.3–2.4 μm, the distal poly-Ig region and the unique N2-B insertion at ∼2.6 μm. Substantial lengthening of the unique N2-B insertion began near 2.15 μm SL, and at 2.3–2.4 μm SL, this region reached 60–70 nm extension. When sarcomeres became highly stretched, the segment elongated to >100 nm. The inset in Fig. 5 a shows extension of the N2-A–PEVK domain and that of the Ig-segment flanked by T12 and S54/56. Whereas the Ig segment extended steadily at all SLs, the extension curve for N2-A–PEVK eventually reached a plateau at ∼2.6 μm SL. An important unresolved question is whether individual Ig domains are able to unfold at the stretch forces acting on titin filaments in normally functioning cardiac muscle . That is, can a segment of Ig-modules be stretched to beyond the contour length predicted from its sequence structure assuming folded domains? To answer this question, we used the available sequence data for cardiac titin to predict the contour length of both the proximal and the distal tandem-Ig segments of the N2-B isoform ( Table ). A value of 4.6 nm was taken as the maximum spacing between folded domains . Because only incomplete sequence information is available for cardiac N2-A titin, we could not predict the contour length of the Ig segment flanked by T12 and S54/56 . However, contour length predictions could be made for all other I-band segments in N2-B–titin and for the N2-A–PEVK domain ( Table ). By relating extension of a given titin segment to predicted contour length, the fractional extension of each segment was calculated . We found that the distal poly-Ig region stretched up to its predicted contour length but not beyond. The proximal Ig domain region of N2-B–titin exceeded the contour length value at a SL of ∼2.5 μm, perhaps as a result of domain unfolding. Thus, proximal N2-B Ig domains might potentially be able to unfold in highly stretched sarcomeres, but are unlikely to do so in normally functioning heart where maximum SLs are presumably below 2.4 μm. For all other titin segments, the end-to-end lengths were predicted to either reach or stay below the assumed contour lengths . Interestingly, when we assumed that the PEVK segments and the unique N2-B insertion are capable of complete unfolding (maximum residue spacing, 0.38 nm), only the curve for N2-B–PEVK was predicted to reach the contour length value . The unique N2-B insertion and the N2-A–PEVK domain at best extended to half the predicted maximum value. On the other hand, because both segments did approach a plateau in their extension curves, we argue that these regions may not unfold entirely. Rather, they may adopt some permanent structural fold, which would lead to a shorter contour length; the resulting curves are shown in Fig. 5 b (solid blue curve) and Fig. 5 b, inset (solid yellow curve), respectively. We aimed to determine the SL range within which cardiac titins can be extended and released in a completely reversible manner. Therefore, nonactivated single cardiac myofibrils were repeatedly stretched and released in steps while passive force was recorded . Between steps, the specimens were rested for 2–3 min, to allow force to drop to a quasi steady state level. In each cycle, the maximum SL was increased and between cycles the slack SL was monitored. Thus, we could study the maximum length from which cardiac sarcomeres can be released to a completely recoverable slack SL. Results obtained with four different myofibrils demonstrated that sarcomeres could be extended to ∼2.5 μm while still returning to their initial slack length after release (cycle 1). However, when the maximum stretch clearly exceeded 2.5 μm, the force-stretch/release curves exhibited large hysteresis and the specimens' slack SL was increased (cycles 2 and 3). This finding implies that cardiac titin filaments can be stretched reversibly to well above physiological length. Further stretch likely results in irreversibly increased contour lengths of titin. This, in turn, leads to dramatic changes in the passive length–tension relation of cardiac myofibrils . To gain further insight into the functional significance of the N2-B region of cardiac titin in cells, we overexpressed this region in primary cultures of chick cardiac myocytes in the hope that the expressed fragment would compete with the endogenous titin domain to generate dominant-negative effects on sarcomere structure. Initially, we transfected cells with a plasmid encoding the entire region of titin N2-B. A GFP tag was used to distinguish the recombinant protein from endogenous protein. Although no significant disruption of actin filaments was observed in cells overexpressing GFP alone, substantial disruption of thin filament structure was observed in ∼70% of all transfected cells where N2-B–titin was overexpressed, as seen by a marked loss in actin filaments . Moreover, when the transfected cells overexpressing titin N2-B were stained for tropomyosin , a similar disruption of thin filament structure was observed (data not shown). In contrast, preliminary data (not shown) suggested that overexpression of the entire N2-A region of cardiac titin has no effect on sarcomere structure. To identify the specific domain(s) within titin N2-B responsible for the disruption of thin filaments, we also transfected cells with plasmids encoding GFP-tagged subdomains of N2-B specific to the 25-kD NH 2 terminus (N-N2-B), a 70-kD middle region (M-N2-B), and the 25-kD COOH terminus (C-N2-B) . Cells overexpressing N-N2-B revealed severe disruption of actin filament structure in all transfected cells . In fact, the number of live myocytes that were transfected with N-N2-B declined drastically as the levels of this fusion protein increased; the cells were observed to round up and detach from the coverslips. This observation was unique to this construct. Remarkably, in cardiac myocytes in which M-N2-B was overexpressed, no obvious disruption of thin filaments was observed . Similarly, thin filaments showed no detectable disruption in cells expressing C-N2-B . In this regard, it is important to note that it is well documented that the process of myofibril assembly in primary cultures of cardiac myocytes is temporarily irregular; thus, numerous structures representing different stages of assembly are observed within the same cell, as well as within different cells in a culture dish . Thus, ∼20% of nontransfected cells, cells transfected with GFP alone, M-N2-B, or C-N2-B demonstrate a disruption-like distribution of actin filaments (data not shown). The striking thin filament disruption observed in cardiac myocytes as a result of the overexpression of titin N2-B, and specifically, N-N2-B, suggested that other sarcomeric components might also be disrupted. To study this, the distribution of the major Z-line component, α-actinin, and the thick filament–associated proteins, myosin and MyBP-C, were analyzed in the transfected myocytes. As demonstrated in Fig. 7 d, mature myofibrils in cardiac myocytes probed with α-actinin antibodies exhibited a sharp repeating (∼2 μm apart) staining pattern. As found for actin filaments, overexpression of N2-B or N-N2-B resulted in disruption of Z-lines also , but to a somewhat lesser degree. That is, Z-disks could still be detected in the transfected cells, although they were frequently distributed in a less ordered fashion . Again, consistent with the observations on thin filament disruption, overexpression of M-N2-B, C-N2-B or GFP alone did not significantly disrupt α-actinin staining patterns . On the other hand, when cells overexpressing N2-B or N-N2-B were stained for thick-filament proteins, including MyBP-C, no altered distribution was detected . Since it was difficult to imagine such well-formed sarcomeres visible by MyBP-C staining in the absence of thin filaments, we performed triple labeling experiments to visualize this phenomenon within identical myofibrils. Costaining cells overexpressing N2-B for actin and myosin confirmed our results: thin filament structure was disrupted in the same myofibrils that demonstrated intact thick filaments . Furthermore, analysis of identical cells overexpressing titin N2-B stained for myosin and several epitopes of titin along the length of the molecule demonstrated that the titin filaments (data shown using anti–N2-A antibodies) also remain intact . Similarly, when cells overexpressing GFP-N2-B were triple stained for GFP, actin, and N2A, the distribution patterns of titin looked unperturbed in the identical myofibrils that demonstrated a disrupted staining pattern for actin (data not shown). Finally, we note that overexpression of GFP alone, titin N2-B, or any of the three N2-B fragments resulted in no observable alteration to the actin-containing stress fibers in the fibroblasts contaminating our cultures, as determined by phalloidin staining (data not shown). We conclude that the NH 2 -terminal region of chick cardiac N2-B–titin possesses structural properties necessary to maintain the integrity of thin filaments. We would also like to stress that similar results were obtained in transfection experiments with primary cultures of neonatal rat cardiac myocytes. Recently, progress has been made in understanding the nature of titin elasticity in skeletal muscle sarcomeres . In contrast, cardiac titin is expressed in different length isoforms, N2-A and N2-B, which makes an investigation of the protein's mechanical properties less straightforward. Insights into the function of cardiac I-band titin have been obtained in mechanical and immunolabeling studies on isolated cells and single myofibrils . It was proposed that in unstretched cardiac sarcomeres, titin's elastic section is in a contracted state . The small passive forces developing upon low stretch to ∼2.0 μm SL were suggested to be entropic in nature and to arise from straightening of I-band titin. With further stretch and exponential tension rise, the molecular domains within titin were thought to unfold. In a later report, in light of new experimental evidence, the SL for the onset of unfolding (of titin's Ig domains) was proposed to be ∼2.2 μm . Similarly, it was concluded in an immunofluorescence microscopical study of stretched single myofibrils stained with sequence-assigned antibodies , that cardiac titin's Ig domains may begin to unfold above 2.2–2.3 μm SL. . These predictions were made based on the hypothesis that titin contains two structurally distinct extensible elements, poly-Ig chains and the PEVK domain . We now demonstrate in this study that this property is indeed a characteristic of the N2-A isoform, but not of the N2-B isoform. The extension properties of cardiac titin isoforms were investigated by immunolabeling techniques, using isoform-specific antibodies. Perhaps most striking was our observation on the extensibility of I-band segments in N2-B–titin. We found that this isoform contains, besides poly-Ig chains and the PEVK segment, a third extensible element, the middle N2-B sequence. The results led us to propose a novel model for titin isoform extension in cardiac muscle . We suggest that in both isoforms, low sarcomere extension is brought about by elongation of tandem-Ig-segments and the PEVK domain . Above 2.15 μm SL, also the 572-residue N2-B region in the middle of I-band titin begins to elongate substantially and may compensate for the relatively short length of the N2-B–PEVK segment (163 residues), whose extensibility would soon be exhausted. Elasticity of the unique N2-B sequence may help the shorter N2-B–titin isoform to adjust its range of extension to that of the longer N2-A isoform. The model in Fig. 10 also predicts that elongation of poly-Ig segments is brought about by straightening, rather than by domain unfolding. Moreover, cardiac titin extension may still be fully reversible after stretch to 2.5 μm SL . Then, complete reversibility of stretch appears to be a characteristic of all three elastic elements, the middle N2-B region, the PEVK segments, and the poly-Ig chains (with folded domains). The model suggests that titin's tandem-Ig modules, earlier thought to unfold above 2.2 μm SL, may not do so within the normal working range of cardiac muscle. Rather, they are likely to remain in a folded state even at high physiological stretch, just as recently suggested also for skeletal muscle titin . These findings, which confirm and extend previous conclusions on the extensibility of the distal Ig domain segment of cardiac titin , address an intriguing issue arising from the results of earlier single-molecule mechanical experiments on titin . In these studies, Ig domains were found to unravel at high stretch forces of 20–300 pN (the exact value depends on stretch speed). However, refolding of the modules occurred relatively slowly and only when the external force was lowered to a few piconewtons, resulting in enormous hysteresis; thus, the Ig domain behaves like a spring that loses considerable energy in each stretch (unfold)–release cycle. It seems unlikely that such an ineffective spring exists in the heart, which undergoes millions of stretch-release cycles during a lifetime. Our results provide strong evidence that the experimentally inducible mechanical denaturation/renaturation of Ig repeats does not take place during normal heart function. If at all, unfolding may be found perhaps in the proximal Ig domain region and only in pathologically overstretched cardiac muscle. Although our understanding of the elastic properties of cardiac N2-A titin is still incomplete, mainly due to limited availability of sequence data, this study nevertheless provides interesting insights. For example, our findings indicated that the ∼700-residue-long N2-A–PEVK domain does not unravel completely even at the highest sarcomere stretch applied (2.8 μm SL). This hints at the fact that this domain may well be able to adopt some structural fold , the nature of which remains to be identified. Also, since our analysis of N2-B–titin extensibility excluded that the proximal and distal Ig domains unfold at physiological stretch , we find it also unlikely that the titin segment NH 2 -terminal to the N2-A–PEVK domain elongates by unfolding of Ig domains. Support for this conclusion comes from earlier observations that in different regions of the titin molecule, the thermodynamic and mechanical stabilities of Ig domains are comparable . Furthermore, our results clearly show that the segment flanked by the T12 antibody epitope and the I17 epitope extends much less than that located between T12 and S54/56 . Then, all evidence taken together, we reason that cardiac N2-A titin should contain more (folded) Ig modules than the N2-B isoform in the proximal tandem-Ig region . It will be interesting to see whether sequence data will eventually confirm this prediction. The elastic properties of cardiac titin have previously been modeled according to entropic polymer elasticity theory, by assuming that two different elastic elements, the Ig domain segments and the PEVK domain, act as worm-like chains (WLCs) in series . With the knowledge that a third type of elastic element exists in the cardiac N2-B isoform, it is obvious that the WLC modeling for cardiac titin needs modification. A complication is that our data on N2-B expression in myocytes raise the possibility that part of the N2-B–titin region might be involved in protein–protein interactions . Thus, titin may not be allowed to move freely in the sarcomere, which could affect the elastic properties of the protein chain. Because it is unsettled how this possible association will change the (hypothesized) WLC behavior of cardiac titin, we did not perform a detailed WLC analysis at this stage. A fascinating observation that emerges from our results is that some portions of cardiac N2-B–titin appear to have properties necessary to maintain an ordered thin filament structure. Although this is, to our knowledge, the first direct evidence for a structural role of I-band titin in cardiac muscle, preliminary, indirect evidence existed before. From the study of deep-etch replica electron micrographs prepared to analyze the filament network present in cardiac I-bands before and after selective removal of actin, it was proposed that the elastic titin could associate laterally with thin filaments . However, despite several reports that titin interacts with actin in vitro , potential actin–titin interaction sites could not be identified in intact sarcomeres, except within a region near the Z-disk . Thus, the nature of the interaction of I-band titin with the thin filaments suggested by Funatsu et al. 1993 remained elusive. In this study, we reasoned that a most characteristic segment of cardiac titin is the central I-band region of the N2-B isoform. Therefore, we investigated the functional significance of this region of titin in the context of cardiac myocytes. The central N2-B–titin region, as well as distinct subfragments of it, were overexpressed in primary cultures of cardiac myocytes. Using transfection techniques, overexpression of the entire N2-B region or its NH 2 -terminal segment encompassing the Ig domains I16 and I17 resulted in severe thin filament disruption . In contrast, overexpression of the middle N2-B region or the COOH domains I18 and I19 resulted in more or less normal phenotypes. Interestingly, whereas the Z-lines were also affected in cells overexpressing the full N2-B region or the NH 2 -terminal segment of this region, the disruption appeared less severe. This observation suggests that the disruption of the Z-lines was a secondary effect of the thin filaments being disrupted. The picture emerging from these results is one in which the region bounded by the N2-B–specific Ig domains I16 and I17 is important for the stability of thin filaments. Also, the integrity of N2-B–titin appears to be a prerequisite specifically for thin filament structure, because overexpression of N2-B–titin segments did not affect the thick filaments . The latter finding lends further support to the hypothesis that nucleation and assembly of I-Z-I bodies (containing several Z-line and thin filament components and titin) occurs independently of the formation of sarcomeric thick filaments, in primary cultures of heart and skeletal muscle cells, as well as in heart development in vivo . Moreover, the observation that the titin filaments were unaffected in cells overexpressing N2-B, is consistent with the idea that titin may be the structural element keeping the A-bands aligned, in the absence of thin filaments. A possible explanation for the severe disruption of the thin filaments is that a dominant-negative phenotype occurred. That is, overexpressed titin N2-B (or the NH 2 -terminal domains of this region) competed for the interaction of endogenous N2-B–titin with an intracellular ligand, thus preventing native titin from taking on its usual intracellular role. A speculative idea is that titin N2-B may interact directly or indirectly with tropomyosin, a protein that assembles head-to-tail, forming two polymers along the sides of the actin filaments and stabilizes them . Alternatively, based on its sarcomeric location, titin N2-B may interact directly or indirectly with nebulette in cardiac muscle. Nebulette is an ∼100-kD protein of unknown function that is related to the giant protein nebulin in skeletal muscle, the predicted ruler of thin filament assembly . Taken together, the interaction between N2-B–titin domains and their ligand could be functionally relevant in several ways: (a) it may be critical for the maintenance of sarcomeric structure and (b) may participate in the sequential assembly of I-Z-I bodies; (c) it may constrain the elastic behavior of cardiac titin, thereby affecting myofibril stiffness; and (d) it could also affect the relative sliding of thick and thin filaments past one another when the sarcomere changes length. An intriguing possibility is that the binding properties of cardiac N2-B–titin might be different in pathologically altered myocardium, resulting in dramatic changes in the heart's mechanical performance. It shall therefore be interesting to investigate how the I-band titin interaction affects the mechanical properties of cardiac myofibrils, both under physiological and pathophysiological conditions. Future studies will also focus on elucidating whether the NH 2 -terminal region of N2-B–titin directly binds to tropomyosin, nebulette, actin, to other thin filament components, and/or to some still unidentified sarcomeric protein(s). In summary, two main conclusions seem especially noteworthy. First, the N2-B region in the center of I-band titin contains, at its NH 2 terminus, a cardiac-specific segment that is directly or indirectly critical for the stability of thin filament structure. Second, the N2-B region also contains an elastic element, the unique sequence insertion, which extends toward the high end of the physiological sarcomere-length range. Cardiac titin thus represents a molecular spring consisting of three elements: the middle N2-B region, the PEVK domain, and poly-Ig regions with folded modules. N2-B–titin must be viewed as a unique isoform critical for both reversible extensibility and structural maintenance of cardiac myofibrils. | Study | biomedical | en | 0.999998 |
10444072 | The primary specific antibodies used in this study were: mAb anti-CK19 (RCK 108) (Accurate Chemical & Scientific Corp.); mAb anti-CK18 (B23.1) (Biomeda Corp.); mAb anti-CK8 (B22.1) (Biomeda Corp.); rabbit polyclonal antibody against a synthetic polypeptide comprising amino acids 38-53 (EEFATEGTDRKDVFFY) of the NH 2 -terminal region of human γ-tubulin (Sigma Chemical Co.); mAb against the same synthetic peptide of human γ-tubulin (Accurate Chem. & Scientific Corp.); mAb against α-tubulin (DM 1A) (Sigma Chemical Co.); and mAb against all actin isoforms (C4) (ICN Biomedicals Inc.). All secondary antibodies were affinity-purified and had no cross-reactivities with immunoglobulins of other species (Jackson ImmunoResearch Laboratories). F-actin was labeled with FITC-phalloidin (Molecular Probes). Low molecular mass Fab goat anti–rabbit IgG coupled to peroxidase was purchased from Protos Immunoresearch. Synthetic phosphorothioate oligodeoxy nucleotides have been used extensively to reduce the synthesis of specific proteins. Four 21-mer oligonucleotides, two with antisense sequences for CK19 mRNA (A19: 3′-TACTGAAGGATGTCGATAGCG, and A19/2: 3′-AGGAAGTCATGGCGAGGCGGA) and their corresponding scrambled sequences (random: 3′-GAAGCTATTGAGACTGGGATC and random/2: 3′-GGGAGAAGAGTGGTGCCGAAC) were synthesized and extensively purified as described before . CACO-2 cells were obtained from American Type Culture Collection and grown in flasks, 24-mm or 6-mm Transwell-Clear™ filters (for experiments), or roller bottles (Corning Costar) as described before . For experiments with anticytoskeletal agents, the cells, at 9 d after seeding, were incubated in DME-F12 supplemented with 33 μM nocodazole, 2 μM cytochalasin D, or 5 mM acrylamide (all from Sigma Chemical Co.) for 7 h before fixation or extraction. Metabolic labeling was performed with 0.15 mCi/ml [ 35 S]methionine and [ 35 S]cysteine (EXPRE Protein Labeling Mix; NEN) in DME with only 15 μM methionine and cysteine for 24 h. For antisense downregulation, cells were maintained in the standard tissue culture media containing 2–10 μM phosphorothioate oligonucleotides as described before . Immunofluorescence procedures were described as before . The cells were fixed in 100% methanol at −20°C. We have previously demonstrated that the morphology of the apical IF cytoskeleton is similar under methanol, formaldehyde or formaldehyde/glutaraldehyde fixations . Likewise, Stearns et al. 1991 has shown that the method of fixation does not affect the localization of γ-tubulin. Double indirect immunofluorescence was performed using a monoclonal antibody against CK19 and a rabbit polyclonal antibody against γ-tubulin together, and two secondary antibodies raised in goats with no cross-reactivity, and labeled with FITC (anti-rabbit) or CY3 (anti-mouse). Transwell™ filters holding the cell monolayers were mounted in polyvinyl alcohol as described before . Laser confocal microscopy was performed with an Odyssey XL (Noran Instruments) microscope, using an Omnichrome laser source. For colocalization experiments (FITC/CY3), a second detection channel was used separating the light with a custom made 540 nm secondary dichroic using a 565 LP emission filter in the red channel and a 535 ± 20 nm emission filter in the green channel (all from Omega Optical Inc.). To increase resolution in the z-axis, a 15-μm slit was routinely used. The images were collected using Intervision software (Noran Instruments). Each confocal section was obtained as the average of 32 frames to filter noise. The sections were collected at 0.3-μm intervals in the z-axis for low magnification images comprising the entire thickness of the monolayer or at 0.1-μm intervals for high-resolution imaging of the apical domain with 100-nm pixels (nearly cubic voxels for high resolution images) through a 63× oil immersion objective. Usually, each field comprised 60–90 confocal sections. Before three-dimensional reconstruction, the images were subjected to three-dimensional deconvolution using Intervision software for nine neighboring voxels. Convergence was usually achieved at or before 10 iterations. To experimentally adjust the parameters of the deconvolution, test the coalignment of the channels, and assess the final limit of resolution of the system after deconvolution, single or double fluorescent 1-μm beads (Molecular Probes) were used. To adjust the point spread function parameter, the same confocal stacks from preparations in the standard mounting medium were processed using different parameters until the distortion in the z-axis was abolished. Three-dimensional reconstructions were performed using Intervision software on deconvoluted stacks of confocal images. Pixel intensity histograms over single cell areas were used to assess efficiency of the antisense targeting. For z-sections (perpendicular to the plane of the monolayer), the deconvoluted images were cut in 9-pixel-thick volumes (in the x-axis) at the desired level, reconstructed, and rotated 90° to obtain a view of the apico-basal axis. For immunolocalization at the EM level, we combined the procedures of Nanogold™ (Nanoprobes, Inc.) and immunoperoxidase for EM . Both first antibodies (mAb anti-CK19 and rabbit anti–γ-tubulin) were added together. To avoid interactions between the peroxidase and the silver enhancer of Nanogold, we completed all the steps of the Nanogold procedure first, and then incubated the cells with the Fab anti–rabbit IgG-peroxidase and followed the diaminobenzidine reaction as described before . Cytoskeletal preparations were obtained as described before . In brief, the monolayers were washed in PBS, extracted in PBS containing 1% Triton X-100, 2 mM EDTA (extraction buffer, EB) and a cocktail of antiproteases (Sigma Chemical Co.) for 10 min at room temperature, and centrifuged for 9 min in an Eppendorf type centrifuge (14,000 rpm). Alternative extractions, in a more physiologic condition, were performed in 70 mM KCl, 80 mM Pipes (pH 6.5), 5 mM EGTA, 2 mM MgCl 2 supplemented with 0.1% saponin (KEB buffer), and 2 nM caliculyn, 5 μM okadaic acid, and 0.5 mM sodium orthovanadate in addition to the standard cocktail of antiproteases. Cytokeratin preparations were obtained by Triton extraction in 1.5 M KCl followed by cycles of 9 M urea solubilization and repolymerization as described by Steinert et al. 1982 . To obtain fragments of the cytoskeletal preparations, the pellets from the first centrifugation were resuspended in 1 ml EB (or KEB) in Eppendorf tubes. Then, the pellets were sonicated while immersing the tubes in ice-water for a total of 3 min (actual sonication time), with intervals of 10-s sonication (∼250 watts, 5 on a scale of 10) and 15-s gaps to allow for heat dissipation. The suspensions of cytoskeletal fragments were immediately loaded on top of a preformed 10-ml continuous sucrose gradient (20–60% sucrose in EB or KEB) and centrifuged in a Beckman ultracentrifuge at 15,000 rpm for 50 min in a swinging bucket rotor (Beckman SW-40) at 4°C. The gradients were fractionated from the top by gently pipetting ten 1-ml fractions from the surface. No sedimentation standards are available for values well >100 S, except for viral particles that provide an approximate calibration for the gradient. To determine the shape and size of the cytoskeletal fragments we used two methods: (a) 1 μl of the corresponding gradient fractions were spread on carbon-coated EM grids and stained with 2% uranyl acetate; and (b) the fractions were fixed in 2% glutaraldehyde, pelleted, embedded in Spur and sectioned. The EM samples were observed and photographed under a JEM 100-CX II (JEOL) transmission electron microscope. Single dimension SDS-PAGE was performed as described by Laemmli 1970 and two-dimensional (IEF and SDS-PAGE) electrophoresis was performed as described by O'Farrell 1975 . Immunoblot and nitrocellulose reprobing were performed as described before . For immunoprecipitation, fractions 1–5 of the gradients described above were diluted with 1 ml EB (or KEB), divided into two equal 1-ml aliquots, and one of the aliquots was mixed with a 1:100 dilution of the purified IgG polyclonal anti–γ-tubulin antibody , while the other was supplemented with 15 μg/ml purified nonimmune IgG . All the samples were supplemented with 0.1% globulin-free albumin (Sigma) and incubated for 2 h with gentle rotation. Then, 10 μl of protein A–agarose beads (Pharmacia Biotech Inc.) preincubated in 0.1% albumin, were added to each sample and incubated overnight with gentle rotation at 4°C. Next, the beads were centrifuged through a 0.5-ml 30% sucrose cushion and washed four times in EB (or KEB) for 1 h each time. All centrifugations were done at 14,000 rpm for 2 s to minimize unspecific copelleting of unbound filaments. After the last wash, the beads were eluted in 1 ml 2% SDS, and 4 M urea for 2 h. The eluates were TCA precipitated and the pellets washed twice in 100% acetone at −20°C. The pellets were resuspended for 2 h in SDS sample buffer and analyzed by SDS-PAGE and immunoblot/chemiluminescence or autoradiogram in a PhosphorImager (Molecular Dynamics). Because CACO-2 cells undergo a complex differentiation process as they become confluent and polarized, we first determined the types of IF as a function of time after confluency. Purified IF proteins were analyzed by two-dimensional gels at 3 d (nondifferentiated, poorly polarized), 7 d (onset of the development of brush border), 9 d (brush border complete), and 15 d (fully differentiated). The CKs were essentially the same and comprised type I CKs 19 and 18, and CK8 as the only type II partner available . Therefore, CACO-2 cells express IF composed of pairs CK18-CK8 and CK19-CK8 at all the stages of differentiation. For the sake of brevity, these IF will be referred to as CK18 or CK19, respectively, hereafter, and were identified with specific monoclonal antibodies against these two type I cytokeratins. Next, CACO-2 monolayers at the 9-d stage were fixed and analyzed by double immunofluorescence, using a polyclonal antibody against a polypeptide in the COOH-terminal region of γ-tubulin and monoclonal antibodies against CKs 18 and 19. Raw (nondeconvoluted) confocal images in the XY plane indicated that centrosomes were in the same confocal plane of apical IF. Noncentrosomal γ-tubulin signal was also abundant in the same plane. Low magnification XZ sections (perpendicular to the monolayer) showed a difference in the distribution of CK19 IF and CK18 . The former was mostly restricted to the apical cortical cytoskeleton, with small (<1 μm) extensions into the lateral domain. The specificity of this distribution of CK19 has been tested before with a panel of four different monoclonal antibodies and one polyclonal antibody against CK19 in three different cell lines . CK18 signal was also present in the apical cortical region but, in addition, extended throughout the lateral cortical region and, in some cells, even under the basal domain. This result is similar to previous observations in MCF-10A mammary epithelial cells and MDCK cells . In the vast majority of the cells, the centrosomes were observed in the apical domain, colocalizing with the apical cortical layer of IF . In <10% of cells the centrosomes were found >0.3 μm away from the cortical IF network ( Table ). These results are consistent with the findings of Karsenti and coworkers and other investigators in MDCK cells . For better resolution, the images were subjected to three-dimensional deconvolution, thus filtering minimal nonconfocal contributions of out-of-focus fluorescence. The image acquisition, the coalignment of the red and green channels, and the deconvolution parameters were calibrated using fluorescent beads. Under these conditions the image resolution approached the pixel size (∼100 nm per side). Although we cannot colocalize CK19 and CK18, both types of IF displayed a similar distribution under the apical domain. The network of CK19 IF bundles was denser than its CK18 counterpart . In both cases, the apical IF network was ∼1 μm thick in the z-axis . At this resolution level, centrosomes, identified as the largest (∼400 nm) spots of γ-tubulin signal, often observed in pairs, were easily distinguishable , and always colocalized with IF in the 3 axis of space. The abundant noncentrosomal γ-tubulin signal under the apical domain described by Meads and Schroer 1995 , was observed as discrete spots smaller (∼170 nm) than centrosomes. A fraction of these spots was also perfectly colocalizing with apical IF . This colocalization was better seen in XY confocal sections than in XZ reconstructions (9 voxel thick). Most of the rest of the noncentrosomal γ-tubulin signal, however, was observed within 3 μm of the apical IF network (z-axis in the basal direction), while a few were also scattered in the rest of the cytoplasm and in the cortical region underneath the basolateral domain. At large, however, the majority of the γ-tubulin signal was observed at or underneath the apical cortical IF network . Because F-actin is also a component of the terminal web, it was important to assess whether or not the γ-tubulin signal also colocalized with apical microfilaments. In CACO-2 cells stained with FITC-phalloidin and indirect immunofluorescence for γ-tubulin (CY3), we found that 81% of the centrosomes appeared disconnected from the phalloidin signal both in XY sections , or in XZ sections . Only 9% of the centrosomes were found colocalizing with F-actin signal at the apical domain. The centrosomes at the lateral domain (10%), on the other hand, always colocalized with cortical actin. In early experiments, we had found that, while there is a general colocalization of F-actin and IF, the microfilaments occupy mostly the apical side of the terminal web, and the CKs are the major components at the nuclear side of the terminal web (not shown). The centrosomes, therefore, usually attached to that nuclear face appeared separated from the F-actin signal. In a previous publication we showed downregulation of CK19 by continuous incubation of expanding cell populations in media supplemented with 2–10 μM antisense phosphorothioate oligodeoxy nucleotides (A19 or A19/2) targeting two different sequences around the origin of the open reading frame of CK19 mRNA. Both antisense oligonucleotides gave similar results, clearly contrasting with their corresponding randomized sequences used as a control. The downregulation of CK19 is only partial and temporary, but, at day 9 of CACO-2 cells confluency is sufficient to show a phenotype. As reported before, fractions of the cells were not targeted at all (36–39%, Table ), some cells were partially downregulated (54–62%, arbitrarily defined as cells displaying <50% of maximum total CK19 signal in three-dimensional reconstructions, Table ) and some were fully targeted, not showing CK19 at all (2–7%, cells displaying <10% of maximum CK19 signal, Table ). Although some variability in the CK19 signal was observed in control monolayers (either under no treatment or incubated in parallel with random or random/2 oligonucleotides), no cells with these low levels of CK19 (<50% of maximum) were observed in the controls . Examples of cells downregulated in CK19 with A19 antisense oligonucleotide are shown in Fig. 3 . The top panels (a–c) in Fig. 3 are three-dimensional projections of the whole stack of confocal sections in the XY plane, and, therefore, show the total CK19 content of the entire cell. Each panel shows at least one example of a fully targeted or partially targeted cells , surrounded by neighbor nontargeted cells shown as an internal control. Examples of partially targeted cells are shown in Fig. 3b and Fig. c (arrows), while a fully targeted cell is shown in Fig. 3 a (arrow). To analyze the position of centrosomes in all these cells , two XZ sections of each three-dimensional image were taken at the level of the centrosomes. The second row of panels are the XZ sections at the level of the centrosomes pointed with arrows, while the third row are XZ sections at the level of the centrosomes pointed with arrowheads. Totally or partially targeted cells are shown in the second row , while their nontargeted neighbors are shown in the third row with the exception of the cell on the left hand side of i. The position of the apical domain and its cortical apical network of IF was hinted in targeted cells by the few remnant IF, that, in addition, were at the same level as those clearly visible in the nontargeted neighbors. In clear contrast with the control cells or the nontargeted cells in the same monolayers treated with A19, most cells downregulated in CK19 showed centrosomes 2–3 μm below the IF network . In some cases, when the centrosomes were at the level of the apical IF network, they were always colocalizing with one of the remnant CK19 bundles . Likewise, there was a correlation between the proportion of centrosomes not localized to the apical domain and the degree of success of the antisense treatment. In partially downregulated cells, 33–36% of the centrosomes were still apical, while none was found in totally downregulated cells ( Table ). In all cases, however there was an increase in the proportion of centrosomes localized at the lateral boundaries of the cells (as identified by the vicinity to a neighbor nontargeted cell) ( Table ). In the nontargeted cells, centrosomes were always colocalizing with the apical IF network . Noncentrosomal γ-tubulin was also delocalized in cells downregulated in CK19 . Since half of the noncentrosomal γ-tubulin is not in direct contact with the IF, but within a 2–3-μm range, this suggests that CK19 IF may also play an indirect role in the localization of this signal to the apical pole of epithelial cells. Both 21-mer A19 and A19/2 antisense oligonucleotides showed similar results ( Table ), suggesting that the probabilities that these results are due to the downregulation of other, unrelated, mRNAs are very low. In all cases, the success of antisense downregulation was checked in parallel monolayers by immunoblot. Usually, a 50–70% decrease of CK19 signal was observed , while CK18, CK8 or actin were not affected. Similar results were observed with A19/2 (not shown). To dissect the molecular mechanisms connecting γ-tubulin–containing structures to apical IF, I reanalyzed the distribution of γ-tubulin in CACO-2 cells treated with anticytoskeletal agents. Nocodazole and cytochalasin D were chosen because of their well known effects on microtubules and microfilaments, respectively. Unfortunately, there is no equivalent anti-IF drug available. A toxic effect reported for IF is that of acrylamide, that does not necessarily depolymerize IF, but may rather detach them from their anchors, for example, the desmosomes . In all cases, long incubations (7 h) with these agents were used to allow for redistribution of the relatively large MTOC that may have exceedingly slow diffusion rates in the cytoplasm . Neither nocodazole or cytochalasin D treatments resulted in a noticeable delocalization of apical centrosomes. In fact, none was observed detached from IF in our samples. A fraction of the centrosomes (33%), on the other hand, was observed within the cytoplasm away from CK19 IF after incubation of the cells in acrylamide ( Table ). Interestingly, this particular effect of acrylamide was almost completely reversible after 24 h ( Table ). Likewise, a count of 1,309 noncentrosomal γ-tubulin spots in the apical-most 2.4 μm of the cytoplasm in cells subjected to treatment with anticytoskeletal agents showed no differences between control cells, and cells treated with nocodazole or cytochalasin D. In all three cases ∼40% of the noncentrosomal γ-tubulin colocalized with IF , and the treatments did not cause major changes in the distribution profile . In cells treated with acrylamide, on the other hand, the percent of γ-tubulin spots decorating apical CK19 IF fell to <10% . It is suggestive that the fraction of γ-tubulin colocalizing with IF is similar to the percent of insoluble γ-tubulin. A similar study was attempted using colocalization experiments of γ-tubulin and CK18. Unfortunately, the tight codistribution of CK19 and CK18 IF in the apical cortical cytoskeleton made this analysis very difficult, as we cannot determine whether a spot of γ-tubulin signal in that region is attached to a bundle of CK19 IF or to a neighboring bundle of CK18 IF, or both. However, an analysis conducted on the relatively less common γ-tubulin signal attached to the extensions of CK18 IF under the lateral domain yielded an intriguing observation. In cells treated with acrylamide, the number of sparse γ-tubulin spots colocalizing with lateral CK18 IF did not change. Instead, only in cells treated with cytochalasin D we observed a dramatic decrease in the γ-tubulin decorating lateral CK18 IF. This result, a preliminary suggestion that attachment to both types of IF may be mediated by different mechanisms, will be further analyzed in the next section using coimmunoprecipitation procedures. To analyze the codistribution of CK19 IF and γ-tubulin at the ultrastructural level, the former was localized with Nanogold, extremely small colloidal gold particles coupled to Fab affinity-purified anti–mouse IgG, that have extensive accessibility to antigens in cells fixed in toto. The size of gold particles was then increased with a silver enhancer that deposits around preexisting gold. To make them visible at low magnifications, the silver was allowed to deposit for 20 min, rendering particles of 200–400 nm. The apical distribution of CK19, with some extensions to the lateral domain that we reported before using immunoperoxidase was fully confirmed. In addition, it was verified that, according to specifications, Nanogold particles can diffuse within fixed/permeabilized cytoplasms . For colocalization, γ-tubulin was localized using an immunoperoxidase reaction in cells embedded in cross-linked albumin, that restricts the diffusion of the diaminobenzidine product. Centrosome images were found in ∼1 out of 30 cells in nonserial sections, always in the apical pole of the cells. The immunoperoxidase reaction for γ-tubulin was found, as expected, in the pericentriolar material, observed at high magnification . The diameters of the peroxidase-positive images were always <400 nm, indicating that the peroxidase reaction product had not significantly diffused beyond the boundaries of centrosomes. For colocalization, the silver enhancing of Nanogold was kept to 5 min, rendering particles in the 15–20-nm range. This CK19 signal was observed on filamentous material in the apical region around the centrosomes. The minimum gold particle-to-peroxidase signal distances was found to be ∼10 nm . Gold particles were never observed within the pericentriolar material nor peroxidase reaction product decorating filaments. Immunoprecipitation of CKs poses a challenge, since these proteins are highly insoluble when polymerized in IF. Standard methods of immunoprecipitation of CK involve denaturation in SDS/urea, followed by dilution in Triton, which are obviously not amenable to test coimmunoprecipitation. Instead, we have previously developed a technique to demonstrate coprecipitation of apical membrane proteins and IF by fragmenting Triton X-100 insoluble cytoskeletal preparations with extensive sonication under nondenaturing conditions. Sonication has been widely used to fragment filamentous structures such as nucleic acids , actin microfilaments or IF . Fragments with a random distribution of sizes are then separated by size by rate centrifugation in sucrose gradients. 10 fractions of one of these gradients were analyzed by immunoblot for their content in various cytoskeletal components. Cytoskeletal fragments of CACO-2 cells containing CK18 and CK19 were present in nearly all fractions. Actin was present in fractions 1–7 and α-tubulin only in the top fraction . The latter result was expected since the extraction in the cold was not devised to preserve microtubules. Interestingly, the γ-tubulin signal extended to the 6th fraction (approximately up to 1,100 S). This distribution in a gradient of insoluble γ-tubulin contrasts with that of soluble γ-tubulin complexes that are typically 28 S. To further analyze possible binding of γ-tubulin containing structures to IF, the fractions from this type of gradient were immunoprecipitated with a polyclonal antibody against the COOH-terminal region of γ-tubulin. The immunoprecipitates were analyzed sequentially by immunoblot using a monoclonal antibody anti-CK19 , and then by stripping off the first set of antibodies and reprobing the same nitrocellulose sheets with an antibody against CK18 . It should be noticed that the order of reprobing did not affect the results, as described in Fig. 7 . The immunoprecipitation was carefully controlled in parallel aliquots with a nonimmune rabbit IgG . It was early noted that nonspecific precipitation increased from none in the first three fractions, to very significant levels in the bottom of the gradient, presumably because very large fragments of the cytoskeleton pellet together with the agarose beads. Therefore, we restricted our analysis to the top five fractions of the gradients where the specific, antibody-dependent signal was clearly larger than nonspecific precipitation . To compare these data with the previous results using anticytoskeletal agents, some monolayers were also treated with nocodazole, acrylamide, or cytochalasin D at the same concentrations and times described in Table , before Triton extraction. In control monolayers, CK19 coimmunoprecipitated with γ-tubulin in the top 5 fractions of the gradient. Interestingly, when the same immunoprecipitates were analyzed for CK18, only the top 1 or 2 fractions were reactive , even when CK18 was abundantly present in fractions 4 and 5 as well . This result strongly supports the notion that insoluble γ-tubulin–containing structures are bound to IF. An extensive treatment with nocodazole (7 h) did not affect the coimmunoprecipitation with CK19 or CK18. The acrylamide treatment, on the other hand, erased the specific coimmunoprecipitation with CK19, but not the coimmunoprecipitation with CK18. Conversely, the cytochalasin D treatment abolished coimmunoprecipitation with CK18, while CK19 was still present in the same immunoprecipitates . These results correlate well with the morphological colocalization data described in the previous section, and suggest that γ-tubulin–containing structures bind to CK18 and CK19 IF via two different mechanisms, one cytochalasin D sensitive (perhaps mediated by actin) and the other acrylamide sensitive. Because there is always the possibility that binding between two structures may be artefactually induced during detergent extraction, a control was done changing the extraction buffer to a different more physiological buffer (KEB), replacing Triton X-100 by 0.1% saponin, that permeabilizes membranes but does not extract material, and adding a cocktail of phosphatase inhibitors in addition to the protease inhibitors. Coimmunoprecipitation of CK19 with γ-tubulin was observed again in sonication fragments obtained under these milder extraction conditions. However, as expected from a preparation that now contained membranes, the distribution of the specific signal in the gradient was different than in the Triton X-100 fragments . To control the possibility that the co-i.p. may be due to simple physical trapping of insoluble γ-tubulin into IF meshworks or cages, we analyzed the cytoskeletal fragments in the gradients under EM. In fraction 1 the fragments were fibrous, with an average caliper diameter of 30 nm and length of 105 nm . Only in a few fields small granule-like structures were seen (not shown). In fraction 5, the vast majority of the fragments were rod-like, with an average caliper diameter of 49 nm and average length of 180 nm . Similar results were observed in the material spread on carbon-coated grids (not shown). No meshwork-like structures were observed at all. Clearly, the size and simple geometry of these fragments cannot sustain physical trapping of much larger (∼300 nm) centrosomes. Next, the composition of the multi-protein complexes in which γ-tubulin and CK19 coimmunoprecipitate was analyzed by metabolic labeling with [ 35 S]methionine/cysteine for 24 h. Sonication fragments of the cytoskeletal preparation were separated in sucrose gradients and pools of the top two fractions were immunoprecipitated with anti–γ-tubulin antibody. The autoradiograms showed z10 antibody-specific bands and 3 unspecific bands . The lower four specific bands had been identified in immunoblot as CK8, CK18, and CK19 . The dense 50-kD band corresponded to γ-tubulin . There were still approximately six unidentified peptides in the complexes. To distinguish which ones belong to the MTOC, a parallel set immunoprecipitates, still bound to the beads, was washed for 2 h in EB supplemented with 0.7 M NaCl, a condition known to disassemble soluble γ-tubulin complexes but not IF-associated proteins. The unspecific bands and two of the specific bands disappeared from these preparations, suggesting that the remaining four specific polypeptides (90, 105, 110, and 180 kD) may be IF-associated proteins or part of the insoluble scaffold that holds γ-tubulin. To test the hypothesis that γ-tubulin–containing structures are attached to IF under the apical pole of epithelial cells we have used three independent approaches: morphological colocalization, analysis of the effect of antisense downregulation of CK19 on centrosome localization, and coimmunoprecipitation in multiprotein cytoskeletal fragments obtained under nondenaturing conditions. Each one of these techniques has advantages and potential problems. The data of the three approaches, however, is complementary and consistent with the notion that centrosomes and a substantial fraction of noncentrosomal γ-tubulin containing structures bind to apical IF. The largest of the γ-tubulin structures were easily identified as centrosomes. All the noncentrosomal γ-tubulin signal was observed in discrete spot-like images. It should be noted that, because these images are the result of indirect immunofluorescence, they represent a slight overestimation of the real size. In fact, given the Stokes radius of the IgG (∼7 nm) in the two layers of antibody covering a structure, one should add a total of ∼28 nm to the original diameter of the structure. The smaller noncentrosomal spots, therefore, after subtracting the contribution of the IgG must be ∼140 nm in diameter and are difficult to identify with previously described structures. This diameter represents nearly five times the diameter of the ring-shaped soluble γ-tubulin complexes described by Zheng et al. 1995 . However, because these structures are at the limit of resolution of our instrument, they must be considered as points without any further estimation of their real size. Furthermore, neither of our anti–γ-tubulin antibodies recognized the epitope in aldehyde fixed cells, and were amenable to use only under methanol fixation, so that I cannot be sure if this discrete γ-tubulin signal is a fixation artefact. Therefore, I am not drawing any conclusion about the structure of noncentrosomal γ-tubulin signal, except that its localization in the apical pole of epithelial cells is consistent with the localization of noncentrosomal MTOC described by others . Resolution of scanning laser confocal microscopy coupled to appropriate deconvolution analysis of three-dimensional images has been proven to yield resolutions at or below the 100-nm level in the XY plane . Deconvolution processes can yield sub-pixel resolution, even for standard (nonlaser) epi-illumination . We experimentally verified this fact in our system, together with the coalignment of the channels using fluorescent beads. The resolution in the z-axis, on the other hand, was slightly less reliable, and we experimentally verified it at ∼300 nm. It must be noted that the colocalization of centrosomes and noncentrosomal γ-tubulin with IF at this level of resolution is more accurate than most colocalizations reported in the literature with standard epifluorescence microscopy, where resolution is 250 nm at best. It can be argued that IF in the apical network are very crowded and that any structure in the same region will randomly touch an IF bundle. While this may be true for centrosomes due to their size, this argument is unsustainable for noncentrosomal γ-tubulin. The surface occupied by IF signal at any XY confocal section is <50% of the image, and the gaps are significantly larger than the noncentrosomal γ-tubulin spots . Yet, <10% of the γ-tubulin signal was not in direct contact with CK signal, indicating a high degree of colocalization. In the case of centrosomes, there was also a perfect colocalization in the z-axis within the ∼1 μm thickness of the apical IF network. Considering that differentiated CACO-2 cells grown on filters are 10–20 μm tall, the position of centrosomes exactly at the level of the apical IF network can be considered a bona fide colocalization. The colocalization with F-actin with γ-tubulin, on the other hand, was good in the lateral domain, but poor in the terminal web, a result consistent with the possibility that different mechanisms of anchoring operate at different domains. Additional evidence was provided by colocalization experiments at the EM level. While there is consensus in the precision of the localization with gold particles, some concerns may arise in regards to the peroxidase/diaminobenzidine reaction product that, potentially, may diffuse away from the site of the antigen. This diffusion can be efficiently confined within ∼10 nm of the site of the antigen by performing the peroxidase reaction within a matrix of glutaraldehyde cross-linked albumin as described by Brown and Farquhar 1984 , and also shown for membrane proteins . Given this possible margin of error and the size of the gold particles themselves, the minimum distance between CK19 and γ-tubulin may be in the range of 10–20 nm. Because no overlapping of signals was observed, the binding interface between centrosomes and IF may be located at the outer boundary of the pericentriolar material (as labeled by γ-tubulin). In addition, it is possible that one or more intermediary proteins may be intercalated between CKs and γ-tubulin. To our knowledge, this is the first report of colocalization of γ-tubulin with cytokeratin IF. Trevor et al. 1995 , however, have reported colocalization of centrosomes with vimentin IF in cells that normally do not express IF, transfected with a vimentin-expressing vector, suggesting that this may be a more general function of IF. In general, coimmunoprecipitation is accepted as evidence of binding between two proteins. In this case, however, we attempted the difficult task of analyzing the protein interactions of the ∼50% of γ-tubulin that is insoluble and has not been previously characterized. To avoid denaturing conditions, we resorted, as in a previous publication , to a fine homogenization of the Triton insoluble cytoskeletal preparation by sonication that yields multi-protein fragments. To apply this method for immunoprecipitation purposes we faced the limitation that the largest fragments (fractions 6–10 in our gradients) are heavy enough to pellet together with the agarose beads. Our original approach to this problem was to separate the beads from the unbound fragments by filtration through a 15-μm nylon mesh. Although this method successfully washes the beads, it has been our experience that, during the backwash of the filter, to recover the specific signal, sometimes there is loss of material, presumably beads retained in the filter or the filter holder. To avoid this and to ensure that the results would enable us to compare gradients from monolayers under different treatments, we decided to study only those fractions (1–5) that were amenable for standard immunoprecipitation, and to do so with careful parallel controls for nonspecific precipitation . The results indicate that we were able to precipitate multi-protein complexes in a specific antibody-dependent fashion, and which contained insoluble γ-tubulin and also either CK19 or CK18. Interestingly, the results indicate that the complexes of γ-tubulin with the two types of IF were different in their migration in the gradient and their stability after treatments with anticytoskeletal agents. The CK19–γ-tubulin complexes displayed a broader range of sizes and were sensitive to pretreatment of the cells with acrylamide, while the CK18–γ-tubulin complexes were smaller, and sensitive only to pretreatment with cytochalasin D. These data are consistent with the morphological results and suggest the possibility that MTOC may be bound to CK18 and CK19 via different mechanisms. Because we were immunoprecipitating fragments of the cytoskeletal preparation or naturally occurring discrete particles, and not soluble proteins, careful thought has to be given to the possibility that Triton insoluble γ-tubulin–containing structures may be physically trapped within IF networks. An obvious prediction of a caging model is that structures of similar sizes, from the same domain must be caged together. If the CK19 cages can physically trap 300-nm centrosomes, they should also trap bundles of CK18-8 IF, especially since both sets of bundles are closely intermingled in the apical network. This was not the case in our experiments. If the γ-tubulin containing structures were physically trapped in fractions 2–5 of the gradients, it is inconceivable that no CK18 IF were trapped in the same cages , even in the relatively large fragments of fractions 4 and 5. It must be remembered that CK18 was present in fractions 4 and 5 of the gradient before the immunoprecipitation . In fact, the different behavior of CK18 and CK19 coimmunoprecipitation with γ-tubulin should lead us to conclude that two perfectly distinct sets of cages were generated by sonication: a small one (fractions 1 and 2) made of CK18 IF that can be opened with cytochalasin D but not with acrylamide, and another one, of a wider range of sizes made only of CK19 IF, sensitive to acrylamide but not to cytochalasin D. Such an exquisite specificity seems extremely unlikely for randomly generated three-dimensional lattices trapping structures inside. A second line of experimental evidence against physical trapping comes from the visualization of the cytoskeletal fragments separated by the gradient under EM . No evidence of three-dimensional lattices capable of trapping centrosomes was found. The structure of insoluble γ-tubulin is unknown and it has been tentatively equated to that associated with centrosomes . There is a possibility, however, that a fraction of the rather abundant noncentrosomal γ-tubulin may also be found in the insoluble cytoskeletal preparation. One can only speculate that the structure of this putative noncentrosomal insoluble γ-tubulin may be similar to that of the ring-like γ-tubulin particles in the soluble fraction, or, perhaps a multimer of them. In this regard, it is unlikely that the noncentrosomal γ-tubulin exists in pieces smaller than 28 nm. In this scenario, therefore, it is also unlikely that 40–50 nm fragments of the cytoskeleton in fraction 1 can sustain physical trapping of 28 nm (or bigger) γ-tubulin complexes or vice versa. Finally, because coimmunoprecipitation was also observed under totally different, more physiological, extraction conditions in the presence of phosphatase blockers , and resistant to high salt , we believe that an artefactual attachment of IF to MTOC due to detergent extraction is also very unlikely. On this basis, the coimmunoprecipitation results indicate that there is binding of γ-tubulin and cytokeratins to the same multiprotein complexes. These complexes comprised γ-tubulin, the CKs, and about six other unidentified proteins. γ-tubulin was clearly more abundant than CK8 , a result that suggests that some of the MTOC fragments, not associated with IF, are also being immunoprecipitated. However, it must be noted that CKs display turnovers of 100 h or more . Therefore, the 24 h labeling may be tagging 30% or less of the CKs. Two of the other proteins disassembled from the complexes in high salt buffer while the association with CKs was maintained. Because of the complex nature of the MTOC , this suggests that the sonication fragments contain only part of the MTOC, perhaps corresponding to the outer boundary of the pericentriolar region. The remaining five proteins were resistant to high salt extraction. Any of these proteins may be intermediaries in the attachment of γ-tubulin to IF, or alternatively the binding between γ-tubulin and CKs may be direct. Further research is necessary to establish the architecture of the protein-protein interactions at the centrosome–IF interface. The effects of anti-CK19 antisense phosphorothioate oligodeoxy nucleotides on the polarity and cytoskeletal organization of CACO-2 cells have been documented before . Here we used the antisense-mediated downregulation of CK19 to analyze the effects of tampering with the subpopulation of IF that are specifically apical on the localization of potential MTOC. It should be noted that we and others determined that the uptake of oligonucleotides decreases as the monolayer becomes confluent, thus providing a possible explanation of why the effect is transitory and heterogeneous. A relatively narrow time window to observe the effects in well differentiated CACO-2 cells, around 9 d after seeding, was previously determined. We took advantage of this fact, however, by using the nontargeted cells as an internal control for the distribution of centrosomes. The heterogeneity in the effect may arise from the decreased uptake of oligonucleotides after the monolayers become confluent . Additional controls were provided by cells subjected to identical treatment with oligonucleotides with the same bases, in a randomized sequence, synthesized at the same time and with the same reagents as the antisense. In contrast with control cells, >60% of the cells downregulated in CK19 showed centrosomes either totally disconnected from the cortical region or on the lateral domain. It is important to highlight that, in the 36% of the partially targeted cells, where centrosomes were still in their normal position, there was always a perfect colocalization with one remnant CK19 bundle of IF. We have extensively documented that these cells have their normal content and localization of CK18 IF. Therefore, it can be concluded that CK19 IF can successfully compete with a large excess of CK18 IF to bind the centrosomes. It can be argued that CK19 downregulation may cause indirect catastrophic effects on other components of the cytoskeleton. We have previously characterized those effects. The most striking consequence of CK19 downregulation is the loss of apical F-actin, while lateral cortical actin and basal stress fibers remain undisturbed . If the effects of CK19 downregulation on the localization of centrosomes were a consequence of the disruption of the apical F-actin, they should be mimicked by cytochalasin D, which was obviously not the case. Second, downregulation of CK19 also induces a slight redistribution of the apical microtubule network. Again, if this effect was responsible for the displacement of centrosomes, it would have been mimicked by nocodazole either with the morphological or biochemical analyses. In summary, three independent methods showed that the apical IF bind centrosomes and a fraction of the noncentrosomal γ-tubulin. Given the well established role of γ-tubulin in the organization of MTOC, it seems safe to conclude that part of all this insoluble γ-tubulin must represent the MTOC that predictably exist in the apical domain of epithelial cells. This as yet unsuspected interaction between IF and MTOC may yield a better explanation of how the cytoskeleton is organized, and warrants the need for future investigations in search for the molecular basis of the MTOC-IF binding. | Study | biomedical | en | 0.999998 |
10444073 | Worms were grown on bacterial strain OP50 spread on NGM (nematode growth media) agar plates according to Brenner 11 . The Bristol variety of the N2 strain of C . elegans is used as the wild-type strain, and the genotypes of the mutant strains used are as follows: RW3625, let-805(st456)/qC1 ; BC4534, unc-45(rh450)let-805/sC1 ; and let-805(st456)/dpy-1(e1)daf-2 . A clone encoding an MH46 epitope was isolated from a genomic library made from randomly sheared C . elegans DNA cloned into the vector λgt11 4 . The screen was performed according to Maniatis 36 with a few exceptions. Filters were washed in TTBS (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) and blocked in TTBS containing 1% normal goat serum (GIBCO BRL) and 5% fish gelatin (Sigma). The MH46 primary antibody was detected using alkaline phosphatase-conjugated goat anti–mouse IgG (Tago) diluted 1:2,500 in block buffer and the secondary antibody was localized using 0.137 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma), 0.33 mg/ml nitro blue tetrazolium (Sigma) and 0.013 mg/ml phenazine methosulfate (Sigma) according to Ey and Ashman 19 . One clone (λG-cc9) was obtained from a screen of 10 6 PFU. The genomic fragment from λG-cc9 was labeled using the Prime-It-II kit (Stratagene) and α-P 32 -labeled ATP (Amersham) according to Stratagene. The labeled DNA was used to screen a randomly primed cDNA library made from RNA of mixed stage worms and cloned into the pACT vector. The screen was performed according to Maniatis 36 except hybridizations were done in 6× SSC, 1% SDS, and 100 mg/ml salmon sperm DNA and washes were done in 10 mM Tris, pH 7.5, containing 1% SDS. The largest clone (pC-cc1A) was sequenced and shown to be identical to the sequence of λG-cc9 in the region of overlap. Using the sequence of pC-cc1A, probes were designed to screen the library for clones containing sequences 5′ and 3′ to pC-cc1A. Similar screens were performed until we had evidence we had cloned the 5′ and 3′ ends of the message. The overlapping cDNA clones were sequenced by fluorescent sequencing techniques using dye-labeled primers or dye-labeled dideoxynucleotides (ABI). The reactions were performed according to the manufacturer and reactions were run on an ABI 373A gel analyzer. Nested deletions of the clone pC-cc1A were made using Erase-a-Base (Promega) according to the manufacturer, and the deletion clones were sequenced. The templates used to obtain the sequence of the ends of each cDNA isolated subsequent to pC-cc1A were amplified by PCR. The sequence of the primers used was as follows: primer 1, 5′-GATGATGAAGATACCCCACC-3′; primer 2, 5′-AGTTGAAGTGAACTTGCGGG-3′; primer 3, 5′- GCGTGTAAAACGACGGCCAGT GATGATGAAGATACCCCACC-3′; primer 4, 5′- GCGTGTAAAACGACGGCCAGT AGTTGAAGTGAACTTGCGGG-3′. The underlined bases in primers 3 and 4 contain a universal priming site. Primers 1 and 3 are complementary to the same sequence 5′ of the pACT cloning site, and 2 and 4 are complementary to the same sequence 3′ of the pACT cloning site. Primer pairs 1 and 4, or 2 and 3, were used to amplify the cDNA insert from each clone and to add the universal priming site to the 5′ or the 3′ end of the amplification products. The products were treated with 0.2 units exonuclease I (United States Biochemicals) and 0.2 units shrimp alkaline phosphatase (United States Biochemicals) for 45 min at 37°C to degrade the unused primer 57 and the enzymes were inactivated for 20 min at 80°C. The products were then sequenced directly. This technique was also used to obtain the sequence of exons 6–9 and 13. These exons were not encoded by any of the cloned cDNAs but were thought to be expressed due to the high degree of homology between the C . elegans and C . briggsae genomic sequences in these regions. In these cases, templates were amplified from the random-primed library using gene-specific primers complementary to the sequence of the exons flanking the region of interest. As above, one primer used in each reaction was fused to the universal priming site. Finally, libraries from each cDNA clone were constructed in the m13 vector to obtain templates to completely sequence each cDNA clone. Each insert was amplified using PCR and primers 1 and 2 (see above). A pool of amplification products was fragmented using a nebulizer and 30 psi nitrogen gas 9 , treated with mung bean nuclease to generate blunt ends, and then cloned into the SmaI site of m13. DNA was isolated from random m13 clones from each library and sequenced. The sequence obtained was edited for quality using the trace editor TED 26 and assembled into contigs using XBAP (a sequence assembly program for X windows; reference 13 ). The identity of each base was confirmed by obtaining the sequence of both strands, by obtaining the base using two different chemistries (dye-labeled primers or dye-labeled dideoxy-terminators) or by comparison to the genomic sequence generated by the C . elegans genome project. The genomic clone was placed on the physical map by hybridization of a P 32 -labeled DNA probe (Prime-It II kit; Stratagene) to YAC clones representing ∼95% of the C . elegans genome 12 . The YAC clones are spotted on a nitrocellulose sheet in a grid pattern. Each YAC represented on the grid has been ordered into contigs by fingerprint analysis and the contigs placed into linkage groups representing each C . elegans chromosome. The st456 mutation (see Results) was mapped by standard three factor mapping techniques. Recombinants were cloned from st456/dpy-1(e1)daf-2 strain, and their progeny were scored to determine where the recombination event took place. Recombination took place between dpy-1 and st456 6 times and between st456 and daf-2 twice, placing the mutation between dpy-1 and daf-2 . The st456 mutation failed to complement a mutation, let-805 , isolated in an unrelated lethal screen (Baillie, D., personal communication) performed as described by Stewart et al. 49 . This result suggests st456 is an allele of the let-805 gene. Protein similarities were identified using the program BLAST (version 2.0; reference 2) to search the nonredundant protein database and the est database (dbest) through the NCBI server . Searches were also done of an ftp site containing data from the two C . elegans Genome Sequencing Centers (http://genome.wustl.edu). FNIII repeats were defined using hidden Markov models (HMM) profile 18 32 . The model used is the fn3 HMM from the Pfam protein domain database (release 1.0; reference 47). This HMM is based on a multiple alignment of 456 FNIII domains from Swissprot 33. The search was performed using the programs hmmls and hmnfs from the HMMER hidden Markov model software package 17 . Alignment scores over 15 bits (a log odds score, base two) were considered significant. The fn3 model identifies only the first six of the seven β-strands of the FNIII repeat. Therefore, a new model was generated to identify the amino acids that form the seventh strand in each repeat, and to produce the alignment shown in Fig. 5 . The new model was generated using the program hmmb 17 , and starting with a seed alignment of the four FNIII repeats of fibronectin 33 . The seed alignment was expanded to include repeats identified in the myotactin sequence that had significant scores (in this case >20 bits). The expanded alignment was used to generate a new HMM model. These steps were repeated until no new repeats could be identified in the myotactin sequence. The 32 repeats identified in this way were then reanalyzed using the fn3 model and 30 of the 32 repeats had significant scores of >15. The exceptions are repeats 8 and 27 which had scores of 13.19 and 13.94, respectively. Secondary structure predictions were made using the program PHD 43 44 . In situ hybridizations to mixed stage C . elegans embryos were done according to the protocol of Seydoux and Fire 45 with a few exceptions. Embryos were obtained by alkaline hypochlorite treatment of gravid adults 52 and then treated in batch in microfuge tubes rather than on microscope slides. After each incubation the embryos were collected by gentle centrifugation. Embryos were fixed in 3% formaldehyde and 0.25% glutaraldehyde in 85 mM K 2 HPO 4 , pH 7.2 31 . The probes used were made by PCR amplification according to Seydoux and Fire 45 . Embryos were prepared for antibody staining and staged as described in Hresko et al. 30 . Fluorescence was viewed on a Bmax-60F microscope (Olympus) equipped with Nomarski and fluorescence optics. Images were taken with a Pentamax camera containing a Kodak KAF-1400 chip (1,317 × 1,035) with 6.8-mm pixels (Princeton Instruments). The camera was controlled by an Optiplex GXPro200 computer (DELL Computer Corp.) running WinView software (v. 1.6.2.1, Princeton Instruments). After acquisition the raw 12-bit WinView images were transferred to a Power Macintosh computer, linearly scaled to 8-bits ([max pixel value − min pixel value]/256), inverted to conform to the Macintosh intensity scale, reduced to 256 shades of gray, and saved as TIFF files using a batch file conversion program (Spe2Tiff; written by and freely available on request from waddle@hamon.swmed.edu). Images were assembled and annotated using Adobe Photoshop 3.0.5 (Adobe Systems, Inc.) and printed on a Tektronix Phaser 440 printer (Tektronix). Adult worm fragments were prepared for antibody staining using a French press as described by Francis and Waterston 21 . After fragmenting the worms, the pieces were extracted three times for 30 min in 0.5% NP-40 in low salt buffer (7.5 mM Na 2 PO 4 , pH 7.0, 40 mM NaCl, 1 mM EDTA, and 1 mM PMSF), for 15 min in 0.5 M KSCN in low-salt buffer and for 15 min in low-salt buffer 22 . The extracted fragments were then fixed for 20 min in −20°C MeOH. The samples were viewed using a Zeiss Axioplan microscope equipped for confocal imaging with a Bio-Rad MRC 1000 laser. Bodywall muscle precursor cells were ablated at the 28-cell stage using a nitrogen pulse laser (Laser Sciences, Inc.) as described 3 . The beam was directed through a Zeiss Axioplan microscope with the 100× objective and laser intensity was adjusted by the use of neutral density filters. In one set of experiments MS.ap and MS.pp were ablated and in another set C.ap was ablated. MS.ap and MS.pp collectively give rise to six anterior bodywall muscle cells from each dorsal quadrant and three from each ventral quadrant 51 . C.ap gives rise to nine posterior bodywall muscle cells from the left dorsal quadrant and seven from the left ventral quadrant 51 . After ablations were performed, the embryos were allowed to develop to a specific development stage, fixed by freeze fracture 50 and stained with appropriate antibodies. Confirmation that the correct cell(s) was ablated was obtained by staining the embryos with an antibody against bodywall muscle myosin. The myotactin genomic fragment (from pG-cc9) maps in the physical interval between the par-2 and ben-1 genes, corresponding to the region of the genetic map containing the dpy-1 gene. We reasoned mutations in the myotactin gene might be lethal and screened for lethal mutations linked to the dpy-1 gene. Homozygous dpy-1(e1) hermaphrodites were mutagenized with 50 mM EMS (ethylmethanesulfonate) 52 . Mutagenized hermaphrodites were mated to wild-type males (N2 strain) and individual L4 hermaphrodites were cloned from the F1 progeny. The progeny of each cloned hermaphrodite were examined for the loss of dpy animals, suggesting a mutagenesis event causing a lethal mutation linked to the dpy-1 gene had occurred in that strain. Embryos from strains segregating few or no dpy animals were stained with MH46 to identify mutants negative for the antibody. One mutant was identified. The mutant strain was outcrossed six times to eliminate other mutations that might be in the background. Germline transformation was carried out according to the protocol of Mello et al. 39 . The DNA fragment used is ∼30 kb and contains the C . briggsae myotactin gene (95% identical to the C . elegans protein), including 8 kb upstream of the translational start site and ∼2 kb downstream of the stop codon. This plasmid, along with a plasmid containing the rol-6 gene carrying a dominant mutation, was injected into dpy-1(e1)let-805(st456)/qC1 hermaphrodites. The qC1 chromosome contains a rearrangement that suppresses recombination between dpy-1 and let-805 . Rolling L4 hermaphrodites were picked 3 d later and their progeny were examined for the presence of rescued animals by looking for animals with the dpy phenotype. Two templates were made from each clone (pC-cc307 and 406) using the enzyme PWO (Boerhinger Mannhiem Biologicals) and primers homologous to the vector. The T7 polymerase binding site was added to the 5′ end of the coding (template 1) or the noncoding (template 2) strand of the templates during the amplification 41 . Sense and antisense RNA's were synthesized from templates 1 and 2, respectively, using mCAP (Stratagene) according to the manufacturer. Equimolar amounts of sense and antisense RNA were annealed to yield double-stranded RNA. The double-stranded RNA was injected into hermaphrodites. Hermaphrodites were allowed to lay eggs for 36 h, then the hermaphrodites were transferred to new plates. The plates were examined for non–wild-type animals to determine the phenotype caused by injection of the RNA. To determine if the phenotype caused by RNA-mediated interference was similar to that of the let-805(st456) homozygotes, embryos were collected 10–15 h after transfer of injected hermaphrodites to new plates and the arrested embryos were fixed by freeze fracture 50 and stained with antibodies. Myotactin and fibrous organelle–associated intermediate filaments colocalize in adult C . elegans worms. Adult worm fragments were fixed and double labeled with MH46 to localize myotactin and a rabbit polyclonal antibody against intermediate filaments 22 , and viewed by confocal microscopy. The distribution of myotactin and intermediate filaments in adult worms is similar . Both proteins are organized in thin bands running circumferentially around the worm, and both are restricted to regions of the hypodermis adjacent to muscle and some mechanosensory neurons. The myotactin- and intermediate filament-dependent staining within each band is not uniform, but rather consists of a large number of discrete punctate structures. Merging of the rhodamine and fluorescein images shows the punctate staining patterns seen with the two antibodies are coincident as evidenced by the yellow fluorescence . The correlation between the distribution of myotactin and intermediate filaments suggests myotactin localizes close to fibrous organelles. As a first step in understanding the role myotactin plays in signaling between muscle and the hypodermis, we cloned a genomic fragment (pG-cc9) encoding an MH46 epitope from an expression library 4 . We used the cloned genomic fragment to start cloning a set of overlapping cDNAs extending from the likely 5′-end to the stop codon. The 5′ most sequence obtained encodes eight bases identical to the eight 3′ bases of the SL1 spliced leader, which is trans-spliced to the 5′ end of many C . elegans messages, indicating this sequence represents the 5′ end of the myotactin message. Typically, the first ATG after SL1 encodes the initiator methionine, and in >90% of the messages examined to date, the initiator methionine is <30 nucleotides from the 3′ end of SL1 8 . In the sequence presented here, there are 12 nucleotides separating SL1 and the first ATG which we have designated as the initiator. The 3′ most clone, pC-cc307, was isolated from an oligo dT primed cDNA library, and contains sequence identical to that of a 3′ expressed sequence tag (CEMSD21; reference 37 ), suggesting the sequence encoded by pC-cc307 is close to the 3′ end of the message. Furthermore, the sequence encoded by pC-cc307 and CEMSD21 contains a stop codon at the end of a long open reading frame beginning with the initiating ATG. This stop codon is followed by sequence consistent with 3′ noncoding region with multiple stop codons in all three reading frames. While the cDNAs were being sequenced, the genomic sequence of the Caenorhabditis elegans gene (Chissoe, S., personal communication) and a Caenorhabditis briggsae homologue (Marra, M., personal communication) became available (the C . elegans genome sequencing project) which allowed us to define the intron–exon boundaries over most of the gene . The exon boundaries are conserved between the C . elegans and the C . briggsae genes with one exception: exons 15 and 16 of the C . elegans gene are encoded by only one exon in the C . briggsae gene. However, the intron sizes are very different, with the C . elegans introns generally being larger than the corresponding C . briggsae introns. Myotactin is predicted to be a large transmembrane protein. A Northern blot of total RNA from mixed stage worms probed with the insert from pG-cc9 shows three RNAs hybridize to the probe, the smallest being ∼15 kb . Furthermore, the longest open reading frame encoded by the DNA fragments depicted in Fig. 3 predicts a protein of 4,450 amino acids with a calculated molecular mass of ∼498 kD. Analysis of the hydrophobicity identifies three regions of the myotactin sequence sufficiently hydrophobic to insert into the membrane. One at the amino terminus (amino acids 1–20) probably represents a signal sequence suggesting the protein is secreted or is a transmembrane protein. The other two regions, encoded by exons 19A and B, respectively, are long enough to span the membrane, and therefore are likely to be transmembrane sequences. These exons are used alternatively suggesting at least two protein isoforms are expressed, each with a different transmembrane domain. The putative extracellular domain of myotactin contains at least 32 repeats of ∼100 amino acids in length with homology to fibronectin type III repeats as identified by a HMM for FNIII domains ( 47 ; see Materials and Methods). Additional sequences contained in the putative extracellular domain may represent another 5 FNIII repeats. These sequences are not identified as FNIII repeats by the fn3 model, but are each ∼100 amino acids in length and are predicted to contain 5–7 β-strands by the secondary structure program PHD 43 44 . This is similar to predictions concerning the secondary structure of the 32 predicted FNIII repeats and is consistent with the crystal structure of the FNIII repeat 33 . The putative cytoplasmic domain of myotactin has two serine-rich regions suggesting this sequence might be modified in response to condition or stimuli. The amino acids encoded by exons 22–23 and those encoded by exons 26 and 27 are each 14% serine (10/69 and 8/55, respectively), and those encoded by differentially expressed exons 24 and 25 collectively, are 18% serine (32/173), while the average protein is only 7% serine 15 . C . elegans myotactin and the presumed orthologue from C . briggsae appear to be novel transmembrane proteins. To date, a protein consisting of multiple FNIII repeats and lacking other known repeat motifs in its extracellular domain has not been reported. Furthermore, the cytoplasmic domain of the protein is unique. The only proteins in the databases with significant similarities to the cytoplasmic domains of the C . elegans and C . briggsae proteins, are proteins rich in serine and an est entry from Onchocerca volvulus , another nematode. Two lines of evidence suggest myotactin is the MH46 antigen. First, the expression of myotactin message is tightly correlated with the presence of the MH46 epitope. Myotactin message is first detected by in situ hybridization in embryos before elongation . Signal is detected in two groups of cells: one group on the dorsal surface of the embryo flanking the dorsal midline, in the position of the dorsal hypodermal cells ; and a second group on the lateral edges of the embryo about midway between the dorsal and ventral surfaces, in the position of the ventral hypodermal cells . These two groups of cells are also positive for the MH46 antibody 30 . Later in development, at about the 1.5–1.75-fold stage, myotactin message is also detected in the pharynx (data not shown), a tissue also positive for the MH46 protein 22 . Similarly, the MH46 protein is not detected in bodywall muscle (although it appears to colocalize with muscle specific antigens during some stages of development) and the myotactin message could not be detected in muscle cells. In situ hybridizations were done using a myosin heavy chain probe to mark the position of the muscle cells and to show that the muscle cells are accessible to DNA probes under the conditions used. When myosin message is first detected, the positive cells are positioned midway between the dorsal and ventral surfaces, away from the lateral edges of the embryo , indicating these cells are not those detected by the myotactin probe at a comparable developmental stage . Later in development, the myotactin probe is detected in hypodermal cells at the dorsal and ventral edges of the embryo . At this stage, myosin is detected in cells positioned more interiorly . We found no evidence that myotactin is expressed in bodywall muscle cells at any developmental stage. The second line of evidence that myotactin encodes the MH46 protein is that both the MH46 epitope and an MH46-negative mutant ( st456 ; see below) map to the same 30 kb of the genome. In wild-type embryos, MH46 staining is seen along the length of the embryo adjacent to the muscle quadrants . In contrast, homozygous st456 mutants do not show MH46 staining, although the embryos are positive for myosin and for MH27 , an antibody that recognizes the boundaries between hypodermal cells 22 . To confirm the st456 mutation maps to the region of the genome encoding the myotactin gene, we used a 30-kb fragment of the C . briggsae genome which encodes a C . briggsae homologue (95% identical to C . elegans myotactin at the amino acid level) to rescue the st456 phenotype. All aspects of the phenotype described below are rescued by this fragment of DNA. A second allele of the gene, s2764 , was isolated (Baillie, D., personal communication) as described by Stewart et al. 49 , which also fails to stain with MH46 and fails to complement the allele isolated in this study. The gene has been designated let-805 (Baillie, D., personal communication). The phenotype of let-805(st456) and let-805 homozygotes (described below) is likely to be the loss-of-function phenotype of the let-805 gene. Both mutations are fully recessive, but more importantly, embryos with a similar phenotype can be produced by a technique referred to as RNA-mediated interference 20 29 41 . RNA-mediated interference involves injection of wild-type hermaphrodites with double-stranded RNA homologous to a gene of interest, and results in progeny with the loss-of-function phenotype for that gene (see 41 and references therein). Wild-type hermaphrodites injected with double-stranded RNA homologous to pC-cc406 or pC-cc307, neither of which encodes the MH46 epitope, produced some progeny that were negative for MH46 suggesting myotactin was not made. Furthermore, these MH46-negative embryos were indistinguishable from st456 homozygotes with respect to all the characteristics described below. These data suggest st456 is a loss-of-function allele. Time-lapse video microscopy shows myotactin mutant embryos have movement and elongation defects. Wild-type embryos begin to elongate at ∼300 min after the first cleavage and elongate three- to fourfold by the time of hatching at 800 min. At the 1.75-fold stage bodywall muscle contractions begin and are first seen as twitches but soon the embryo begins to roll vigorously within the eggshell 51 . Like wild-type embryos, myotactin mutants also begin to twitch at about the 1.75-fold stage, but unlike wild-type embryos, the mutants never roll within the eggshell. The mutants fail to elongate beyond the twofold stage, and die before hatching. Since many, if not all mutations that result in failure of embryos to roll also result in arrest of elongation at the twofold stage 5 28 42 54 56 59 , the elongation defect associated with myotactin mutations is probably a secondary consequence of the motility defect. The abnormal movement associated with the mutant myotactin phenotype apparently results from defects in muscle–cell adhesion. In the homozygous mutants, before contractions begin, bodywall muscle quadrants extend from the anterior to the posterior of the embryo , and the sarcomeric structure of the muscle appears wild-type. However, at about the time when muscle contractions begin (1.75-fold stage) the muscle quadrants in the mutants no longer extend the length of the embryo, indicating that the muscle cells have detached . As embryogenesis continues, the detachment worsens until muscle cells are seen only in the mid-section of the embryo and the normal structure of the contractile apparatus is lost . Without connection of the muscle to the exoskeleton, movement is severely impaired. To ascertain more precisely where the attachment between the muscle cells and the hypodermis is disrupted in the mutants, we examined the localization of other components of muscle attachment structures. At the time in development when the muscle cells detach, fibrous organelle–associated intermediate filaments and the MH5 protein (an immunologically defined protein that localizes close to fibrous organelles [22 and data not shown]), appear to localize as in wild-type . By contrast, perlecan, a basement membrane heparin sulfate proteoglycan synthesized by bodywall muscle 42 , remains associated with the muscle cells upon their detachment in myotactin mutants (data not shown). These data suggest the detachment is associated with a break at the interface between hypodermal cells and the basement membrane. Since myotactin in later stages of embryogenesis localizes near fibrous organelles, we examined the effect of the myotactin mutations on the localization of intermediate filaments as well as on the localization of the MH5 protein (data not shown). During early developmental stages, the localization of both components appears wild-type in the mutants . Both are concentrated in regions of the hypodermis adjacent to muscle, and both appear to be organizing into the banded distribution observed for these proteins in older embryos. However, in mutant embryos some time after elongation arrest and muscle cell detachment, both intermediate filaments and the MH5 protein become dispersed throughout the dorsal and ventral hypodermis . The staining seen with either MH4 or MH5 (data not shown) extends all the way to the boundaries between dorsal or ventral hypodermis, and seam cells. The precise correlation of this developmental stage with a wild-type stage is made difficult by the elongation arrest (elongation is a ready marker of developmental events in wild-type embryos). Nonetheless, given the timing of events after elongation arrest, this is likely equivalent to the wild-type threefold stage where MH4- and MH5-dependent staining is restricted to regions of the hypodermis adjacent to muscle . It is also around this time that the MH46 staining pattern reflects that of the fibrous organelle intermediate filaments. Although after muscle cell detachment intermediate filaments and the MH5 protein do not remain restricted to the regions of the hypodermis previously contacted by muscle, they do appear to maintain their association with the cuticular annuli since the staining pattern seen is regularly spaced, circumferentially oriented bands . As discussed earlier, each fibrous organelle contains two membrane plaques, one associated with the hypodermal membrane adjacent to the muscle basement membrane and one associated with the membrane adjacent to the cuticle. The resolution of the light microscope is not sufficient to distinguish between the two sets, and therefore it is not clear if both sets, or only the set adjacent to the cuticle, is present and/or delocalized in the mutants. To understand more about how the muscle acts to establish or maintain the correct spatial relationship between the contractile apparatus and the fibrous organelles, we looked at the localization of fibrous organelle intermediate filaments and myotactin in embryos lacking groups of muscle cells. We previously suggested that the initial recruitment of these proteins to regions of the hypodermis adjacent to muscle was initiated by contact between muscle and dorsal or ventral hypodermis 30 . To test this idea, we ablated muscle cell precursors in 28-cell embryos, allowed the embryos to develop for some time, and then fixed and stained the embryos with MH4 (to localize intermediate filaments) or MH46 (to localize myotactin). We ablated either the MS.ap and MS.pp cells, or the C.ap cell, resulting in the loss of 18 anterior or 16 posterior bodywall muscle cells, respectively. To assess the success of the ablations, the embryos were stained for myosin . Myotactin protein was absent in regions of the hypodermis that normally contact the missing muscle cells, but was present in regions of the hypodermis adjacent to the remaining muscle cells . Where present, myotactin appears organized in the obliquely striated wild-type pattern . The same phenomenon is seen when ablated embryos are stained for intermediate filament subunits (data not shown). That is, intermediate filaments in the ablated embryos are only in regions of the hypodermis contacting muscle, and appear to be organized as in wild-type embryos. These results are consistent with the hypothesis that intermediate filament proteins, myotactin, and possibly other hypodermal proteins organize in response to a signal produced by bodywall muscle cells. We do not believe the lack of fibrous organelle staining in ablated regions of the embryo is due to a lack of expression of intermediate filament proteins and myotactin. In wild-type embryos, both proteins are expressed before contact is made between muscle and dorsal or ventral hypodermis 30 , making it unlikely that muscle cells are required for proper expression of these proteins. Instead, we think the results of the ablation experiments suggest muscle cells are required for recruitment to and/or stabilization of these proteins in this region of the hypodermis. Myotactin is a novel transmembrane protein. The sequences of the DNA fragments diagrammed in Fig. 3 potentially encode two alternative transmembrane domains (encoded by exons 19A and B). The putative extracellular domain consists mainly of at least 32 FNIII repeats and can be expressed in multiple forms due to differential splicing. Two alternate cytoplasmic domains are also encoded by the gene. The larger predicted cytoplasmic domain (encoded by exons 19B–27) is 519 amino acids in length, and includes an additional 173 amino acids (encoded by exons 24 and 25) not found in the shorter form, 18% of which are serines. A search of the nonredundant protein database using BLASTX identifies many FNIII repeat–containing proteins, but all of these contain other known extracellular repeat motifs. Furthermore, the only database entries that show any significant homology to the putative cytoplasmic domain are proteins rich in serine, the presumed orthologue from C . briggsae (see Results) and an est entry from Onchocerca volvulus , another nematode. Analysis of myotactin mutants shows myotactin is required for muscle–cell adhesion . Myotactin may help mediate the attachment by forming part of a link between specific muscle substructures, such as dense bodies or M-lines, and the hypodermal membrane. During some embryonic stages the distribution of myotactin, a hypodermal transmembrane protein, mirrors that of the forming contractile apparatus 30 . This raises the possibility that a specific muscle substructure may be linked, in part through myotactin, to specific sites in the hypodermal membrane. Myotactin, which is anchored in the hypodermal membrane, has the potential to span the basement membrane and interact with a muscle membrane protein. Based on crystallographic data, the length of a FNIII repeat is ∼35Å 33 . The 32 predicted FNIII repeats of the extracellular domain of myotactin could extend over 110 nm, or long enough to span the basement membrane, which has been estimated from electron micrographs to be only ∼20 nm wide 58 . If myotactin does indeed interact with the muscle membrane, it is of interest to identify the muscle-associated protein to which it binds. Although the distribution of integrin and perlecan make them likely candidates to interact with myotactin, we do not believe this is the case. Integrin (encoded by the pat-3 gene; reference 25 , 59) is a transmembrane receptor positioned at the base of each dense body and M-line 21 , and perlecan is a basement membrane proteoglycan concentrated at these same sites 21 . However, in integrin and perlecan mutants, myotactin is associated with muscle cells, suggesting myotactin–muscle interactions are not dependent solely on either integrin or perlecan 30 . Although myotactin is not organized in oblique striations in integrin and perlecan mutants 30 , we think this reflects the loss of muscle structure in the two mutants rather than the loss of a myotactin-binding protein. Thus, early myotactin organization depends on muscle organization. The initial association between muscle and fibrous organelles is evident when muscle cells migrate onto dorsal or ventral hypodermis and hypodermal proteins organize in response to the contact. During wild-type development, upon contact between muscle and dorsal or ventral hypodermis, intermediate filaments and myotactin accumulate in the region of the hypodermis adjacent to muscle . Embryos missing certain groups of muscle cells, due to ablation of one or more muscle precursor cells, fail to accumulate these proteins adjacent to the region the missing muscle cells should occupy. This suggests a signal from the muscle cells is required to recruit certain proteins, including some fibrous organelle–associated proteins, to regions of the hypodermis adjacent to muscle. Myotactin is not involved in receiving the signal from muscle that establishes the association between muscle and fibrous organelles. In myotactin mutants, fibrous organelle–associated intermediate filament proteins and the MH5 protein, both accumulate in regions of the hypodermis adjacent to muscle upon contact between muscle, and dorsal or ventral hypodermis. Only later in development, after the time contraction normally begins, do the hypodermal proteins in the mutants become delocalized and no longer restricted to the regions overlying muscle. The fibrous organelles do however remain associated with the cuticular annuli. Fibrous organelles become delocalized in myotactin mutants. In embryos homozygous for the st456 mutation, the localization of intermediate filaments and the MH5 protein appears wild-type until late in embryogenesis when both are seen all through the dorsal and ventral hypodermis. We believe the distribution of intermediate filaments and the MH5 protein in myotactin mutants reflects the distribution of fibrous organelles for two reasons. First, although unlike in wild-type embryos the two proteins are not restricted adjacent to muscle, they do codistribute with one another, and both remain associated with cuticular annuli as in wild-type embryos. Second, the distribution of the two proteins appears wild-type through the embryonic stages when the fibrous organelles form. We suggest that in the myotactin mutants, the fibrous organelles at least partly assemble as in wild-type, but in the absence of myotactin may be allowed to move within the membrane. Movement of another membrane-associated, intermediate filament anchoring structure, the vertebrate hemidesmosome, has been shown by treating 804G cells with cytochalasin D to disrupt the actin cytoskeleton 40 . Hemidesmosomes were located subjacent to the nucleus before cytochalasin treatment, but in as little as 30 min after addition of the drug, hemidesmosomes were found at the cell periphery in cell projections. These peripheral hemidesmosomes were associated with cytoplasmic intermediate filaments and with the transmembrane hemidesmosome-associated protein BP180. The speed of the effect suggests relocation of hemidesmosomes is due to movement of a complex of proteins rather than to disassembly and reassembly of the structures 40 . One possibility is that muscle cell detachment is responsible for the delocalization of fibrous organelles in myotactin mutants. However, observations of fibrous organelle–associated proteins in contractile mutants argue this is probably not the case. Examination of myosin A and tropomyosin mutants, where muscle cells cannot contract and appear to remain attached to the hypodermis at the anterior and posterior of the embryo, revealed that fibrous organelles also become delocalized (Hresko, M., and R.H. Waterston, unpublished results). Delocalization of fibrous organelles is clear even in the regions of the hypodermis where the muscle cells appear to remain attached. Interestingly, in both myotactin and contractile mutants, delocalization of fibrous organelles occurs during what might be equivalent to the wild-type threefold stage. This is the stage when the distribution of myotactin first reflects that of fibrous organelles. Perhaps the localization of myotactin to the hypodermal membrane at or near fibrous organelles is important for restricting fibrous organelles at this stage. The delocalization observed in the myotactin mutants would therefore occur because myotactin is absent. An alternative possibility is that, in the myotactin mutants, the delocalization of fibrous organelles occurs because only the membrane plaques adjacent to the cuticle are present. As discussed earlier, each fibrous organelle consists of two membrane plaques, one at the membrane adjacent to muscle and one at the membrane adjacent to the cuticle. Possibly, in the absence of myotactin, the plaques adjacent to the muscle either do not assemble, or assemble but then disassemble. Due to the absence of the plaques adjacent to muscle, the ones adjacent to the cuticle are not restricted to regions of the hypodermis adjacent to muscle and become delocalized. Since in the myotactin mutants fibrous organelle proteins initially concentrate in regions of the hypodermis adjacent to muscle, and in fact fibrous organelles appear to assemble in these regions of the hypodermis, we think it is unlikely that the plaques adjacent to muscle do not form. Therefore, it seems more likely that myotactin helps in maintaining the correct spatial relationship between muscle and fibrous organelles either by maintaining the integrity of the fibrous organelle structures and preventing their disassembly, or by restricting the movement of fibrous organelles within the hypodermal membrane. Therefore, we favor a model in which myotactin is essential to maintain the correct spatial relationship between the contractile apparatus of the bodywall muscle and the fibrous organelles of the hypodermis. In this model, during wild-type development, myotactin is synthesized and secreted by the dorsal and ventral hypodermal cells, but remains attached to the hypodermis through a transmembrane domain. As muscle cells migrate and contact the dorsal and ventral hypodermis, the extracellular domain of myotactin extends and interacts with a muscle-associated protein. The interaction between muscle cells and the hypodermal membrane, made through myotactin, is required to prevent detachment of muscle cells when contractions first begin. At some time during development, the distribution of myotactin becomes correlated with the position of fibrous organelles. Myotactin, due to its association with muscle and close proximity to fibrous organelles, would help maintain the correct spatial relationship between the muscle contractile apparatus and the fibrous organelles. This might serve to strengthen the connection between the hypodermis and the muscle. The resolving power of the light microscope does not allow us to determine if myotactin is a fibrous organelle component. However, if myotactin does interact with fibrous organelles, it may do so only later in development (during the threefold stage), suggesting the association is regulated in some way. Based on the sequence presented in this paper, there are at least two ways in which this regulation might occur. First, regulation might occur through the use of different isoforms. The gene encodes eight differentially used exons which allow for myotactin isoforms that differ in the extracellular, the transmembrane, and the cytoplasmic domains. The sequence encoded by these exons might be important for positioning myotactin near fibrous organelles. The second mechanism by which an association between myotactin and fibrous organelles might be regulated is through phosphorylation. The carboxyl end of the putative cytoplasmic domain is enriched in serine residues. Phosphorylation of these regions might alter the function of the myotactin molecule. Interestingly, a vertebrate transmembrane hemidesmosome-associated protein, BP180 27 34 , that shares no sequence similarity to myotactin, may serve a role similar to that of myotactin. A human patient with GABEB (generalized atrophic benign epidermolysis bullosa) has been described who has a different mutation in each allele of his/her BP180 gene 38 . In this patient, hemidesmosomes form as do the anchoring filaments that secure the hemidesmosomes to the connective tissue below. However, the lamina lucida, the junction between the epithelium and the dermis, is widened as if the strength of the connection between the hemidesmosomes and the anchoring filaments was not sufficient to keep the epithelium attached 38 . This result suggests BP180, like myotactin, may be responsible for strengthening the connection between the membrane at or near a cell adhesion structure, and the basement membrane. While it is known that β4α6 integrin forms a link between hemidesmosomes and the basement membrane 16 24 46 53 , the existence of mutations in transmembrane proteins (BP180 or myotactin) other than integrin which result in detachment at epidermal-basement membrane junctions suggests there are multiple links between cell adhesion structures and the basement membrane or dermis. Furthermore, in mice lacking β4 16 53 or α6-integrin 24 , there are some regions of the skin where the basal epithelial cells have not detached from the dermis, again suggesting β4α6 integrin–independent links exist between the epithelium and the basement membrane. Whether BP180 and myotactin perform analogous functions of anchoring cell adhesion complexes in specific regions of the membrane awaits further study as does elucidation of mechanisms by which these types of proteins might function. | Study | biomedical | en | 0.999998 |
10444074 | The C32 human melanoma cells (American Type Culture Collection) and the human ovarian carcinoma OV10 cells were maintained as described . The following mAbs were used in this study: 2D3, B6H12, 2B7, 1F7, and 10G2 ; MAR4 (anti-β1, CD29; PharMingen); 7G2 and 1A2 ; P1F6 ; and anti-Gβ (Upstate Biotechnology Inc.). Vn was prepared as described . The amino acid sequence of the IAP-binding TSP-derived peptide 4N1K peptide is KRFYVVMWKK; it was synthesized as previously described . mAbs were iodinated using Iodobeads (Pierce Chemical Co.). Cells were preincubated with saturating levels of 125 I-labeled mAbs in RPMI-1640 with 10% FCS for 30 min on ice, followed by extensive washing to remove excess antibody. Preliminary experiments including 200-fold excess unlabeled antibody showed that >98% of the bound radioactivity represented specific binding. Cells were lysed in 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, 2 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mM PMSF, and 0.5% vol/vol Brij58 for 10 min on ice, homogenized using 10 strokes of a Dounce homogenizer, then lysed 20 min more on ice. The resulting lysate was adjusted to 40% wt/wt sucrose and applied onto a 60% wt/wt sucrose cushion. A sucrose step-gradient consisting of 25% wt/wt sucrose and 5% wt/wt sucrose were layered on top of the lysate. Gradients were centrifuged 16–20 h at 170,000 g at 4°C in a SW55 rotor (Beckman Instruments, Inc.). Fractions (0.5 ml) were taken from the top of the gradient using an auto densi-flow gradient harvester (Labconco). The amount of 125 I present in each fraction was measured using a Packard Crystal II γ counter. Sucrose solutions were made in buffer containing 20 mM Tris-HCl, pH 8.2, 140 mM NaCl, and 2 mM EDTA. Sucrose density was determined by refractive index using a refractometer. The amount of protein in each fraction was determined using the BCA Protein Assay Kit (Pierce Chemical Co.). Vn or peptides were coupled to 4.5-μm tosyl-activated magnetic beads and mAb were coupled to sheep anti–mouse 4.5-μm magnetic beads (Dynal) as described by the manufacturer. Cells were resuspended in HBSS with 0.1 mM MnCl 2 . Substrate-coated magnetic beads were added to the cells and the sample was rotated for 15 min at 37°C. Cells bound to the magnetic beads were collected by placing the sample in a magnet. Lysis buffer (20 mM Hepes, pH 7.5, 0.1 mM MnCl 2 , 250 mM sucrose, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mM PMSF, and 10 mM CHAPS) was added and the sample was incubated with agitation for 10 min at 37°C. Magnetic beads were resuspended and incubated 5 min in HBSS with 3 mM MgCl 2 and 2.5 units DNase I. 2× SDS-PAGE sample buffer was added and the samples were heated for 10 min at 65°C. The samples were analyzed by SDS-PAGE and Western blotting. Cells were resuspended at 2.5 × 10 5 cells/ml in RPMI/0.1% fatty acid-free BSA (Sigma Chemical Co.) with or without 5–15 mM methyl-β-cyclodextrin (MβCD; Aldrich Chemical Co.) and incubated 15 min at 37°C. After washing, cells were added to the prepared tissue culture wells and allowed to spread 30 min to 1 h at 37°C. Cholesterol was reintroduced into cells using 1.33 mg/ml cholesterol-MβCD inclusion complexes in RPMI/0.1% fatty acid-free BSA, which results in a 0.1 mM solution of cholesterol . To quantitate the amount of cholesterol in membranes after these treatments, cellular lipids were extracted by the method of Bligh and Dyer 1959 , and cholesterol content was assayed by the cholesterol oxidase method (Wako Chemicals USA). Approximately 30% of cellular cholesterol was removed in OV10 cells by this treatment. Cholesterol-MβCD inclusion complexes were prepared as described . In brief, a solution of 30 mg cholesterol (Sigma Chemical Co.) in 400 μl 2:1 vol/vol methanol/chloroform was added drop-wise to a stirred solution of 1 g of MβCD in 11 ml PBS prewarmed in an 80°C waterbath. The solution was stirred at 80°C until a clear solution resulted. The cholesterol-MβCD inclusion complexes were used immediately or freeze-dried. Inclusion complexes of the steroid analogues were made similarly using 30 mg 5-cholestene-3-one, 20 mg 5-cholestene, or 24.7 mg pregnenolone. IAP-enhanced spreading of C32 cells on suboptimal doses of Vn was performed as described . In brief, 12-well plates (Costar Corp.) were coated for 2 h at room temperature with 0.125 μg/ml Vn in HBSS. Plates were blocked with 1% BSA/PBS for 2 h at room temperature and washed three times with PBS. After MβCD treatment, C32 cells were plated in HBSS with 1 mM CaCl 2 and 1 mM MgCl 2 with or without the addition of 20 μM 4N1K peptide. MβCD inclusion complexes made with cholesterol or the steroid analogues were added to some wells. Cells were allowed to spread for 30 min at 37°C. Indirect immunofluorescence was analyzed on OV10 cells with or without pretreatment with MβCD as described . The primary antibodies used were 10G2, a murine mAb that detects a subset of IAP on some cells , and 1F7, a murine mAb that detects all IAP . FITC-labeled antimurine μ chain and antimurine γ chain (Sigma Chemical Co.) was used to detect cell-bound 10G2 and 1F7, respectively. Previous studies using liposomes reconstituted with αvβ3 showed that lipid composition affected ligand binding by this integrin and that a cholesterol-containing environment markedly enhanced ligand binding . Ligand binding by αvβ3 also is influenced by IAP . Because IAP likely contaminated the αvβ3 used to prepare the liposomes, we determined whether cholesterol affected αvβ3 association with IAP. Trimeric G proteins have been identified as a signal transduction component associated with IAP and αvβ3 . αvβ3/IAP/G protein complexes were isolated from OV10 cells expressing both β3 integrin and IAP , and treated in vitro with the cholesterol-chelating agent MβCD . Treatment with MβCD markedly decreased association of both IAP and Gβ with the purified αvβ3 . Anti-Gα Western blots showed a similar decrease in Gα association with αvβ3 . Thus, removal of cholesterol compromised association of αvβ3 with IAP and trimeric G protein. No caveolin was identified in these isolated complexes, although OV10 cells do express caveolin (data not shown). No αvβ3, IAP, or G protein was precipitated with YIGSR-coated beads . These control beads bind to a nonintegrin laminin receptor expressed on OV10 cells. Cholesterol also was required for complex assembly in intact cells, since little associated IAP or G protein was copurified with αvβ3 isolated from OV10 cells treated with MβCD . Whereas MβCD removes cholesterol from cell membranes, MβCD–cholesterol inclusion complexes mediate the incorporation of cholesterol into membranes . Treatment of OV10 cells with MβCD decreased cell cholesterol content 30–40%; subsequent incubation with MβCD-cholesterol inclusion complexes restored cellular cholesterol to basal levels (data not shown). In cells treated first with MβCD to disassemble the integrin/IAP/G protein signaling complexes and then incubated with cholesterol-charged MβCD to restore membrane cholesterol, IAP/αvβ3/G protein complexes were isolated to the same extent as in untreated cells . To examine the structural requirements for cholesterol in complex formation, inclusion complexes were made with several cholesterol analogues. Steroids with minimal structural differences to cholesterol were chosen, all having the hydrophobic, planar, core ring structure . 5-cholestene lacks the 3β-hydroxyl group. Pregnenolone contains a methyl ketone group instead of the aliphatic tail. A carbonyl replaces the 3β-hydroxyl group in 5-cholestene-3-one. These compounds yield an equilibrium between MβCD-complexed steroid and steroid incorporated into the plasma membrane, similar to what is observed with cholesterol itself . Neither 5-cholestene nor pregnenolone was able to restore association of IAP/αvβ3/G protein complexes in MβCD treated cells . However, 5-cholestene-3-one was even better than cholesterol (∼2.3-fold better by densitometry) in restoring the IAP/αvβ3 association. Thus, there is structural specificity in the lipid requirement for complex formation, with both the aliphatic tail and an oxygen in the 3 position of the cholestene ring apparently required. To determine the role for the cholesterol-dependent supramolecular complex in IAP/αvβ3 signaling, we examined the role of the complex in TSP modulation of αvβ3 function in C32 cells. This is an excellent model to test signaling by the complex since the COOH-terminal domain of TSP (TSP-1) has been shown to modulate αvβ3 integrin-mediated adhesion and spreading of these cells through interaction with IAP, and this effect requires activation of a heterotrimeric G protein . Moreover, cholesterol depletion resulted in no loss of viability and no obvious morphologic change in these cells (data not shown). Only when C32 cells were treated with the IAP-binding agonist peptide from TSP-1 did they spread on surfaces coated with low concentrations of Vn , an effect abolished by cholesterol chelation with MβCD . Cholesterol repletion using MβCD inclusion complexes restored IAP-dependent spreading . In contrast, MβCD had no effect on C32 spreading on high density Vn , which is known to be IAP-independent . None of the cholesterol analogues were able to restore 4N1K-induced spreading in MβCD treated cells . The failure of 5-cholestene-3-one, which restores complex formation, to restore TSP induction of C32 cells spreading, resulted from a general inhibition of cell spreading by this cholesterol analogue, since C32 spreading on high concentration Vn was inhibited in cells repleted with this cholesterol analogue (data not shown). In contrast, neither 5-cholestene nor pregnenolone affected C32 spreading on high Vn substrates. Thus, cholesterol is required for cooperation between IAP and αvβ3 in C32 cells, but not for cell spreading in general. While cell spreading in response to 4N1K likely requires IAP-dependent signal transduction, the biochemical mediators of this signaling are not known . Therefore, we measured 4N1K-mediated inhibition of adenylate cyclase in prostaglandin E 1 (PGE1)-treated resting platelets, an event known to require heterotrimeric G protein signaling . 4N1K caused an 80% decrease in cAMP levels in platelets (81.4 ± 5.2 fmol/2 × 10 7 cells to 16.0 ± 3.2). In MβCD-treated platelets, the cAMP was unchanged by 4N1K (33.4 ± 3.3 fmol without and 31 ± 3.2 with 4N1K). Repletion of cholesterol in MβCD-treated platelets allowed 4N1K to again induce a drop in cAMP to 18 ± 2.4 fmol, not different from the cAMP in 4N1K-treated platelets without cholesterol perturbation. The reason that MβCD caused a decrease in the basal level of cAMP in resting platelets is unknown, but may reflect a failure of PGE1 signaling, since it binds to a seven transmembrane Gs-coupled receptor that likely resides in DIGs . Removal of cholesterol did not abolish all cell signaling since tyrosine phosphorylation in response to both 4N1K treatment and adhesion to high concentrations of Vn was normal in MβCD treated cells . Thus, while cholesterol depletion by MβCD does not affect integrin-dependent tyrosine phosphorylation, it does block 4N1K-initiated spreading and signaling, which are dependent on heterotrimeric G proteins. To determine how cholesterol affected assembly of the supramolecular signaling complex, we determined the role of the IAP multiply membrane spanning (MMS) domain and extracellular domain in complex formation. αvβ3 integrins were isolated from transfected OV10 cells expressing normal IAP, IAP in which the MMS domain had been replaced either by a single pass CD7 transmembrane domain (IAP/CD7) or by a glycan phosphoinositol anchor (IAP/GPI), or IAP in which the extracellular domain of IAP was replaced with a FLAG epitope (MC2). The IAP Ig domain was expressed equivalently on OV10 cells transfected with each of the Ig domain-expressing constructs and the MMS domain was equivalently expressed in wild-type and IAP/MC2 transfected cells, as assessed by antibody against the IAP cytoplasmic tail (data not shown). While the complex was easily isolated using either the αvβ3 ligand Vn or anti-β3 mAb (1A2) from cells expressing normal IAP, minimal IAP or G protein was copurified with integrin from cells expressing either IAP/CD7, IAP/GPI, or IAP/MC2 . Furthermore, the IAP MMS domain influenced cholesterol association with αvβ3, since there was 1.7 ± 0.85-fold ( P < 0.03, n = 4) more cholesterol associated with anti-β3 1A2 immunoprecipitates from OV10 cells expressing IAP than from cells deficient in IAP. In addition, there was 2.1 ± 0.46-fold ( P < 0.03, n = 3) more cholesterol associated with anti-IAP immunoprecipitates from OV10 cells expressing wild-type IAP than from cells expressing either IAP/GPI or IAP/CD7. Thus, both the MMS and the Ig domains of IAP are required for complex assembly, and the MMS domain enhances cholesterol association with the αvβ3 integrin. Because cholesterol and heterotrimeric G proteins are concentrated in DIGs in many cell types , it was possible that the IAP-dependent signaling complex was preferentially formed in these domains. Sucrose density ultracentrifugation demonstrated that both αvβ3 and IAP were enriched in DIGs, which are found at the interface of the 25 and 5% sucrose layers . Approximately half of IAP was isolated in this low density membrane fraction, together with about one-fourth of the αvβ3 . In contrast, the closely related αvβ5 integrin localized predominantly to the bulk membranes , together with >95% of total cellular protein . To determine whether complex formation occurred preferentially among the IAP, αvβ3, and G proteins in DIGs, αvβ3 was purified from individual fractions of the sucrose gradient and coassociation of αvβ3, IAP, and heterotrimeric G proteins determined . Together with 15% of the bead-bound αvβ3 (as determined by densitometry), ∼40% of the associated IAP and Gβ were coprecipitated in the DIGs. This represents a minimum estimate of DIGs-associated complex, since the purification procedure was presumably not 100% efficient. Thus, αvβ3 in DIGs was at least four times more likely to be involved in complex formation than αvβ3 in the bulk membrane fractions of the cell, demonstrating a preferential association of the complex with DIGs. To determine whether complex formation was required for localization of IAP or αvβ3 to DIGs, fractionation studies were performed on cells lacking IAP or αvβ3. In OV10, αvβ3 localized to DIGs similarly whether or not IAP was present . In the Jurkat T lymphoid line, which expresses little if any αvβ3 and as a consequence have no detectable αvβ3/IAP complex, >60% of the IAP was in low density fractions (data not shown). Thus, IAP and αvβ3 localize to DIGs independent of complex formation. It is already known that trimeric G proteins localize to DIGs because of acylation and/or interaction with caveolin . Thus, each of the three protein components of the complex is targeted to DIGs independent of complex formation, suggesting that DIGs are the membrane sites where these signaling complexes form. To determine the effect of cholesterol removal on localization of the protein components of the complex to DIGs, OV10 were treated with MβCD before sucrose density gradient centrifugation. The localization of IAP was minimally affected by MβCD treatment . However, both αvβ3 and Gβ localization were substantially diminished . To determine if DIGs localization of each of the components was sufficient for complex formation, the membrane localization of IAP/CD7 and IAP/GPI , both of which failed to form complexes, was determined. While less IAP/CD7 was in DIGs, IAP/GPI localized to the DIGs to a similar extent to wild-type IAP . This is consistent with the known propensity of GPI-linked proteins to be enriched in DIGs . Since complexes did not form in cells expressing IAP/GPI despite the ability of the IAP Ig domain to mediate interaction with αvβ3 , this result demonstrates that localization of the IAP Ig domain to DIGs is not sufficient for complex formation and that the MMS domain of IAP must serve another function in complex formation. Furthermore, this experiment demonstrates that the protocol for isolating the αvβ3/IAP/G protein complex does not simply nonspecifically copurify all proteins found in DIGs. However, DIGs localization of each component of the complex may be necessary for complex formation. Since cholesterol is an essential component of the complex, DIGs localization may provide a mechanism for focusing the proteins of the complex at a site where an adequate concentration of cholesterol exists for complex assembly. A previous study suggested that the interaction between IAP and αvβ3 could occur through the IAP Ig domain . Thus, it was possible that cholesterol interaction with the IAP MMS domain could affect the IAP conformation to facilitate complex formation. To determine whether any cholesterol-dependent conformation of IAP could be discovered, FACS ® analysis was performed with 10G2, a mAb that recognizes only a subset of IAP on many cells . 10G2 recognizes the IAP Ig domain, as demonstrated by ELISA and Western blotting with purified Ig domain (data not shown). On OV10 cells, 10G2 recognized wild-type IAP approximately fourfold better than IAP/CD7 and did not bind IAP/GPI at all . This difference in detection was not due to differences in expression, as detected by the conventional anti-IAP mAb B6H12, which detects equivalent expression of the three constructs . When OV10 expressing wild-type IAP were treated with MβCD, 10G2 binding increased another sixfold . Repletion of cellular cholesterol with MβCD/cholesterol complexes returned 10G2 binding to the level of untreated cells. MβCD did not affect 10G2 binding to IAP/CD7 or IAP/GPI (data not shown). Furthermore, MβCD did not affect the binding of the anti-IAP mAbs 2D3 or 2B7 . These data demonstrate that the availability of the 10G2 epitope on the IAP Ig domain on OV10 cells is markedly influenced by the MMS domain and is significantly modulated by cholesterol. Its ability to modulate 10G2 binding to the Ig domain suggests that cholesterol binding to the IAP MMS domain affects IAP conformation. IAP is a plasma membrane protein first isolated by copurification with αv integrins. Subsequent studies have demonstrated a functional association of IAP with β3 integrins in leukocytes and endothelial cells and coimmunoprecipitation of IAP with αvβ3 from several cells and tissues . Moreover, IAP is required for Vn bead binding by both αvβ3 and αvβ5 integrins in OV10 cells . However, IAP appears to be unnecessary for adhesion of these same cells to Vn-coated surfaces and IAP-deficient mice develop normally, in contrast to αv-deficient mice, demonstrating that IAP is not absolutely required for αv integrin function. This has led to the hypothesis that IAP and αvβ3 form a signaling complex in at least some cells that can influence specific cell functions. Recently, association of αvβ3 and IAP with trimeric G proteins has been demonstrated , suggesting a mechanism by which the αvβ3/IAP complex may signal. However, the molecular mechanisms involved in association of αvβ3 with IAP or of this membrane complex with G proteins have not been resolved. An unusual feature of IAP is its MMS domain, which could allow for enhanced interactions with and regulation by membrane lipids. To determine whether this was the case, we examined whether cholesterol was required for association of IAP with αvβ3 integrin and with trimeric G proteins. Although the removal of cholesterol with MβCD has been shown to disrupt receptor signaling in a variety of cells , a specific role for cholesterol in the formation of supramolecular receptor complexes or in regulation of integrin function has not been demonstrated previously. We found that cholesterol is required both for maintenance of complexes in cells and for maintenance of isolated complexes in vitro. Thus, cholesterol is the fourth molecular and first described nonprotein component of this signaling complex. Likely, the cholesterol interacts with the IAP MMS domain, since replacement with a single transmembrane domain in IAP/CD7 or with a GPI-anchor in IAP/GPI abolished complex formation as efficiently as cholesterol removal. Moreover, removal of cholesterol or replacement of the MMS domain with CD7 also abolished functional responses to IAP ligation in Jurkat T cells and murine fibroblasts (unpublished data). Thus, cholesterol is required not only for physical association of IAP with integrins and G proteins, but for integrin-independent functions of IAP as well. Recently, the understanding has emerged that there are distinct domains within the lipid bilayer in which specific lipids are concentrated . Because they can be purified and studied due to their low density and relative resistance to solubilization by some detergents, domains enriched in glycosphingolipids and cholesterol are the best characterized . These domains are called DIGs, GEMs (for glycosphingolipid-enriched membranes), or rafts, to reflect these properties. In addition to lipids, these domains are enriched in specific integral membrane proteins, of which caveolin is the best known . However, it is now clear that caveolin is not required for organization of the domains and DIGs can exist even in cells lacking caveolin . In addition to caveolin, DIGs contain disproportionately high concentrations of GPI-linked membrane proteins, as well as a variety of cytoplasmic proteins that associate with the plasma membrane through lipid modifications, including NH 2 -terminal myristoylation and/or palmitoylation . In this latter category are a variety of signal transduction molecules, such as heterotrimeric G proteins and some src family kinases, which have been shown to localize to these domains . However, the significance of domain localization is somewhat controversial. While specialization of these domains as sites for initiation of signal transduction has been proposed , so has the opposite, that these are sites where signal transduction proteins can be sequestered in inactive form . IAP and αvβ3 both preferentially localize to DIGs and their association is greatly enhanced in these domains. This may imply that the functional signaling complex localizes to these membrane domains and that DIGs are essential for signal propagation across the membrane, perhaps because of proximity to other cytoplasmic molecules required for the signaling cascade. Alternatively, the localization to DIGs may simply reflect the tight association with cholesterol that apparently characterizes the complex. However, it is clear that localization to DIGs is not sufficient for stable physical association, since IAP/GPI, which localizes to DIGs equally as well as wild-type IAP does not participate in complex formation. Moreover, removal of cholesterol fails to disrupt localization of IAP to DIGs, even though the functional association among integrin, IAP, and G proteins is disrupted, and IAP no longer functions in integrin-mediated spreading. It is possible that a particularly tight or specific association of IAP with cholesterol would require more extensive depletion of cholesterol than is achieved with 10 mM MβCD to affect localization. It is interesting that localization of IAP to DIGs and formation of stable complexes with αvβ3 and G proteins appears not to be required for IAP enhancement of αv integrin bead binding, since both IAP/CD7 and IAP/GPI can mediate this effect of IAP . This suggests that IAP's regulation of αvβ3 ligand binding does not depend on its signaling capacity, but relies instead on direct interaction of its Ig domain with αv integrins, potentially altering the Vn-binding ability of these integrins. It is possible that this effect of IAP does not require stable association with the integrin and that once Vn binding is activated, the αvβ3 integrin can bind ligand tightly without IAP . Nonetheless, these experiments suggest that the IAP Ig domain is an important component of the interaction of IAP with αvβ3. Consistent with this, no complex formation can be demonstrated in cells transfected with a chimeric molecule in which the IAP Ig domain has been replaced with an irrelevant domain. Based on these observations, we propose a model in which the conformation of IAP is influenced by the MMS domain and further influenced by the tight binding of cholesterol to the MMS domain. This is consistent with the pattern of binding of the 10G2 mAb to OV10-expressed IAP, for which the MMS domain is required and mAb binding enhanced by cholesterol removal. In this model, the cholesterol-replete conformation of IAP can interact with αvβ3, and for this, the Ig domain is required. This entity, in turn, associates with the trimeric G protein to form the complete signaling complex. Since there is specificity in the ability of cholesterol analogues to mediate IAP-αvβ3 complex formation, it will be interesting to determine whether the failure of some analogues to mediate complex formation reflects failure to interact with IAP or failure to induce the change in IAP conformation and function. In summary, we have demonstrated that cholesterol has a critical role in assembly of the αvβ3/IAP/G protein signaling complex. This is a novel role for a membrane lipid and suggests a new and direct mechanism for regulation of signal transduction by supramolecular complexes in the plasma membrane. It has been shown that cell activation signals can regulate the association of the high affinity Fc∈ receptor with DIGs . It is possible that IAP association with these cholesterol rich domains also is regulated and that this, in turn, affects formation and maintenance of the signaling complex containing αvβ3 and heterotrimeric G proteins. | Study | biomedical | en | 0.999996 |
10444075 | Plasmids containing cDNAs encoding either human occludin or cx32 (a gift of Dr. David Paul, Harvard Medical School, Cambridge, MA) were used as templates in PCRs to construct chimeric cDNAs. All cloned PCR products were confirmed by dideoxy sequencing in both directions. The vesicular stomatitis virus glycoprotein (VSV-G) epitope–tagged human occludin cDNA (HOC) has been described previously . All constructs were expressed in MDCK or NRK cells using the mammalian expression vector pCB6 (Karl Matter, University of Geneva, Switzerland). The connexin-occludin chimera (CX/OC) was constructed in three steps. First, primers 19985 (5′-GTACTAGTAGGCAGGATGAACTGGACAGGT-3′) and 19984 (5′-ATCATCCGGGCCTGTGCCCGCGCGGCCGC-3′) were used to amplify the NH 2 -terminal 219-residues encompassing all four transmembrane regions of connexin 32 using Vent polymerase (New England Biolabs Inc.). The product was digested with Spe1 and Not1 and cloned into pSK-Bluescript (Stratagene). Second, using primers 19983 (5′-cgcggccgcgaaaactcgaagaaagatggac-3′) and 19986 (5′-GACTATGATAGACAGAAAACAGCCTACACCGACATCGAGATGAACAGGCTGGGCAAGTGAGAGCTC-3′), the entire COOH-terminal cytoplasmic tail of occludin was amplified and a VSV-G tag was added. The resulting fragment was digested with NotI and SacI and inserted into pSK-Bluescript. Third, PCR products were subsequently excised and ligated into pCB6. The distal COOH-terminal residues of occludin (DOC) chimera were made by ligating the transmembrane region of connexin 32 to the distal 150 amino acids of occludin (residues 373–522), as amplified and VSV-G tagged using primer 19986 combined with 21063 (5′-CGCGGCCGCGAACTTTGAGACAGGTTGAAAAACA-3′). The resulting fragments were digested as above and subcloned as above into pCB6. The proximal residues 266–372 of the occludin cytoplasmic tail (POC) chimera were made by ligating the transmembrane region of connexin 32 to the membrane proximal portion of the occludin tail (residues 266–372), which was amplified and VSV-G tagged using primers 19983 and 24008 (5′-CCTCGTTACAGCAGCGGTGGTGCCTACACCGACATCGAGATGAACAGGCTGGGCAAGTGAGGATCC-3′). The resulting fragments were digested with SpeI, NotI and NotI, and BamHI, respectively, and subcloned into pCB6. MDCK and NRK cells were obtained from the American Type Culture Collection and grown in DME supplemented with 10% FBS (Atlanta Biologicals), 5 mM L -glutamine (GIBCO BRL), 5 mM penicillin, and 5 U/ml streptomycin, hereafter known as DME-complete. To generate MDCK cells stably expressing the constructs, cells were transfected via lipofection (GIBCO BRL) or calcium-phosphate coprecipitation and selected using DME-complete containing 0.8 mg/ml G418 (GIBCO BRL). Resistant colonies were isolated and maintained in DME-complete containing 0.25 mg/ml G418. cDNAs were introduced transiently into NRK cells plated on coverslips by calcium phosphate coprecipitation . MDCK clones stably expressing chimeric proteins were plated on coverslips and cultured 2–6 d before being fixed. 12–16 h after transient transfection, NRK cells were fixed and prepared for immunofluorescence as described in Fanning et al. 1998 . VSV-G–tagged constructs were detected with a rabbit polyclonal anti–VSV-G antibody (MBL Laboratories) or mouse mAb P5D4 (a gift of Dr. Thomas Kreis, University of Geneva, Switzerland), both used at 1:200. To visualize the endogenous ZO-1, cells were labeled using rat mAb 40.76 or a rabbit polyclonal antibody 4476 (Zymed Labs, Inc.). Rat connexin 32 was visualized with mouse mAb M12.13 (a gift of Dr. David Paul, Harvard Medical School, Cambridge, MA) or mouse anti–rat connexin 32 mAb (Chemicon International, Inc.). For epifluorescent microscopy, labeling was visualized using a Nikon Microphot FX microscope with a 60× PlanApo objective and images were captured using either T-MAX 400 film or a Sensys cooled CCD camera (Photometrics). These images were processed using Adobe Photoshop 4.0 or Image Pro Plus 2.0 (Media Cybernetics), respectively. MDCK cells were plated in 10-cm diameter tissue culture plates and grown to confluence. Cells were fixed in 1% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 15 min at 4°C for SDS-digested freeze-fracture replica labeling or in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 30 min at 4°C for conventional freeze-fracture. The cells were washed in the respective buffers, scraped from the substrate with a plastic cell scraper and infiltrated with graded amounts of glycerol in 0.1 M cacodylate buffer for 50 min at 4°C. Cell pellets were frozen in liquid nitrogen slush and freeze-fractured at −115°C in a Balzers 400 freeze-fracture unit (Balzers, Liechtenstein). SDS digestion and immunolabeling of replicas with polyclonal anti–VSV-G antibody (1:50 or 1:100) followed by protein A gold (10 nm) was performed as described . Replicas were examined using a Philips 301 electron microscope. To quantitate gold particle distribution on micrographs, the distance from the center of a gold particle to the center of the nearest fibril was measured on photographic enlargements of micrographs using dial calipers (Small Parts, Inc.) that accurately measure in increments of 0.02 mm. The number of junctions and gold particles analyzed was as follows: 13 junctions and 343 gold particles for HOC, 8 junctions and 735 gold particles for CX/OC, and 2 junctions and 9 gold particles for control cells. The distance of a gold particle from fibril versus the number of occurrences was plotted using Microsoft Excel. To investigate the role of the cytoplasmic COOH-terminal domain of occludin in targeting occludin, we fused regions of the tail of human occludin to the membrane-spanning portions of rat connexin 32 (cx32). Occludin and cx32 share similar membrane topogenics, with each predicted to form two extracellular loops, four transmembrane helices, and NH 2 and COOH cytoplasmic termini. Fig. 1 illustrates constructs used in this study. An 11–amino acid tag from the VSV-G protein was engineered at the COOH terminus of each connexin-occludin construct to facilitate immunodetection. MDCK and NRK cells do not express detectable amounts of rat cx32, as determined by immunofluorescence labeling and immunoblotting (data not shown), providing null backgrounds in which to introduce chimeric proteins. Plasmids encoding chimeric proteins were transfected into MDCK cells and multiple clones of each construct were isolated by selection for G418 resistance. Stably transfected cell lines used were morphologically indistinguishable from their untransfected counterparts. At least three clones were analyzed for each experiment and each showed results similar to the representative clones reported in this study. NRK experiments reported in this study were performed by transient transfection. We first investigated the membrane targeting characteristics of full-length cx32 transfected into MDCK cells to interpret the results of subsequent chimeric studies. Published studies have shown that the NH 2 -terminal, membrane-spanning half of cx32 contains information sufficient to target to gap junctions . Indirect immunofluorescence revealed that cx32 transiently transfected in MDCKs localized in the typical punctate pattern of gap junctions . Protein was concentrated on the lateral surfaces of transfected cells regardless of whether adjacent cells also expressed cx32. Colabeling for ZO-1, a protein highly concentrated at tight junctions, demonstrated typical tight junction staining in a focal plane distinct from cx32 labeling . Thus, rat liver cx32 clusters in gap junctionlike structures on the lateral surface of MDCKs and has no inherent ability to localize with ZO-1 at tight junctions. In contrast, when the COOH-terminal tail of cx32 was replaced with the COOH-terminal tail of occludin to form the CX/OC chimera , VSV-G epitope immunolabeling revealed that this chimera localized at tight junctions and not solely at gap junctions, as might be expected because of the gap junction targeting information in the NH 2 terminus . VSV-G staining for CX/OC paralleled that of endogenous ZO-1 staining , with both colocalized in a reticular pattern around the apical surface of cells. Note that expression of the chimera in adjacent cells is not required for localization, as continuous, circumferential VSV-G labeling is seen in cells adjacent to untransfected neighbors. Interestingly, in clones expressing very low levels of CX/OC, the protein localized primarily at tight junctions , whereas clones greatly overexpressing CX/OC localized it at both tight and gap junctions (data not shown). These observations may imply that the incorporation of CX/OC into tight junctions is saturable, and that excess chimeric protein follows a default localization to gap junctions. Thus, the human occludin tail is sufficient to target chimeras containing the transmembrane portions of rat cx32 to the tight junction, even though the NH 2 -terminal half of cx32 possesses gap junction targeting information. In addition, the NH 2 -terminal, membrane-spanning half of human occludin is not necessary for occludin's localization at the tight junction. To ask whether the CX/OC chimera was actually incorporated specifically into fibrils or simply concentrated around them, we employed immunogold labeling of freeze-fracture replicas. Control cells expressing pCB6 vector alone showed no specific labeling at tight junction fibrils . In contrast, cells expressing full-length human occludin (HOC) or CX/OC both showed specific labeling along fibrils. Note that in CX/OC-containing cells, a two-dimensional crystalline lattice reminiscent of gap junctions is also labeled , indicating that chimeras expressed at high levels can also assemble into gap junctionlike aggregates. The increased gold labeling in CX/OC cells versus human occludin cells is probably due to higher protein expression in the CX/OC cells, as documented by immunoblotting (data not shown). It has long been known that fixation of cells with glutaraldehyde will transform the linear junction particles into continuously fused fibrils . Whereas unfixed particles appear on the ectoplasmic face, fixed fibrils appear on the P-face as continuous strands interspersed with occasional hemispherical particles . The proportion of particles to strands varies depending on cell type. In contrast, gap junctions visualized by freeze-fracture in glutaraldehyde fixed cells never fuse but always appear as large, paracrystalline aggregates of discrete connexon particles on the P-face. Interestingly, in CX/OC-expressing cells fixed in 2% glutaraldehyde and processed for conventional freeze-fracture, distinct particles similar in size and shape to connexons are visible within the fused fibrils of the P-face , but are absent in cells transfected with HOC (data not shown) and the control empty vector . These particles, which presumably represent the connexin portion of CX/OC, occur in perfect register with the rest of the fibril. Similar morphological changes are not apparent in the immunogold-labeled replicas because the relatively brief, gentle fixation of 1% formaldehyde used in the gold labeling was not sufficient to fuse the fibrils. Together, these results strongly suggest that the CX/OC chimera is intercalated directly into junctional fibrils. To determine whether the CX/OC chimera was within or just near fibrils, we next quantified the orthogonal distance between individual gold particles and the nearest fibril. Transfected, epitope-tagged occludin was assumed to be incorporated into fibrils and was used as a control reference. Both occludin and the CX/OC chimera show similar distance distributions from fibrils, with the most common distance ranging from 6–12 nm . This distance corresponds roughly to the length of the intervening primary and secondary antibodies used in the labeling procedure . Thus, the CX/OC chimera localized directly within the tight junction fibril, presumably interspersed with endogenous occludin and claudin family molecules, and without obviously disrupting normal junction morphology. We next asked whether the ZO-binding domain is necessary for localization of connexin chimeras at tight junctions. In MDCK cells stably expressing DOC, which contains the ZO-binding domain, VSV-G labeling occurred at the tight junction , as seen by its colocalization with endogenous ZO-1 . However POC, which lacks the ZO-binding domain, localized predominantly in discrete puncta , similar to gap junctionlike structures seen with full-length connexin 32 . ZO-1 at the tight junction was visible in a more apical focal plane than POC immunoreactivity. These results suggest that potential interactions between endogenous occludin and transfected POC in the juxtamembrane region (amino acids 266–372) do not confer tight junction localization to POC. In addition, these results suggest that only the ZO-binding region of occludin is necessary to localize a connexin chimera at the tight junction. If an interaction with ZO proteins is independently sufficient to localize occludin in the absence of endogenous occludin and claudin, we predict that connexin-occludin chimeras will be able to localize to ZO-1–containing cell–cell contacts, even in cells lacking these transmembrane tight junction proteins. We tested this prediction in NRK fibroblasts that express ZO-1 and ZO-2 at cadherin-based contacts but lack immunodetectable occludin, claudin-1 and-2 (data not shown) and lack freeze-fracture fibrils. In support of this prediction is our previously published observation that full-length occludin stably expressed in NRK cells colocalizes with ZO-1 at cell–cell contacts . In contrast, occludin lacking the ZO-binding domain does not localize in NRK cells, yet the same construct localizes to tight junctions in MDCK cells (Van Itallie, C., personal communication). When cx32 is transiently expressed in NRK cells its localization depends on whether adjacent cells also express the protein. In isolated cells that are not in contact with other transfected cells, the protein is detected diffusely in the cytoplasm or in perinuclear aggregates (data not shown). In contrast, when adjacent cells both express cx32, it localizes at cell contacts between them , but not at the contacts with neighboring, nontransfected cells . cx32 contacts in adjacent transfected cells occasionally appeared to overlap ZO-1 immunoreactivity , but were quite different in shape and size. These results suggest that clustering of cx32 at cell–cell contacts in NRK cells requires expression of cx32 on both cells and that cx32 has no inherent tendency to associate with ZO-1 at cell contacts. In contrast, CX/OC colocalized with ZO-1 at cell–cell contacts, implying that endogenous occludin or claudins are not necessary for targeting to ZO-1–containing cell contacts. This behavior is distinct from that seen with cx32, which only localized at contacts between two transfected cells . Consistent with this is the observation that full-length occludin expressed in NRK cells will colocalize with ZO-1 at cell contacts even when the adjacent cell does not express occludin (Van Itallie, C., personal communication). Since endogenous occludin is not necessary for localization of connexin-occludin chimeras in NRK cells, we next tested the requirement for the ZO-binding domain. DOC colocalized with ZO-1 in NRK cells. However, POC , which lacks the ZO-binding domain, did not . Instead, it localized only at contacts between adjacent transfected cells and not between adjacent, nontransfected cells . This is a pattern identical to that seen with full-length cx32. Thus, the region of occludin that binds ZO MAGUKs is sufficient to target connexin-occludin chimeras to cell contacts in cells that do not contain endogenous occludin or claudins. Further, these results suggest that occludin need not bind occludin on an adjacent cell (transcellular interactions) to enter the contact. We present evidence that the ZO-binding domain of occludin's cytoplasmic tail is sufficient to target connexin-occludin chimeras to the tight junction and organize them into the linear fibrils that create the paracellular barrier. In fibroblasts, which lack endogenous occludin and claudins, the ZO-binding domain is also sufficient to localize chimeras to ZO-1–containing cell contacts. Extrapolating these results to occludin suggests interactions with ZO proteins are important for organizing occludin in tight junction fibrils. This conclusion is different from, but not in conflict with, that of previous studies that focused on the intramembrane and extracellular regions of occludin, and concluded that the cytoplasmic tail plays no role in localization. We suggest further study of cytoplasmic scaffolding is important since these connections may ultimately be most responsible for initial fibril assembly and subsequent barrier regulation. We demonstrate using immunogold freeze-fracture labeling that connexin fused to the cytoplasmic tail of occludin intercalates directly into the occludin- and claudin-containing tight junction fibrils. This conclusion is further supported by the observation that tight junction fibrils in cells stably expressing CX/OC appear dotted, even after fixation with glutaraldehyde, with individual particles. This response to fixation is characteristic of connexin, but not of occludin . Although gap junctions have been reported near or in continuity with tight junctions, they are always confined to the regions around the fibrils and are not found in continuity with them. In addition, although cx43 is reported to bind ZO-1 in cardiac myocytes , cx32 is not known to form hemichannels with cx43. Moreover, MDCK cells do not contain cx43 . Further, our chimeric protein approach narrowed this targeting region to occludin's COOH-terminal 150–amino acids, designated the ZO-binding domain; DOC localizes at the tight junction in MDCK cells and at cell contacts in NRK fibroblasts. In contrast, chimeras that contain the membrane proximal region of the occludin cytoplasmic tail (POC) localize in MDCK cells exclusively to gap junctionlike plaques or to intracellular aggregates. In NRK fibroblasts POC localizes, as does full-length connexin 32, to plaques between neighboring transfected cells. Together, these data strongly suggest that the ability to bind ZO proteins is necessary and sufficient to localize connexin-occludin chimeras in tight junction fibrils and fibroblast cell contact sites. Previous studies have demonstrated the ability of occludin lacking the ZO-binding domain to target to tight junctions in cultured MDCK cells and Xenopus embryos . Balda et al. 1996 observed that occludin truncated after the fourth membrane-spanning region, and, thus, lacking the ZO-binding region, was able to localize to tight junctions. Similarly, Chen et al. 1997 reported tight junction localization in Xenopus embryos of an occludin construct missing almost the entire COOH-terminal cytoplasmic domain. These authors demonstrated the coimmunoprecipitation of endogenous and truncated transfected occludin, suggesting the possibility that tight junction targeting of truncated occludin resulted from an association with endogenous occludin through its transmembrane and/or extracellular regions . Interestingly, Furuse et al. 1998b recently demonstrated that claudin-1 and -2 can recruit occludin to fibrillike structures in L-cells; thus, interactions between transmembrane proteins are clearly important for occludin localization. Alternately, it is also possible that the transfected claudins recruited ZO proteins, which then functioned to localize occludin. Nonetheless, the apparent dispensability of the ZO-binding region in the localization of truncated occludin in these previous studies may be due to the ability of truncated occludin to self-oligomerize with full-length occludin or co-oligomerize with claudins, either of which are already interacting with plaque proteins. In contrast to our data, Matter and Balda 1998 recently demonstrated that the entire COOH-terminal tail of occludin targets Fc chimeras to the basolateral surface but is not sufficient to localize them at tight junctions. The chimeras reported by Matter and Balda 1998 and those used in this study differ in two respects: this study used the human occludin sequence, whereas Matter and Balda 1998 used chicken's, and the NH 2 -terminal region of the Fc protein used by Matter and Balda 1998 is a single span protein with no known ability to aggregate, whereas cx32 oligomerizes to form multisubunit connexons. As human and chicken occludin are only 35.3% identical across the entire COOH-terminal tail and 44.9% identical between ZO-binding domains, it is possible that species-specific sequence differences contributed to our contrary results. However, chimeras containing the entire COOH-terminal human occludin tail fused to the extracellular and membrane-spanning domains of glycophorin C, a single span transmembrane protein unable to oligomerize, localized basolaterally, not junctionally, in MDCK cells, and did not colocalize with ZO-1 in NRK cells (Mitic, L., E. Schneeberger, and J.M. Anderson, unpublished data). Similar results were obtained using the COOH-terminal 150–amino acids of human occludin fused to the amino half of glycophorin C. Thus, it is possible that retention of occludin chimeras at tight junctions and cell contacts requires an NH 2 terminus capable of aggregation. cx32 may be able to substitute functionally for occludin in this respect. Our results indicate that connexin can be aligned side-by-side with occludin into ordered, linear fibrils when it is fused to the ZO-binding region, thus, suggesting a scaffolding function for ZO-1, ZO-2, or ZO-3. The ZO proteins are members of the MAGUK protein family, many of whose members have already been shown to function as cytoplasmic scaffolds in organizing membrane proteins into specialized membrane domains . Genetic evidence from invertebrates supports this idea. For example, in Caenorhabditis elegans loss of LIN-2, a MAGUK, results in membrane mislocalization of the LET-23 receptor tyrosine kinase, a subsequent lack of receptor activation during development and a vulvaless phenotype . In Drosophila , the Discs-large MAGUK binds Shaker-type K + channels at neuromuscular junctions . Mutations in Discs-large are associated with a loss of Shaker-type K + channels from the neuromuscular junctions and their redistribution over the plasma membrane . Likewise, a vertebrate MAGUK called postsynaptic density protein-95 kD (PSD-95) has also been demonstrated in vertebrate synapses to bind and cluster both the N -methyl- d -aspartate receptor and the Shaker-type K + channel . Thus, the precedent for MAGUK involvement in organizing membrane proteins at specialized surface domains is well established. MAGUK protein family members are characterized by a multidomain organization that includes one or three PSD-95/ Discs-Large/ZO-1(PDZ) domains . PDZ domains are 80–100-amino acid protein binding cassettes that recognize a three residue peptide motif in the COOH termini of their binding partners . MAGUK proteins also serve as linkers between integral membrane proteins and cytoskeletal networks. For example, p55, a MAGUK protein that contains one PDZ, anchors the transmembrane protein glycophorin C and also binds protein 4.1 . Similarly, the human homologue of the C . elegans protein Lin-2, a MAGUK protein, likely ties the membrane protein syndecan-2 to the cytoskeleton via the actin-binding protein 4.1. Recently, ZO-1 has been reported to bind directly to actin in fibroblasts via its proline-rich COOH terminus . Likewise, ZO-1 itself appears to directly link occludin to actin , suggesting that it may mediate cytoskeletally induced changes in occludin's sealing properties. Of note, members of the claudin family contain PDZ-binding motifs at their COOH termini. Our results together with previously published data suggest the possibility of multiple steps for the assembly of occludin into fibrils. Free occludin in the lateral plasma membrane concentrates at the tight junction via contacts with a preformed, cytoplasmic ZO-containing scaffold. Concomitant self-aggregation, or oligomerization with other transmembrane proteins such as claudins, is necessary to assemble and/or maintain occludin in linear fibrils. Incorporation into a fibril could be regulated at either of these two steps: binding to ZOs or oligomerization. This idea is consistent with the recent observation of a second pool of occludin that is present on the lateral cell surface and not organized into fibrils . The junctional pool of occludin is hyperphosphorylated relative to the nonfibrillar pool, suggesting phosphorylation might be one mechanism for regulating assembly . Lending further support for this model, ZO-1 is localized at cell–cell contacts in the absence of occludin, such as in the NRK fibroblasts used in this study, but no examples of occludin localization in the absence of ZO-1 are known. Several published experimental observations suggest cadherin is the initial organizer of the tight junction plaque. First, extracellular antibodies against cadherin inhibit formation of tight junctions . Second, the tight junction and the adherens junction may be physically linked, particularly during formation of the tight junction, via ZO-1, which binds the cadherin-associated proteins α- and β-catenin . Moreover, cells containing a mutant form of αE-catenin that is unable to bind vinculin do not localize ZO-1 at tight junctions or form continuous fibrils . Lastly, ZO-1 assembles at cell–cell contacts before occludin and coincident with E-cadherin in studies of tight junction formation in the early mouse embryo . These observations, together with those reported here, suggest that occludin is recruited into a preformed ZO-containing structure whose assembly is initiated at the adherens junction. We have demonstrated that the ZO-binding domain is sufficient for localization in fibrils. Such a model implies that regulation of the tight junction barrier may occur through reversible recruitment of occludin into fibrils organized by a ZO-containing scaffold. The occludin–ZO interactions and the overall organization of the cytoplasmic plaque will be important subjects of future inquiry into the assembly and regulation of tight junctions. | Study | biomedical | en | 0.999999 |
10447409 | Cheng et al. 1993 was the first to propose that the spontaneous Ca 2+ spark is the elementary intracellular Ca 2+ release unit that underlies excitation–contraction coupling in cardiac muscle. They estimated that the local Ca 2+ flux underlying the Ca 2+ spark would need to be ∼2 × 10 −17 mol/s, assuming a volume of ∼10 fl (i.e., an ∼2-μm cube), duration of 10 ms (time to peak), and a final [Ca 2+ ] of ∼300 nM \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{resting}}\;[{\mathrm{Ca}}^{2}+]\;=\;100\;{\mathrm{nM}})\end{equation*}\end{document} . This type of calculation predicts that the underlying unitary RyR channel Ca 2+ current would need to be 1–4 pA to generate the observed Ca 2+ spark . An early estimate of the unitary Ca 2+ current through the cardiac RyR channel was 2.5 pA . This lead Cheng et al. 1993 to propose that the Ca 2+ spark may arise from the opening of a single RyR Ca 2+ release channel. The RyR channel, however, is a poorly selective Ca 2+ channel, and thus other ions (e.g., K + and Mg 2+ ) are likely to compete with Ca 2+ for occupancy of the pore. Consequently, the unitary Ca 2+ current must be smaller under more physiological conditions (1 mM lumenal Ca 2+ , 150 mM K + , and 1 mM Mg 2+ ). Tinker et al. 1993 used a RyR permeation model to estimate that the unitary Ca 2+ current was 1.4 pA (at 0 mV, 1.2 mM lumenal Ca 2+ charge carrier in symmetrical 120 mM K + and 0.5 mM Mg 2+ ). This updated estimate lead Blatter et al. 1997 to propose that simultaneous opening of two RyR channels may generate the Ca 2+ spark. If Ca 2+ sparks arise from the opening of one or two RyR channels, then certain pharmacological manipulations that alter single channel properties should be reflected at the Ca 2+ spark level. Cheng et al. 1993 reported that lower amplitude, long duration Ca 2+ sparks occur in the presence of ryanodine. This resembles the ryanodine-induced long-lasting subconductance states observed at the single channel level. Shtifman et al. 1999 reported that prolonged small-amplitude Ca 2+ sparks occurred after application of Imperatoxin A (IpTx A ). This resembles the prolonged subconductance of IpTx A -modified RyR channels in bilayers . In summary, the single channel Ca 2+ spark interpretation is largely based on two lines of evidence: first, the relatively large estimates of unitary RyR channel Ca 2+ current and, second, the parallel pharmacological actions at the spark and single channel levels. The hypothesis that multiple RyR channels open simultaneously to generate the Ca 2+ spark is consistent with the clustered arrangement of RyR channels in heart . It is also consistent with the stereotypic amplitude of the Ca 2+ spark. If Ca 2+ sparks were generated by spontaneous openings of a single channel, then the distribution of Ca 2+ spark amplitudes should be exponential in nature because single channel open times are distributed exponentially. Observed Ca 2+ spark amplitudes, however, are normally distributed. There is also a curious lack of small Ca 2+ spark events that is not easy to reconcile with the single channel spark hypothesis. Recently, Mejía-Alvarez et al. 1999 have directly measured the amplitude of unitary Ca 2+ current through a single cardiac RyR channel under quasi-physiological ionic conditions. The unitary Ca 2+ current was considerably smaller than previously predicted . This suggests that Ca 2+ sparks may arise from 3 to 10 RyR channels opening simultaneously. In summary, the multichannel Ca 2+ spark interpretation is based on three lines of evidence: first, the stereotypic nature of the Ca 2+ spark; second, new smaller estimates of unitary RyR channel Ca 2+ current; and, third, tantalizing correlations with the clear physical clustering of RyR in heart. A published kinetic Markovian scheme of RyR channel gating was used to generate simulated single RyR channel records. The simulated gating reflects single RyR channel measurements made in planar lipid bilayer studies . The unitary Ca 2+ current was fixed at 0.35 pA . To predict free [Ca 2+ ] fluctuations, a multicompartment unidimensional diffusion model was evaluated . The diffusion model includes Ca 2 + binding/unbinding to known buffers and SR Ca 2+ reuptake. The only entity allowed to diffuse is the Ca 2+ ion. The predicted fluorescence (Fluo-3) signals due to the local Ca 2+ fluxes produced by the simulated single RyR channel activity were calculated and are presented in Fig. 1 . At a steady state Ca 2+ concentration of pCa 7, the applied RyR gating scheme predicts that spontaneous single channel events occur at low open probability ( P o ). (The gating scheme does not consider other regulatory factors [e.g., Mg 2+ ] that may impact the stationary P o of the channel.) Most single channel open events are brief and bursts of open events are rare . Every RyR channel opening elevates the local Ca 2+ concentration. However, nearly all local Ca 2+ elevations would not be detected as Fluo-3 fluorescence signals. The largest local Ca 2+ elevations induced by bursts of RyR openings are just barely detectable at the fluorescence level. The same RyR gating scheme was also used to predict the response of five RyR channels to a trigger Ca 2+ pulse (10 μM for 500 μs). The trigger Ca 2+ pulse was applied to synchronize the opening of the RyR channels. Simultaneous opening of multiple RyR channels elevates the local Ca 2+ concentration to levels consistent with that predicted to underlie the Ca 2+ spark . These local Ca 2+ concentrations generate Fluo-3 fluorescence signals reminiscent of the experimentally observed Ca 2+ spark. Our simulations suggest that individual openings of a single RyR channel under steady state conditions at a resting Ca 2+ level are unlikely to generate detectable local Ca 2+ release events. Barely detectable Ca 2+ release events occasionally occur when bursts of open events (lasting many milliseconds) occur. This implies that an abnormally long opening of a single RyR channel would generate a prolonged detectable local Ca 2+ release. Simultaneous opening of multiple RyR channels generated fluorescence signals that were consistent with the observed Ca 2+ spark waveform. We propose that the stereotypical Ca 2+ sparks are generated by the simultaneous opening of multiple RyR channels. This proposition is consistent with our recent estimates of unitary Ca 2+ current, the stereotypical nature of the spark, and the clustering of RyR channels in the diadic space. We also propose that pharmacological manipulations that generate small-prolonged local Ca 2+ fluxes could arise from the opening of single RyR channels. | Study | biomedical | en | 0.999997 |
10447410 | 6 yr and volumes of new data have provided further insight into RyR adaptation. We believe RyR adaptation should be viewed as a physiologically important phenomenon and not as a molecular mechanism. There is now substantial evidence that the adaptation phenomenon is due to a transient, Ca 2+ -dependent shift in the modal gating behavior of the RyR channel . Fast trigger Ca 2+ stimuli drive the channel into a high open probability ( P o ) mode. If the trigger Ca 2+ stimulus is sustained (even at a lower level), RyR activity spontaneously decreases as a new steady state between high and low P o modes is reached. A second rapid elevation of [Ca 2+ ] disrupts the equilibrium again, causing another transient increase in activity. Repeated activations can occur only within a certain range of [Ca 2+ ] because the Ca 2+ binding sites that govern the equilibrium between the high and low P o modes can saturate. It is reasonable to assume that Ca 2+ binding to the same sites that govern the RyR's steady state Ca 2+ dependence may be responsible for this phenomenon . Thus, the activity of single RyR channels may represent a dynamic Ca 2+ -dependent balance between the time spent in high, low, and zero activity modes. This balance would be governed by multiple Ca 2+ binding sites with different affinities and kinetics . The RyR adaptation phenomenon is observed when the channel is activated by a free Ca 2+ waveform generated by laser flash photolysis of DM-nitrophen . The Ca 2+ waveform has a complex time course composed of a fast Ca 2+ step (0.1 to 1.0 μM), with a very fast (∼150 μs), large (∼100 μM) Ca 2+ overshoot at its leading edge . The impact of the fast Ca 2+ spike on data interpretation has been debated . Direct measurement of RyR response to fast Ca 2+ spikes alone showed that channel deactivation after these brief Ca 2+ changes was ∼1,000× faster than the observed adaptation phenomenon . Fast Ca 2+ spikes alone trigger only a single open event, while adaptation is characterized by a prolonged transient burst of channel activity. Additionally, repetitive transient bursts of channel activity can be induced only over a relatively narrow Ca 2+ concentration range, and this Ca 2+ concentration range is defined by the sustained Ca 2+ step, not the properties of the fast Ca 2+ spike . Therefore, it is highly unlikely that the adaptation phenomenon is due to simple deactivation after the fast Ca 2+ spike, or that it artifactually induced the fast Ca 2+ spike. Instead, the impact of the Ca 2+ spike appears to be limited to “super charging” the trigger Ca 2+ signal in that it may accelerate the transition into the high P o mode. When true step Ca 2+ stimuli (without fast Ca 2+ spikes) are composed of single RyR channels in planar bilayers, these step-like Ca 2+ stimuli trigger bursts of RyR channel activity that spontaneously decay over time . In some studies, the spontaneous decay in channel activity was not always observed . In other studies, the spontaneous decay occurred only if the channel was initially in a high activity state . This decay has been interpreted as a conventional “inactivation” mechanism. An alternative interpretation is that smaller, slower Ca 2+ stimuli are simply less efficient at triggering the initial high activity burst. In this latter view, the spontaneous decay is due to a time- and Ca 2+ -dependent shift in the channel's modal gating behavior. Thus, the apparent “inactivation” here and the apparent “adaptation” described above are actually two manifestations of the same underlying mechanism (i.e., modal gating). Fabiato 1985 proposed that Ca 2+ -dependent inactivation is the negative control mechanism that regulates the SR Ca 2+ release process in heart. However, early patch clamp studies of intact ventricular myocytes found no evidence of inactivation (i.e., refractory behavior) of cell-averaged SR Ca 2+ release in experiments using conventional two-pulse protocols . Subsequent studies have shown that SR Ca 2+ release does indeed “turn-off” when activated by a sustained trigger Ca 2+ stimulus . Paradoxically, the apparently inactivated Ca 2+ release process could be reactivated by the suddenly increased trigger Ca 2+ stimulus carried by the tail current upon repolarization. This ability of incremental macroscopic trigger Ca 2+ stimuli to trigger multiple transient SR Ca 2+ releases qualitatively resembles the adaptation phenomenon observed at the single RyR channel level described above. Thus, it was proposed that this reactivation of SR Ca 2+ release is a whole cell manifestation of the RyR adaptation phenomenon . A recent study using confocal Ca 2+ imaging, however, suggests the situation is more complicated and may involve both complex single channel behavior and multichannel interactions . Defining the mechanisms that terminate elementary SR Ca 2+ release events (i.e., Ca 2+ sparks) is an important step towards understanding how release is regulated. The candidate negative control mechanisms include: (a) Ca 2+ -dependent inactivation, (b) adaptation, and (c) use-dependent inactivation . These mechanisms have been viewed as potentially independent and mutually exclusive RyR regulatory entities. This view, however, may not be accurate. Modal RyR gating behavior may provide a framework in which these apparently different mechanisms can be integrated. The hallmark of the adaptation phenomenon is thought to be the ability of apparently “refractory” RyR channels to reactivate in response to a larger Ca 2+ stimulus. This, however, is not likely to be relevant to regulation of CICR in situ, as even small trigger Ca 2+ stimuli in situ may elevate the local free Ca 2+ concentration in the diadic cleft to very high levels representing maximal activating stimuli for the local RyRs. These high Ca 2+ levels should result in maximal occupation of Ca 2+ binding sites that govern the equilibrium between the high and low P o modes, and thus the reactivation by even larger Ca 2+ stimuli would not occur. Perhaps the more physiologically relevant feature of adaptation is the underlying modal gating shift. In the presence of a sustained trigger Ca 2+ signal, a time- and Ca 2+ -dependent shift to the low- and zero- P o mode would cause a decline in channel activity. The implication is that the decreasing RyR channel activity would always appear as a consequence of earlier channel activation. Thus (provided it is sufficiently fast) the shift in gating modes could account for apparent use-dependent properties of Ca 2+ release inactivation in situ . Many vesicle Ca 2+ flux studies and single RyR channel studies demonstrate an inhibition of RyR activity at high (>50 μM) steady [Ca 2+ ]. Traditionally, inactivation by Ca 2+ is thought to be mediated by a Ca 2+ -dependent transition to an absorbing inactivated state . The modal nature of RyR channel behavior suggests an alternative Ca 2+ -dependent mechanism in which channel activity is decreased by stabilizing low- or zero- P o modes. Binding of Ca 2+ to the low affinity inhibition sites could accelerate the rate of shift in gating modes, bringing it to a more physiologically relevant range. Intuitively, this may be analogous to the modal mechanism of Ca 2+ -dependent inactivation proposed for the L-type Ca 2+ channels . In this sense, adaptation and Ca 2+ -dependent inactivation may represent the different aspects of a common underlying mechanism; i.e., time- and Ca-dependent shifts in modal gating. Thus, we propose that the modal RyR gating behavior may represent a common factor that underlies the apparently different mechanisms of Ca 2+ -dependent inactivation, use-dependent inactivation, and adaptation. The implication is that the negative control mechanisms that counter the inherent positive feedback of CICR may be a time- and Ca 2+ -dependent shift in the modal gating behavior of the RyR channel. The intent of our proposition is to simply stimulate discussion. It is clear that additional experimentation and a far more detailed theoretical framework is required to understand termination of the SR Ca 2+ release process in heart. Nevertheless, even a relatively speculative exchange of scientific ideas can generate new and interesting ideas and future directions. | Study | biomedical | en | 0.999997 |
10449515 | For the antineoplastic effects of NSAIDs to be exerted through COX inhibition, COX and PGs must contribute substantially to colorectal carcinogenesis. Evidence that eicosanoids and COX isozymes are important in colorectal cancer development includes: (a) PGE 2 levels are elevated in colorectal tumors; (b) eicosanoids, including PGs, stimulate proliferation and reduce apoptosis in colonocytes; (c) COX-2 is upregulated in colorectal tumors, more frequently in cancers than adenomas; and (d) COX-2 contributes to colorectal tumorigenesis in APC knockout mice 9 where APC Δ716(+/−) COX-2 (−/−) mice develop fewer intestinal tumors than do APC Δ716(+/−) COX-2 (− / +) mice, which in turn bear fewer tumors than do APC Δ716(+/−) COX-2 (+/+) mice (for review see reference 3). Aside from producing several eicosanoids, COX may promote carcinogenesis by activating carcinogens via its peroxidase activity, which can operate on substrates other than PGG 2 ; or by producing either malondialdehyde, a direct-acting mutagen, or peroxyl radicals . Angiogenesis, which is important in carcinogenesis, is related to PGs and COX. PGE 1 and COX-2 induce angiogenesis 10 and NSAIDs inhibit it 11 . Clearly, inhibition of angiogenesis could explain the ability of NSAIDs to regress adenomas in familial adenomatous polyposis (FAP) patients and to inhibit colorectal adenocarcinoma formation. However, like most if not all of the mechanisms discussed here, it has yet to be formally demonstrated in vivo that angiogenesis mediates the chemopreventive effects of NSAIDs in colorectal cancer. It is noteworthy that sulindac sulfone also inhibits angiogenesis 12 . This raises the possibility of PG- or COX-independent mechanisms of angiogenesis as well. Whether COX or eicosanoids are necessary or essential for colorectal carcinogenesis has not been fully assessed. Clearly, some colorectal cancers develop without overexpressing COX or producing high levels of PGs. However, this still does not rule out a central role for COX enzymes, at least for most colorectal cancers. A potentially important means by which NSAIDs prevent colorectal neoplasia is to affect cell turnover in the colorectal epithelium. Cell death and renewal are critical for the regulation of the structural integrity of all tissues. The growth rate of a tissue or a tumor is determined by the rate of proliferation and counterbalanced by the rates of cell loss by apoptosis or necrosis of the cells that comprise them. We and others have shown that several NSAIDs, including SA, ASA, sulindac (and its metabolites sulindac sulfide and sulindac sulfone), indomethacin, piroxicam, naproxen, as well as selective COX-2 inhibitors, retard the proliferation and induce apoptosis in colon cancer cells 3 13 . Human studies assessing the effects of NSAIDs on colonocytic proliferation have generated conflicting results, with sulindac reported either to show no effect upon proliferation or to reduce it 3 . Apoptosis is suppressed in sporadic colorectal adenomas and carcinomas and in the flat mucosa or adenomas of patients with FAP. In FAP patients, sulindac normalizes apoptosis in normal rectal mucosal colonocytes while reducing the size and number of their adenomas (for review see reference 3). Thus, it is possible, but not yet fully substantiated, that cell kinetic effects play a major role in the antineoplastic effects of these compounds. As we have shown previously, and as is now described in additional detail by Zhang et al., these events in vitro are clearly not dependent upon the expression of COX isozymes 5 8 . NSAIDs inhibit cell proliferation by inducing cell cycle quiescence in colonocytes, in part by reducing the levels of several key molecules that catalyze transitions through the various phases of the cell division cycle 3 6 7 . However, the detailed molecular pathways that induce quiescence have yet to be fully elucidated. Several groups contend that they have identified mechanisms by which NSAIDs induce PG- or COX-independent apoptosis. Preliminary reports claim that sulindac sulfone induces this form of cell death by inhibiting cGMP-dependent phosphodiesterase 5 14 . Others have shown that NSAID treatment of colon cancer cells generates the proapoptotic lipid, ceramide 15 . By blocking the biosynthesis of prostanoids, NSAIDs increase the intracellular levels of arachidonic acid, which activates neutral sphingomyelinase, which in turn converts sphingomyelin to ceramide. Strictly speaking, this is a PG-independent mechanism, but, as presented, this model of ceramide formation is dependent upon COX inhibition and therefore may not explain the apoptosis induced by sulindac sulfone or SA, nor that induced in COX null cells, like HCT-15 or those generated by Zhang et al. Aside from Zhang et al., other groups have also reported that NSAIDs inhibit cell transformation. Dong et al. showed that salicylates inhibited phorbol ester (TPA)-induced transformation of mouse epidermal JB6 cells 16 . Hermann et al. demonstrated that sulindac sulfide inhibited transformation of primary rat embryo fibroblasts by activated H-ras and SV40 T antigen or other transformation-inducing stimuli 17 . In both cases, transformation was inhibited at drug concentrations below those required to inhibit cell proliferation or cell viability. Dong et al. proposed that AP-1 activation is important for this effect of salicylates. This process appeared to be independent of PG inhibition, as indomethacin could not inhibit TPA-induced transformation, nor AP-1 activity. Mitogen-activated protein (MAP) kinase was shown not to be involved in this process. Hermann et al. proposed that sulindac sulfide inhibits oncogenic cell transformation by directly inhibiting Ras signaling. They showed that sulindac sulfide binds noncovalently to Ras and inhibits Ras-dependent Raf binding and Raf activation without affecting its GTPase or GTP binding activity. Apart from its role in cell transformation, Ras influences many pathways potentially important for the chemopreventive activity of NSAIDs, and thus it may be a pivotal target molecule integrating several disparate pathways influenced by NSAIDs. Inhibition of Ras signaling may also explain the effects of NSAIDs on proliferation and apoptosis at higher drug concentrations. Ras inhibition may link the effect of NSAIDs to NF-κB and MAP kinase activity (see below) 18 . Modulation of Ras activity by NSAIDs may also relate to the eicosanoid pathway, as arachidonic acid and PGs regulate Ras regulatory proteins such as p120GAP and NF-1GAP 19 . Both ASA and nonsalicylate NSAIDs reduce microsatellite instability in colon cancer cell lines deficient in DNA mismatch repair (i.e., HCT-116, HCT-15, SW48, and LoVo cells) 20 . Mismatch repair–deficient cells with mutations in hMLH1, hMSH2, or hMSH6, but not with a hPMS2 mutation, die selectively by apoptosis in response to NSAID treatment. Given that HCT-15 cells were used in this study, which we found to be deficient in COX expression 8 , this effect is also likely to be COX independent. However, Rüschoff et al. 20 did not directly confirm the absence of COX isozymes or PG production in the cells they used. Patients with hereditary nonpolyposis colorectal cancer have germline mutations in DNA mismatch repair genes. Therefore, it was speculated that NSAIDs could prevent colon cancer in most of these patients. However, the clinical efficacy of NSAIDs in this particular high-risk group remains unknown. SA activates p38 MAP kinase and induces apoptosis in FS-4 fibroblasts, and both are inhibited by the p38 MAP kinase inhibitor SB-203580. p38MAP kinase activation may also be important for inhibition of nuclear factor (NF)-κB by SA 21 . ASA and SA, but not indomethacin, inhibit NF-κB activation 21 22 . There is evidence that these compounds bind to and inactivate IκB kinase β, which in turn prevents the degradation of IκB and the subsequent translocation of NF-κB to the nucleus, where it activates the transcription of a variety of genes 22 . Depending on the cell type and the circumstances, NF-κB augments or inhibits apoptosis. Inhibition of the proliferation of ras -transformed rat fibroblasts by ASA may also be related to inhibition of NF-κB activation 18 . To show that activation of MAP kinases or inhibition of NF-κB activation are COX-independent processes, Yin et al. demonstrated that each is influenced exclusively by salicylates, and not by indomethacin 22 . However, indomethacin and other nonsalicylate NSAIDs prevent colon neoplasias and regress adenomas in FAP patients. As a result, it is unlikely that these two processes, as delineated in these experiments, play a major role in the chemopreventive effects of NSAIDs. The observations that follow relate to processes that do not appear related to PGs or COX inhibition. However, since they have not been examined in detail in cells that lack COX enzymes or PG production, their relationship to COX and/or arachidonic acid metabolism remains uncertain. Lu et al. found that, in addition to apoptosis, Myc was markedly induced in serum-starved chicken embryo fibroblasts after activation of pp60 v-src and NSAID treatment. Apoptosis was markedly inhibited by transfection of antisense myc 23 . Similar results were found in other cancer cell lines. Peroxisomal proliferator-activated receptors (PPARs) are a group of nuclear hormone receptor protein transcription factors that, when stimulated, induce differentiation of fibroblasts to adipocytes. PPAR-γ receptors are expressed in the colon and to an even greater degree in colon tumors 24 . Activation of PPARs in colon cancer cells reduces their growth and induces differentiation in vitro 25 . Interestingly, indomethacin and selected, but not all, NSAIDs bind to and activate PPAR-γ receptors 26 . However, piroxicam, which regresses colorectal polyps in humans and animals, does not bind PPAR-γ as effectively as other NSAIDs 26 and PPAR-γ activators, such as troglitazone, increase colon tumors in APC Min mice 27 28 . These latter data suggest it is unlikely that PPAR activation is important for the chemoprevention of colorectal cancer by NSAIDs. There is ample indirect evidence for a role of NSAIDs, and by extension of COX enzymes, in immune phenomena related to various cancers. A clearly studied case concerns their role in the expression of HLAs, which is altered in many cancers and frequently downregulated in colon cancer 3 . Such abnormalities may adversely affect the clinical course of cancer and the outcome of T cell–based immunotherapy. NSAIDs may boost mechanisms of tumor immune surveillance; tumors are hypothesized to escape from immune-mediated destruction by thwarting mechanisms that detect tumor-associated antigens. Class I and II HLA antigens, participating in antigen presentation, may be critical to this process. The role of NSAIDs in these processes has been assessed in animal models of colon cancer and in cell culture systems. Piroxicam upregulates the expression of MHC genes in the colonic mucosa of rats treated with a carcinogen 3 . PGE 2 reduces the transcription of HLA class II molecules and NSAIDs can increase it 29 . That such effects occur in the presence of PG-rich serum in cell culture systems indicates, along with additional evidence (Rigas, B., unpublished observations), that this is probably another COX-independent effect of NSAIDs. That NSAIDs can induce the expression of the suppressed HLA genes in colorectal neoplasias suggests that these versatile compounds may restore the ability of the immune system to eliminate transformed cells. As this brief overview demonstrates, multiple processes with their attendant molecular pathways have been proposed to mediate the chemopreventive effects of NSAIDs ( Table ). The NSAIDs, highly protein bound molecules, probably interact with and inhibit the function of many proteins and, perhaps, other macromolecules. To complicate matters, it is not clear whether the key in vitro effects of NSAIDs that were discussed earlier, such as cell transformation, cell growth, or angiogenesis, are at all relevant to colorectal cancer chemoprevention. Some of the data reviewed here indicate that COX inhibition by NSAIDs is indeed required for their chemopreventive effect. However, another body of data, including that of Zhang et al. in this issue 5 , make the equally strong case that COX inhibition is not required for certain presumed chemopreventive effects of NSAIDs. This apparent inconsistency is not merely of theoretical interest, but has important implications for the rational design of strategies for colon chemoprevention and for assessing the relative significance of each mechanism in carcinogenesis. In all likelihood this is not a contradiction at all; rather, NSAIDs bring about their chemopreventive effects in the colon through both COX-dependent and -independent mechanisms. Indeed, we have proposed a model that assumes both mechanisms operate to produce the clinical antineoplastic effects of NSAIDs, in which COX-dependent and -independent pathways modulate different stages of the multistep process of colon carcinogenesis or different events regulating each stage 30 . The multiplicity of action of NSAIDs, if confirmed, could in fact explain their high degree of effectiveness in colon cancer prevention in humans. The great challenge will be to determine which of these or other yet unknown mechanisms produce the remarkable anticancer effect of NSAIDs, as well as the relative contribution of each. If and when these key questions are worked out, then a great deal will have been learned about colorectal carcinogenesis. In addition, new tools will have been collected to identify new compounds that hold promise to prevent colorectal cancer more safely and effectively than the conventional NSAIDs. | Review | biomedical | en | 0.999996 |
10449516 | COX-1 +/− or COX-2 +/− pregnant heterozygous female mice 15 16 were killed by CO 2 asphyxiation on day 18 after fertilization. Each embryo was taken from the uterus, separated, minced, and trypsinized twice with 0.1% trypsin-EDTA-DMEM for 15 min. Tissue debris were discarded, and cells were pooled by brief centrifugation and then suspended in DMEM containing 10% fetal bovine serum. Cells from each of the embryos were grown in the same medium at 37°C with 5% CO 2 . PCR analysis was used to determine the genotypes of each embryo. Primers used are the following: for COX-1 genotyping, 5′-AGGAGATGGCTGCTGAGTTGG-3′ and 5′-AATCTGACTTTCTGAGTTGCC-3′ were used to detect the intact COX-1 exon 11; 5′-GCAGCCTCTGTTCCACATACAC-3′ and 5′-AATCTGACTTTCTGAGTTGCC-3′ were used to detect the targeted disruption of COX-1 exon 11 containing the neomycin gene. For COX-2 genotyping, primers 5′-ACACACTCTATCACTGGCAC-3′ and 5′-AGATTGTTGTCAGTATCTGCC-3′ were used to detect the endogenous COX-2 gene (the PCR product extending from exon 8 to exon 10); 5′-ACGCGTCACCTTAATATGCG-3′ and 5′-AGATTGTTGTCAGTATCTGCC-3′ were used to detect the targeted disruption of COX-2 exon 8 containing the neomycin gene. COX-1 −/− /COX-2 −/− deficient cell lines were developed by breeding male COX-1 −/− /COX-2 +/− mice and female COX-1 +/− /COX-2 +/− mice; cells from these animals were isolated by an identical protocol as that described above. For Western blot analysis, proteins were collected and dissolved in protein lysis buffers (10 mM Tris, pH 7.6, 1% Triton X-100, 100 mM NaCl, and 2 mM PMSF). 20 μg of protein was loaded and separated by 10% SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride membranes and blotted with polyclonal antibody specific to murine COX-1 (provided by Dr. William Smith, Michigan State University, East Lansing, MI) and mAb specific to COX-2 (Transduction Labs). Membranes were also probed with anti–β-actin antibody (Santa Cruz Biotechnology) to normalize sample differences between the gel lanes. Immunodetection was done using the Enhanced Chemiluminescence Western blotting detection system (ECL kits) purchased from Amersham Pharmacia Biotech. For Northern blot analysis, total RNAs were extracted from the cell with 4 M guanidium thiocyanate homogenization buffer (4 M guanidium thiocyanate, 0.5% sodium laurel sarcosinate, 1% β-mercaptoethanol in 100 mM Tris-HCl, pH 7.5). 20 μg of the total RNA was loaded and then electrophoresed on formaldehyde agarose gels. The separated RNAs were transferred to Duralon UV membrane and hybridized with COX-1– or COX-2–specific cDNA probes. These membranes were later probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to normalize the RNA loading of each sample. Primary embryonic fibroblast cells were seeded in duplicate in 12-well plates and grown for 24–48 h in DMEM containing 10% fetal bovine serum. Cells were treated with 100 ng/ml phorbol ester (12- o -tetradecanoylphorbol 13-acetate [TPA]) or vehicle for 10 h. PGE2 accumulations in the medium were measured using RIA kits purchased from Amersham Pharmacia Biotech. Cyclooxygenase activities were quantified by adding 30 μM free AA to the culture and measuring PGE2 production after 15 min had elapsed. The quantity of cells per well was counted using a hemacytometer. The experiments were conducted using primary cultured cells from several different cyclooxygenase-deficient embryos, and similar data were recorded. Early passage embryonic fibroblasts were plated in 100- or 60-mm dishes, and 10–20 μg of plasmids containing pEJ- ras and SV40 oncogenes were transfected at a 1:1 ratio into the cells by calcium-phosphate precipitation methods. The cells were allowed to grow for 2 d after transfection, then split in a 1:3 ratio into new tissue culture plates and cultured for an additional 2 wk. Plates were fixed with ethanol and subsequently stained with Giemsa dyes; foci formed on the plate were counted. Growing foci were also picked and allowed to grow to confluence to measure cyclooxygenase expressions. Cells expressing various levels of cyclooxygenase were plated at the same density in 0.33% soft agar with different NSAIDs: indomethacin (Sigma Chemical Co.), NS-398 (Cayman Chemicals), sulindac (Sigma Chemical Co.), ibuprofen (Sigma Chemical Co.), and piroxicam (Sigma Chemical Co.). The drugs were dissolved in DMSO, and identical volumes of top agar containing various concentrations of drugs were replenished at days 2 and 5. Cells were allowed to grow for 2 wk, and foci were counted. Duplicate plates were counted for each type of drug at each different concentration. DNA synthesis was estimated by [ 3 H]thymidine incorporation into cellular DNA. Cells in triplicate were plated in 24-well plates for 24 h and then treated with vehicle or a different NSAID for 24 h. [ 3 H]Thymidine (1 mCi/ml) was added and allowed to label the cells for 24 h. The cells were washed twice with cold DMEM, twice with 10% TCA, and three times with ethanol. The cells were then lysed in NaOH/SDS, and radioactivity was counted. Postconfluent cells were treated with each of the NSAIDs for 72 h. NSAID-treated and vehicle-treated cells were washed with PBS and then lysed in buffer containing 10 mM Tris-HCl (pH 8.0), 1% SDS, and proteinase K. Lysates were digested for 3 h at 55°C and then extracted with phenol/chloroform. The genomic DNA was precipitated in ethanol and resolved on a 1.5% agarose gel at 60 V for 4 h. Cells from each embryo were collected and grown as described in Materials and Methods, the genotype of each cell line was identified by PCR, and total RNA was used for Northern blot analyses. As anticipated , wild-type cells express both TPA-inducible COX-2 and constitutive COX-1 mRNA, whereas cells from COX-1 −/− or COX-2 −/− embryos each lack expression of their respective wild-type COX genes. Cells from COX-1 −/− /COX-2 −/− deficient embryos lack expression of both COX-1 and COX-2 transcripts. A small amount of abnormal COX-2 transcript is detected by Northern blot analysis in COX-2 −/− cells; however, Western blot analyses and measurements of PGE2 production confirm the disruption of the cyclooxygenase genes. Reports have shown that COX-1 and COX-2 isozymes exhibit distinctive selectivity over a different source of arachidonic acid (AA). COX-2 is more efficient at using mitogen-activated endogenous AA, whereas COX-1 is more efficient at using exogenously added AA, or AA released by stimuli such as calcium ionophore 17 18 . Therefore, we investigated the production of PGE2 from these cells by measuring metabolism of either exogenous AA or endogenous AA. In the presence of 30 μM of added AA , both COX-1– and COX-2–deficient cells produced less PGE2 than wild-type cells. TPA stimulation doubled the production of PGE2 in both wild-type and COX-2–containing cells. In the absence of added AA , when 8-h PGE2 accumulation was measured, stimulation by TPA was found to dramatically increase PGE2 production in both wild-type and COX-2–containing (COX-1 −/− ) cells, but the COX-2 −/− cells produced almost undetectable levels of PGE2 even with an active COX-1 enzyme. TPA-activated AA releases were also measured by labeling the cells with 3 H-AA, and all of the cells, regardless of the presence of the enzymes, demonstrated similar induction of AA release in response to TPA or calcium ionophore (data not shown). Also, as expected, the COX-1 −/− /COX-2 −/− cells do not produce detectable PGE2 either in the presence or the absence of AA or TPA. The failure to detect any PG production in the COX-1 −/− /COX-2 −/− cells confirms the inactivation of both genes. Previous studies by Kirtikara et al. 19 showed that COX-1 or COX-2 is increased in the null cells to compensate for the deficiency of PG production. Our data support this finding by showing that in the COX-1 −/− cells, PGE2 production was increased both at the basal and the stimulated level . However, we failed to detect any increased level of PGE2 production in the COX-2 −/− cells compared with the wild-type cells. To examine the role of cyclooxygenases in neoplastic transformation, we conducted transformation assays in primary cultured embryonic cells. The oncogene ras was used in this study because its expression contributes to many malignancies, and several studies, including our unpublished data, have demonstrated that transformation by ras induces COX-2 gene expression 20 . The expression of a mutant ras gene usually does not by itself lead to a transformed phenotype in primary cultured cells; additional mutations in other protooncogenes such as c- myc , or inactivation of tumor suppressor genes such as p53, are required to achieve a full malignant transformation 21 . Thus, we evaluated the contribution of the cyclooxygenases to the transforming activity of activated EJ- ras by adding wild-type SV40 viral gene to complete the transformation. As shown in Table , transfection of EJ- ras alone did not induce any foci formation, and transfection of SV40 alone induced only a few small foci. These foci from SV40 transformation failed to grow in the 0.33% soft agar within 2 wk. In contrast, SV40 plus EJ- ras cooperatively led to dramatically increased foci formation, and these transformed cells developed a fully transformed phenotype, as shown by their loss of contact inhibition and their rapid growth in soft agar and nude mice. By comparing the wild-type cells and the cells deficient in either or both of the cyclooxygenases, our results demonstrate that cells lacking either or both of the cyclooxygenases can be readily transformed by SV40 plus ras at almost equal frequencies. Both cyclooxygenase-null cells and wild-type cyclooxygenase-containing cells showed similar morphology and growth behavior in soft agar. This result indicates that cyclooxygenases are not required for transformation per se, nor specifically for the operation of the ras -dependent transformation pathway. Expression of the cyclooxygenase isozymes in transformed embryonic fibroblasts was analyzed using Western blots. As shown in Fig. 3 a, six of the seven transformed clones showed moderate or marked increases of COX-2 expression, and three of them had increases in the expression of both enzymes. However, one of the clones showed unchanged expression of both cyclooxygenases, again suggesting that fibroblasts can be transformed without altering the cyclooxygenase pathway. Interestingly, we found that the embryo fibroblasts immortalized only by SV40 expressed less cyclooxygenase, both COX-1 and COX-2, than the untransformed cells . Further studies confirmed that the corresponding cyclooxygenase product, PGE2, was dramatically reduced in the cells that were transformed by SV40 alone (data not shown). Isoenzyme expression was also measured in cells lacking either cyclooxygenase . In the cells lacking COX-1, transformation by EJ- ras plus SV40 led to an increased basal expression of COX-2 , and in cells lacking COX-2, the expression of COX-1 was also markedly increased . The results from the Western blot analyses are consistent with our Northern blot analyses, further confirming the genotype of each cell line we used in these studies. Taken together, these experiments illustrate that both cyclooxygenases can be selectively altered by oncogene ras or viral gene SV40; these actions are oncogene specific and might involve the alterations of signal transduction pathways that lead to the expression of both cyclooxygenases. However, despite the changes in cyclooxygenase gene expression, transformation does occur in the absence of both cyclooxygenases. Clinical and experimental evidence strongly suggests that NSAIDs are anticarcinogenic, antiproliferative, and antineoplastic. Although some evidence suggests that the effects of NSAIDs are achieved via their known inhibitory effects on COX-2, recent data from several independent groups demonstrate that some of the actions may be COX independent. Cells in which either or both cyclooxygenase genes have been disrupted offer distinct advantages for evaluation of the antineoplastic activities of these drugs. For this purpose, we examined the actions of several NSAIDs on transformed cells with different deficiencies in the expression of the cyclooxygenase isozymes. The antineoplastic activities of the drugs were evaluated by their influence on colony formation in soft agar, [ 3 H]thymidine incorporation, and by DNA fragmentation analysis. NSAID dosages were varied from 1 to 200 μM, and these dosages are compatible with those used in several studies where antineoplastic effects were achieved 11 22 23 24 25 . The therapeutic plasma concentration for NSAID therapy can be achieved with up to 10 μM indomethacin, 20 μM sulindac, 20 μM piroxicam, or 300 μM ibuprofen 26 . However, it is reported that the local concentration of NSAIDs in certain organs such as intestines can be built up to an even higher level 27 . Therefore, the dosages we used in these studies include both the physiological dosages and the higher dosages that might be achievable in certain organs and tumor tissues in vivo. We evaluated NSAID actions by seeding the same number of cells in soft agar containing various concentrations of different NSAIDs. All of the NSAIDs used (NS-398, sulindac, indomethacin, piroxicam, and ibuprofen) reduced the size and the number of colonies derived from the cells containing either, neither, or both cyclooxygenase genes in a dosage-dependent manner, with the inhibition being statistically significant at the higher dosages . Several cell clones of each cyclooxygenase-deficient cell line were used in this study. Quantitative differences in colony formation were observed in the presence of various NSAIDs, but these differences were not correlated with the presence or the absence of cyclooxygenase expression, as seen in Fig. 4 . Thymidine incorporation was used to examine DNA synthesis of the cells in the presence of different NSAID doses. At the 20 and 100 μM doses of a COX-2–selective NSAID, NS-398, [ 3 H]thymidine incorporation of all four cell lines examined was reduced by 15–68% regardless of the presence or absence of the two isozymes . At these doses, where PGE2 production was reduced by 95% in the wild-type cells and the COX-1 −/− or COX-2 −/− cell lines, further addition of PGE2 up to 10 μM failed to restore DNA synthesis (data not shown). Finally, we examined the apoptotic responses of the various deficient cells upon treatment with the different NSAIDs. At higher doses (indomethacin 200 μM, sulindac 200 μM, piroxicam 200 μM, NS-398 100 μM, and ibuprofen 1 mM), these drugs potently induced apoptosis in postconfluent cells at 48–72 h . The cell death was induced in cells with neither COX-1 nor COX-2 expression. Based on these results, we conclude that the antiproliferative and antineoplastic actions of NSAIDs in these cells at these concentrations are independent of cyclooxygenase inhibition. The activating mutations of ras occur in ∼30% of all human tumors. In squamous carcinomas, these mutations appear at early or intermediate stages of neoplasia. In colon cancers, ras gene mutations were found in 50% of adenocarcinomas and larger adenomas 28 . To initiate studies on the involvement of the cyclooxygenases in transformation and malignancy, we developed cells that lack COX-1, COX-2, or both, and studied ras -induced malignant transformation. We found that transformation by SV40 alone downregulated the expressions of both cyclooxygenases. However, addition of the activated ras oncogene induced malignant transformation accompanied by an increased expression of either or both cyclooxygenase enzymes. Although most of the clones exhibit increased expression of either or both cyclooxygenases, some do not express either of the genes at increased levels. This result by itself suggests that the expression of either cyclooxygenase does not have a direct implication on mechanisms of transformation. Furthermore, our transformation experiments conducted in the cyclooxygenase-null cells provide still stronger evidence that transformation can occur in the absence of either of the cyclooxygenases, and that cyclooxygenase overexpression is not necessary to induce neoplastic transformation. Nevertheless, there is abundant evidence suggesting that high levels of cyclooxygenase activity do play a critical role in some malignancies. Recent reports show that forced overexpression of cyclooxygenase enzymes increases metastatic potential, invasiveness, and angiogenesis of tumor cells both in vitro and in vivo 29 30 31 . Moreover, carcinogenesis is a chronic, sequential, and progressive process that usually involves accumulation of mutations of protooncogenes and tumor suppressor genes in later steps. Mechanisms involving cyclooxygenases in mutagenesis have been suggested 32 33 . COX-2 overexpression also leads to resistance to apoptosis and increases in the cellular life-span 10 11 ; these phenotypic changes may result in the additional development of mutations leading to more malignant behavior. Elevated cyclooxygenase expression is also known to increase malondialdehyde generation, providing another mechanism to foster higher cellular mutation rates 34 . So while activation of ras by itself is not usually sufficient for malignancy, the elevations in cyclooxygenases induced by the activation of ras could well predispose the cells to mutations in p53 or other genes that participate in the later steps towards high-grade malignancy. It is also noteworthy that in addition to COX-2, COX-1 expression is often induced by the activated ras oncogene, suggesting that both cyclooxygenases may serve some overlapping roles in tumorigenesis. Further studies are required to clarify the functional differences and relative importance of the two isozymes in cell malignancy and in the different stages of carcinogenesis. NSAIDs such as sulindac and indomethacin have been shown to be beneficial for the prevention and in some cases for the treatment of certain cancers, although the underlying mechanisms are still unknown. Inhibition of cellular proliferation and/or induction of apoptosis are reported to be the mechanisms that cause the regression of tumors in vivo 35 . The antiproliferative and apoptotic effects of NSAIDs have been documented in a variety of cells and cell lines, including colon cancer cells, breast cancer cells, and fibroblasts 22 23 . Considerable recent data suggest that some of the NSAIDs actions may not be entirely explained by inhibition of cyclooxygenases. First, some NSAID-related compounds, which are not cyclooxygenase inhibitors, can also induce antineoplastic changes in both the cell cycle and cellular apoptotic responses 36 37 . Second, the antineoplastic or anticarcinogenic activity of NSAIDs may not coincide with the inhibition of PG production in some tumor cells and animals 38 . Finally, Hanif et al. 39 found that sulindac and piroxicam inhibit the growth of and induce apoptosis in HCT-15 cells, which were shown to produce neither COX-1, COX-2, nor PGs. Elder et al. 40 also showed that NS-398 induced apoptosis in S/KS cells, which lack COX-2 protein. In contrast, Murphy et al. 41 showed that the growth of the cells expressing COX-2 was not suppressed by COX-2–selective inhibitors. In this study, we further illustrate that a selective knockout of cyclooxygenase activity does not change the ability of NSAIDs to inhibit transformation or to induce apoptosis. These results confirm that inhibition of cyclooxygenase is not the only mechanism responsible for the anticarcinogenic and antineoplastic activity of NSAIDs. NSAID actions that are independent of cyclooxygenase have been reviewed by Abramson and Weissmann 42 . New evidence suggests that many of the traditional NSAIDs have targets other than cyclooxygenases. A recent report by Herrmann et al. 24 demonstrated that sulindac sulfide inhibits the ras signaling apparatus, potentially via an eicosanoid-independent pathway. Lehmann et al. 43 demonstrate that many NSAIDs are ligands of peroxisome proliferation–activated receptors (PPARs) α and γ. Chan et al. 25 demonstrated that the elevation of the PG precursor AA could be involved in the induction of apoptotic response by the NSAIDs (i.e., sulindac and indomethacin) through production of ceramide. However, due to the higher concentration of NSAIDs used in their study, it is not clear whether the induction of AA is a result of cyclooxygenase enzyme inhibition or direct production of AA through activation of phospholipases. NSAIDs are a large group of structurally diverse compounds; therefore, differences in the actions of individual NSAIDs are also expected. For example, aspirin inhibits the activation of the transcription factor NF-κB, whereas indomethacin does not 44 . Recently COX-2–specific inhibitors were demonstrated to have potent antiproliferative effects and to induce apoptosis in cultured cells 20 45 46 . Whether or not actions exerted by these drugs are mediated through inhibition of COX-2 needs additional investigation. We have also observed in our studies that there are differences between cell lines in their sensitivity to NSAIDs that are independent of cyclooxygenase expression . Thus, it seems likely that the differences in the sensitivity of tumor cell lines to NSAIDs as reported in the literature may also be independent of cyclooxygenase expression, particularly when NSAIDs are used at high concentrations. Finally, the effects of NSAIDs on apoptosis, cell growth, and DNA synthesis studied here are those often cited to account for the antitumor activity of this class of compounds, yet in accord with the results of others, our data show that NSAID inhibition of the cyclooxygenases does not account for the antiproliferative and apoptotic actions seen in transformed embryonic fibroblasts. Our results further demonstrate that although cellular transformation by ras and SV40 usually leads to a dramatic increase in the expression of COX-1 and COX-2 when present, neither these inductions nor the presence of either of the enzymes is required for neoplastic transformation per se. These results suggest that the involvement of cyclooxygenase expression in neoplastic growth may occur at steps beyond those involved in transformation. | Study | biomedical | en | 0.999998 |
10449517 | Tg mouse lines carrying either H or L chain gene for the anti-RBC mAb (4C8 mAb) have been established previously 18 . Double Tg (H×L) mice with H and L chain transgenes were obtained by mating H and L chain Tg mice. In this study we generated new lines of 4C8 mAb Tg mice that carried tandem joined H and L chain transgenes. To construct tandem joined transgenes, a 5.6-kb BamHI fragment of pMO-κ4C8 was first subcloned in the BamHI site of pSP73 Vector (Promega Corp.). Next, a 15.5-kb XhoI fragment of pMO-μ4C8 was subcloned between the SalI and XhoI sites. The pSP73 plasmid containing both H and L chain genes was designated as pSP73-κμ4C8. Tandem joined H and L chain Tg (H+L) mice were generated by injecting a 22.0-kb PvuI–XhoI fragment of pSP73-κμ4C8 . Heterozygous mice were mated to get homozygous mice in two representative lines, H+L5, with 4–12 copies, and H+L6, with 1–3 copies of the transgene. The presence of the transgenes and homozygosity of the Tg loci were screened by PCR and Southern blot analysis. Tg mice were maintained under conventional conditions in our animal facility. Mice were typed by PCR analysis of tail DNA with set of primers to the H and L chain genes. PCR was carried out for 30 cycles consisting of denaturation (for 1 min at 94°C), annealing (for 1 min at 55°C), and extension (for 2 min at 72°C). The primer pairs for PCR were as follows: 5′Hc, 5′-CTACGCATTTAGTAGTGACTGG-3′; J H 3, 5′-TGCAGAGACAGTGACCAGAG-3′; IgL5′-2, 5′-CTGCAAGTCCAGTCAGAGCC-3′; IgL3′-2, 5′-CAGCACCGAACGTGAGAAAG-3′. FITC- or Cy-Chrome ® –conjugated anti–mouse CD45R (B220), FITC- or PE-conjugated anti–mouse IgM a (Igh-6 a ), FITC- or PE-conjugated anti–mouse IgM b (Igh-6 b ), PE-conjugated anti–mouse CD11b (Mac-1 α chain), biotin-conjugated anti–mouse λ 1 and λ 2 L chain mAbs, and FITC- or Cy-Chrome ® –conjugated streptavidin (SA) were purchased from PharMingen. FITC- or biotin-conjugated F(ab′) 2 fragments of goat anti–mouse IgM were from ICN Pharmaceuticals (Cappel). PE-conjugated SA was from Dako. Anti-idiotype (Id) mAb (S54) against the transgene-encoded anti-RBC antibody (4C8) 18 was conjugated with N -hydroxysuccinimidobiotin (Pierce Chemical Co.) according to the directions provided by the manufacturer. Single cell suspension from bone marrow was prepared by flashing femur bones with a staining buffer (PBS containing 2% FCS and 0.05% sodium azide), gently pipetted, and washed with the staining buffer. Single cell suspensions from bone marrow, spleen, and peritoneal cavity were prepared with the staining buffer and pretreated with 10% heat-inactivated normal rat serum. After 15 min of incubation, FITC- and/or biotin-conjugated mAbs at appropriate dilutions in the staining buffer were added directly. After 30 min of incubation, PE-conjugated mAbs and/or FITC- or PE-conjugated SA was added. Before and after each step of incubation, cells were washed with the staining buffer. Stained cells were then applied to FACSCalibur ® (Becton Dickinson). After excluding dead cells by propidium iodide staining, cells present in the lymphocyte gate defined by forward and side light scatters were analyzed. To assess whether the levels of transgene expression are higher in homozygous than in heterozygous mice, we analyzed the bone marrow cells of H+L5 and H+L6 mice that carried tandem joined H and L chain transgenes cording for the 4C8 anti-RBC antibody . In H×L mice, B220 + IgM + bone marrow cells almost completely disappeared 18 . In contrast, both lines of H+L mice had significant numbers of B220 + IgM + cells in bone marrow . The majority of these cells were stained both with antiallotypic mAb for IgM a (Tg) and with antiidiotypic mAb (S54) for the anti-RBC antibody. We compared the fluorescence intensity of Id + bone marrow cells in homozygous mice to that in heterozygous mice . The mean fluorescence intensities (MFI) of Id + cells in heterozygous and homozygous H+L5 mice are 481.70 and 717.42, respectively. Similarly, the MFI of Id + cells in heterozygous and homozygous H+L6 mice are 997.76 and 1670.11, respectively. These results indicate that in both H+L5 and H+L6 lines the levels of transgene expression are clearly higher in homozygous than in heterozygous mice. We also examined surface Ig expression on the bone marrow cells of H3 Tg mice that had only H chain transgene. Fluorescence intensity of antiallotypic mAb for IgM a (Tg) of bone marrow cells was also stronger in homozygous (MFI = 908.77) than in heterozygous (MFI = 319.25) mice. In a parallel experiment, anti-IgM a mAb MFI of homozygous H+L5 and H+L6 were 173.07 and 250.72, respectively, indicating that homozygous H+L B cells had not saturated the capacity of surface Ig expression . Taken together, homozygosity of Tg Ig loci enhances the level of transgene expression on B cell surface. To test whether inhibition of endogenous H chain expression is stronger in homozygous than in heterozygous mice, we analyzed lymphocytes in bone marrow and spleen of H+L5, H+L6, and H3 mice by flow cytometry. We stained bone marrow and spleen cells with antiallotypic mAbs for IgM a (Tg) and IgM b (endogenous). In all heterozygous Tg mice, the numbers of IgM a+ IgM b+ cells (allelic inclusion) were small but significant, whereas these cells almost completely disappeared in all homozygous Tg mice. This conclusion is further confirmed by the finding that both bone marrow and spleen B cells with only endogenous H chains (IgM a− IgM b+ ) decreased in homozygous Tg mice as compared with heterozygous Tg mice . In all lines of Tg mice, total numbers of endogenous H chain–expressing (IgM b+ ) spleen cells were lower in homozygous than in heterozygous mice . These results indicate that inhibition of endogenous H chain expression (allelic exclusion) is stronger in homozygous than in heterozygous mice. Taken together, higher levels of Ig transgene expression appear to cause stronger inhibition of endogenous H chain expression, suggesting that a certain level of surface Ig expression is required for allelic exclusion of the H chain locus. Next, we asked whether homozygosity of Tg loci also reduces endogenous L chain expression. Since the allotypic specificity for the Tg Lκ chain is not known, we estimated the degree of endogenous L chain expression by difference in expression of Tg H chain (IgM a ) and Id (the combined epitope of the H and L chains of the 4C8 anti-RBC mAb) that is recognized by the S54 mAb. In the H+L5 line, two populations of IgM a+ cells were observed: one stained with both anti-IgM a and anti-Id mAbs diagonally, and the other stained more weakly with anti-Id mAb than with anti-IgM a mAb . As a control, IgM a+ cells from H3 mice did not contain the population stained with the S54 mAb . We presume that the cells that stained diagonally with both mAbs express the Tg H and L chains equally, and that the IgM a+ Id low cells express the Tg H chain with both endogenous and Tg L chains. In support of this assumption, IgM a+ bone marrow cells in heterozygous H+L6 mice, which consisted of almost exclusively IgM a+ Id + cells and only few IgM a+ Id low cells, contained <1.2% λ + cells, which represent a fraction of B cells that express endogenous L chains . In contrast, IgM a+ bone marrow cells in heterozygous H+L5 mice, which consisted of ∼28% IgM a+ Id + and ∼72% IgM a+ Id low cells, contained >5.5% λ + cells . Furthermore, IgM a+ spleen cells in heterozygous H+L5 mice, which consisted of ∼4% IgM a+ Id + and ∼96% IgM a+ Id low cells, contained >14% λ + cells . The total number of cells expressing endogenous L chains (IgM a+ Id low ) in bone marrow and spleen was less in homozygous than in heterozygous Tg lines, indicating that homozygosity of the Tg loci enhances the inhibition of endogenous L chain expression as compared with heterozygosity. H×L mice show almost complete deletion of autoreactive B cells from bone marrow and spleen, whereas a normal level of B-1 cells is found in the peritoneal cavity 18 . Bone marrow of both lines of H+L mice contained some autoreactive B cells (IgM a+ Id + ), which have B-2 cell phenotypes (CD5 − B220 high IgM low ), whereas autoreactive B cells in spleen almost completely disappeared in H+L mice . We examined whether homozygosity of the Ig transgene loci influences the size of the autoreactive B-1 cell compartment in the peritoneal cavities of H+L5 and H+L6 mice. Peritoneal cells of H+L mice had a limited number of B220 + Id + cells, almost all of which were IgM a+ . Almost all Id + cells in the peritoneal cavity were Mac1 + and therefore belong to the B-1 subset . Thus, IgM a+ Id + cells are most likely to be autoreactive B-1 cells . These B220 + Id + cells in the peritoneal cavity were much more abundant in homozygous H+L mice than in heterozygous H+L mice, indicating that the size of the autoreactive B-1 cell subset is larger in homozygous than in heterozygous H+L mice. The relative size of the B-1 cell compartment is directly proportional to the B-1 cell number because total cell numbers in the peritoneal cavity did not vary between homozygous and heterozygous Tg mice. These results suggest that a higher expression level of the autoantibody facilitates the increase in autoreactive B-1 cells in the peritoneal cavity. To test whether autoreactivity of Ig expressed on B cells is crucial to enlargement of the B-1 cell subset, we carried out similar studies on the peritoneal B cells in H3 Tg mice, the vast majority of which are not autoreactive due to the absence of the Tg L chain. The majority of peritoneal B cells expressed the Tg H chain (IgM a+ ), in agreement with analysis of bone marrow and spleen cells . Indeed, B cells expressing endogenous H chains were less in homozygous than in heterozygous H3 mice , probably because of more efficient allelic exclusion due to stronger expression of the transgene product on homozygous B cells . Nonetheless, the percentage of IgM + Mac1 + cells in the peritoneal cavity did not increase in homozygous H3 mice as compared with those in heterozygous H3 mice . These results indicate that increased surface Ig expression of non-auto-reactive B cells did not facilitate enlargement of the B-1 cell subset in the peritoneal cavity. Taken together, enhancement of autoreactive surface Ig expression appears to be crucial to increase the B-1 cell subset in the peritoneal cavity. In this study, we generated new lines of the anti-RBC antibody Tg mice that carry tandem joined H and L chain transgenes, and compared B cell phenotypes between heterozygous and homozygous Tg mice to evaluate quantitatively the effect of surface Ig level on allelic exclusion and B-1 cell development. Higher levels of surface Tg Ig expression resulted in (a) stronger clonal deletion of B-2 cells from bone marrow and spleen; (b) reduction of B cells expressing endogenous H or L chains; and (c) enlargement of the autoreactive B-1 cell subset in the peritoneal cavity. Several lines of evidence suggest that surface expression of the μ chain is critical for H chain allelic exclusion 2 4 5 6 . In addition, pre-BCR, which is the H chain paired with the surrogate L chain, is suggested to mediate H chain allelic exclusion through downregulation of recombination-activating genes 42 43 44 45 46 . Igα and Igβ, which associate with surface-expressed H chains, have also been shown to trigger the signals inducing allelic exclusion 47 48 49 . Taken together, expression of the H chain as a pre-BCR on the cell surface may induce signals mediated by Igα and Igβ, resulting in H chain allelic exclusion. In this study we have demonstrated that homozygosity of the transgene Ig loci increases surface Ig expression on bone marrow B cells and causes stronger allelic exclusion, as compared with heterozygosity. Since allelic exclusion is all or none in each B cell, the present results suggest that there is a threshold of the pre-BCR signal intensity that induces allelic exclusion. There are several other possibilities to explain our results. First, B cells with allelic inclusion may be negatively selected. However, Sonoda and Rajewsky 50 have shown that B cells with allelic inclusion can normally expand in the periphery using double Ig knock-in mice, indicating the absence of negative selection against allelic inclusion B cells. In addition, we have shown that endogenous only B cells (IgM a− IgM b+ ) are also reduced in homozygotes . Second, reduction of allelic inclusion B cells could be due to selective expansion of higher surface Ig-expressing cells by a self-antigen. However, the self-antigen (RBC) can kill self-reactive B cells 19 . In fact, Id + B cells of H+L mice decreased in homozygotes as compared with heterozygotes in spleen as well as in bone marrow . In addition, we have shown that non–self-reactive (H chain alone expressing) Tg (H3) B cells also show suppression of endogenous Ig expression . These results cannot be explained by positive selection of self-reactive Ig expressing B cells by the self-antigen. Third, excess Tg Ig expression on surface may inhibit detection of endogenous Ig expression. It is unlikely that endogenous and Tg Ig molecules compete for surface expression in H+L5 and H+L6 spleen cells. This is because the surface Ig levels of homozygous H3, H+L6, and H+L5 cells are 909, 251, and 173 (MFI using the anti-IgM a Ab), respectively , indicating that H+L5 and H+L6 B cells have not hit the ceiling of the surface Ig expression level. Sonoda and Rajewsky 50 have shown that there is no inhibitory mechanism for the H chain on the surface as long as it can associate with the L chain. It is inconceivable that the surface expression efficiency differs between H chains derived from the transgene and endogenous gene. The possibility that a lower detection efficiency by anti-IgM b Ab staining in the presence of large amounts of IgM a is also unlikely because the FACS ® profiles of H3 spleen cells expressing large amounts of IgM a clearly show the presence of IgM a IgM b double-positive cells even in homozygotes . Finally, the suppression efficiency of the endogenous locus may be increased in homozygotes simply because the frequency of silencing the transgene locus is reduced by doubling the number of the transgene locus in homozygotes. Assuming that this is the case, the efficiency of silencing of the transgene locus can be calculated from the endogenous H chain only cells shown in Fig. 2 A: H3, 0.06 / (0.06 + 0.11 + 6.50) = 0.009 in heterozygotes and 0.00144 = 0.01 / (0.01 + 0.03 + 6.9) in homozygotes. The value in homozygotes is ∼18 times of the expected value based on the simple statistics. Similar discrepancy was seen in the H+L5, H+L6, and H3 spleen cells . In addition, homozygosity alters the number of Tg Id + B cells in spleen and peritoneal cavity to the opposite direction. These considerations make the final possibility unlikely. We have shown that H+L Tg mice have B cells expressing endogenous L chains together with the Tg H chain. These cells, expressing endogenous L chains in H+L mice, may be generated either by incomplete allelic exclusion or by receptor editing because the B cells which express the transgenes are self-reactive 51 52 . Nemazee and colleagues 53 54 55 have suggested that interaction with autoantigens leads IgM low IgD − bone marrow cells to undergo receptor editing but IgM high IgD + cells to undergo rapid apoptosis. On the other hand, Rusconi et al. 16 generated B cell hybridomas from anti-trinitrophenol antibody (anti-non-self) Tg mice that carried tandem joined H and L chain transgenes and demonstrated that the B cell hybridomas secreting the Tg antibody expressed the Tg L chain at about one-tenth of the level of coexpressed endogenous L chains. Endogenous L chain expression in their Tg mice is probably due to incomplete allelic exclusion because these B cells are unlikely to receive BCR stimulation by self-antigens to trigger receptor editing. Although we cannot exclude the possibility that receptor editing is involved in the appearance of B cells with endogenous L chains in our Tg mice, we think it less likely because of the following reason. If the expression of endogenous L chain is due to receptor editing induced by stimulation with the self-antigen, the mechanism to reduce the number of B cells with endogenous L chains by enhanced expression of self-reactive Ig should be clonal deletion. However, when B cells expressed more self-reactive Ig on surface, B cells with both Tg and endogenous L chains (IgM a+ Id low ) are more efficiently reduced than Tg only B cells (IgM a Id + ), which are most likely eliminated by clonal deletion . This observation is somewhat opposed to the expected efficiency of clonal deletion by the self-antigen because stronger BCR signaling will be induced in IgM a Id + cells than IgM a Id low cells. Although it is still controversial whether B-1 cells belong to an ontogenetically different B cell lineage from conventional B cells 34 35 36 37 38 39 40 , B-1 and B-2 cells clearly constitute different subsets of B cells. In this study we have shown that the size of the Tg B-1 cell compartment is larger in homozygous than in heterozygous H+L mice . Since Tg B-1 cells in heterozygous and homozygous mice show the same antigen specificity, our results suggest that the level of surface Ig expression directly influences the size of the Tg B-1 cell compartment. Our findings are consistent with the previous observations that defects of BCR signaling cause reduction of the B1 cell 56 57 , and loss of a BCR inhibitory molecule, SHP-1, increases the B1 cell number 58 59 60 61 62 . It is important to note that increased levels of autoreactive BCR induced augmented clonal deletion of B-2 cells in bone marrow and spleen but expansion of B-1 cells in the peritoneal cavity . Increased expression of nonautoreactive Ig (H3) enhanced neither clonal deletion of B-2 cells nor expansion of B-1 cells. These results suggest that there are at least three levels of BCR signaling that regulate self-reactive B1 and B2 cell differentiation. At a lower level, self-reactive B-2 cells can be stimulated to induce receptor editing or to become anergic 53 54 55 63 . At an intermediate signaling level, B-2 cells are clonally deleted and B-0 40 41 and/or B-2 cells are induced to differentiate into B-1 cells, which migrate into the peritoneal cavity. At a strong signaling level, B-1 cells are also clonally deleted 19 . It is tempting to speculate that at least a sizable fraction of peritoneal B-1 cells originate and expand from autoreactive B cells that are stimulated by self-antigens to a level strong enough to be activated but weak enough to avoid apoptosis. | Study | biomedical | en | 0.999997 |
10449518 | The B1-8f 11 and glD42i 10 IgH insertion mice were generated in the laboratory in Cologne and in collaboration with D. Eilat's group at Hebrew University (Jerusalem, Israel), and have been described previously (references 10 and 11, as indicated behind each mouse strain). The generation of the V H 12f mice will be described elsewhere (Lam, K.-P., and K. Rajewsky, manuscript in preparation). The conventional Vκ4 transgenic mice 8 were obtained from Stephen Clarke (University of North Carolina, Chapel Hill, NC). Mice used were 2–4 mo old and maintained in a conventional animal facility. The following mAbs used in this study were produced and conjugated to fluorochromes in our laboratory: anti-B220 (RA3-6B2); anti-IgM (R33-24.12); anti-IgD (1.3-5); anti-CD43 (S7); anti-μ a (RS3.1); anti-μ b (MB86); anti–V H B1-8 (Ac146); and anti-V H 12 (5C5). The anti-CD5 and anti-CD23 mAbs were purchased from PharMingen. Tissues and cell preparations for flow cytometric analyses and cell sorting were prepared as previously described 12 . In brief, spleen cells were prepared by dissociation between frosted glass slides. Peritoneal cavity and bone marrow cells were obtained by injecting staining medium (PBS containing 3% FCS and 0.1% NaN 3 ) into the peritoneal cavity and femurs and tibia, respectively, using a 1-ml syringe with a 26-gauge needle. All cells were treated with RBC lysing solution (0.15 M NH 4 Cl, 1 mM KHCO 3 , and 0.1 mM Na 2 EDTA) to eliminate erythrocytes. For FACS ® analyses, cells were stained with optimal amounts of FITC-, PE-, and biotin-conjugated mAbs for 10 min on ice and washed three times with staining medium. Biotin-conjugated mAbs were revealed with streptavidin-Cychrome. Flow cytometry analyses were performed on a FACScan™ (Becton Dickinson) and cell sorting was done on a FACStar PLUS™ . Genomic DNA was prepared from mouse livers and sorted splenic B cells 13 , digested with BamHI and fractionated on a 1% agarose gel. After capillary transfer, the membrane was hybridized with a random-primed α-P 32 –labeled specific probe, as shown in Fig. 5 . We used gene targeting to generate a series of IgH insertion mice in which the J H locus was replaced by distinct V H D H J H segments 14 . These segments were taken from the 4-hydroxy-3-nitrophenyl acetyl–binding antibody, B1-8 9 ; the antibody glD42, which is a reduced affinity mutant of the DNA-binding antibody, D42 10 ; and the anti-PtC antibody, V H 12 8 . The corresponding mice were designated B1-8f, glD42i, and V H 12f respectively. The B1-8f 11 and glD42i 10 mice have been described previously, whereas the generation of the V H 12f mice will be described elsewhere (Lam, K.-P., and K. Rajewsky, manuscript in preparation). Flow cytometric characterizations of the B cell populations in the spleens of wild-type, glD42i, B1-8f, and V H 12f mice are depicted in Fig. 1 . The majority of the B cells present in glD42i and B1-8f mice are B-2 cells in that they express high levels of CD23 (shown for glD42i mice), IgD (shown for B1-8f mice), and B220, the pan-B cell marker. In addition, they do not express CD5, a marker found on T and B-1 cells. In contrast, V H 12f mice generate cells predominantly of the B-1 phenotype in that they express intermediate levels of CD5, low levels of B220 and IgD, and no detectable CD23. These data are consistent with a previous report that showed the preferential generation of B-1 cells in the lymphoid organs of conventional V H 12-transgenic mice 8 . Thus, different V H gene specificity seems to bias the generation and/or selection of different B cell subsets in the mouse. B cell development under the condition of H chain allelic inclusion had previously been analyzed in mice bearing V H B1-8 and V H glD42 15 . In these double IgH insertion mice, B cells expressing two functional V H alleles are readily generated in the bone marrow and are not counter-selected in the peripheral lymphoid organs. Here, we cross V H 12f mice with glD42i and B1-8f mice to examine the developmental potential of B cells bearing two distinct BCRs, one that is preferentially expressed in B-1 and the other in B-2 cells. Expression of the V H 12 and glD42 alleles in B cells can be identified by the expression of their constant regions as the former is of the a and the latter, of the b allotype. Flow cytometric analyses of the B cells in double V H 12f/glD42i mice revealed that the majority of the cells in the spleen , bone marrow, and lymph nodes (data not shown) of these mice coexpress both V H genes. Similar results were also obtained when V H 12f mice were crossed with B1-8f mice. Expression of V H 12 and V H B1-8 can be distinguished by staining with the anti-idiotype (Id) mAb 5C5 8 and Ac146 9 respectively. The 5C5 mAb recognizes V H 12 independent of the L chains 8 whereas the Ac146 mAb recognizes V H B1-8 in association with λ and the majority (∼80%) of the κ L chains 9 . As shown in Fig. 1 B, the majority of the splenic B cells in V H 12f/B1-8f mice coexpress both Ids, indicating that they are double-IgH expressors. This is also true for the B cells in the bone marrow and lymph nodes of these mice (data not shown). Taken together, these data indicate that V H 12-expressing B cells can coexpress another V H gene. Surprisingly, phenotypic characterization of the IgH “double-producers” in V H 12f/glD42i and V H 12f/B1-8f mice revealed that these B cells express high levels of B220 and IgD ; and the majority of them are also CD23-positive . In addition, these double-producers do not express CD5 on their cell surfaces. Thus, in contrast to B cells that express V H 12 only, B cells that coexpress V H 12/V H glD42, or V H 12/V H B1-8 assumed a phenotype that is characteristic of B-2 cells. The specificity of the BCR is determined by the variable regions of the H and L chains. Thus, the loss of the B-1 phenotype in cells coexpressing V H 12 and V H B1-8 or V H 12 and V H glD42 may be due to altered Ig L chain usage. It is conceivable that the L chains that associate with both V H 12 and V H B1-8 or V H 12 and V H glD42 under the condition of H chain allelic inclusion are different from those that normally associate with V H 12 alone. This altered L chain usage could affect the specificity of the V H 12 receptor and thus could influence the generation and/or selection of B-1 cells. To examine this possibility, we crossed Vκ4 L chain transgenic (tg) mice 8 with V H 12f/+, B1-8f/+, and V H 12f/B1-8f mice. The Vκ4 gene used in the generation of the transgenic mice was initially isolated from a CD5 + B lymphoma cell line that together with V H 12 recognizes PtC 8 . In addition, this Vκ4 L chain can also associate with the B1-8 H chain to form a BCR of innocuous specificity. Association of the Vκ4 L chain with either or both V H 12 and V H B1-8 is demonstrated in Fig. 2 . We had previously shown that the bone marrow pre-B cell compartment is absent in Ig transgenic mice whose H and L chains pair to form a BCR of an innocent specificity 16 . This probably reflects the fact that precursor cells carrying functional Ig H and L chain transgenes rapidly differentiate into IgM + B cells. We have used this phenomenon to examine the association of Vκ4 with both V H 12 and V H B1-8. As shown in Fig. 2 , B220 + CD43 − pre-B cells are present in wild-type and in the various single and double IgH tg mice and represent ∼8% of the cells present. However, this population is fivefold reduced in the B1-8f/+, V H 12f/+, and B1-8f/V H 12f H chain tg mice that also carry the Vκ4 L chain transgene. This suggests that the Vκ4 L chain can associate efficiently with both V H B1-8 and V H 12. Association of Vκ4 with V H B1-8 is also evident in the splenic B cell population of B1-8f/+, Vk4tg mice, as the cells expressing this H and L chain combination are all Ac146 Id + . Phenotypic analyses of splenic B lymphocytes in B1-8f/+, Vκ4tg mice indicate that these cells are predominantly B-2 cells as they are B220 high(hi) , IgD hi , and CD5 + . In comparison, splenic B cells present in V H 12f/+, Vκ4tg mice are of the B-1 phenotype as indicated by their lower levels of B220 and IgD expression. Furthermore, these cells are CD5 + , in agreement with previous published data 8 . Interestingly, two populations of B cells are present in the spleens of V H 12f/B1-8f, Vκ4tg mice . The Ac146 + 5C5 + population (fraction a) represents double producers that coexpress V H 12, Vκ4, and V H B1-8, Vκ4 receptors. The Ac146 − 5C5 + population (fraction b) seems to have lost the surface expression of the B1-8 H chain and appears to express only V H 12, Vκ4. In contrast, Ac146 + 5C5 − cells (fraction c) that express only V H B1-8, Vκ4 are not seen in these mice. FACS ® analyses of the Ac146 + 5C5 + double producers indicate that these cells are conventional B cells in phenotype, as they express high levels of B220 and IgD and lack CD5 expression. They are indistinguishable from the B cells found in B1-8f/+; B1-8f/+, Vκ4tg; or V H 12f/B1-8f and V H 12f/glD42i mice. Thus, the development of allelically included V H 12-expressing B lymphocytes into conventional B cells is not likely to be due to altered L chain usage, as it occurs also in the presence of the Vκ4 transgene. The Ac146 − 5C5 + B cells present in the spleens of V H 12f/B1-8f, Vκ4tg mice appear to have lost the surface expression of the B1-8 H chain. Thus, they are de facto single V H 12, Vκ4 expressors and, not surprisingly, have a B-1 phenotype . Further analyses revealed that B cells expressing V H 12 only can also be found in the peritoneal cavities of V H 12f/B1-8f and V H 12f/glD42i (data not shown) mice that do not carry the Vκ4 L chain transgene. As shown in Fig. 4 , the Ac146 − 5C5 + cells comprise a large fraction (>90%) of the B cells present in the peritoneal cavity of these mice and possess, as expected, a B-1 cell phenotype (data not shown). Although we cannot exclude the possibility that cells bearing V H B1-8 but not expressing the Ac146 Id (due to pairing with certain L chains) are also included in this population, such cells should represent a minor fraction. This is supported by FACS ® analysis of control B1-8f/+ mice , which suggests that the majority of the B cells (>80%) in the peritoneal cavity express the Ac146 Id. To determine the nature of the lack of B1-8 gene expression in these V H 12-only cells, we first sorted 5C5 + Ac146 − cells from the spleen of V H 12f/B1-8f, Vκ4tg mice and analyzed the targeted IgH loci by Southern blotting using a probe located 5′ of the DQ52 region. The wild-type, targeted V H 12f and B1-8f alleles should yield fragment sizes of 2.3, 3.4, and 13.5 kb , respectively. In the 5C5 + Ac146 − B cells sorted from the spleen of V H 12f/B1-8f, Vκ4tg mice, the band corresponding to the targeted B1-8f allele is missing , suggesting that the gene has been replaced. Similar results were also obtained from 5C5 + Ac146 − B cells isolated from the peritoneal cavities of V H 12f/B1-8f mice (data not shown). Thus, 5C5 + Ac146 − B cells in the spleens and peritoneal cavity of V H 12f/B1-8f, Vκ4tg or in the peritoneal cavity of V H 12f/B1-8f have lost V H B1-8 and consequently express only V H 12. Loss of the B1-8f allele could occur by rearrangement of upstream V or D gene segments into the B1-8 VDJ and resulting in a nonfunctional allele 17 18 19 . It is not known presently at which developmental stage the replacement of the B1-8f allele occurs. FACS ® analyses of the bone marrow of V H 12f/B1-8f or V H 12f/B1-8f, Vκ4tg mice suggest that the immature and mature B lymphocytes in this compartment are predominantly double producers (data not shown). Assessment of the number of B cells in wild-type and various IgH insertion mice revealed that V H 12-expressing B lymphocytes undergo pronounced cellular expansion. As shown in Table , 5 H 12f/+ mice kept in a conventional animal facility generally have 2- and 20-fold more B cells in the spleen and peritoneal cavity respectively, compared with wild-type, B1-8f/+, or glD42i/+ mice. Interestingly, the number of splenic B cells in mice coexpressing V H 12 and either V H B1-8 or V H glD42 is similar to that in wild-type, B1-8f/+, or glD42i/+ mice, suggesting that the expansion of V H 12-expressing B cells is curtailed in these mice. However, the number of peritoneal B cells in V H 12f/B1-8f or V H 12f/glD42i mice is similar to that in V H 12f/+ mice. This probably reflects the fact that the B cells that accumulate in the peritoneal cavities of the double IgH insertion mice are mainly V H 12 expressors that have lost expression of the other H chain . The presence of a Vκ4 L chain transgene leads to an even greater increase in the number of splenic B cells as V H 12f/+, Vκ4tg mice have threefold more cells than do V H 12f/+ mice and eightfold more cells than do either Vκ4tg or B1-8f/+, Vκ4tg mice. This is probably due to ligand-mediated clonal expansion, as V H 12 together with Vκ4 recognizes PtC 8 . Again, this expansion is modulated in V H 12/B1-8f, Vκ4tg mice ( Table ). The three- to fivefold increase in the number of splenic B cells in this mouse strain compared with Vκ4tg or B1-8f/+, Vκ4tg mice is probably due to the fact that >50% of these cells in V H 12f/B1-8f, Vκ4tg mice are single V H 12 expressors . V H 12 insertion mice, like conventional V H 12 transgenic mice 8 , generate mostly B-1 cells, whereas V H B1-8 and V H glD42 insertion mice produce predominantly conventional, or B-2, cells. This is in line with the concept that BCR specificity is a major determinant in B-1 versus B-2 cell development. The novel findings in this study are that the coexpression of V H 12 with either of the two other V H region genes in double IgH insertion mice (which express wild-type κ chains or a Vκ4 transgene) results in the generation of a population of double-producing B-2 cells. In addition, in such animals a population of single-producing B cells appears, namely, B-1 cells expressing only V H 12. Why do V H 12-expressing B cells that coexpress a second H chain not differentiate into B-1 cells? This can perhaps, best be explained by postulating that signaling via a BCR of a certain specificity, expressed at the cell surface at high density, is required to drive the differentiation of B cells into the B-1 subset. In our experiments, the provision of a second H chain of a different specificity presumably acts in a “dominant-negative” manner to dilute out the V H 12-containing BCR complexes on the cell surface. Assuming equivalent production of H chains from the various inserted V H D H J H segments and equivalent pairing of the H and L chains involved, only 25% of the Ig molecules on the cell surface of double-producing cells would carry V H 12 regions on both H chains. This reduced density of B-1–specific BCRs may not provide sufficient signal for the development of B-1 phenotype. The hypothesis that BCR signaling is responsible for the development of the B-1 cell phenotype has been supported by experiments that show that under certain conditions the cross-linking of sIg on splenic B cells may lead to development of a B-1 cell phenotype on B-2 cells 20 . The skewed development of B-1 and B-2 cell subsets in many gene-targeted mice with mutations in specific signaling molecules is also consistent with this hypothesis. For example, CD19-deficient mice 21 22 and xid 23 mice that have a mutation in the btk gene have reduced numbers and a lack of B-1 cells, respectively. In contrast, lyn-deficient mice 24 25 and motheaten mice that have a mutation in SHP-1 26 have increased numbers of B-1 cells. More significantly, the introduction of the xid defect into V H 12 conventional transgenic mice leads to the predominance of V H 12-expressing B cells with a B-2 cell phenotype 27 , compared with wild-type V H 12 transgenic mice that generate mostly B-1 cells. The appearance of large numbers of V H 12-expressing B-1 cells in the double mutants that have lost expression of the second H chain is of particular interest. Such loss variants are rare in V H B1-8/V H glD42 mice 15 , emphasizing the stability of these targeted IgH loci in B cell development. This suggests that the V H 12-only cells in the present system are strongly selected, in accord with the concept that B-1 cell development is driven by BCR signaling. It will be of interest to determine at which stage of development the loss of the second IgH allele occurs in these cells; and whether its loss in mature (B-2) double producers will change their phenotype to that of B-1 cells. It is apparent that B-2 cell development depends to a lesser extent than that of B-1 cells on density of BCRs of certain specificities at the cell surface (reference 15 and the data presented here). This might reflect a lesser dependence of B-2 cells on positive selection by (self)-antigens. The requirement of BCR expression for B-2 cell survival 11 would then largely be a cell-autonomous phenomenon. | Study | biomedical | en | 0.999997 |
10449519 | Methods for maintenance of SCID-hu mice and harvest of thymocytes from SCID-hu Thy/Liv organs were identical to those previously published 17 . In some cases, SCID-hu Thy/Liv organs were harvested and placed in RPMI 1640 media (Life Technologies) supplemented with 10% FCS (Summit Biotechnology) and transported overnight at 4°C before harvest of thymocytes. After isolation, thymocytes were resuspended in PBS supplemented with 2% FCS and kept on ice before staining with mAbs for flow cytometric analysis or cell sorting. All procedures and practices were approved by the University of California, San Francisco Committee on Human Research (CHR) or Committee on Animal Research. Whole blood samples from human subjects were collected by phlebotomy into EDTA collection tubes (Becton Dickinson). PBMCs were isolated from whole blood by density–gradient centrifugation (Life Technologies). PBMCs were washed twice with PBS before resuspension in PBS supplemented with 2% FCS before staining with mAbs for flow cytometry or cell sorting. Human umbilical cord blood cells were obtained (with CHR approval) from healthy delivery specimens and placed in heparinized collection tubes (Becton Dickinson) under sterile conditions. Cord blood mononuclear cells (CBMCs) were isolated as described above for whole blood specimens and resuspended at a concentration of 2 × 10 6 cells/ml in RPMI 1640 supplemented with 10% human AB serum (Ultraserum; Gemini Bio-Products). CBMCs were then cultured (at 37°C in 5% CO 2 ) for 48, 72, or 96 h or 9 d (time points encompassed in two different experiments) and stimulated with 5 μg/ml of PHA (Sigma Chemical Co.) and 10 U/ml purified IL-2 (Boehringer Mannheim). The supplemented medium was changed every 3 d. Cell culture controls did not receive PHA or IL-2 stimulation but were cultured for 72 h in the same medium. Aliquots of the cell cultures at different time points were analyzed by flow cytometry for the expression of the cell surface markers CD45RA and CD62L. PBMCs, thymocytes from SCID-hu mice, or CBMCs were stained with fluorescent-conjugated mABs specific for cell surface markers at a concentration of 10 7 cells/ml at 4°C for 30 min. After staining, cells were washed with PBS supplemented with 2% FCS and sorted on either a FACStar™ or FACS Vantage™ cell sorter (both from Becton Dickinson). The cells were stained with one of the following antibody combinations: (a) anti-CD8–FITC and anti-CD4–PE (Becton Dickinson); (b) anti-CD45RA–FITC or anti-CD45RO–FITC (Immunotech), anti-CD62L–PE (Becton Dickinson), and anti-CD4–ECD (Coulter Immunology); (c) anti-CD62L–FITC, anti-CD45RA–PE (PharMingen), and anti-CD4–tricolor or anti-CD4–allophycocyanin (Caltag Labs., Inc.). Sort purities were checked after each sort and were ≥97%. For analysis of cord blood CD45RA and CD62L expression, CBMCs were stained with anti-CD45RA–FITC (Immunotech) and anti-62L–PE (Becton Dickinson) and analyzed using a FACScan™ cytometer and CELLQuest™ software (both from Becton Dickinson). Total DNA from distinct cell populations was extracted and purified via a standard protocol 18 before spectrophotometric quantitation at 260 and 280 nm. The freshly isolated DNA was stored at 4°C for further processing. Thermal cycling was performed for 30 cycles (1 min at 94°C, 1.5 min at 65°C, and 1.5 min at 72°C) for each round of a seminested PCR protocol designed to detect VβDβ-specific deletion circles (DCs) generated by TCR-β recombination. All first and second round primers were generated to fully hybridize with noncoding regions of the TCR-β locus 19 located next to the recombination signal sequences . Four PCR replicates were performed on each total DNA serial dilution to ensure a precise readout for each experiment. Concentrations of total DNA were adjusted so that a constant volume of 3 μl was added to each 50-μl PCR reaction (200 μM dNTPs, 1× PCR buffer [Boehringer Mannheim], 100 ng of each primer, and 2 U of Taq polymerase [Boehringer Mannheim]). From the first PCR amplification, 3 μl of the first PCR product was used as template for the second (seminested) PCR reaction (under the same conditions) using the “Circle” primer and the DC-Dβ1 primer. Second round PCR products were visualized with ethidium bromide on 1.25% agarose gels and digitally photographed. Individual amplifications were scored as positive or negative by two observers. The highest dilution returning a positive amplification was taken as the endpoint for each dilution series. Dilution series with more than two “skipped” wells (i.e, a failed amplification followed by a successful amplification at higher dilution) were omitted from the analysis. The abundance of DCs was estimated by the Reed-Muench method 20 21 . This method uses information from replicate dilution series to estimate an endpoint (measured in terms of nanograms of input DNA) in which 50% of samples were positive for DCs (the 50% DC endpoint). The DC frequency (DCF) was arbitrarily defined as the reciprocal of the 50% DC endpoint × 100. Alternatively, the seminested PCR data were analyzed by a maximum likelihood estimated method of dilution endpoint with a parametric method 22 . Unlike the Reed-Muench method, this method returns an estimate of goodness of fit of the data to the estimated endpoint. Endpoints estimated by the two methods were highly correlated ( r 2 = 0.929), and the choice of method did not alter the conclusions drawn from the data. The degree of inter- and intraassay variation was assessed by performing two independent experiments on two different samples from the same individuals ( n = 3) and ranged on the order of two- to threefold (data not shown). To determine whether the CD4 + CD45RA + CD62L + subpopulation of circulating human T cells contains RTEs, we devised an assay to detect physical evidence of recent TCR gene rearrangement. We chose to focus on rearrangements at the β locus because the complete sequence of this locus has been obtained 19 , permitting the construction of a panel of Vβ-specific primers to assess the diversity of rearranged TCRs. Moreover, allelic exclusion is more complete at the TCR β locus than at the TCR-α locus 23 24 . Rearrangements at this locus are a salient feature of intrathymic T cell production and require expression of the recombination activating genes ([RAG]-1 and RAG-2) and recognition of conserved heptamer and nonamer RSS flanking each V, D, and J gene segment . As the coding segments are brought together, excision–ligation of the heptamer–heptamer signal joint creates an episomal TCR rearrangement DC 25 30 bearing two identifiers: first, each Vβ-Dβ DC has a precise molecular mass determined by the length of intervening, noncoding DNA; second, a unique DNA sequence bridges the signal joint. Using the known nucleotide sequences of the noncoding DNA regions adjacent to Vβ2, Vβ17, Vβ5.1, and Dβ1 19 , primers were designed such that a PCR product would only be amplified if the primers were facing each other within a closed DC ( Table ). As shown in Fig. 1 B, the product amplified for a Vβ2/Dβ1 rearrangement would have a predicted size of 439 bp, with characteristic restriction enzyme sites. In the case of DCs specific for Vβ17/Dβ1 and Vβ5.1/Dβ1 rearrangements, the corresponding molecular masses would be 445 and 442 bp, respectively. The specificity and reliability of this strategy was first assessed in developing human thymocytes, expected to have a high frequency of DCs 31 . DNA was extracted from two different samples of human CD4 + CD8 + thymocytes (harvested from Thy/Liv organs of SCID-hu mice) 17 . After amplification using the primers specific for Vβ2/Dβ1 DCs, all were found to generate the expected 439-bp PCR product. As shown in a representative case , this product carried predicted restriction enzyme recognition sites for Sac1, PvuII, and ApaL1 and was not observed with PCR performed on DNA from Jurkat cells (a Vβ8.1 T cell line that should not carry Vβ2/Dβ1 DCs). Nucleotide sequence analysis of the PCR product confirmed its identity to the predicted sequence spanning the signal joint of the Vβ2/Dβ1 DC (not shown). Within a population of cells, the fraction bearing DCs should be proportional to that which has recently undergone TCR rearrangement. To directly compare this fraction among different cell populations, a semiquantitative assay was developed to measure a dilution endpoint of DC DNA within a given amount of total cell DNA. DNA was diluted in four replicate series, and PCR was carried out to determine whether a given well was positive or negative for the DC PCR product. The 50% DC endpoint, measured in terms of nanograms of input DNA, was calculated using either the Reed-Muench method 20 21 or a maximum likelihood estimate 22 (see Materials and Methods). The 50% DC endpoint represents the median minimal amount of DNA from which a DC may be amplified by nested PCR; the DCF was arbitrarily defined as the reciprocal of the 50% DC endpoint × 100 and is proportional to the number of DCs that can be amplified from 100 ng of input DNA. A representative experiment using the assay to quantitate DCs is shown in Fig. 2 A. Four replicate dilution series of DNA from CD3 + CD8 + single positive (SP) thymocytes were amplified with primers specific for Vβ2/Dβ1 DCs, and these yielded a positive PCR signal for DCs at final (highest) dilutions of 16, 16, 16, and 3.2 ng input DNA. This corresponds to a 50% DC endpoint of 5.47 ng (as determined by the Reed-Muench method) and a DCF of 18.3 (100/5.47). Assuming typical recovery of DNA and amplification sensitivity, this would return a minimum estimate of 1 DC in 547 SP8 thymocytes, or, as 2–5% of the total express a Vβ2/Dβ1 TCR, 11–22 Vβ2/Dβ1 SP8 thymocytes. Similar frequencies of DCs were noted in sorted populations of CD3 + CD4 + and CD4 + CD8 + thymocytes, yielding DCFs of 8.4 and 11.7, respectively . As prior studies indicated that DCs were detectable within chT1 + RTEs in chickens 3 and in human children and adults 14 32 , the Vβ DC assay was used to determine whether Vβ DCs were present in various populations of human peripheral blood T cells. Reasoning that the frequency of RTEs in the peripheral blood would be highest early in life, T cells in cord blood were examined first. Flow cytometric analysis revealed that >95% of CD4 + T cells in unstimulated cord blood carried the “naive” CD45RA + CD62L + phenotype , and all of these cells were “bright” for CD45RA staining. The frequency of DCs within unstimulated cord blood was higher than that observed for SP thymocytes (with DCFs approximating 43.1 and 41.8 in the two cord blood specimens compared with values of 18.3 and 8.4 for SP8 and SP4 thymocytes, respectively) . After 9 d of stimulation in vitro with PHA and IL-2, the percentage of CD4 + cord blood T cells with the naive (CD45RA bright CD62L + ) phenotype dropped to negligible levels, and most cells were instead negative for CD62L and/or dimly positive for CD45RA . Within this same time frame, the frequency of DCs dropped from an average of 42.5 to 0.85 DCF, a 50-fold decrease over a 9-d period . These results indicated that DCs could be detected in circulating T cells and that their detection was correlated with the presence of cells bearing the naive CD45RA + CD62L + phenotype. DCs were then quantitated in the peripheral blood of 17 adult individuals, ranging in age from 22 to 76 yr. In each, naive CD4 + CD45RA + CD62L + and memory/effector CD4 + CD45RO + CD62L − cells were quantitated by flow cytometry and sort-purified for determination of DC frequency. Results are shown in Fig. 3 C. Within the population of circulating CD4 + CD45RA + CD62L + T cells, DCs were observed with a frequency that was higher than that found in the CD4 + CD45RO + CD62L − population (which had nondetectable levels of DCs in these 17 individuals; data not shown). As a function of age, there was a consistent decrease in the frequency of DCs within the CD4 + CD45RA + CD62L + subpopulation , even though individuals across this age range had equivalent percentages of CD45RA + CD62L + within their CD4 + T cells . These data suggest that RTEs exist within the circulating population of CD4 + CD45RA + CD62L + T cells of adults, that their proportion decreases with age, and that the DC assay appears to provide a much more reliable estimate of de novo–generated T cells than that provided by phenotypic cell surface markers such as CD45RA and CD62L. To determine whether other subpopulations of circulating CD4 + T cells might harbor TCR-β rearrangement DCs, cells were sort-purified into subpopulations that were CD4 + CD45RA + CD62L + , CD4 + CD45RO + CD62L − , CD4 + CD45RO + CD62L + , and CD4 + CD45RO − CD62L + . In eight individuals ranging in age from 22 to 76 yr, the highest frequency of DCs was found in the CD45RA + CD62L + subpopulations and the lowest in the CD45RO + CD62L − subpopulation ( Table ). DCs were also found in the CD45RO + CD62L + subpopulation in four out of eight individuals tested, albeit at a lower frequency. Finally, DCs were detected in T cells with the phenotype CD45RO − CD62L + (data not shown) and CD45RO + CD62L − , although only one out of nine individuals showed detectable levels of DCs in the latter compartment. These cells may possibly represent direct progeny of RTEs in the CD45RA + CD62L + subpopulation; alternatively, DCs may be present within them as a consequence of extrathymic TCR rearrangements 1 33 . Previous studies have demonstrated the presence but not the degree of TCR diversity of RTEs in adult humans 14 32 . To address this parameter of diversity, we generated primers that could amplify DCs issued from three different TCR Vβ-Dβ rearrangements (Vβ2/Dβ1, Vβ5.1/Dβ1, and Vβ17/Dβ1). Flow cytometric analyses (not shown) revealed different percentages of circulating T cells bearing these three Vβs (Vβ2: 8–10%; Vβ5.1: 3–4%; and Vβ17: 3–4%). Results illustrated in Table clearly show that DCs detectable in circulating human T cells encompass several (at least two) Vβs and were present not only in the CD45RA + CD62L + but also in the CD45RO + CD62L + subpopulations of CD4 + T cells. Interestingly, the relative frequency of DCs from different Vβ regions, as measured by flow cytometry, did not correlate with the proportion of PBLs expressing these TCR Vβ products. For instance, Vβ2 + T cells were always at least twofold more abundant in PBLs from normal individuals compared with Vβ5.1 + or Vβ17 + T cells (data not shown). Yet analysis of DCF values (shown in Table ) indicated that, in the two individuals tested (aged 31 and 32 yr), Vβ5.1/Dβ1 or Vβ17/Dβ1 DCs were two- to fivefold more abundant than Vβ2/Dβ1 DCs. These differences in the relative abundance of Vβ DCs compared with the expected frequencies of their parental cell populations could reflect a relative dilutional effect on some Vβ DCs due to varying degrees of peripheral expansion in Vβ-specific subsets, as well as a relative overestimate of some subpopulations due to the detection of DCs from nonproductive rearrangements that might be more prevalent in certain Vβ subsets. In sum, these experiments demonstrate that TCR-β DCs can be detected within thymocytes and circulating human CD4 + T cells with a naive (CD45RA + CD62L + ) phenotype. Detection of such circles is specific, reliable, and quantitative; our method also indicates that they are generated upon rearrangement of multiple Vβ coding segments. Finally, DCs in CD4 + CD45RA + CD62L + T cells are observed in a pattern that is consistent with known parameters of intrathymic maturation: their frequency decreases as cord blood T cells are stimulated to divide in vitro and in older individuals who have less active thymi, as measured in autopsy series or by noninvasive radiography. As such, quantitation of DCs within human peripheral blood CD4 + CD45RA + CD62L + T cells appears to represent a measure of RTEs and, hence, thymic function. These results serve to directly confirm previous inferences about thymic function. First, the finding of DCs within the CD4 + CD45RA + CD62L + population of adult individuals aged 23–76 yr underscores the premise that the thymus, though less functional, is nonetheless operative into adulthood 2 5 9 14 32 . Secondly, the fact that the frequency of DCs decreases in the CD4 + CD45RA + CD62L + population as a function of age demonstrates that this population is heterogeneous 11 12 and that its composition is age dependent. It may not be useful, in other words, to assume that the presence (or reappearance) of such cells is synonymous with “immune reconstitution” 34 35 36 37 . Finally, the finding of DCs within other populations of circulating T cells raises the possibility that extrathymic sources (e.g., gut or liver) may contribute to formation of the circulating TCR repertoire 1 33 . Although further work is required to optimize the quantitative precision of the DC assay and enhance its applicability for comprehensive studies of human thymic function, it is now applicable to important contemporary questions about thymic function and immune reconstitution in humans. Most immediately, it will be of interest to determine the extent of thymic dysfunction at different stages of HIV infection and after bone marrow reconstitution postmyeloablation. It will also be interesting to determine the extent of de novo rearrangement in lymph nodes, which might be induced by chronic viral replication, as recently suggested in a murine model of persistent antigen exposure 38 . This measure of thymic function may also facilitate the design of studies aimed at augmenting intrathymic T cell production. | Study | biomedical | en | 0.999997 |
10449520 | A total of 21 different patients with hypereosinophilia associated with skin diseases, allergy, hypereosinophilic syndromes, or myeloproliferative disorders were selected for this study, after informed consent. Five normal donors were also included in the study. The characteristics of eosinophil donors and eosinophil preparations are summarized in Table . Anti–human CD16 and CD3 magnetic beads and the magnetic cell separation system (MACS ® ) were purchased from Miltenyi Biotec. Percoll was obtained from Amersham Pharmacia Biotech. RPMI 1640 medium, glutamine, penicillin, streptomycin, and FCS were obtained from GIBCO BRL. The FITC-conjugated and nonconjugated anti-CD28 (clone B-T3), anti-CD86, and mouse IgG1 mAbs were purchased from Diaclone. The FITC-conjugated and nonconjugated anti-CD80 were from Immunotech. The anti-CD28 Ab (9.3 mAb) was a gift from Dr. C. June (University of Pennsylvania School of Medicine, Philadelphia, PA). The anti-CD28 (clone CD28.2) and the anti–mouse IgG F(ab′) 2 were from Sigma Chemical Co. The FITC-conjugated anti–IFN-γ, anti–IL-2, and anti–IL-10 mAbs and isotype-matched Abs were obtained from Diaclone. The nonconjugated anti–IL-2 was from Genzyme Corp. The anti–IFN-γ, anti–IL-10, and isotype control Abs were from Diaclone. The recombinant human (rh)IL-2 was purchased from Boehringer Mannheim, and the rhIFN-γ was from Diaclone. Human secretory IgA was obtained from Sigma Chemical Co. Anti–human IgA mAb was from Immunotech. The Quantum Simply Cellular quantification kit and the mouse alkaline phosphatase anti-alkaline phosphatase (APAAP) 1 detection system were purchased from Dako. The goat anti-CD28 (C-20) was from Santa Cruz Biotechnology. The mouse IgG control Ab and the horseradish peroxidase (HRP)-conjugated anti–goat IgG were obtained from Jackson ImmunoResearch Laboratories. Eosinophils were isolated from the venous blood of patients by the method described previously 23 with minor modifications, using immunomagnetic beads and the magnetic cell separation system (MACS ® ). Diluted whole blood (1:1) was layered onto a Percoll gradient ( d = 1.082 g/liter) and centrifuged at 1,800 rpm for 20 min. The granulocyte pellet, mainly neutrophils and eosinophils, was harvested and depleted of erythrocytes by hypotonic saline lysis. In brief, the granulocyte pellet was incubated for 30 min at 4°C with anti-CD16 and anti-CD3 immunomagnetic beads in order to remove neutrophils and contaminating lymphocytes, respectively. Eosinophils were eluted by passage of the cells through the field of a permanent magnet. After isolation, eosinophil preparations were cytocentrifuged, and the cytospins were stained with May-Grünwald-Giemsa (RAL 555; Rieux). The purity of eosinophil preparations usually reached >97% for patients ( Table ). Freshly purified eosinophils were resuspended at 3 × 10 6 /ml in PBS/1% BSA. Aliquots of 50 μl were incubated with FITC-conjugated anti-CD28, anti-CD80, or anti-CD86 mAb or FITC-conjugated isotype-matched Ab at a final concentration of 5 μg/ml for 1 h at 4°C in round-bottomed 96-well plates. After two washes in PBS, the cells were resuspended in PBS/0.5% BSA before analysis. For intracellular staining, eosinophils were fixed with 2% paraformaldehyde in PBS for 10 min. After washing in PBS, the cells were resuspended at 4 × 10 6 /ml in PBS containing 1% BSA and 0.5% saponin (permeabilization buffer) for 10 min at room temperature. The samples were then preincubated for 10 min with 5 μl normal mouse serum in order to block nonspecific binding, and were incubated for an additional 30 min with FITC-conjugated anticytokine mAb or isotype-matched Ab at a final concentration of 5 μg/ml in permeabilization buffer. After washing in permeabilization buffer, followed by washing in PBS, the cells were resuspended in PBS/0.5% BSA. Samples were analyzed on a FACSCalibur™ using CellQuest software (Becton Dickinson). 10 4 events were usually acquired per sample. Thresholds were set on control stains (included for every sample at every time point). The antigen density at the cell surface was quantified with the Quantum Simply Cellular quantification kit, in which the median values of fluorescence intensity were converted into Ab binding capacity (ABC) units using calibrating microbeads with specific Ab binding capacity. In parallel to staining of the samples, the goat anti–mouse Ig–coated microbeads were incubated with FITC-conjugated anti-CD28, anti-CD86, or FITC-conjugated isotype-matched Ab. The lower quantification limit for CD28 and CD86 expression was 7,300 ABC units. Cytospins of freshly purified eosinophils were fixed in acetone at −20°C for 10 min. After air drying, slides were stored at −20°C until use. The APAAP method was used for immunostaining. In brief, after saturation with 10% normal rabbit serum diluted in Tris-buffered saline (TBS) containing 1% BSA, cytospins were incubated with anti–human cytokine or isotype control mAbs at 30 μg/ml in TBS/1% BSA overnight at 4°C. The slides were washed twice for 15 min in TBS, then incubated with rabbit anti–mouse Igs (1:25) in TBS/1% BSA for 1 h at room temperature. After washing as above, they were incubated with APAAP complex (1:40) for 1 h, and the reaction was developed with New Fuchsin substrate (Dako). The slides were counterstained with Mayer's hematoxylin and mounted with Fluoroprep (BioMérieux). For IFN-γ and IL-10 detection, the intensity of the reaction was increased by performing a second round of APAAP reaction. Freshly purified eosinophils (2.5 × 10 7 ) and Jurkat cells (2 × 10 7 ) were lysed on ice for 30 min with 0.5% Triton X-100 in borate-buffered saline (BBS) buffer, pH 8.0, containing 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin. The lysates were centrifuged at 12,000 g at 4°C. Supernatants were sequentially incubated with 30 μl of protein G–Sepharose beads (1 h, 4°C; Sigma Chemical Co.), 30 μl protein G–Sepharose-bound mouse IgG (1 h, 4°C), and then 30 μl protein G–Sepharose-bound anti-CD28 (9.3 mAb) for 2 h at 4°C. The beads were washed five times with lysis buffer, and immune complexes were resuspended in reducing Laemmli sample buffer. The samples were heated to 95°C for 5 min and then run on 8% SDS-PAGE. The separated proteins were transferred onto nitrocellulose membrane. The membrane was incubated in a blocking buffer containing 5% BSA and 0.1% Tween 20 in PBS for 2 h, followed by incubation with a goat anti-CD28 at a 1:500 dilution in PBS/1% BSA/0.1% Tween 20 for 2 h. After washing in PBS/0.1% Tween 20, the blot was incubated for 1 h with HRP-conjugated anti–goat Ab at a 1:5,000 dilution in PBS/0.1% Tween 20. Immunoblot signals were detected using Renaissance Western Plus reagent from NEN Life Sciences. Culture medium consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin. Cross-linking experiments were performed in 24-well culture plates. Wells were first coated with 40 μg/ml anti–mouse IgG F(ab′) 2 for 2 h at 37°C in 5% CO 2 . After washing, wells were incubated with 10 μg/ml mouse anti-CD28 (B-T3 mAb), anti-CD86 mAb, or isotype-matched control mAb for 2 h at 37°C in 5% CO 2 . The wells were then washed twice with PBS, and 2 × 10 6 eosinophils in 1 ml culture medium were added per well. After 18 h culture, supernatants were collected and analyzed for cytokine secretion. For stimulation with IgA and anti-IgA, highly purified eosinophils were first incubated with secretory IgA at a final concentration of 15 μg/ml. After 1 h incubation at 37°C, cells were transferred into either 24-well plates or anti-CD28–coated plates and stimulated with 20 μg/ml anti-IgA mAb at 37°C in 5% CO 2 . After 18 h culture, cell viability was determined by trypan blue exclusion, and supernatants were collected and analyzed for cytokine secretion. Eosinophil cytolysis was followed by measuring the cytoplasmic marker lactate dehydrogenase (LDH) in the supernatants of eosinophils incubated for 18 h with medium alone, immobilized anti-CD28 mAb, IgA immune complexes, or with Triton X-100 as positive control. LDH was evaluated by colorimetric assay (Boehringer Mannheim) 24 . IL-2, IFN-γ, and IL-10 were assayed in eosinophil supernatants using specific ELISA kits (Diaclone) according to the manufacturer's instructions. The lower detection limit was 10 pg/ml for IL-2, 5 pg/ml for IFN-γ, and 5 pg/ml for IL-10. The biological activity of IL-2 released in eosinophil supernatants was measured by the conventional CTLL-2 assay. In brief, 10 4 CTLL-2 cells per well were cultured in 96-well microtiter plates with serial dilutions of rhIL-2 or eosinophil supernatants (at a 50% dilution) in a final volume of 100 μl, for 24 h at 37°C in 5% CO 2 . 6 h before harvesting, cells were pulsed with 0.5 μCi [ 3 H]thymidine. All assays were performed in duplicate. To determine the biological activity of IFN-γ released by eosinophils, the capacity of IFN-γ to induce expression of MHC class II (HLA-DR) protein on the human colon carcinoma cell line Colo 205 (American Type Culture Collection) was used 25 . In brief, 2 × 10 4 cells in a final volume of 200 μl were cultured in 96-well plates in the presence of variable concentrations of rhIFN-γ (0.1–10 U/ml) or eosinophil supernatants (at a dilution of 20%), in the presence or absence of neutralizing anti–IFN-γ mAb (Diaclone). After 48 h culture at 37°C in 5% CO 2 , cells were harvested and analyzed for HLA-DR expression by flow cytometry, using PE-conjugated anti–HLA-DR mAb (Becton Dickinson). Freshly isolated eosinophils from eosinophilic patients were used to examine the intracellular expression of IL-2, IFN-γ, and IL-10. Results from flow cytofluorimetric analysis presented in Fig. 1 A, which illustrates one representative experiment, showed that human eosinophils could express IL-2 and IFN-γ, as well as IL-10. The specificity of intracellular staining was controlled by incubation of the cells with FITC-conjugated mAb in the presence of an excess of the corresponding recombinant cytokine. For the three cytokines under study, this procedure led to an almost total inhibition of the signal (data not shown). To confirm the intracellular expression of the cytokines observed by flow cytometry analysis, cytospin preparations of purified eosinophils were incubated with anticytokine mAb from different clones than those used for flow cytometry. The preparations were then processed for immunohistochemical analysis using the APAAP method, followed by counterstaining with hematoxylin. As shown in Fig. 1 B, eosinophils exhibited positive staining for IL-2, IFN-γ, and IL-10. No staining was observed when cells were incubated with an irrelevant isotype control Ab. Until now, most studies have examined the cytokine content of human eosinophils by nonquantitative approaches such as reverse transcription PCR for mRNA expression and immunocytochemistry or electron microscopy for intracellular protein detection 2 3 4 5 . By using cytofluorometry, we showed clearly that a large proportion of human eosinophils have the capacity to express both type 1 (IL-2 and IFN-γ) and type 2 (IL-10) cytokines. To investigate the expression by eosinophils of the costimulatory molecules CD80, CD86, and CD28, freshly isolated human eosinophils from 17 hypereosinophilic patients and 5 normal donors were stained with FITC-conjugated anti-CD80, anti-CD86, or anti-CD28 (B-T3) mAb or isotype-matched Ab and analyzed by flow cytometry. Results presented in Fig. 2 A show clearly that, among the B7 family, eosinophils could express CD86 (B7-2) but not CD80 (B7-1). Unexpectedly, the presence of the T cell–associated molecule CD28 was also detected on eosinophils. Both CD86 and CD28 were detected in all donors, but their expression was variable among individual patients, ranging from 6 to 63% and from 4 to 25%, respectively . For both CD86 and CD28, the mean fluorescence intensity (MFI), which reflects the level of binding of the Abs, was low, suggesting a low level of membrane expression. Indeed, using a technique for direct quantification of cell surface molecules, we detected 8,003 ± 231 and 9,039 ± 644 Ab binding sites per cell for CD28 and CD86, respectively ( n = 4). Preliminary experiments performed on a limited number of healthy donors indicate that normal eosinophils could also express membrane CD86 and CD28, but at a lower level . Due to the small number in each group of eosinophilic patients, it was difficult to observe any correlation with the etiology of the disease, as reported for other membrane molecules 26 . To further confirm the expression of CD28 by human eosinophils, immunoprecipitation followed by Western blotting was performed. As shown in Fig. 3 , a 50-kD protein was specifically immunoprecipitated with anti-CD28 mAb (9.3 mAb), whereas no band appeared when lysates were immunoprecipitated with mouse IgG–bound Sepharose beads. Under the same conditions, immunoprecipitation using Jurkat cells as positive control showed a much stronger band, indicating that CD28 expression in human eosinophils is lower than in T cells, as already suggested by flow cytometry . Although CD28 is a homodimeric molecule, comprising a 44-kD subunit, the variation in molecular mass observed between eosinophils and Jurkat cells likely arises as a result of different glycosylation patterns. Such an observation has already been made for a T cell leukemia cell line 27 . The demonstration that human peripheral blood eosinophils could express CD86 and CD28 led us to investigate the functional significance of these costimulatory molecules in eosinophil activation. Since it has been reported that CD28 ligation in T cells resulted in IL-2 secretion and proliferation, cross-linking experiments of purified eosinophils with immobilized anti-CD28 or anti-CD86 mAb were performed, and the release of cytokines was assayed in supernatants. Eosinophils from six individual patients were added to plates coated with anti-CD28 or anti-CD86 mAb, and supernatants were collected after 18 h and analyzed. As shown in Fig. 4 , CD28 ligation induced IL-2 and IFN-γ secretion, whereas in the same activation conditions, no release of IL-4, IL-5, or IL-10 was detected (data not shown). Contrary to T cells, stimulation via the CD28 molecule did not appear to require a second signal for optimal eosinophil stimulation, since the addition to anti-CD28 mAb of various stimuli such as LPS, Ca 2+ ionophores, or PMA did not lead to an increase in cytokine secretion (data not shown). Fig. 4 shows that the secretion of IL-2 and IFN-γ was variable according to individual patients, with a massive production of IL-2 (up to 700 pg/ml) observed for some donors after CD28 stimulation. The levels of IFN-γ were lower than those of IL-2, with a maximum of 180 pg/ml and a minimum of 30 pg/ml. It is interesting to notice that the maximum secretion for IL-2 and IFN-γ was reached for the same eosinophil donors. In contrast to CD28 cross-linking and although CD86 was expressed on these eosinophil preparations, CD86 ligation did not induce the secretion of IL-2 or IFN-γ or the release of IL-4, IL-5, or IL-10. Nonstimulated eosinophils or eosinophils cross-linked with isotype-matched Ab produced very low amounts of IL-2 and IFN-γ (<10 pg/ml). In parallel to the anti-CD28 mAb from clone B-T3, we also used anti-CD28 mAb from a different clone. Immobilized 9.3 mAb was also able to induce IL-2 and IFN-γ secretion, although to a lesser extent (data not shown). To exclude the possibility that some lymphocytes could contaminate the eosinophil preparation and therefore be responsible for the IL-2 and IFN-γ secretion observed, lymphocytes isolated from hypereosinophilic patients were stimulated in the same conditions as purified eosinophils with immobilized anti-CD28. No secretion of IL-2 and IFN-γ could be detected, in the absence of additional CD3-TCR triggering. These findings indicate that activation of the costimulatory molecule CD28 expressed by human eosinophils is able to induce the release of IL-2 and IFN-γ. In contrast, CD86 cross-linking did not induce a similar cytokine release by eosinophils. In spite of the numerous studies published on cytokine secretion by eosinophils, very few have evaluated the biological activity of the cytokines released. Therefore, appropriate bioassays were performed in order to evaluate the functional activity of IL-2 and IFN-γ in eosinophil supernatants. The biological activity of IL-2 in the supernatants of CD28-activated eosinophils was evaluated in a proliferation assay using the IL-2–dependent mouse cytotoxic T cell line CTLL-2. The results presented in Fig. 5 indicate that eosinophil supernatants induced the proliferation of CTLL-2 cells, with large variations according to individual eosinophil donors. Comparison with the results obtained for purified rhIL-2 indicated that between 100 and 250 pg/ml of biologically active IL-2 could be detected in eosinophil supernatants after CD28 ligation . Similarly, the bioactivity of IFN-γ secreted by eosinophils was assessed according to its capacity to enhance MHC class II expression on the Colo 205 cell line 25 . In parallel with the effect of rhIFN-γ , supernatants derived from eosinophils activated upon CD28 ligation were able to induce MHC class II, as shown by flow cytometric analysis . This effect was significantly inhibited by the addition of neutralizing anti–IFN-γ mAb . In contrast, supernatants from eosinophils incubated with immobilized anti–mouse IgG1 did not induce the release of biologically active IFN-γ. Since previous studies have shown that secretory IgA immune complexes could induce eosinophil degranulation, as well as IL-5 and IL-4 secretion 3 4 , we have investigated whether the activation of eosinophils with IgA could influence the CD28-induced cytokine release. As shown in Fig. 6 , the addition of secretory IgA and anti-IgA mAb together with anti-CD28 ligation strongly inhibited the secretion of IL-2 (82 ± 32% inhibition) and IFN-γ (74 ± 23% inhibition) ( n = 3). These results indicate that secretory IgA–dependent activation of eosinophils could induce a downregulation of the CD28 activation pathway and prevent type 1 cytokine secretion. To ascertain that the secretion or lack of secretion of cytokines observed was not due to eosinophil death during incubation, the release of LDH, a sensitive marker of cell lysis 24 , was measured in the supernatants of eosinophils incubated with medium alone, immobilized anti-CD28, or IgA–anti-IgA for 18 h. Depending on the patient, LDH release ranged from 13.1 ± 1.5 to 32.2 ± 4% compared with a positive control represented by Triton X-100–lysed cells (data not shown). The trypan blue exclusion method confirmed ∼96% cell viability at the end of the culture, in all conditions. These results indicate that cytokine secretion after CD28 and/or IgA complex ligation is not due to a massive cytolysis. To further elucidate the mechanism of inhibition of the release of type 1 cytokines by IgA complexes, we evaluated the role of IL-10, which is the more potent cytokine involved in the negative regulation of type 1 cytokines. Activation of eosinophils with IgA–anti-IgA induced IL-10 secretion, in contrast to CD28 ligation . These results favor the view that IgA complexes, by inducing IL-10 release, could have an inhibitory effect on CD28-mediated release of IFN-γ and IL-2. Moreover, the addition of immobilized anti-CD28 at the time of IgA cross-linking led to a significant inhibition of IL-10 secretion . These results indicate that IFN-γ and IL-2 on one hand, and IL-10 on the other, could be released by human eosinophils upon different signaling pathways. In conclusion, our results showed that human eosinophils can express both CD86, classically expressed by APCs, and its CD28 ligand, a potent signaling molecule expressed by T cells. The detection on eosinophils of T cell–associated molecules has already been shown for CD4 28 . The coexpression, on the same cell population, of two molecules acting as ligands for each other has also been reported, for example by eosinophils in the case of CD40–CD40L 13 14 and by T cells which could, in addition to CD28, express B7 29 . Further experiments are needed to ascertain whether such pairs of molecules are really expressed by the same individual cell, and if this coexpression might be of some significance for cell activation in vivo. However, these results suggest that eosinophils might provide costimulation of T cells (specially type 2 cells) via CD86, and conversely that they might respond to stimulation via CD28 engagement. In this study, only stimulation of the CD28 molecule, not CD86 ligation, triggered cytokine release by highly purified human eosinophils. In contrast to CD28, able to provide a strong costimulatory signal on T cells, the main function of CD86 is to bind to CD28 and not to act as a signaling molecule in APCs, although the structure of the cytoplasmic tail (three potential phosphorylation sites) indicates a potential signaling role for this molecule 17 . The precise function of CD86 has yet to be investigated in other pathways of eosinophil activation. Although CD28 mainly participates in T cell activation as a second signal, it has been recently shown that stimulation through CD28 could induce early signaling events, such as cytokine synthesis, in resting T cells, without further requirement for TCR engagement 30 . Experiments are now in progress to investigate the patterns of signaling events induced by CD28 activation of eosinophils. An important outcome of this study is the release of type 1 cytokines by eosinophils after CD28 ligation, suggesting that interactions between CD28-expressing eosinophils and B7-expressing cells could induce previously unsuspected functions of eosinophils in the immune response. Until now, IL-2 was believed to be produced mainly by T lymphocytes. However, two groups recently reported the production of IL-2 by human eosinophils, its storage in the crystalloid granule, and IL-2 secretion after GM-CSF and A23187 stimulation 10 31 . The fact that eosinophils can produce IL-2 after CD28 ligation suggests that these cells could induce lymphocyte activation and consequently sustain the inflammatory processes, particularly in chronic diseases associated with eosinophils, such as asthma, skin diseases, or inflammatory bowel diseases. The release of biologically active IFN-γ by eosinophils, which has never been reported, led us to speculate that eosinophils might exert antiviral properties, and prompted consideration of strategies aiming toward the demonstration of this new effector function of eosinophils in innate as well as acquired immunity. Although at this stage it is not possible to draw definitive conclusions about the mechanism of inhibition of the CD28-dependent activation induced by secretory IgA–anti-IgA complexes, our results indicating that activation with secretory IgA but not with anti-CD28 could induce the release of IL-10, a potent inhibitory cytokine of the Th1 pathway, suggest the existence of cross-regulatory signals involved in the release of type 1 versus type 2 cytokines by eosinophils. Further experiments are needed to elucidate the precise role of IL-10 in this mechanism and the possible intervention of IL-10 receptor expressed by eosinophils. Interestingly, the inhibitory role of IL-10 on eosinophil activation as well as its role in the decrease of type 1 cytokine production by T cells have already been reported (for a review, see reference 32 ). Thus, it will be of interest to investigate whether this inhibitory function of secretory IgA on the CD28-mediated release of type 1 cytokines, which would favor the Th2-mediated response, could be also detected for other cell populations, such as T cells, and whether it could play a role in the low dose oral tolerance phenomenon or other clinically relevant disease situations. | Study | biomedical | en | 0.999995 |
10449521 | The patient is female, born November 1990, the only child of healthy, unrelated parents. At birth, the patient's absolute neutrophil count was 0 × 10 9 /liter. During the first 18 mo of life, the patient experienced four episodes of cellulitis and abscesses. From June 1992 to October 1993, the patient was treated with a series of escalating daily subcutaneous doses of G-CSF from 10 to 70 μg/kg, with no response; peripheral polymorphonuclear cells remained at 0 × 10 9 /liter, and bone marrow smears revealed a maturation arrest at the myelocyte stage, indicative of severe congenital neutropenia. In January 1994, a continuous intravenous 24-h infusion of 150–200 μg/kg G-CSF was administered to the patient for 10 d consecutively with no response and persistent marrow maturation arrest. The patient continued to have recurrent bacterial infections. Total RNA was isolated from bone marrow mononuclear cells using acid guanidinium isothiocyanate extraction 31 , and reverse-transcribed using oligo(dT) primers and MMLV reverse transcriptase (GIBCO BRL). PCR amplification was performed using the primer pairs GRFR3 (5′-TCGGAAAGGTGAAGTAACTTGTCC) and GRRV5 (5′-TCCATGGGATCAAGACACAG) to amplify nucleotides 106–832, and GRFR5 (5′-TGCAGGCAGAGAATGCGCTG) and GRRV4 (5′-AGGGTC-CCATGGTGTCCTGGTACA) to amplify nucleotides 772–1707 of the GCSFR cDNA. The products were cloned into the TA cloning vector (Invitrogen) and sequenced using the dideoxy chain termination method with T7 polymerase (Amersham Pharmacia Biotech). The GCSFR mutation identified in patient AR introduces a new BsiHkAI site (G A / t GC A / t C) . Therefore, to analyze for the presence of the mutation, genomic DNA was prepared from fibroblast and blood cells using standard techniques, and subjected to PCR with the primer pair GRFRI6 (5′-ACCTAGAGAGAAACAAAGAC) and GRRV15 (5′-AGCGGATGCAGCGTATCT). The resultant products were digested with BsiHkAI (New England Biolabs) before separation on a 3% agarose gel. The subline of the IL-3–dependent murine myeloid cell line 32Dcl3, called 32D.cl8.6 29 , and the IL-3–dependent murine pro-B cell line Ba/F3 32 were maintained in RPMI 1640 medium supplemented with 10% FCS and 10 ng/ml of murine IL-3, at 37°C and 5% CO 2 . Cloning of the wild-type (WT) GCSFR cDNA into the eukaryotic expression vector pLNCX 33 has been described previously 25 . To clone the mutant receptor (mAR) into this vector, a multistep procedure was required. First, the region spanning the extracellular region of the mutant receptor was obtained by joining the 5′ and 3′ regions obtained using reverse transcription (RT)-PCR via a common BsmI site at position 782, to produce plasmid pTAR1600. From this construct, the region encoding the mutation was obtained as an AvaI fragment which was used to replace the equivalent region of the WT GCSFR cloned into pBlueScript. The entire receptor coding region was then subcloned into pLNCX as a HindIII-ClaI fragment. The pLNCX expression constructs were linearized by PvuI digestion and transfected into 32D.cl8.6 or Ba/F3 cells by electroporation. After 48 or 24 h of incubation, respectively, cells were selected with G418 (GIBCO BRL) at a concentration of 0.8 or 1.2 mg/ml, respectively, with multiple clones expanded for further analysis. For retroviral infections, the WT and mAR GCSFR cDNAs were cloned into pBabe, as described 25 . The resulting plasmids were transfected into the Phoenix A amphotropic packaging cell line (Clontech) to generate recombinant retrovirus, following standard protocols. Virus supernatants were used to infect 32D cells harboring the pLNCX.GCSFR(WT) vector (32D[WT Neo ]) using RetroNectin™ (Takara Biomedicals), as described by the manufacturer. After 48 h, puromycin was added to 1 μg/ml to select for stably transduced cells. To determine G-CSF-R expression levels, cells were incubated at 4°C for 60 min sequentially with 10 μg/ml of biotinylated mouse anti–human G-CSF-R mAb LMM741 (PharMingen), 5 μg/ml of PE-conjugated streptavidin, 5 μg/ml of biotinylated antistreptavidin antibody, and finally 2 μg/ml of PE-conjugated streptavidin, with washing between each antibody step. Samples were analyzed by flow cytometry using a FACScan™ (Becton Dickinson). Several independently derived cell lines of each construct were selected on the basis of homogeneous receptor expression. The GCSFR mutation identified in patient AR introduced a new BsiHkAI site (G A / t GC A / t C). Therefore, as confirmation of mutant receptor expression, RT-PCR was performed with primers GRFR5 and GRRV11 (5′-ACTGTCCTGGGGTCCAGCTG), and the resultant products were cut with BsiHkAI before electrophoresis on an agarose gel. For routine analysis of G-CSF binding, biotinylated G-CSF protein was used, followed by 5 μg/ml of PE-conjugated streptavidin, as described 28 . To estimate the affinities of the G-CSF binding sites, G-CSF binding experiments and Scatchard analysis were performed using 125 I–G-CSF (800–1,500 Ci/mmol; Amersham Pharmacia Biotech), as described 34 . DNA synthesis was assessed by [ 3 H]-thymidine ( 3 H-TdR) incorporation. Cells (5 × 10 4 ) were incubated in triplicate in 100 μl of 10% FCS/RPMI medium supplemented with titrated concentrations of human G-CSF, or with 10 ng/ml murine IL-3, in 96-well plates for 24 h. 16 h before cell harvest, 0.1 μCi of 3 H-TdR (2 Ci/mmol; Amersham Pharmacia Biotech) was added to each well. 3 H-TdR incorporation was measured by harvesting cells on glass fiber filters followed by counting on a Packard Top Count scintillation counter. To determine proliferation, cells were incubated at an initial density of 1–2 × 10 5 cells/ml in 10% FCS/RPMI medium supplemented with 100 ng/ml of human G-CSF, 10 ng/ml of murine IL-3, or without growth factors. The medium was replenished every 1–2 d, and the cell densities were adjusted to 1–2 × 10 5 cells/ml. Viable cells were counted on the basis of trypan blue exclusion. To analyze the morphological features, cells were spun onto glass slides and examined after May-Grünwald-Giemsa staining. Cells were deprived of serum and factors for 4 h at 37°C in RPMI 1640 medium at a density of 10 6 /ml, and then stimulated with either RPMI 1640 medium alone or in the presence of 100 ng/ml human G-CSF. At different time points, 10 vol of ice-cold PBS supplemented with 10 μM Na 3 VO 4 were added. Subsequently, cells were pelleted and resuspended in ice-cold hypotonic buffer (20 mM Hepes, pH 7.8, 20 mM NaF, 1 mM Na 3 VO 4 , 1 mM Na 4 P 2 O 7 , 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 0.2% Tween-20, 0.125 μM okadaic acid, 1 mM Pefabloc SC, 50 μg/ml aprotinin, 50 μg/ml leupeptin, 50 μg/ml bacitracin, and 50 μg/ml iodoacetamide) 35 . Cells were vortexed for 10 s, and the nuclei were pelleted by centrifugation at 15,000 g for 30 s. Nuclear extracts were prepared by resuspension of the nuclei in high-salt buffer (hypotonic buffer with 420 mM NaCl and 20% glycerol) and extraction of proteins by rocking for 30 min at 4°C. Insoluble materials were removed by centrifugation at 4°C for 15 min at 15,000 g . Nuclear extracts of ∼0.5 × 10 6 cells were incubated for 20 min at room temperature with 0.2 ng of 32 P-labeled double-stranded oligonucleotide (5–10 × 10 3 cpm) and 2 μg of poly(dI-dC) in 20 μl of binding buffer (13 mM Hepes, pH 7.8, 80 mM NaCl, 3 mM NaF, 3 mM NaMoO 4 , 1 mM dithiothreitol, 0.15 mM EDTA, 0.15 mM EGTA, and 8% glycerol) 36 . The oligonucleotide probes used in this study were m67 (5′-CATTTCCCGTAAATC), a high-affinity mutant of the sis-inducible element (SIE) of the human c- fos gene 37 , which binds STAT1 and STAT3, and β-cas (5′-AGATTTCTAGGAATTCAATCC), derived from the 5′ region of the β-casein gene 38 , which binds STAT5 and STAT1. The DNA–protein complexes were separated by electrophoresis on 5% polyacrylamide gels containing 5% glycerol in 0.25× TBE. The gels were dried and subsequently analyzed by autoradiography. Patient AR represents an unusual SCN patient, being totally unresponsive to standard and high-dose G-CSF therapy. Subsequently, the patient responded to combined prednisone therapy in combination with G-CSF, the details of which will be published elsewhere (Ward, A.C., I.P. Touw, and M.H. Freedman, manuscript in preparation). We had previously reported that no mutations were present in the sequence encoding the cytoplasmic domain of the G-CSF-R of this patient 27 . However, the unusual phenotype of this patient prompted us to analyze the complete sequence of the GCSFR . This identified a C→A mutation at nucleotide 850 of the GCSFR cDNA, which produces a Pro→His substitution at position 206 (P206H) of the mature receptor . This change lies in the joining peptide “hinge” between the NH 2 - and COOH-terminal “barrels” (BN and BC domains) of the CRH region of the G-CSF-R extracellular domain 39 . Along with the WSXWS sequence, this short Pro-rich stretch is highly conserved among cytokine receptors, with the position equivalent to 206 usually a Pro, or otherwise an Ala residue 12 40 . This mutation was found in ∼50% of clones sequenced, indicating that essentially all bone marrow cells possessed one mutant GCSFR allele. This was confirmed by restriction digestion of cDNA produced in an independent RT-PCR with the enzyme BsiHkAI, as described in Materials and Methods. In addition, a fragment covering the mutation site was amplified from genomic DNA isolated from fibroblast cells of the patient. Restriction enzyme analysis with BsiHkAI revealed that both mutant and wild-type GCSFR alleles were also present in these cells . Thus, unlike other GCSFR mutations reported 25 , the mutation in patient AR is not restricted to cells of the hematopoietic system, with the patient apparently heterozygous for the mutation. Unfortunately, a full pedigree for the patient was not available to determine if the mutation was inherited, although the mother was found to possess a wild-type GCSFR allele and normal blood neutrophil counts (data not shown). To study the functionality of the P206H mutant G-CSF-R, designated mAR, we replaced the extracellular region of the wild-type G-CSF-R with that of the mutant form. Expression vectors containing the WT and mAR GCSFR cDNAs were introduced into the IL-3–dependent murine myeloid cell line 32D.cl8.6, which does not express endogenous G-CSF-R. Expression levels of the different G-CSF-R proteins in the transfectants were determined by flow cytometry using anti–G-CSF-R antiserum . Several independent clones were obtained expressing approximately equivalent levels of wild-type and P206H mutant receptors (32D[WT] and 32D[mAR], respectively), and used in subsequent analyses. To directly test whether the P206H mutation could contribute to the G-CSF hyporesponsiveness observed in the patient, we analyzed the sensitivity of the WT and mAR clones to G-CSF by measuring DNA synthesis in 3 H-TdR incorporation assays in response to titrated doses of cytokine . This revealed a large right shift in dose–response for the 32D[mAR] clones. However, even at maximal G-CSF concentrations, 32D[mAR] clones failed to reach the same level of 3 H-TdR incorporation as those expressing the wild-type receptor. These data establish that the P206H mutation causes hyporesponsiveness to G-CSF in myeloid cells. We next sought to determine the effect of the P206H mutation on long-term proliferation, survival, and neutrophilic differentiation responses to G-CSF. Therefore, 32D cell clones were switched from IL-3– to G-CSF–containing medium after extensive washing to remove residual IL-3. Without IL-3 or G-CSF, all transfectants died within 1–2 d and showed no signs of neutrophilic differentiation. Parental 32D.cl8.6 cells and cells transfected with empty LNCX vector also died within 1–2 d in G-CSF–containing medium. However, the 32D[WT] cells proliferated in response to G-CSF for 6–7 d . After 6–8 d, these cells developed into terminally differentiated neutrophils, showing an enlarged cytoplasm/nucleus ratio, lobulated nuclei, and neutrophilic cytoplasm . In contrast, 32D[mAR] cells showed an almost complete block in G-CSF–mediated proliferation and reduced survival . Since the bulk of the 32D[mAR] cells fail to survive past 6 d, it is difficult to accurately quantitate the effect of the P206H mutation on differentiation. However, most 32D[mAR] cells surviving until day 7 showed clear signs of neutrophilic differentiation . This suggests that differentiation signals per se are not severely affected by the receptor mutation. To further investigate the selective abrogation of G-CSF–mediated proliferation and survival signals by the mutant receptor, we analyzed STAT activation, since STAT3 and STAT5 have been strongly implicated in control of G-CSF–mediated differentiation and proliferation/survival, respectively 23 24 . In addition, STAT activation represents a sensitive measure of receptor activation. This analysis revealed that, compared with 32D[WT] control cells, 32D[mAR] cells showed reduced activation of all STAT proteins and a delay in the time of peak activation from 10–15 min to 30 min, indicating defective receptor activation . Interestingly, however, the quantitative effect on STAT1 and STAT5 activation was significantly greater than on STAT3. Examination of the dose–response of STAT activation showed a decrease in sensitivity for STAT activation from the mutant receptor, with again a larger effect on STATs 1 and 5 than on STAT3 . We also examined activation of the Ras–mitogen-activated protein kinase (MAPK) pathway by examining extracellular signal regulatory kinase (Erk) phosphorylation. This was also both delayed and reduced in cells expressing mutant receptors (data not shown), suggesting that different pathways are altered to varying degrees by the receptor mutation. Given the drastic effects of the P206H mutation on both signaling pathways and biological responses from the G-CSF-R, as well as its proximity to the ligand binding sites on the receptor 41 42 43 , we investigated whether altered ligand binding could explain our results. Scatchard analyses on 32D[WT] and 32D[mAR] clones showed that ligand binding affinity ( K d ) was unaffected by the mutation ( Table ). However, it was apparent in the analyses that the 32D[mAR] clones showed decreased total ligand binding. To quantitate this more precisely, we analyzed a pair of clones almost exactly matched for receptor levels as determined by FACS ® with anti–G-CSF-R antiserum. This revealed a clear reduction in total binding sites on mAR receptors, to approximately half that on wild-type G-CSF-Rs ( Table ). We also performed similar analyses on Ba/F3 clones expressing WT and mAR receptors, and obtained similar results ( Table ). As a further independent test of the relative ligand binding properties of the wild-type and mutant G-CSF-Rs, we compared binding of biotinylated G-CSF to those 32D clones that expressed nearly identical levels of WT or mAR receptors by anti–G-CSF-R binding. Again there was a clear reduction in the quantitative binding of ligand to mAR receptors . The data above suggested that mutant mAR receptors have an altered stoichiometry of ligand–receptor complex formation, which may be responsible for their altered signaling properties. To directly test this hypothesis, we stimulated 32D[WT] cells with saturating and nonsaturating G-CSF levels, which should favor formation of different receptor complexes (see Discussion). This analysis revealed that the use of nonsaturating G-CSF concentrations (0.3 ng/ml, which is >30 times lower than the receptor K d ) elicits responses that largely mimic the P206H mutant phenotype. At this concentration, cells showed decreased proliferation and survival , although again some differentiated cells were observed (data not shown), while STAT activation, particularly of STATs 1 and 5, was decreased and delayed . Since the patient harbors a heterozygous mutation in the GCSFR gene, we sought to coexpress both wild-type and mutant receptors in the same cells in order to more accurately recapitulate the clinical situation. To achieve this, we recloned both WT and mAR GCSFR cDNAs separately into the retroviral expression vector pBabe, which harbors a puromycin-resistance gene. Infectious retrovirus was subsequently produced using the Phoenix A amphotropic packaging line, and used to infect 32D[WT Neo ] cells, which express the wild-type G-CSF-R from the neomycin-resistance encoding pLNCX vector. Subsequent bulk selection on puromycin yielded cells expressing just wild-type receptor (32D[WT Neo /WT Puro ] cells) or coexpressing wild-type and mutant receptors (32D[WT Neo /mAR Puro ] cells). FACS ® analysis of these bulk cultures revealed an increase in G-CSF-R expression due to the introduction of the pBabe retrovirus . Northern blot analysis confirmed the presence of GCSFR transcripts from both pLNCX and pBabe expression vectors (data not shown). When these cultures were switched from IL-3 to G-CSF, the 32D[WT Neo /WT Puro ] cells showed substantially more proliferation than 32D[WT Neo /mAR Puro ] cells, especially at low G-CSF concentrations . Finally, analysis of G-CSF–induced STAT5 activation in these cells showed that the presence of the mutant receptor also suppressed the wild-type receptor signal in this regard, producing an altered dose–response of activation . SCN is a heterogeneous disorder defined by a severe reduction in blood neutrophil count—the result of a maturation arrest at the promyelocyte/myeloid stage defect in the bone marrow 1 2 3 . The molecular mechanisms leading to SCN remain largely unknown. However, G-CSF treatment can restore granulopoiesis in the majority of SCN patients, which suggests that defects in G-CSF signal transduction might be important in the etiology of the disease. Indeed, in a subset of these G-CSF–responsive SCN patients, mutations have been identified in the cytoplasmic domain of the G-CSF-R that generate truncated receptors defective in G-CSF maturation signaling 25 26 27 28 34 . Although these mutations correlate well with leukemic progression from SCN, their exact contribution to neutropenia is still controversial. The mutation described in this report represents a novel G-CSF-R mutation affecting the extracellular domain of the receptor in an SCN patient who was hyporesponsive to G-CSF treatment. Introduction of this P206H mutant receptor into myeloid cells reproduces the hyporesponsiveness to G-CSF, severely affecting proliferation and survival, although not differentiation, responses to G-CSF. These data further highlight the importance of GCSFR mutations in the pathophysiology of SCN, and also for the first time implicate such mutations in hyporesponsiveness to G-CSF therapy. In addition, the mutation also severely, but selectively, abrogates signaling from the G-CSF-R, apparently due to altered ligand–receptor complex formation, which contributes to our understanding of cytokine receptor function. How does the P206H mutation lead to the observed defects in receptor function? It is clear that the mutation affects ligand–receptor stoichiometry, such that the number of ligand binding sites per receptor is reduced, suggesting that the mutation alters the molecular architecture of the receptor complex. Using purified G-CSF-R, Fukunaga et al. have shown that high-affinity G-CSF-Rs consist of oligomers 44 . From studies with isolated recombinant domains, it appears that G-CSF can bind to the isolated BN and BC subdomains of the G-CSF-R CRH region in a 1:1 stoichiometry 42 43 . However, unlike other receptors, the G-CSF-R requires its Ig domain in addition to its CRH domain for high-affinity binding and oligomerization 45 46 . From these and other studies, Hiraoka et al. have proposed a model for the interaction of G-CSF with its receptor—at low ligand concentrations, an asymmetric 2:1 receptor–ligand complex is formed, whereas at high G-CSF concentrations, this converts to a 4:4 receptor–ligand tetramer 45 . In contrast, Horan et al. have implicated a conversion instead from a 2:1 to a 2:2 receptor–ligand complex depending on G-CSF concentration 47 48 . Regardless of the exact composition, this ligand concentration–dependent transition to a higher order complex involving the Ig domain contrasts with other hematopoietin receptors, such as growth hormone receptor, which only requires its CRH region to form a simple, symmetrical 2:1 receptor–ligand complex 13 49 . In addition, we have recently shown that the ligand sensitivity for STAT3 activation is ∼1 log higher than for activation of STATs 1 and 5 from the wild-type G-CSF-R 22 . This suggests that different intracellular signaling complexes are also formed depending on ligand concentration: at low concentrations, one that can activate STAT3, and at high concentrations, one that can activate all three STATs. The P206H mutation we have identified lies in a short connecting loop or hinge between the BN and BC subdomains of the CRH region 39 . From the structures of the CRH region from the closely related gp130 receptor component 50 and the isolated BC domain of the G-CSF-R 51 , we can expect this mutation to impact on the β-sheet angles as well as on the relative orientation of the BN and BC domains. In addition, we have shown that this mutation leads to a reduction in ligand binding sites, and produces a drastic effect on signaling and biological responses. In light of this data and the studies detailed above, we would propose the following model for the signaling complexes formed with wild-type and P206H mutant receptors at different ligand concentrations . At low ligand concentration, wild-type receptors form a 2:1 complex that can activate a subset of signaling pathways, including STAT3 (weak signal). At high ligand concentration, there is a shift to a higher order complex (2:2 or 4:4), which can activate all pathways, including STATs 1 and 5 (strong signal). In contrast, the mAR receptor is able to form the asymmetric 2:1 complex normally, but the P206H mutation perturbs the receptor in a manner that blocks higher order binding. This would explain the 50% reduction in binding sites per mutant receptor in spite of an unaltered receptor K d , as well as the ability of the mutant to elicit only weak intracellular signals (such as STAT3), i.e., equivalent to the complex formed when the wild-type receptor is stimulated at low G-CSF concentration, with similar biological outcomes. Consistent with this model, a G-CSF-R mutant in which the Ig domain was deleted, which could presumably form a 2:1 but not a higher order complex, also retained a very weak signal transducing ability 41 . Recently, the X-ray structure of the EpoR extracellular domain has been solved 52 53 . These studies have revealed that the EpoR is present as a dimer in the absence of ligand, with ligand binding inducing a conformational change of the dimer necessary for signal transduction. Importantly, this is accompanied by an alteration in the interdomain angle between the BN and BC subdomains. Our data are consistent with this model, and further suggest that there may indeed be a two-step conformational change in the G-CSF-R as more ligand binds to the receptor complex, the latter of which is impaired by the P206H mutation, which lies in a prime position to perturb the interdomain angle. Our suggestion of a ligand concentration–dependent switch in complex formation of the wild-type receptor may also have important consequences for understanding the control of basal versus “stress” granulopoiesis from the G-CSF-R. Recent data have implicated STAT3 activation in the control of differentiation from the G-CSF-R 22 23 , whereas STAT5 seems important in the control of G-CSF–mediated proliferation and survival 24 . Thus, we would envisage a switch from a differentiation function (STAT3) at basal G-CSF levels, to a proliferative/survival function (STAT5, and other pathways?) when G-CSF levels are elevated to facilitate a rapid expansion of the granulocytic compartment, such as in response to infection. The identification of a mutation in the extracellular domain of the G-CSF-R in an SCN patient unresponsive to normal G-CSF treatment has important ramifications. First, PCR screening for GCSFR mutations in SCN is currently confined to the cytoplasmic region of the receptor. Our data suggest the need for a more complete analysis, particularly in “nonresponders” to G-CSF. Second, as our understanding of the biological consequences of different G-CSF-R or other mutations in SCN increases, it would seem appropriate in the future to prescribe different treatments to different categories of SCN patients. In this regard, it will be of interest to determine whether the use of prednisone in combination with G-CSF may be of benefit for other patients hyporesponsive to standard G-CSF therapy. | Clinical case | clinical | en | 0.999995 |
10449522 | pEGFP-N1 (Clontech) encodes EGFP, a bright red–shifted variant of GFP containing the amino acid substitutions phenylaline-64→leucine and serine-65→threonine of GFPmut1 19 . An mCD14–EGFP gene fusion was constructed in pEGFP-N1 in two steps. First, a 1.2-kb BamHI fragment coding for the entire CD14 protein minus the last eight COOH-terminal residues was inserted into BamHI-digested pEGFP-N1, yielding pCD14–EGFP. Second, a 116-bp BsrGI–NotI fragment, encoding the 36 COOH-terminal residues of decay accelerating factor (DAF) and a stop codon, was inserted into BsrGI- plus NotI-digested pCD14–EGFP, yielding pCD14–EGFP–GPI. The amino acid sequence of the fusion protein coded by this construct is shown . Similarly, an mEGFP gene fusion was constructed by first inserting a 116-bp BsrGI–NotI fragment, encoding the 36 COOH-terminal residues of DAF and a stop codon, into BsrGI- plus NotI-digested pEGFP-N1, yielding pEGFP–GPI. Then, a 134-bp SalI–BamHI fragment coding for the first 19 NH 2 -terminal residues, the signal peptide (SP) of CD14, was inserted into SalI- plus BamHI-digested pEGFP–GPI, yielding pSP–EGFP–GPI. The sequence of the fusion protein coded by this construct is shown . These constructs placed the mCD14–EGFP and mEGFP chimeras under the control of the cytomegalovirus promoter/enhancer and permitted the selection of stable clones using geneticin. The 1.2-kb BamHI fragment encoding most of CD14 was synthesized by PCR using pcDNAI-neo-CD14 as a template 20 and the primers 5′-GAG atg gat cca cca tgg agc gcg cgt cct gc-3′ and 5′-GAG ATG GAT CCA GCA CCA GGG TTC CCG A-3′. The 116-bp BsrGI–NotI fragment encoding part of DAF was synthesized by RT-PCR using total RNA from human monocytes as a template and the primers 5′-aat atg tac aat aaa gga agt gga acc ac-3′ and 5′-taa agc ggc cgc taa gtc agc aag ccc at-3′. The 134-bp SalI–BamHI fragment was synthesized by RT-PCR using total RNA from human neutrophils as a template and the primers 5′-ACG CGT CGA CGC CGC TGT GTA GGA AAG-3′ and 5′-CGC GGA TCC GCA GAG ACG TGC ACC Aat-3′. All syntheses were followed by digestion with the appropriate restriction enzymes and gel purification. Both RT-PCR amplifications were performed using the Gene Amp RNA PCR kit purchased from Perkin-Elmer Corp. The PCR insertions were sequenced to confirm the absence of PCR synthesis errors. U373 cells were grown as monolayers in RPMI (BioWhittaker, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Inc.), penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively), and 2 mM glutamine. For making stable transfectants, 10 5 cells from a confluent culture of U373 cells were seeded on a 35-mm cell culture dish and grown to subconfluence for 24–48 h before transfection with either pCD14–EGFP–GPI or pSP–EGFP–GPI. For each dish, 1–2 μg of highly purified expression plasmid was used for transfection with 6 μl lipofectamine (GIBCO BRL) according to the manufacturer's instructions. The DNA–lipofectamine mixture remained on the cells for 6 h at 37°C and was then replaced by RPMI with 10% FBS and 2 mM glutamine without antibiotics. 72 h after transfection, the cells were trypsinized, plated at clonal density, and selected with 0.5 mg/ml geneticin (GIBCO BRL). After 3 wk, surviving cell colonies were visually screened for fluorescence. Several positive clones were identified, isolated using cloning rings, and expanded into cell lines for further analysis. U373–CD14 cells were obtained by selecting clones of U373 cells stably transfected with pcDNAI-neo-CD14 as described elsewhere 20 . Recombinant human sCD14 was purified from conditioned medium of Schneider-2 insect cells transfected with cDNA encoding human CD14 as previously described 21 . LPS from Salmonella minnesota R595 was purchased from List Biological Labs. The fluoroprobe BODIPY 558/568 (Molecular Probes, Inc.) was conjugated to unlabeled LPS micelles as previously described 22 . LPS–sCD14 and BODIPY–LPS–sCD14 complexes were formed by incubating LPS or BODIPY–LPS (20 μg/ml), respectively, with sCD14 (500 μg/ml) overnight at 37°C in Dulbecco's PBS (BioWhittaker, Inc.) with 0.5% pyrogen-free human serum albumin (Centeon, Armour, and Berring Pharmaceutical Co.). Previous work has shown that under these conditions all of the LPS forms stoichiometric complexes with monomeric sCD14 and that these complexes efficiently stimulate cells and deliver LPS to the plasma membrane 2 3 . BODIPY–LPS aggregates were prepared by incubating BODIPY–LPS at 1 μg/ml in FBS for 10 min at 37°C. The aggregation state of LPS was verified by monitoring its fluorescence emission at 568 nm before and after the addition of detergent, as described elsewhere 22 . Adding 2% SDS to the BODIPY–LPS aggregates led to a 10-fold increase in fluorescence due to the loss of self-quenching as monomers were released from the aggregates. In addition, aggregates observed directly by fluorescence microscopy exhibited a pointillistic pattern of fluorescence, rather than the very diffuse fluorescence seen with BODIPY–LPS–sCD14 complexes. U373 transfectants were cultured for 24–48 h before experiments in RPMI without phenol red (BioWhittaker, Inc.) supplemented with 10% FBS, antibiotics, and 2 mM glutamine on glass chamber slides (Nunc, Inc.) precoated with 0.5% gelatin (Sigma Chemical Co.). The cells were washed twice in HAP buffer (Dulbecco's PBS, 0.05% human serum albumin, and 3 mM d -glucose, containing 0.5 U/ml of aprotinin) and incubated in HAP at 37°C with or without LPS–sCD14, BODIPY–LPS–sCD14, or BODIPY–LPS aggregates. Slides were washed twice with HAP and further incubated at 37°C. At the end of the incubation, the plastic chamber and silicone gasket were removed, and the slide was mounted in HAP for immediate microscopic observation. For removal of cell surface mCD14–EGFP with phosphatidyl inositol phospholipase C (PI-PLC), cells on slides were incubated for 1 h in 20 mM Hepes, pH 7.4, and 150 mM NaCl on ice with 5 U/ml of PI-PLC (Boehringer Mannheim). When anti-CD14 mAb 26ic or 60b 23 was used, cells were incubated with the antibody at 10 μg/ml in HAP buffer at 4°C for 30 min and washed twice with ice-cold HAP and once with HAP at 37°C just before adding the BODIPY–LPS–sCD14 complexes. Confocal scanning laser microscopy was performed using a Nikon Optiphot-2 microscope with a ×60 objective (NA 1.4) and Bio-Rad MRC 1024 instrumentation with a krypton/argon laser. Unless otherwise noted, each image represents a single Kalman averaged (6–10 scans) optical section collected with a 2–3-mm-diameter iris aperture. Optical sections were collected digitally and analyzed using LaserSharp software (Bio-Rad Labs.). For two-color images, each color was acquired sequentially. This was necessary because EGFP has a broad peak of fluorescence, and some signal bleedthrough was observed in the BODIPY–LPS channel when simultaneous collection was attempted. Quenching of cell surface fluorescence by trypan blue was employed to both quantitate and observe the distribution of intracellular mCD14–EGFP and mEGFP. U373 transfectants were grown to confluence in a 96-well culture plate and, after the experiment, the total fluorescence associated with the cells was measured using a Cytofluor 4000 (PE Biosystems) (excitation 485 nm, emission 530 nm). Trypan blue (200 μg/ml, ambient temperature) was added to the wells to quench fluorescence from cell surface EGFP, and the remaining fluorescence from intracellular mCD14–EGFP or mEGFP was immediately measured. Intracellular fluorescence is expressed as a percent of total fluorescence from triplicate samples. Trypan blue was also used to quench cell surface EGFP on U373 transfectants before observing the cells by confocal microscopy. After the experimental manipulation, U373 transfectants cultured on glass chamber slides were washed once and mounted in HAP containing 200 μg/ml trypan blue before immediate observation. U373 transfectants grown in 96-well cell culture plates were washed extensively with AIM-V serum–free medium (GIBCO BRL) and incubated as indicated in AIM-V medium containing 0.5 mg/ml human serum albumin. After 16 h at 37°C, the overlying medium was collected from each well and assayed for IL-6 by ELISA as described 24 . For each condition, 10 6 cells were lysed by incubation on ice for 20 min in 300 μl of 100 mM Tris/HCl, pH 8.0, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.3 U/ml aprotinin, 2 mM PMSF, 3 mM diisopropyl fluorophosphate, 50 μg/ml benzamidine, and 5 μg/ml each of antipain, leupeptin, chymostatin, and pepstatin A. Lysates were centrifuged for 15 min at 12,000 g , and the supernatants were prepared for SDS-PAGE under reducing conditions. SDS-PAGE was run on a 4–20% gradient Tris–glycine gel (Novex). Proteins were electrotransferred to nitrocellulose membranes and detected with either an anti-EGFP rabbit pAb (Clontech) or an anti-CD14 pAb 25 . Horseradish peroxidase–conjugated goat anti–rabbit IgG was used as the secondary antibody, and the enzymatic reaction was detected with an ECL kit (Amersham Corp.). Purified rEGFP was obtained from Clontech. To generate a fluorescently tagged CD14 that was attached to the membrane via a GPI anchor, we fused a sequence coding for CD14 to one coding for EGFP and added the sequence for the 36 COOH-terminal residues of DAF. The chimeric protein resulting from the gene fusion pCD14–EGFP–GPI, shown in Fig. 1 , contains the NH 2 -terminal signal peptide of CD14 and the COOH-terminal signal peptide of DAF. Thus, when it was expressed and processed, it would translocate normally into the endoplasmic reticulum and have a GPI anchor attached. For a control, we also engineered a chimeric EGFP with a GPI anchor that contained the NH 2 -terminal signal peptide (SP) of CD14 and the COOH-terminal signal peptide of DAF. This second chimera was used to compare the distribution of EGFP-tagged CD14 (mCD14–EGFP) with a generic GPI-anchored protein (mEGFP). The two gene fusion products were transfected into the U373 astrocytoma cell line, which does not express mCD14. Stable transfectants for mCD14–EGFP and mEGFP were designated U373–CD14–EGFP and U373–EGFP, respectively. One clone of U373–CD14–EGFP was used for the results presented, but a second clone gave identical results. Expression of mCD14–EGFP as an intact polypeptide in U373–CD14–EGFP was tested in Western blots of cell lysates run in parallel with rsCD14 and EGFP. Antibodies against either CD14 or EGFP recognized the appropriate control protein and the same single band at 80 kD in U373–CD14–EGFP . The band corresponded to a protein of the expected mass for mCD14–EGFP and was not present in cell lysates of untransfected U373. Thus, mCD14–EGFP was expressed in U373 cells as an intact polypeptide, and we can be confident that by observing EGFP fluorescence we are also observing the location of mCD14. We further confirmed by confocal microscopy that both mCD14–EGFP and mEGFP were expressed on the surfaces of U373 cells. Optical sections of live U373–CD14–EGFP revealed fluorescence associated with the plasma membrane that was relatively uniform in distribution . Fluorescence was also observed on fine, filamentous projections from the cell surface. Intracellular mCD14–EGFP was observed in a juxtanuclear reticulum, a structure characteristic of the Golgi apparatus. A similar labeling pattern was observed with U373–EGFP (data not shown), suggesting that this is a normal distribution of GPI-anchored proteins in U373 cells. To confirm that the mCD14–EGFP was attached to the plasma membrane via a GPI anchor, we digested U373–CD14–EGFP cells with PI-PLC, which cleaves GPI-anchored proteins from their GPI anchors, before observing them by confocal microscopy. After 1-h treatment at 4°C with PI-PLC, cell surface fluorescence on U373–CD14–EGFP was barely detectable (not shown), suggesting that most of the cell surface associated mCD14–EGFP was removed. U373 astrocytoma cells do not respond to LPS alone, but when LPS is added in the presence of sCD14, they produce IL-6 26 . Transfection of U373 cells with mCD14 allows responses to LPS in the absence of sCD14. More importantly, it greatly increases the sensitivity of these cells to LPS–sCD14 complexes and allows more rapid responses to LPS–sCD14. This is consistent with observations that mCD14 on neutrophils and monocytes is necessary for responses to LPS in the absence of sCD14 and greatly increases the sensitivity of their responses to LPS–sCD14 complexes 2 9 . To show that the mCD14–EGFP chimera was functional, we measured secretion of IL-6 by U373–CD14–EGFP and U373–EGFP cells in response to either LPS or LPS–sCD14 complexes. The U373–EGFP cells did not respond to LPS alone up to 100 ng/ml . In contrast, expression of mCD14–EGFP in U373 cells led to IL-6 production in response to concentrations of LPS of 1 ng/ml or higher. Similarly, U373–CD14–EGFP cells responded to concentrations of LPS–sCD14 ∼100-fold lower than those required to elicit the same response in U373–EGFP cells . The enhancement of sensitivity by mCD14–EGFP was quantitatively similar to that previously observed with mCD14. Thus, the presence of EGFP does not affect the function of mCD14–EGFP on cells. To determine the time of exposure required to elicit a response to LPS, U373–CD14–EGFP and U373–EGFP cells were given a fixed concentration of LPS (40 ng/ml), either alone or in sCD14 complexes, for increasing intervals of time before washing and incubation to allow synthesis of IL-6. U373–EGFP cells were essentially unresponsive to LPS alone after up to 2 h of exposure . As previously seen with mCD14, the presence of mCD14–EGFP dramatically enhanced the magnitude and speed of cellular responses to LPS. In particular, after as little as 5 min of exposure to LPS, U373–CD14–EGFP cells responded with IL-6 production to both LPS–sCD14 complexes and LPS aggregates (data not shown). Taken together, these results suggest that mCD14–EGFP was able to bind LPS and mediate cellular responses to it. All of the properties exhibited by U373–CD14–EGFP are the same as those of mCD14 expressed in U373, indicating that mCD14 expressed as a chimera with EGFP was still a functional entity. These results further suggest that the distribution and trafficking pattern of mCD14–EGFP in U373 would reflect that of a fully functional protein. The association of LPS with mCD14–EGFP was confirmed by confocal microscopy. BODIPY–LPS in the form of monomeric complexes with sCD14 was incubated with U373–CD14–EGFP for 2–3 min at 37°C before washing and observation. Fluorescence from BODIPY–LPS was seen associated with the plasma membrane of U373–CD14–EGFP , indicating that it was successfully transferred from sCD14. There was colocalization of BODIPY–LPS with mCD14–EGFP on the cell surface, as demonstrated by the overlap in fluorescence signals . The same cell surface distribution of BODIPY–LPS was observed with U373 transfectants expressing mCD14 without EGFP attached . In contrast, U373–EGFP cells incubated briefly with BODIPY–LPS–sCD14 complexes did not have detectable BODIPY–LPS fluorescence associated with their cell surfaces . To confirm that the transfer of BODIPY–LPS from sCD14 was mCD14 dependent, we pretreated U373–CD14–EGFP cells with a blocking anti-CD14 mAb, 60b 3 . The antibody prevented binding of BODIPY–LPS, and no colocalization with mCD14–EGFP was observed . Colocalization of BODIPY–LPS and mCD14–EGFP was still observed when the cells were pretreated with 26ic, a nonblocking anti-CD14 antibody 3 . Thus, colocalization of BODIPY–LPS and mCD14 required binding of LPS to mCD14. To determine whether LPS remained bound to mCD14 after internalization, we observed the trafficking of mCD14–EGFP and BODIPY–LPS simultaneously in live cells. U373–CD14–EGFP cells were incubated with BODIPY–LPS–sCD14 complexes for 2–3 min at 37°C, the complexes were removed by washing, and the cells were incubated further at 37°C before observation. Particular attention was devoted to observations made 5–10 min after the incubation began. At the earliest time points, BODIPY–LPS was clearly visible in small intracellular vesicles . These vesicles were visible at times as early as 5 min (data not shown). However, the vesicles containing BODIPY–LPS did not contain mCD14–EGFP, as evidenced by the lack of overlap in fluorescent signals . This indicates that in the brief time required for LPS to reach a vesicular location, LPS had separated from mCD14. At later times, vesicles containing BODIPY–LPS accumulated predominantly in the perinuclear area . Although by 60 min a punctate localization of mCD14–EGFP in the same vicinity was observed , repeated observations with LPS concentrations between 10 and 200 ng/ml demonstrated that colocalization of BODIPY–LPS and mCD14–EGFP in this area did not occur (not shown). LPS concentration therefore had no effect on its localization. Thus, not only did LPS leave mCD14 upon entering the cells, but it also did not reassociate with mCD14 in any intracellular compartment. The kinetics of BODIPY–LPS internalization in U373–CD14–EGFP were identical to those in U373 expressing mCD14 without EGFP (data not shown). Furthermore, the distribution of intracellular BODIPY–LPS in vesicles and their subcellular localization was the same in both cell types (data not shown). This indicates that the presence of EGFP on mCD14 did not disturb the normal trafficking pattern of LPS in U373 cells. Although there was no apparent change in the distribution of mCD14–EGFP in response to LPS in the colocalization studies, the bright cell surface fluorescence prevented observation of any changes in the distribution of intracellular mCD14–EGFP. To better observe the distribution of intracellular mCD14–EGFP, we quenched the cell surface fluorescence on U373–CD14–EGFP cells with trypan blue . In addition to the bright fluorescence emanating from the Golgi apparatus area, numerous fluorescent vesicles of ∼50–100-nm average diameter were distributed throughout the cytoplasm. The vesicles were particularly evident near the basal aspects of the cells . A similar intracellular localization of mEGFP in the Golgi complex and cytoplasmic vesicles was also observed (data not shown). Thus, the vesicular compartment may represent either a component of the secretory pathway en route to the cell surface or a recycling compartment for GPI-anchored proteins. The effect of LPS on the intracellular distribution of mCD14–EGFP was examined by adding LPS–sCD14 complexes to U373–CD14–EGFP cells and observing mCD14–EGFP localization by confocal microscopy after trypan blue quenching. The localization of mCD14–EGFP in the Golgi complex was unaffected by LPS stimulation . However, after addition of LPS, we observed a steady decrease in the number of mCD14–EGFP-containing vesicles from the earliest times observed (2–5 min), with almost no mCD14–EGFP-containing vesicles remaining after 45–60 min , suggesting that the compartments containing mCD14–EGFP were exocytosed upon exposure of the cells to LPS. Although the resolution of these experiments does not allow us to rule out the possibility that a small fraction of mCD14–EGFP was internalized, the decline in the number of intracellular vesicles suggests that exocytosis was the preferred route. A similar loss of fluorescent vesicles was observed in U373–EGFP cells exposed to LPS–sCD14 complexes for 45–60 min (data not shown), indicating that the vesicle pool in question is not defined by the presence of CD14 per se. These results suggest that LPS either induces the release of a compartment in U373 cells that contains GPI-anchored proteins or that it prevents the reinternalization of a recycled pool of membrane containing GPI-anchored proteins. Thus, rather than being internalized with LPS from the cell surface, mCD14 apparently moved to the cell surface from an intracellular store in response to LPS. Exocytosis of the mCD14-containing vesicles was confirmed by quantitative measurements of the time course. U373–CD14–EGFP cells were grown in 96-well tissue culture plates, and both total and intracellular fluorescence was measured before and at various times after exposure to LPS–sCD14 complexes (see Materials and Methods). Stimulation with LPS did not induce any change in total fluorescence associated with the cells (data not shown) but did induce a rapid decrease in intracellular mCD14–EGFP . A decrease of ∼20% in the intracellular fluorescence was observed after a 15-min incubation of U373–CD14–EGFP cells with LPS–sCD14 complexes. A similar decrease in intracellular fluorescence associated with U373–EGFP cells was observed in response to LPS–sCD14 complexes , indicating that the compartments released contained GPI-anchored proteins in addition to mCD14–EGFP. LPS aggregates are formed when LPS micelles and LBP are incubated with little or no sCD14. These LPS–LBP aggregates can bind to mCD14 on the surfaces of cells, and they are subsequently internalized 27 28 29 . However, binding of LPS–LBP aggregates by mCD14 differs from binding of monomeric LPS presented as a complex with sCD14. Aggregates bind at 4°C, but monomeric LPS cannot be transferred from sCD14 to mCD14 at this temperature. In addition, although internalization of monomeric LPS correlates with intracellular signaling 13 14 , internalization of LPS aggregates can be dissociated from the generation of signals 27 . These results suggest that LPS aggregates may have a pathway for internalization that is distinct from that of monomeric LPS. To determine whether mCD14 traffics with LPS aggregates during internalization, we incubated U373–CD14–EGFP with BODIPY–LPS aggregates at concentrations between 40 and 100 ng/ml and followed both BODIPY–LPS and mCD14–EGFP fluorescence by confocal microscopy. After incubation for 5–10 min at 37°C with BODIPY–LPS aggregates, the cells were washed and incubated further at 37°C. The aggregates bound to the cell surface and colocalized with mCD14–EGFP . At least 15 min at 37°C was required to detect BODIPY–LPS in intracellular vesicles, suggesting that internalization of LPS aggregates was somewhat slower than internalization of LPS monomers in U373–CD14–EGFP. The mCD14–EGFP colocalized with BODIPY–LPS in intracellular vesicles detected at the earliest times, although not all of the vesicles that contained BODIPY–LPS also contained mCD14–EGFP. These results suggest that, in contrast with LPS monomers, LPS aggregates can remain bound to mCD14 during internalization. This supports the idea that there is more than one pathway for internalization of LPS. Here we have used a chimeric construct of mCD14 with EGFP transfected into the astrocytoma cell line U373 as a probe for observing the distribution of mCD14 in living cells. Labeling mCD14 directly with a fluorescent tag avoided any perturbation of the distribution of mCD14, a GPI-anchored protein, that might be caused by antibody cross-linking 30 . A variety of functional studies confirmed that the mCD14–EGFP chimera was an intact and functional protein that was attached to the cell surface via a GPI anchor. Using this method, we observed that mCD14 was not only expressed on the cell surface but was also present intracellularly both in the perinuclear area and in small vesicles near the basal aspect of the cells. These vesicles might represent compartments in which mCD14, and perhaps other GPI-anchored proteins, traffic from the Golgi apparatus to the plasma membrane. They may also represent a recycling compartment for GPI-linked proteins that are internalized from the plasma membrane and then returned to it 31 . Most of these vesicles were exocytosed when U373–CD14–EGFP cells were exposed to LPS–sCD14 complexes, indicating that they are capable of fusion with the plasma membrane. The rapid exocytosis of the vesicles containing mCD14–EGFP upon stimulation of U373–CD14–EGFP cells with LPS was reminiscent of the exocytosis of secretory vesicles containing mCD14 and other GPI-anchored proteins upon stimulation of neutrophils with an agonist 25 . Secretory vesicles are thought to be an endocytic compartment, as they also contain the serum proteins albumin and tetranectin 32 33 . In response to formyl peptide, secretory vesicles are brought rapidly to the neutrophil surface, augmenting the expression of GPI-anchored proteins on the plasma membrane 25 . Additional studies will be required to determine whether any of the mCD14-containing vesicles in U373–CD14–EGFP cells represent a recycling compartment for GPI-anchored proteins similar to the secretory vesicles of neutrophils. Expression of mCD14–EGFP on the plasma membrane enabled uptake of BODIPY–LPS and cellular responses to LPS . CD14 binds LPS 34 and, not surprisingly, added LPS was found to colocalize with plasma membrane mCD14–EGFP . After association with the membrane, monomeric LPS is known to be rapidly internalized 13 14 . Here we show that when LPS was internalized, it moved into intracellular vesicles that did not contain mCD14. Thus, we have demonstrated in living cells that LPS, once it is bound by mCD14 on the cell surface, is internalized without being accompanied by its receptor. Together with our previous observations that each mCD14 on the surfaces of human monocytes enables the uptake of 15 LPS molecules in 30 min 3 and that this uptake depends on an additional cell surface protein 3 , these observations support a model for LPS trafficking that involves transfer of LPS from mCD14 to another cell surface protein or to the lipid bilayer of the plasma membrane. In addition to our studies with monomeric LPS, we have also observed the trafficking of LPS aggregates. Aggregated LPS may engage multiple copies of mCD14 on the cell surface at the same time, and it may be more difficult for mCD14 to transfer LPS from aggregates to the plasma membrane. Using the mCD14–EGFP chimera, we observed that mCD14 was internalized with aggregated LPS in U373 cells. This behavior is opposite to that of LPS monomers. It is, however, consistent with a variety of other studies documenting different fates of monomeric and aggregated LPS. After internalization, primarily through noncoated structures 35 , LPS aggregates move over the course of several hours into a compartment that is likely to be lysosomal in nature. There, acyloxyacyl hydrolase deacylates and thus detoxifies LPS 18 . Internalization of aggregates can be disassociated from signaling 27 29 and thus appears more relevant to the detoxification and clearance of LPS rather than signaling. Several observations indicate a close correlation between signaling and LPS transit to the Golgi complex. For example, inactive structural analogues of LPS are not transported to the Golgi complex 15 , and cells from Lps d mice, which exhibit a defect in LPS signaling, fail to transport LPS to the Golgi complex 14 . Recent work has shown that Lps d mice are defective in Toll-like receptor (TLR)4, a member of the IL-1 receptor family 36 . In this regard, it is interesting to note that the ligated IL-1 receptor type I (IL-1RI) may require trafficking to an intracellular compartment to generate signals 37 . A thymocyte cell line has been identified that is defective in its responses to IL-1 and also does not internalize IL-1RI. The defect can be overcome by intracellular delivery of IL-1 38 or by transfection with IL-1R accessory protein (IL-1RAcP), which restores both IL-1RI internalization 39 and IL-1 responses 39 . Interaction of IL-1 with IL-1RI and IL-1RAcP triggers a cascade of signaling events, including activation of the stress-activated, mitogen-activated protein (MAP) kinase pathways and transcription factor NF-κB. Of interest in this regard is that TLR4 shares homology in its cytoplasmic domain with the IL-1R family 40 , suggesting the possibility that it may also share similar directions of intracellular trafficking. Whether TLR4 colocalizes with LPS before or after transport to the Golgi complex, however, will have to await further studies. | Study | biomedical | en | 0.999998 |
10449523 | Aprotinin, defatted (DF)–BSA, and PMA were purchased from Sigma Chemical Co. Pyrogen-free human serum albumin (HSA) was obtained from Centeon, Armour, and Berring Pharmaceutical Co. TNF-α was purchased from Genzyme Corp. The mAbs used were 26ic (anti-CD14; reference 9 ), 3C10 (anti-CD14; reference 10 ), and 44a (anti-CD11b; reference 11), available from American Type Culture Collection (ATCC), and were purified from ascites fluid by chromatography on protein G. The anti-CD14 mAb MY4 was purchased from Coulter Immunology. Dulbecco's PBS, DME, AIM-V serum-free medium, FCS, penicillin, and streptomycin were purchased from BioWhittaker. Brefeldin A (BFA) was purchased from Epicentre Technologies. TRITC-labeled cholera toxin B subunit (TRITC–CTB) and unlabeled LPS from Salmonella minnesota R595 (LPS) were purchased from List Biological Labs. LysoTracker™ Red DND-99, BODIPY 558/568–BFA, DiOC6(3), TR–dextran, BODIPY–fluorescein-like (FL) ceramide, BODIPY 558/568 ceramide, TR–Con A, and TR–transferrin (Tf) were purchased from Molecular Probes, Inc. BODIPY–LPS was prepared using LPS from S. minnesota using BODIPY–FL and BODIPY–558/568 amine labeling kits (BODIPY–LPS; Molecular Probes, Inc.) as previously described 12 . The ratio of BODIPY/LPS molecules was estimated at 1:5. A 1:1 complex of BODIPY–C5-ceramide with DF–BSA was prepared as described 13 . The complex (5 μM) was prepared in acid-buffered Eagle's MEM, pH 7.4, without color indicator. Recombinant human sCD14 was purified from conditioned medium of Schneider-2 insect cells transfected with cDNA encoding human CD14 and was provided by Dr. R. Thieringer (Merck Research Laboratories). To deliver LPS as a monomer, preformed complexes of BODIPY–LPS with sCD14 were used. To prepare LPS–sCD14 complexes, 100 μg/ml sCD14 was incubated with 5 μg/ml LPS for 16 h at 37°C. Previous work has shown that under these conditions, all of the LPS forms stoichiometric complexes with monomeric sCD14 and that these complexes efficiently stimulate cells and deliver LPS to the membrane 2 5 . Heparinized blood was obtained by venipuncture from human healthy volunteers and PMN were purified on neutrophil isolation medium (Cardinal Associates, Inc.) according to the manufacturer's directions. Cells were suspended in HAP buffer (Dulbecco's PBS with 0.5 mg/ml HSA, 0.3 U/ml aprotinin, and 3 mM glucose). Human epithelial HeLa cell line was obtained from ATCC and were cultured in DME supplemented with 10% heat-inactivated FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. HeLa cells were plated in 96-well plates at a density of 100,000 cells/well 24 h before stimulation. The cells were washed extensively with AIM-V serum-free medium and then incubated with various stimuli in AIM-V medium containing 0.5 mg/ml of HSA. After 5 h, the supernatants were collected and stored at −20°C. Samples were assayed for the presence of IL-8 using a commercially available human IL-8 ELISA kit (Endogen, Inc.). Results are the mean values of triplicate wells ± SEM. Purified PMN were washed in HAP buffer and plated on glass microslides (Carlson Scientific, Inc.) for 20 min on ice and then exposed to LPS (100 ng/ml) and fluorescent probes for organelles for 30 min at 37°C. LPS was added complexed to sCD14. After the incubation, cells were washed in HAP and examined live by confocal microscopy. HeLa cells (10 6 cells/ml) were washed in DME containing 0.5 mg/ml HSA, incubated with the fluorescent markers at 37°C, and processed for microscopy. Analysis of BFA treatment was performed using HeLa cells incubated with TRITC–CTB to visualize the Golgi complex. BFA (1 μg/ml) was added after labeling of cells for 1 h at 37°C. Cells were viewed unfixed with a confocal laser scanning system. Confocal scanning laser microscopy was performed using a Nikon microscope equipped with a ×100 objective (NA 1.4) and Bio-Rad MRC 600 or MRC 1024 instrumentation. A dual wavelength laser was used to excite green (BODIPY or FITC) and red (BODIPY 558/568, TRITC, or TR) fluorochromes at 488 and 568 nm spectral line of an Ar-Kr laser, respectively. The fluorescence signals from the two fluorochromes were recorded sequentially. Confocal images presented were single optical sections. Images were analyzed using NIH image 1.6 (National Institutes of Health) and LaserSharp (Bio-Rad Labs.) software and were processed for presentation with Adobe Photoshop 3.0 (Adobe Systems, Inc.) and Corel Draw 6.0 (Corel Corp.). Lysosomes are acidic vesicles rich in hydrolytic enzymes and represent the site of degradation of extracellular macromolecules internalized by pinocytosis or phagocytosis. It has been shown that LPS aggregates move at least transiently into an acidic intracellular compartment of PMN, and deacylation by acyloxyacyl hydrolase occurs over several hours 14 15 . We have determined if LPS is internalized into lysosomes using a fluorescent, freely permeant probe with a high selectivity for acidic organelles, LysoTracker™ Red DND-99 16 . PMN were incubated with BODIPY–LPS (presented as BODIPY–LPS–sCD14) for 30 min at 37°C in the presence of LysoTracker™ Red DND-99, and live cells were observed by confocal microscopy. As previously reported, LPS appeared in a perinuclear area in a punctate or tubular pattern . In contrast, the fluorescent lysosomes were distributed throughout the cytoplasm of PMN, consistent with the numerous azurophilic granules contained in these cells. The compartment containing the internalized LPS was readily distinguishable from lysosomes. We cannot exclude the possibility that a small amount of BODIPY–LPS might be localized within lysosomes. However, our methods could not distinguish LPS fluorescence above background autofluorescence in positions coincident with lysosomes. According to these observations, the vesicular transport from the plasma membrane does not appear to deliver LPS to lysosomes. Activation of PMN with PMA or TNF-α induces fusion of specific granules with the cell membrane and upregulation of extracellular matrix receptors (laminin receptor, vitronectin receptor, and CD11b/CD18 antigens) on the cell surface, leading to leukocyte adhesion and extravasation 17 . PMA-stimulated PMN release the contents of both their specific and azurophilic granules. When PMN were incubated with both PMA and BODIPY–LPS–sCD14 complexes, the autofluorescence of the cells was increased . However, no modification in the punctate pattern of LPS fluorescence was observed, suggesting that LPS does not accumulate in specific granules. Endosomes are a structurally diverse population of vacuoles and tubules serving as sorting intermediates along both the biosynthetic and endocytic pathways, accumulating intraluminal membrane as they mature. Fluorescent dextran, an hydrophilic polysaccharide with poly-(α- d -1,6-glucose) linkages, is rapidly taken up by an endocytic process and moves to early and late endosomes 18 . We have studied the internalization of a fluid-phase marker (TR–dextran) and LPS in neutrophils. After 30 min at 37°C, TR–dextran appeared in an heterogeneous assortment of internalized vesicles as expected . BODIPY–LPS, on the other hand, was concentrated in an organelle distinct from TR–dextran , suggesting that LPS is not localized in late endosomes after 30 min. Nevertheless, there was partial colocalization of LPS vesicles with dextran, as manifested by the punctate yellow staining in the merged images in some cells. This partial colocalization was detected most often near the plasma membrane, suggesting that LPS may traffic via endosomes containing dextran. Vesicular traffic involves several steps with distinct kinetics, and intermingling of endocytic markers with LPS after sequential endocytic uptake may be dependent on the length of incubation. To further resolve the localization of LPS in endosomes, we examined the intracellular transport of endocytosed LPS in PMN using Tf to probe the recycling pathway. Fluorescent Tf (TR–Tf), which reaches sorting and recycling endosomes via clathrin-coated pits 19 , was localized in a compartment clearly different from the structure containing BODIPY–LPS. After 30 min of incubation at 37°C, TR–Tf was in fact preferentially excluded from the regions labeled with BODIPY–LPS–sCD14 . These observations indicate that the trafficking and sorting of LPS in neutrophils may occur in endosomes containing dextran and but not Tf. The endoplasmic reticulum (ER) is the largest endomembrane system within eukaryotic cells and performs a wide variety of functions, including calcium uptake and release, lipid and protein synthesis, protein translocation, folding, glycosylation, concentration, and export to the Golgi complex. In live cells, the flattened membranous sacs of the ER can be stained with DiOC6(3), a lipophilic fluorescent dye 20 . AS DiOC6(3) fluoresces green, we performed colocalization studies using LPS labeled with a BODIPY probe that fluoresces in the red part of the spectrum (BODIPY 558/568–LPS). Green or red staining thus illustrates the respective distribution of DiOC6(3)– and BODIPY 558/568–LPS. Fluorescent LPS was observed in vesicular structures distinct from the ER , suggesting that there was no direct transport of LPS to the ER from the cell surface. We have confirmed the absence of LPS in ER using a second marker. The fluorescently labeled fungal metabolite BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions 21 . After incubation of PMN with BODIPY– BFA, LPS was observed in vesicular structures clearly distinct from those stained with BFA . Con A binds with high affinity to immature structures that terminate in glucosyl, mannosyl, or mannosyl and N -acetyl glucosaminyl residues. Con A can label internal structures by uptake and trafficking and stains the rough ER and the dilated cisternae of the cis-Golgi apparatus side 22 . Fig. 1 F depicts confocal images of PMN that were treated with TR–Con A and BODIPY–LPS–sCD14 for 30 min at 37°C. There was partial colocalization of LPS with Con A in most of the cells, as manifested by the punctate yellow staining in the merged images. As there was no colocalization of LPS with the previous ER probes, these observations were consistent with the finding that LPS concentrates in the proximal side of the Golgi complex. The Golgi apparatus can be selectively stained with fluorescent ceramide, which tends to associate preferentially with the trans-Golgi complex 23 . After rapid transport to the Golgi apparatus, ceramide is metabolized to sphingomyelin, glucosylceramide, and further glycosphingolipids, suggesting that the pattern of distribution of fluorescently labeled ceramide may change over time in live cells. To stain cells with this probe, PMN were incubated first with BODIPY–LPS–sCD14 complexes for 20 min at 37°C and then BODIPY–ceramide–BSA was added for 5 min at room temperature. Cells were washed and subsequently warmed for 2 min at 37°C. Confocal microscopy studies showed that the punctate pattern of labeled cells was very similar with LPS or ceramide and that the brightly labeled LPS vesicles colocalized with ceramide fluorescence . This strong colocalization suggests that LPS accumulates in the Golgi region. Various glycolipid-binding toxins are internalized from the cell surface to the Golgi complex, and we have used CTB as marker of the Golgi apparatus 24 . CT consists of a pentameric B subunit, which binds with high affinity to ganglioside GM1, and an A subunit, which stimulates adenylate cyclase, resulting in the elevation of cAMP. CTB is internalized by vesicular transport from the plasma membrane to the Golgi apparatus and persists in this compartment. As previously observed with ceramide, the punctate pattern of TRITC–CTB was similar to that seen for fluorescent LPS. Merging magnified areas of optical sections of fluorescently double-labeled PMN confirmed that the brightly labeled LPS vesicles colocalized with fluorescent CTB . The specific Golgi subcompartment(s) containing LPS (or CTB) could not be identified, as they cannot be resolved by confocal fluorescence microscopy. Transport of LPS was quantified by counting fluorescent LPS vesicles, which overlap with specific probes of the intracellular compartment. Using LysoTracker™ Red DND-99 and TRITC–CTB as probes of lysosomes and the Golgi apparatus, respectively, we observed that 74% of LPS vesicles colocalized with CTB probe in neutrophils . In contrast, most LPS vesicles failed to colocalize with the lysosomal marker. These results suggest that, as seen in Fig. 1 , most LPS is directed to the Golgi apparatus. Although LPS appeared to be transported to the Golgi complex, which is also stained with CTB and ceramide, it was possible that the probes were in separate compartments that were unresolvable using confocal microscopy. This is a particular concern in the centers of the cells, where many organelles are concentrated. It is even more of an issue with neutrophils, which are small (12–14 μm in diameter) and have compact, spheroidal and juxtanuclear Golgi complexes. In contrast, epithelial cells exhibit an extensive, loose, and perinuclear Golgi apparatus, forming a heterogeneous ribbon-like structure connected to several networks of anastomosed membranous tubules or trans-Golgi network (TGN). The Golgi structure has been thoroughly investigated in epithelial HeLa cells, which are 100–140 μm in diameter 25 26 , and we therefore used this cell type to confirm the localization of LPS within the cell. HeLa cells take up a low amount of BODIPY–LPS–sCD14 after 30 min at 37°C, as detected by FACScan™ (Becton Dickinson; data not shown). Although the uptake of LPS from LPS–sCD14 complexes is relatively low, HeLa cells do respond to LPS–sCD14 complexes by secretion of IL-8 . This secretion was LPS dose– and CD14-dependent, as the cytokine response was completely inhibited by two neutralizing anti-CD14 mAbs, 3C10 27 and MY4 . IL-8 production was not inhibited by two control mAbs, 26ic, which recognizes CD14 without inhibiting binding of LPS or responses towards LPS 4 28 , and 44a, which binds CD11b molecules 11 . We have tested the ability of HeLa cells to internalize LPS. Cells were labeled for 60 min at 37°C with BODIPY–LPS and washed before observation by confocal microscopy. Fluorescent LPS was detected in a collection of elongate and punctate structures consistent with the location of the Golgi . Cells contained either a compact juxtanuclear reticulum, a structure characteristic of the Golgi apparatus, or dispersed tubulovesicular membranes, suggesting mitotic disassembly, fragmentation, and redistribution of the structure in living HeLa cells. We analyzed in parallel the location of LPS and Golgi apparatus labels. We observed that the distribution of LPS overlapped with that of both CTB–FITC and BODIPY–ceramide , confirming that most of the internalized BODIPY–LPS accumulated in the Golgi apparatus. It should be noted that dots of fluorescent LPS not overlapping with Golgi apparatus probes were also detected, suggesting that a fraction of internalized LPS may reside in endosomes, resembling the pattern of BODIPY–LPS observed in PMN . These results are consistent with the Golgi apparatus being the primary delivery site of LPS by endocytic membrane movement from the plasma membrane. These observations suggest that the trafficking and sorting of LPS in HeLa cells follows the same general scheme as in PMN: LPS internalization may occur through endosomes, initially accessible to the fluid phase marker dextran, and is further directed into vesicular/tubular parts of the Golgi apparatus. Treatment of cells with BFA is known to interfere with coat assembly and results in retrograde merging of Golgi complex membranes with the ER 29 . BFA blocks membrane export out of the ER and inhibits vesicle formation. This is due to BFA's inhibition of nucleotide exchange onto ADP ribosylation factor, a low-molecular-mass GTP-binding protein that prevents assembly of cytosolic coat proteins (including COP I components) onto target membranes. At the same time, extensive retrograde transport of Golgi complex components to the ER mediated by growth of Golgi tubules occurs with BFA, leading to the dilation of the ER and the complete loss of Golgi apparatus structure. In this study, we investigated the effect of BFA-induced retrograde membrane transport from Golgi apparatus to ER on LPS vesicles. HeLa cells were first labeled with both LPS and CTB probes for 1 h at 37°C and washed and then incubated with BFA for 1 h at 37°C before observation by confocal microscopy. In BFA-treated cells, the fluorescent LPS probe was found in scattered/fragmented cytoplasmic patches . The disassembly of the Golgi complex was visualized by the dispersed CTB fluorescence after BFA treatment. Fluorescent LPS frequently colocalized or was located in proximity to the dispersed CTB-positive structures. Our finding indicates that BFA induced disassembly of the Golgi complex and, in parallel, redistribution of LPS vesicles. These results confirm our conclusion that LPS localizes in the Golgi compartment. Here we show that biologically active LPS, delivered to cell membranes as monomers, rapidly colocalizes with vital stains of the Golgi apparatus. Furthermore, the LPS localization was disrupted after BFA-induced disruption of the Golgi complex. The colocalization was observed in two cell types , suggesting that trafficking of LPS to the Golgi complex is not unique to leukocytes and may be a general property of cells. A major function of the ER and Golgi apparatus is to package proteins for export, and membranes of these organelles must therefore move with their protein cargo toward the plasma membrane. Retrieval of the vesicular membrane is achieved by the process of “retrograde transport,” and we hypothesize that LPS utilizes this process to achieve movement to the Golgi complex. The pathway of retrograde transport involves the endosomal compartment 30 31 and, in keeping with this hypothesis, we have observed that LPS shows partial overlap with the endosomal marker, dextran . It is interesting to note that certain bacterial toxins such as cholera toxin, diphtheria toxin, Pseudomonas exotoxin A, Shiga toxin, or plant toxins such as ricin also utilize retrograde transport to reach their target intracellular compartments 24 30 . Surface-bound toxin enters cells by endocytosis, with the precise endocytic route depending on the nature of the receptor. The toxins are then carried on membranes that recycle between the plasma membrane and the TGN and between the TGN and the ER. After reaching the ER, the toxins cross the membrane and introduce a toxic enzyme into the cytoplasm. In a similar fashion, retrograde transport of LPS (endotoxin) via sorting endosomes may be responsible for its Golgi complex localization and perhaps its biological activity as well. As BFA disrupts the Golgi apparatus, it would be of interest to determine if it affects LPS signaling. Unfortunately, BFA is also a potent inhibitor of protein secretion and, therefore, we could not evaluate a potential effect of BFA treatment on cytokine production in response to LPS. Furthermore, previous work 32 has shown that BFA treatment strongly enhances nuclear factor (NF)-κB translocation in HeLa cells, thus precluding a study on LPS-induced NF-κB activation in BFA-treated cells. We wish to stress that the process observed here with monomeric LPS differs critically from that studied by a range of prior authors using LPS aggregates or whole Gram-negative bacteria 33 34 35 36 37 38 39 40 . Indeed, it appears that there are two distinct routes of LPS trafficking with two distinct biological outcomes: monomeric LPS is delivered to the Golgi apparatus, a process associated with cell stimulation, whereas LPS aggregates are delivered to lysosomes, a process not associated with cell stimulation. LPS aggregates and whole bacteria are clearly delivered to lysosomes 38 41 42 . Like other particulates, LPS aggregates and whole bacteria are carried as cargo in the lumen of endosomes or phagosomes. Delivery to lysosomes results in degradation of LPS, and the action of one particular lysosomal enzyme (acyloxyacyl hydrolase; reference 14) results in the transformation of LPS to a form that antagonizes the cell-stimulating capacity of complete LPS 43 . Receptors for IgG on macrophages are known to deliver antigen to lysosomes, and anti-LPS antibodies are known to hasten clearance and decrease cellular responses to LPS 44 . Finally, recent work indicates that during uptake of LPS aggregates, mCD14 accompanies LPS into the cell 45 . sCD14 delivers LPS to cells not as aggregates but as monomers. In contrast to aggregates, endocytosis of monomeric LPS occurs after dissociation from mCD14 45 , and it appears that this LPS is not carried in the lumen of a vesicle but rather as part of the membrane bilayer. Monomeric LPS is clearly not transported to lysosomes but moves instead to the Golgi complex. Finally, it is clear that LPS delivered as a monomer is biologically active. Responses to LPS are optimized when LPS is delivered as monomers with CD14 (references 2 and 4 and Le Grand, C.B., N. Lamping, T. Sugiyama, S.D. Wright, and R. Thieringer, manuscript submitted for publication) or when fused with the plasma membrane after incorporation into a viral envelope 46 . The studies reported here use LPS–sCD14 complexes designed to optimize the efficacy of LPS in cell stimulation and have resulted in predominant delivery of LPS to the Golgi complex. At the opposite pole, LPS may be delivered to cells in complex with IgG in such a way as to minimize cell stimulation 44 and presumably maximize delivery to lysosomes. We wish to stress that neither condition closely mimics infection. It appears likely that during phagocytosis of a live Gram-negative bacterium, the bulk of LPS will be delivered as vesicular cargo to the lysosome, and a smaller fraction of LPS may fuse with the plasma membrane, either by diffusion or the action of the lipid transfer proteins LBP and CD14. Under these circumstances, the bulk of LPS may be destroyed in the lysosomes, while the cells may simultaneously receive an optimal stimulus from LPS that has fused with the membrane. As described in the Introduction, several lines of evidence suggest that internalization of LPS may be necessary for at least some types of signal transduction by LPS. The precise mechanism by which Golgi apparatus internalization affects signal transduction, however, is not clear at the time of this writing. Two hypotheses may be entertained. In the first, LPS may have its effects through modulation or interruption of vesicular transport per se. It is clear that the lipid composition of membranes may direct their fate. Transformation of sphingomyelin to ceramide in the plasma membrane causes vesiculation 47 , and after traffic to the Golgi complex, ceramide may be converted to glucosyl ceramide which, in turn, is brought back to the plasma membrane 48 . LPS differs from endogenous lipids in that it cannot be metabolically transformed in the Golgi complex and may thereby interrupt traffic. In support of this hypothesis, accumulation of unfolded proteins in the ER is known to initiate a stress response called the “ER stress response” 49 or “unfolded protein response” 50 . Activation of NF-κB 51 is a well known signal induced by LPS and has been reported to occur during ER stress 52 . A second hypothesis is that proteins involved in sensing infection (pattern recognition receptors) may be located in the Golgi apparatus or may need to move to the Golgi apparatus to signal. An example of the latter case is seen in the proteins that sense cholesterol concentration 53 . SCAP (SREBP cleavage-activating protein) senses cholesterol concentration and initiates a cascade of events that activates transcription of genes for cholesterol synthesis. In the presence of adequate levels of cholesterol, SCAP does not initiate this cascade, and recent data suggests that under these conditions it is sequestered in a pre-Golgi compartment in an endo H–sensitive form. Low cholesterol levels trigger both translocation to the Golgi apparatus and initiate the steps leading to cholesterol synthesis. Mutations in SCAP delete in parallel the ability to both move to the Golgi complex and initiate cholesterol synthesis. In this regard, recent studies have implicated both Toll-like receptor (TLR)2 and TLR4 as proteins involved in signaling by LPS 54 55 56 57 58 59 . The subcellular distribution of TLR protein before and after LPS stimulation will be of substantial interest. Whether or not responses are initiated by either of the mechanisms described above, it is now increasingly clear that LPS is recognized not as a free monomer but in the context of its packing in a membrane. E5531 is a synthetic LPS antagonist that resembles LPS but blocks its action in cells 60 . A mirror image of E5531 was synthesized by inversion of all 13 optically active sites in the molecule, and the enantiomeric form was an equally active antagonist . This observation argues against the recognition of LPS by a stoichiometric interaction with a stereospecific binding site and rather suggests that the colligative properties of LPS may play a key role in recognition. In keeping with this view, we have observed that the ability of another LPS antagonist ( Rhodobacter sphaeroides [Rs]LPS) to stimulate cells is dramatically altered by membrane-active agents. The antagonist activity of RsLPS may be transformed to agonist by the addition of the membrane-active agent, chlorpromazine, suggesting an important role for membrane packing in recognition of LPS. From these considerations, we are directed to seek recognition proteins that sense LPS in a bilayer or the properties of a bilayer containing LPS. The LPS trafficking process we have observed here represents a cellular response to LPS as part of a membrane and contributes to an emerging picture of key steps in the innate recognition of LPS. | Other | biomedical | en | 0.999995 |
10449524 | RNA extracted from peritoneal macrophages (PMs) was reverse-transcribed to cDNA using an oligo-dT primer (Promega). Two pairs of PCR primers were synthesized 22 to generate the complete open reading frame. The first pair: upstream primer 5′-GGAGGCAAAAATGATGCTATC-3′, downstream primer 5′-CCGAGCACGAGTCCAGAGCCG-3′; and the second pair: upstream primer 5′-CACCATGAAGTCCTGCGGCCT-3′, downstream primer 5′-GGCGCCAATGTCAGGGATCAG-3′. cDNA amplifications were performed using a Perkin-Elmer PCR kit. 10 μl of the PCR product using the first primer pair was reamplified with the second primer pair. PCR products were resolved on an agarose gel, transferred to a nitrocellulose membrane, and probed with murine SLPI (mSLPI) cDNA. The cDNA fragment that hybridized to mSLPI was subcloned and used to probe a rat macrophage cDNA library in Lambda ZAP II (Stratagene). Inserts of the positive clones were subcloned and sequenced with T3 and T7 primers at the National Institute of Dental and Craniofacial Research DNA core facility. The EMBL, Swissprot, and GenBank molecular biology databases were searched using the network service (National Center for Biotechnology Information, NLM, Bethesda, MD) and the FASTA program from the Genetics Computer Group (GCG) Wisconsin Sequence Analysis Software Package (University of Wisconsin). Multiple sequence alignments were performed using ClustalW alignment in MacVector software (Oxford Molecular Group, Oxford, UK). PBMCs and polymorphonuclear neutrophils (PMNs) were isolated from female Lewis rats as previously described 25 . Resident PMs were collected by PBS lavage of the peritoneal cavity. Total RNA was isolated by the RNeasy protocol (Qiagen). 8 μg of total RNA was then subjected to Northern blot analysis using [ 32 P]dCTP-labeled rSLPI and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as the probes. The rSLPI cDNA encoding the mature secreted protein was generated by PCR with an upstream primer 5′-GGCAAAAATGATGCTATC-3′ and a downstream primer 5′-TTACACTGGGGGAAGGCAGA-3′ with BamHI and SalI adaptors, respectively. The BamHI–SalI cDNA fragment was subcloned into a prokaryotic expression vector pQE30 (Qiagen Inc.) with an in-frame ATG and the hexahistidine tag to the NH 2 -terminus. Bidirectional sequencing verified the correct sequence and reading frame. Escherichia coli strain M15 was transformed with the rSLPI expression construct and grown in a 10-liter fermentation system at 37°C with ampicillin (100 μg/ml) and kanamycin (25 μg/ml) until an OD of 10 was reached. rSLPI expression was induced by addition of 1 mM IPTG for 5 h at 37°C. The cells were then harvested and the pellet was resuspended in 50 mM Na-phosphate buffer (pH 8.0) containing 300 mM NaCl, and in 10 mM imidazole. After sonication and RNase A and DNase I treatment, sequential 15,000 and 50,000 g centrifugations were performed. The supernate was mixed with pre-equilibrated Ni-NTA resin and incubated at 4°C for 1 h before being packed into a chromatographic column. Step-elution with 50, 75, 100, 125, and 150 mM imidazole was then carried out. The eluate from 75 mM imidazole, which showed anti-elastase, anti-cathepsin G, and anti-chymotrypsin activities, was dialyzed against sodium phosphate buffer (PBS, 10 mM sodium phosphate, 0.9% saline, pH 7.4), concentrated, filter-sterilized, and stored in aliquots at −80°C. 25 mg of purified rrSLPI protein was isolated from 50 g of wet weight of E. coli , which constituted 0.6% of the total soluble protein. rrSLPI was sequenced by Edman degradation (CBER, FDA, Bethesda, MD) to confirm identity. The molecular weights of the purified proteins were determined by mass spectrometry (Dr. Lewis Pannel, NIDDK, NIH, Bethesda, MD). An 8.4-kD truncated HIS-rSLPI recombinant protein (the COOH-terminus ends at Arg 64 of the mature rSLPI protein) was obtained using the same purification scheme, which has no protease reactive site and lacked anti-elastase, anti-cathepsin G, and anti-chymotrypsin activities. Endotoxin levels of both the full-length and truncated rrSLPI protein preparations were found to be ≤25 pg/ml (detection limit). Rabbit polyclonal antibodies generated against the rSLPI peptide EGGKNDAIKIGAC (Quality Controlled Biochemicals, Inc.) were affinity purified and used for immunoblotting, ELISA, and immunohistochemical studies. Elastase activity was measured as the amidolytic effect of human neutrophil elastase (15 nM; Calbiochem) on pyroGlu-Pro-Val-pNA (0.5 mM; Chromogenix) after 10 min at 37°C 26 . Trypsin activity was determined by measuring the amidolytic effect of l -1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated bovine pancreatic trypsin (Sigma Chemical Co.) on the chromogenic substrate N -α-benzoyl- l -Arg-pNA (Boehringer Mannheim, Inc.) 27 . Chymotrypsin (12.5 nM) and cathepsin G (32 nM) (Calbiochem) activities were measured using the chromogenic substrate N -suc-Ala-Ala-Pro-Phe-pNA (Sigma Chemical Co.) at 0.1 and 0.4 mM concentrations for 15 and 20 min, respectively 28 29 . In all assays, the respective enzymes were incubated with or without different concentrations of purified rrSLPI at 37°C for 20 min. Then the substrate for each enzyme was added and the residual activity was measured as the change in absorbence at 405 nm after an appropriate incubation time at 37°C. Arthritis was initiated in female Lewis rats (Charles River Breeding Labs., Inc.) by an intraperitoneal injection of group A SCW (Lee Labs.) 25 . The severity of arthritis (AI) was determined by blind scoring of each ankle and wrist joint, based on the degree of swelling, erythema, and disfigurement on a scale of 0–4 and adding the scores for all four limbs during the course of the study (26–28 d). Rats were randomly assigned to five groups for two separate experiments (I and II) as follows: control animals, which received PBS on day 0 ( n = 6 for both experiments); SCW-injected animals ( n = 6 for both experiments); SCW-injected animals treated with rrSLPI on days 1 and 9 ( n = 6 for each dosage group in experiment I, and n = 12 for experiment II); SCW-injected animals treated with rrSLPI on day 13 ( n = 3 for each dosage group for experiment I, and n = 12 for experiment II); and SCW-injected animals treated with truncated rrSLPI on days 1 and 9 ( n = 3 for each dosage group for experiment I, and n = 9 for experiment II). In experiment I, 100 μg or 1 or 5 mg of purified full-length or truncated rrSLPI was injected intraperitoneally at the times specified for each group of animals; in experiment II, 100 μg of full-length or truncated rrSLPI was used. Statistical significance was ascertained using the analysis of variance followed by Scheffe's post-hoc test. Radiographs were taken as previously described 25 . Excised ankle joints were processed for histopathology 25 , and sections were stained with anti-rSLPI antibody (5 μg/ml) using Vectastain-ABC kit with DAB substrate (Vector Labs.) and counterstained with methyl green 25 . Rat plasma samples (1:2) were diluted in the appropriate dilution reagent and the levels of TNF-α were measured by ELISA (Biosource International). The plasma levels of rrSLPI were measured by our ELISA assay. In brief, Immulon 4 HBX microtiter plates (DYNEX Technologies, Inc.) were coated with 200 μl of anti-tetra-HIS antibody (3 μg/ml; Qiagen), washed four times in PBS, and incubated in blocking buffer (2% sucrose, 0.1% BSA, and 0.9% sodium chloride) for 2 h. Standard ELISA procedure was then performed using rabbit anti-rSLPI (5 μg/ml) as the primary antibody, alkaline phosphatase–conjugated anti–rabbit antibody as secondary antibody (1:4,000; Boehringer Mannheim Biochemicals), and p -nitrophenyl phosphate as substrate (Sigma Chemical Co.). Joint samples were obtained at the indicated times and processed in a freezer/mill (SPEX CertiPrep) in the presence of liquid nitrogen 25 . Nuclear proteins were isolated from the powdered joint tissue by homogenization in lysis buffer (20 mM Tris, pH 7.6, 120 mM NaCl, 1% NP-40, 10% glycerol, 10 mM NaPPi, 100 mM NaF, 2 mM Na-orthovanadate, 1 mM AEBSF, and 5 μg/ml leupeptin). Electromobility shift assay (EMSA) for nuclear factor (NF)-κB was performed using a radiolabeled NF-κB consensus oligonucleotide probe (Promega) as previously described 30 . Rat plasma samples were collected by tail bleeding or intracardiac puncture. Plasma levels of type II collagen cleavage product Col2-3/4C long neoepitope were monitored by a solution phase ELISA assay based on a published method 31 . The Col2-3/4C long neoepitope resides at the COOH terminus of the TC A piece produced by cleavage of the α1(II) chain by collagenase. The mouse mAb used in the assay is distinct from the rabbit antibody described previously 31 in that it recognizes only the cleaved α1(II) chain and thus is absolutely specific for type II collagen (Billinghurst, R.C., M. Ionescu, M.-A. Fitzcharles, E. Keystone, and A.R. Poole, manuscript in preparation). rSLPI mRNA was identified by reverse transcriptase PCR using nested primers designed against the mSLPI open reading frame 22 . A 400-bp cDNA fragment was isolated and used to probe a cDNA library from rat PMs. A 490-bp cDNA fragment was isolated, sequenced, and found to contain an open reading frame encoding a predicted translation product of 131 amino acids with an estimated molecular mass of 14 kD. The putative rSLPI coding sequence shares 88% homology with murine SLPI (mSLPI) and 75% with hSLPI. At the amino acid level, rSLPI exhibits 83 and 58% identity to mSLPI and hSLPI, respectively . 16 cysteine residues are conserved in the mature SLPI and the serine protease binding site in hSLPI (Leu 72 of the mature protein) is identical in rSLPI . However, by Northern blot analysis, distinct tissue distribution patterns compared with humans were observed (Song, X.y., L. Zeng, W. Jin, and S.M. Wahl, manuscript in preparation). Moreover, although no detectable hSLPI mRNA has yet been observed in human mononuclear cells and macrophages, rSLPI was constitutively present at very high levels in naive PMs, less abundant in PBMCs, and essentially absent in resting PMNs . When rSLPI mRNA was monitored during the course of synovial inflammation and compared with the low constitutive SLPI mRNA levels in control joints, a striking biphasic expression pattern was observed during the course of the disease. The increase in rSLPI mRNA coincided with the peak acute (day 4 after SCW injection) and chronic (day 30) inflammatory responses , but decreased significantly at the onset of the remission phase (day 7), and was negligible at the onset of chronic disease (day 14). These findings suggest that failure to produce sufficient SLPI or to maintain the initial elevated levels of the protease inhibitor preceding chronic disease may facilitate joint destruction. Assessment of the origins of SLPI in the inflamed synovium revealed that rSLPI protein was found in infiltrating inflammatory cells consistent with SCW-induced mRNA expression , and was also associated with some chondrocytes and cells in the bone marrow . Because SLPI levels were minimal during the interval before joint destruction becomes most pronounced, we attempted to restore levels of the protease inhibitor by exogenous delivery of active rrSLPI. For this purpose, a mature rSLPI–HIS fusion protein was expressed, purified to homogeneity , and found to contain a polypeptide recognized by both anti-rSLPI and anti-HIS tag antibodies (data not shown). This full-length fusion protein exhibited antiprotease activity toward human neutrophil elastase , cathepsin G, and bovine chymotrypsin, but not trypsin (data not shown), and was used in the subsequent in vivo study. A COOH-terminally truncated form of the rrSLPI protein without anti-elastase, anti-cathepsin G, or anti-chymotrypsin activities was used as the control agent in the following animal experiments. Based on the SLPI expression profile , full-length or truncated rrSLPI (0.1, 1, and 5 mg) was administered intraperitoneally before the clinically evident acute response to SCW or preceding the chronic inflammatory response, when endogenous SLPI was at a low ebb. As early as 2 d after the first full-length SLPI-injection (0.1 mg), a reduction in AI was evident that was more significant by day 4 of the acute response . The effects of 0.1, 1, and 5 mg of rrSLPI were similar based on AI (data not shown). However, the impact of a single injection of only 0.1 mg of full-length rrSLPI was transient, as the clinical and histopathological symptoms reappeared during the late chronic disease stage, albeit at reduced levels (data not shown). Importantly, joint swelling, erythema, and disfigurement, the hallmarks of chronic arthritis, a response typically considered T cell– and macrophage- rather than neutrophil-dependent, were drastically curtailed after a second rrSLPI injection 9 d after SCW and remained suppressed for the duration of the experiment. Radiographic analysis revealed that compared with SCW-injected animals, which exhibit overt erosion of cartilage and bone , full-length rrSLPI-treated animals had minimal pathological changes with only mild soft tissue swelling . In contrast, treatment of SCW-injected animals with the antiproteolytically inactive truncated rrSLPI caused no significant changes in disease severity . Moreover, a single injection of full-length rrSLPI at the beginning of chronic arthritis (day 13) appeared to reduce the disease severity, achieving statistical significance within 10 d . When plasma levels of rrSLPI were examined in rrSLPI-treated (1 mg) and untreated SCW animals, 2,652 pg/ml of rrSLPI was detected in rrSLPI-treated animals 2 h after injection, which declined over the next 16 h. SLPI levels were not detected in control or untreated arthritic animals. Finally, no apparent adverse effects were observed in rrSLPI-treated (1 mg) animals, which maintained their body weight at a level similar to the nonarthritic control animals (body wt = 175.7 ± 1.8 g in rrSLPI-treated arthritic animals vs. 158.6 ± 1.76 g in untreated SCW arthritic animals and 184.5 ± 2.4 in nonarthritic control animals, day 26). Circulating type II collagen collagenase–generated cleavage products reflect the increase in cleavage in degenerate articular cartilage in human osteoarthritis 31 , but have not been analyzed in experimental animal models. A new assay to detect a hidden type II collagen epitope in the COOH-terminal three-quarter–length fragment (Col2-3/4C long neoepitope), which is exposed only when native triple helical type II collagen is cleaved by collagenase, enabled us to quantify cartilage degradation in SCW arthritic rats. We demonstrate that Col2-3/4C long neoepitope increased in plasma with disease progression , paralleling the evolution of cartilage destruction observed in these animals 25 . Interestingly, during the remission phase, when inflammation and swelling typically decline, collagen cleavage appears to persist with the spill-over of cleavage products into the circulation. After a single full-length rrSLPI treatment on day 1 after an arthritogenic injection of SCW, a significant reduction (two- to threefold) in Col2-3/4C long neoepitope plasma levels was detected even during acute arthritis . The lack of significant decrease in cleavage products in rrSLPI-treated arthritic animals during remission (day 10) is consistent with the clinical and pathological data in which a single treatment with full-length rrSLPI does not fully sustain suppression of the arthritis through the chronic phase. Although a second rrSLPI injection was administered on day 9, this probably represents an inadequate interval of time to effect a further reduction in enzymatic profile on day 10. However, the injection of SLPI on day 9 did have a profound impact on the subsequent evolution of cartilage and matrix destruction associated with chronicity. In fact, full-length rrSLPI appeared to nearly eliminate the joint destruction as quantified by multiple parameters, including AI, radiologic assessment of joint destruction, and the release of type II collagen cleavage products . In addition to proteolytic blockade of matrix degradation, histopathological analysis revealed reduced evidence of synovial inflammation in rrSLPI-treated arthritic animals. It has been shown that TNF-α can induce SLPI production in epithelial cells 32 , and SLPI can in turn inhibit TNF-α production in macrophages 21 22 . To determine whether exogenously administered active rrSLPI could reinforce the endogenously produced SLPI to antagonize SCW-induced TNF-α 25 , we compared plasma levels of TNF-α in full-length rrSLPI-treated and untreated arthritic animals over the course of the disease. Plasma levels of TNF-α in arthritic animals were elevated during the acute phase, decreased in remission, and markedly increased in the chronic phases of the disease . However, after administration of full-length rrSLPI on days 1 and 9, there was a rapid and sustained decrease in detectable levels of circulating TNF-α . Since SCW 30 and TNF-α both activate NF-κB, which is responsible for the transcription of multiple inflammatory mediators including TNF-α 34 , the consequences of rrSLPI injection on NF-κB activity in active rrSLPI-treated and untreated animals during both acute and chronic (data not shown) arthritis were examined. By EMSA, NF-κB was strongly activated in SCW-induced arthritic joint tissue as compared with PBS-injected control animals, and this activation was suppressed by rrSLPI . These data implicate a potential role for rrSLPI in breaking the positive feedback loop between TNF-α and NF-κB activation, thereby dampening SCW-induced joint inflammation. SLPI, a serine protease inhibitor with potent neutralizing activity for leukocyte enzymes such as neutrophil elastase, is upregulated in response to bacteria and bacterial products 21 22 24 . Here we demonstrate that, in contrast to hSLPI, rSLPI, like its murine counterpart 24 , is expressed in monocytes and PMs and, apparently, other cells exposed to bacterial cell wall components. Our identification of SLPI in articular chondrocytes is consistent with recent reports, although it is still controversial whether SLPI is produced by these cells in situ or is sequestered into the cartilage matrix 35 36 37 . Moreover, we have expressed biologically active recombinant rSLPI, and show that when delivered to animals in which bacterial products induce arthritic lesions, SLPI can profoundly inhibit proteolytic tissue destruction and joint inflammation. This effect of rrSLPI on SCW-induced arthritis and the parameters monitored implies that SLPI inhibits multiple pathways either directly as a consequence of its antiprotease activity or via yet to be determined regulatory pathways. SCW-induced arthritis presents a biphasic disease process with a neutrophil-dependent acute phase and a prolonged T cell–dependent, macrophage-mediated chronic phase that manifests ∼2 wk after SCW injection 25 . Although the acute phase is considered reversible, the chronic destructive disease presents irreversible changes with progressive pannus invasion of the cartilage and bone, and eventual loss of normal joint structure and function. The increasing differences between nonarthritic and SCW arthritic animals in type II collagen cleavage as disease progresses is, in part, due to a gradual decrease in normal type II collagen cleavage in young control animals as they age, associated with the closure of the growth plate 38 . Parallel to the biphasic disease manifestation, the expression of endogenous SLPI is substantially upregulated at both mRNA and protein levels. The unexpected increases in endogenous SLPI observed during both acute and chronic arthritis most likely represent an attempted defense against the elevated serine proteases triggered by SCW activation. However, expression of SLPI nearly vanishes in the interval preceding chronic destructive disease, suggesting that this loss of SLPI activity may contribute to the joint destruction typical of chronic arthritis. In this regard, the therapeutic effect of exogenously delivered rrSLPI might result from direct inhibition of the serine protease cascade, initiated not only by neutrophil elastase, but also by that derived from both lymphocytes and macrophages 39 40 41 . Consistent with this hypothesis, denaturation of type II collagen in articular cartilage as reflected by quantifiable circulating levels of a neoepitope resulting from cleavage of collagen by collagenase, the Col2-3/4C long neoepitope, was significantly suppressed by exogenously delivered rrSLPI during both the acute and chronic phases of the disease. The persistent collagen cleavage during remission, when inflammatory activity is low, indicates that the devastating cartilage destruction associated with chronicity is the consequence of smoldering proteolysis. Consequently, the therapeutic effect of exogenous SLPI on chronic arthritis may also involve suppression of protease activities existing at the transition between acute and chronic arthritis. By providing exogenous active rrSLPI, the severity of disease at the clinical, histological, and molecular levels can be reduced. By neutralizing elastase, which cleaves not only elastin, but also collagen, fibronectin, and proteoglycans leading to cartilage and matrix degradation, this protease inhibitor may have multiple beneficial actions 8 9 . Moreover, because elastase 3 5 and the plasminogen–plasmin system 3 can activate latent matrix metalloproteinases, SLPI may also indirectly block matrix metalloproteinase–mediated matrix cleavage. Recent evidence indicates that SLPI inhibits synthesis of macrophage collagenase and impairs PGE synthesis 42 , implicating SLPI in the interruption of a proteolytic cascade of inflammatory events that evolve to tissue destruction 43 44 . In addition to inhibiting proteolytic tissue destruction, SLPI appears to have multiple antiinflammatory actions. SLPI inhibits TNF-α production, probably through its ability to block NF-κB activation 22 45 . Our demonstration of SLPI's effect on TNF-α production and NF-κB activity in vivo complements the in vitro findings that SLPI is upregulated by proinflammatory stimuli including LPS, TNF-α, IL-6, and IL-1β, and subsequently inhibits TNF-α and nitric oxide production. These pathways may underlie SLPI's antiinflammatory functions in vivo 22 24 32 . Based on the similarities between human rheumatoid arthritis and SCW-induced experimental arthritis, and the shared protease reactive site in hSLPI and rSLPI, our study provides initial preclinical evidence in support of the use of this serine protease inhibitor in alleviating arthritic pathology. SLPI may represent a prototype therapeutic approach for multiple inflammatory destructive diseases. | Study | biomedical | en | 0.999998 |
10449525 | BALB/c mice were obtained from The Jackson Laboratory. Transgenic (Tg) BALB/c mice express the 14.3d TCR-α/β specific for hemagglutinin (HA)110-120 in the context of I-E d class II molecules. The CD4 T cell epitope HA110-120 of influenza virus PR8/A/8/34 HA 22 and NP147-154 of influenza virus PR8/A/8/34 23 were prepared by solid phase Fmoc technology and purified by reverse phase HPLC on a C2/C18 column (Amersham-Pharmacia Biotech). Purity of the synthetic peptides was assessed by amino acid sequencing in the Protein Core Facility at Mount Sinai School of Medicine. The DEF molecule consists of the I-E d α and I-E d β extracellular domains that were dimerized through a murine Fcγ2a fragment at the COOH termini of I-E d β chain 20 . The HA110-120 (SFERFEIFPKE) CD4 T cell epitope of HA of influenza virus A/PR/8/34 was covalently linked at the NH 2 terminus of I-E d β chains as previously described and designated DEF 20 . Recombinant DEF protein was produced in the baculovirus/SF9 insect cell system and purified by affinity chromatography on goat anti–γ2a–Sepharose column as described 20 . MOPC 173 myeloma cells secrete an IgG2a that was used in experiments as an isotype control for the Fcγ2a portion of the DEF molecule. A BSA–HA110-120 conjugate was prepared using the heterobifunctional cross-linker 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (Imject Immunogen EDC Conjugation kit; Pierce Perstorp Biotech Co.) according to the manufacturer's instructions. The conjugate was purified by size exclusion chromatography on a Superose-6 column (Amersham-Pharmacia Biotech). Influenza A/PR/8/34 virus was used for immunization of BALB/c mice and for measurement of the antiviral isotypes in these mice by RIA. The virus was grown in the allantoic fluid of chicken eggs and then purified on sucrose gradient according to standard procedures. Chemicals were purchased from Sigma Chemical Co. unless noted otherwise. TCR-HA (TCR recognizing HA110-120 peptide in context of I-Ed class II molecules) T cells were purified from the spleens of Tg mice by Ficoll-Hypaque gradient centrifugation followed by two passages on nylon wool columns (Unisorb T&B; Nycomed Pharmaceuticals) and incubation for 2 h at 37°C in plastic dishes. The nonadherent cells were collected and measured for purity by FACS™ analysis (Becton Dickinson) using 2C11 anti-CD3∈ mAb (American Type Culture Collection [ATCC]) conjugated with PE and 6.5.2 clonotypic anti–TCR-HA mAb conjugated with FITC. Such preparations contained 95% CD3 + cells, of which 32% expressed the TCR-HA transgene. P-815 is a mouse mastocytoma cell line expressing MHC class I H-2 d and lacking class II molecules (ATCC). 14-3-1 T cell hybridoma (TcH) expresses the 14.3d TCR-α/β that recognizes HA110-120 peptide in the context of class II I-E d molecules 24 . The proliferative response of T cells upon incubation with various antigens was measured by a thymidine incorporation assay. In brief, splenic cells or purified TCR-HA T cells (5 × 10 5 ) were incubated for 72 h at 37°C in the presence or absence of various antigens. Tritiated thymidine (1 μCi) was added for the last 24 h, cells were harvested on filter papers (Skatron Inc.), and radioactivity was measured in a β-scintillation chamber (Amersham-Pharmacia Biotech). Spleen cells from PR8-immunized BALB/c mice treated or untreated with antigens on days 7, 8, and 9 after immunization were collected on day 10 after immunization and then stimulated for 5 d in vitro with NP147-154 peptide (2 μg/2 × 10 6 cells/ml) and then incubated for 4 h at 37°C in 96-well V-bottomed plates at various ratios with P-815 target cells labeled with 51 Cr and coated with NP147-154 peptide (20 μg/10 6 cells/ml). Cell culture supernatants were measured for the amount of 51 Cr release in a gamma counter (Amersham-Pharmacia Biotech), and the results were computed as percentage of cytotoxicity in triplicate samples for each E/T ratio as follows: (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100. Noncoated P-815 target cells were used to determine 51 Cr spontaneous release. Maximum 51 Cr release refers to the amount of 51 Cr liberated from noncoated target cells upon lysis with 5% Triton X-100. 10 6 cells in 1% PBS/BSA were singly or doubly stained with various mAbs conjugated with fluorochrome (1–2 μg/10 6 cells/ml; PharMingen) for 30 min on ice, washed with 3% BSA in PBS, and then fixed with 1% paraformaldehyde in PBS. The fluorescence intensity was analyzed in an EPICS PROFILE II Analyzer (Coulter Corp.). The effects of DEF were determined in vivo using BALB/c mice inoculated with 3.0 × 10 7 TCR-HA Tg T cells that were injected 24 h later with 130 μg, i.v. of DEF or MOPC 173 protein control (single dose injection) or with 390 μg of DEF or MOPC 173 (three injections at 1-d intervals) in 200 μl PBS. Spleen cells (10 6 ) were isolated 1 and 6 d after the last injection. The proliferative response to HA110-120 peptide and the amount of IL secreted in culture supernatants were determined by [ 3 H]TdR incorporation and ELISA (Cytoscreen Immunoassay; Biosource International, Inc.), respectively. To analyze DEF specificity in vivo, BALB/c mice were immunized i.p. with 100 μg of OVA in PBS. 9 d later, mice were injected intravenously at 24-h intervals with three 130-μg doses of either DEF or MOPC 173 protein, and spleen cells were collected 3 d after the last injection to measure the proliferative response and cytokine production upon in vitro challenge with OVA (10 μg/ml). The effect of DEF on Ab response was investigated in BALB/c mice previously immunized with 5 μg, i.p. of live influenza virus PR8/A/8/34 in PBS and then injected on days 7, 8, and 9 after immunization with 130 μg, i.v. of DEF or PBS. Blood samples were collected on days 0, 7, 8, 9, and 14 after immunization, and the titers of anti-PR8 isotypes were determined by RIA on individual mice sera. In brief, 96-well plates coated with 5 μg/ml PR8 virus were incubated for 2 h at 37°C with serum samples diluted 1:100 with 1% BSA in PBS. Plates were washed with PBS/0.05% Tween 20 and incubated for 2 h at 37°C with 50 μl of rabbit anti–mouse isotypes (mouse isotyping kit; Bio-Rad Labs.), washed with PBS/0.05% Tween 20, and then incubated for 2 h at room temperature with 125 I-goat anti–rabbit IgG Ab (5 × 10 4 cpm/well). Bound Abs were measured as cpm in a gamma counter (Pharmacia LKB). Phosphorylation of p56 lck , ZAP-70, STAT4, and STAT6 proteins was analyzed in total cell lysates. To detect phosphorylated p56 lck and ZAP-70, 2.0 × 10 7 TcH cells were treated for 5 min with 5 μg/ml soluble DEF or medium alone. DEF was then removed by centrifugation, and the cell pellets were lysed for 30 min on ice with 2 ml of RIPA buffer, pH 7, containing 1% NP-40, 10 mM Tris, 150 mM NaCl, 5 mM NaF, 2 mM Na 3 VO 4 , 5 mM Na pyrophosphate, and a cocktail of protease inhibitors (Complete™; Boehringer Mannheim). To detect phosphorylation of STAT4 and STAT6, TCR-HA Tg spleen cells (6.0 × 10 7 ) were incubated for 48 h at 37°C with 5 μg/ml of HA110-120 peptide, soluble DEF, anti-CD3∈ mAb 2C11, or clonotypic anti–TCR-HA 6.5.2 mAb. T cells were purified to >95% CD3 + T cells as described and stimulated for 30 min at 37°C with either mouse recombinant IL-4 (40 ng/ml −1 ) or IL-12 (1 ng/ml −1 ) (Sigma Chemical Co.). Furthermore, TcH or TCR-HA Tg cells were washed with PBS and lysed with RIPA buffer, and 100 μl of total cellular lysates (0.2 mg protein) was immunoprecipitated overnight at 4°C with 10 μg of one of the following rabbit Abs: anti-p56 lck , specific for the COOH terminus; anti–ZAP-70 (LR), specific for an amino acid sequence mapping within the “linker” region of ZAP-70 in the case of TcH; anti-STAT4 (C-20), specific for a region at the COOH terminus of mouse origin; or anti-STAT6 (M-200), specific for the amino acid region 280–480 of mouse origin (Santa Cruz Biotechnology) in the case of purified TCR-HA T cells. The immunoprecipitates were incubated for 2 h at room temperature with 20 μl of 50% slurry of agarose–protein A/G (Santa Cruz Biotechnology). The agarose–protein A/G–Ab–protein complexes were washed twice with lysis buffer and then boiled for 5 min in 1% SDS buffer and 5% 2-ME. Samples were electrophoresed in a 10–15% gradient of polyacrylamide, electrotransferred onto PVDF (polyvinylidene difluoride) membranes, blocked with 3% BSA, and probed with RC20 antiphosphotyrosine mAb–horseradish peroxidase (HRP) conjugate (1:10,000; Transduction Labs.). Bound Ab–enzyme conjugates were revealed by chemiluminescence using the Supersignal ® substrate for HRP (Pierce Chemical Co.) as indicated by the manufacturer. To identify the protein of interest, PVDF membranes developed with RC20–HRP conjugate were stripped with 0.1% SDS/1% guanidine hydrochloride for 30 s and reprobed for 2 h at room temperature with 10 μg/ml of rabbit Abs specific for p56 lck , ZAP-70, STAT4, or STAT6. Membranes were washed with 0.1 M Tris/HCl/PBS/0.05% Tween 20, pH 7.5, and bound Abs were revealed by chemiluminescence using goat anti–rabbit IgG Ab–HRP conjugate (Santa Cruz Biotechnology). We have previously shown that a soluble, dimeric HA110-120–I-E d /Fc chimera (DEF) binds stably and specifically to HA110-120–specific 14.3d TCR-α/β expressed by 14-3-1 TcH cells 20 . Herein, we investigated the T cell response to soluble DEF molecules. Using a protocol aimed at determining the immunopotency of HA110-120 peptide expressed by various protein carriers 25 , we found that soluble DEF is ∼88 times more potent in stimulating cognate T cells than the nominal peptide. Thus, half-maximal proliferation of spleen cells from naive TCR-HA Tg mice as determined by [ 3 H]thymidine incorporation assay was obtained with 4.5 nM peptide carried by DEF or with 400 nM synthetic peptide. Inhibition of antigen processing with 50 μM chloroquine did not significantly reduce the stimulatory capacity of HA110-120 synthetic peptide (12%) and DEF (17%) (data not shown). This was expected in the case of HA110-120 peptide, because stimulation of T cells by synthetic peptides does not require processing. As antigen processing did not significantly affect the stimulatory capacity of soluble DEF, we next analyzed the nature of DEF interaction with the cognate T cells using a two-step in vitro culture system. In the first step, purified resting T cells were incubated for 12 h with various doses of soluble DEF, BSA–HA110-120 conjugate, soluble anti-TCR clonotypic 6.5.2. mAb, or medium alone. Only T cells treated with soluble DEF secreted IL-2 . Lack of IL-2 secretion upon incubation with BSA–HA110-120 conjugate in the purified T cell preparation confirmed the absence of APCs, which are required for the processing of conjugate and presentation of peptides to T cells. It is noteworthy that the same magnitude of IL-2 secretion was observed with 0.05–5 μg/ml DEF but not with 0.005 μg/ml DEF. Also, lack of IL-2 secretion upon incubation with a soluble clonotypic anti-TCR mAb revealed its inability to stimulate T cells . Removal of antigens from the cell culture and addition of irradiated syngeneic APCs (2,200 rads) for 72 h in the second step of the assay augmented IL-4 secretion and fostered proliferation only in 5–0.05 μg/ml of DEF-pretreated T cells. In contrast, T cells pretreated with BSA–HA110-120 conjugate, clonotypic anti-TCR mAb, or the lowest dose of DEF (0.005 μg/ml) did not secrete cytokines and did not proliferate . Addition of HA110-120–pulsed APCs to the cultures enhanced proliferation and secretion of IL-4 up to two to three times only in DEF-pretreated T cells . Under the same culture conditions, T cells pretreated with BSA–HA110-120 conjugate, clonotypic anti-TCR mAb, or 0.005 μg/ml DEF secreted mainly IL-2 and proliferated . These results indicate that the interaction of resting T cells with soluble DEF, but not with a soluble clonotypic anti-TCR mAb, induced efficient IL-4–dependent proliferation. IL-4 is a potent mitogen in T cells via IL-4 nuclear activating factor 26 . As little as 0.005 μg/ml DEF was unable to trigger a Th2 response, as no proliferative response or cytokine production was detected at this dose. Most likely, the Th1 response obtained in T cells treated with 0.005 μg/ml DEF and then challenged with HA110-120–pulsed APCs was the result of further stimulation with HA110-120–pulsed APCs. To determine the effect of soluble DEF on resting T cells in vivo, BALB/c mice were inoculated with TCR-HA T cells from nonimmunized Tg mice and then injected with 390 μg, i.v. of DEF or MOPC 173 as a control administered at 24-h intervals as 130-μg injections. Spleen cells from mice injected with DEF proliferated upon challenge in vitro with HA110-120 peptide and showed the following kinetics of cytokine secretion: IL-2 followed an ascending pattern in control animals, while descending on day 3 in all DEF-treated mice ; IL-4 was barely detected on days 2 and 3 in control animals and significantly elevated on day 2 in all DEF-treated animals , and IL-10 was considerably increased in DEF-treated mice on day 3 as compared with the control mice . Similar results were obtained with a single dose of DEF (130 μg), although the magnitude of the proliferative response and cytokine production was slightly lower (data not shown). The results clearly indicate that a quite wide range of DEF doses (5–0.05 μg/ml) was able to induce differentiation of resting, peptide-specific T cells toward a Th2 phenotype. We next analyzed the fate of activated T cells upon interaction with soluble DEF. TCR-HA T cells stimulated with HA peptide and then restimulated with DEF or HA peptide showed a strong proliferative response, but only restimulation with DEF reversed the IL-2/IL-4 ratio, with an order of magnitude of ∼7 in favor of IL-4 ( Table ). When BALB/c mice were immunized intraperitoneally with live influenza PR8 virus and then injected intravenously with DEF or MOPC 173 protein control, the HA110-120–specific proliferative response in mice treated with DEF was accompanied by a 30-fold increase in IL-4 production as compared with control mice ( Table ). These data demonstrate that DEF polarized HA110-120–activated T cells into proficient IL-4 producers. As revealed in BALB/c mice immunized with OVA and then treated with DEF or MOPC 173 protein control, DEF-induced Th2 differentiation was restricted to HA110-120–specific T cells. The spleen cells from OVA-immunized mice that were or were not treated with DEF mounted a vigorous proliferative response to OVA in vitro and secreted comparable amounts of IL-2 and IL-4 (data not shown). Together, these data indicate that soluble, dimeric peptide/MHC II/Fc molecule (DEF) can polarize resting and activated peptide-specific T cells toward the Th2 immune response. The recruitment and differentiation of CD8 T cells depends greatly on the availability of cytokines secreted by CD4 Th1 cells, particularly IL-2. We thus investigated the extent to which DEF-induced Th2 response in vivo may affect the cytolytic function of CD8 T cells. BALB/c mice immunized with live PR8 virus and then treated with DEF showed a 40% reduction of CTL activity against target cells coated with NP147-154 peptide . NP147-154 is a dominant CD8 T cell epitope of the nucleoprotein of influenza virus A/PR/834 in H-2 d mice 23 . Reduced CTL activity was correlated with a double bystander effect: IL-2 deprivation and increase of IL-4 production. Th1-derived IL-2 is the major growth factor required for differentiation and activation of CD8 T cells 27 . Indeed, we previously found that depletion of CD4 T cells in naive TCR-HA Tg mice resulted in lack of differentiation of CTLs 28 . Increase of IL-4 after DEF treatment could also deprive CD8 T cells of IL-2, as it is known that IL-4 and IL-10 are potent suppressors of IL-2 synthesis 29 . Th1 cells mediate IgG2a, and Th2 cells mediate IgG1 Ab response 30 . We thus sought to analyze the effect of DEF-induced Th2 polarization on the IgG1 and IgG2a Ab responses in BALB/c mice immunized with live influenza PR8 virus. BALB/c mice immunized with live PR8 virus develop a mixed Th1/Th2-mediated Ab response 31 . The HA of influenza PR8 virus bears major B cell epitopes 32 and the HA110-120 T cell immunodominant epitope that is recognized by CD4 T cells in the context of I-E d class II molecules 22 . Mice immunized with PR8 virus and treated with DEF developed higher IgG1 and lower IgG2a anti-PR8 responses than the control mice . This can be explained by a double bystander effect mediated by elevated IL-4: enhanced transcription of γ1 constant region in the germline and inhibition of Th1-mediated IgG2a synthesis 30 . It is likely that DEF inhibited virus-specific CTL activity in PR8-immunized BALB/c mice by a double bystander effect (IL-2 deprivation and IL-10–induced suppression), whereas it had no apparent bystander effect on the proliferative response of T cells nor on the cytokine profile in OVA-immunized BALB/c mice. This can be explained by a very low frequency of HA-specific T cells in OVA-immunized mice, demonstrating once more that DEF targeting ability is specific to T cells recognizing HA110-120 peptide in association with I-E d αβ class II molecules. We previously demonstrated that DEF binds stably and specifically to 14.3d TCR-α/β, which recognizes HA110-120 peptide in the context of class II I-E d αβ molecules, and this binding was efficiently inhibited by anti-TCR clonotypic 6.5.2 mAb 20 . We also showed that soluble DEF binds to FcγRIIβ on the surfaces of APCs 20 . Herein, we investigated the role of DEF on the HA110-120–specific response upon its interaction with TCR, CD4, and FcγRIIb. Blocking of TCR with clonotypic 6.5.2 mAb or CD4 with GK1.5 mAb inhibited DEF-induced proliferation of TCR-HA Tg spleen cells. We and others have found that soluble 6.5.2 and GK1.5 mAbs have no effect on signaling T cells 20 33 . Therefore, inhibition of DEF-induced proliferation by these Abs was the result of hindering DEF access to TCR and CD4 molecules. In contrast, blocking of FcγRIIβ on APCs with 2.4G-2 mAb showed a weak inhibitory effect on DEF-induced proliferation . This indicated that the stimulation of T cells by soluble DEF molecules depends on its interaction with TCR and CD4 molecules and is little influenced by the binding of DEF to FcγR on APCs. To analyze the nature of the interaction between soluble DEF and TCR/CD4, we investigated the phosphorylation of p56 lck and ZAP-70 tyrosine kinases. p56 lck associates with the cytoplasmatic domain of CD4 molecules 34 and undergoes autophosphorylation upon engagement of CD4 and CD3–TCR complex with the peptide/MHC ligands expressed on APCs 35 . Phosphorylated p56 lck translocates to the ITAM (immune receptor tyrosine-based activation motifs) of the CD3/TCR ζ chain and mediates recruitment and phosphorylation of ZAP-70, a tyrosine kinase expressed exclusively in T cells 35 . Phosphorylation of p56 lck and ZAP-70 is critical for triggering downstream stimulatory events in T cells 36 . We found that 5-min exposure of 14-3-1 TcH to soluble DEF sufficed for phosphorylation of p56 lck and ZAP-70, which persisted 30 min after treatment . The results demonstrated that interaction of soluble DEF with the cognate TCR and CD4 molecules induced early transductional events in T cells. Among the signaling pathways mediated through the cytokine receptors, activation of the cytosolic latent STATs can rapidly modulate expression of genes responsible for Th differentiation 37 . We investigated the extent to which critical STAT proteins involved in Th1 and Th2 differentiation may be affected upon stimulation with DEF. Naive TCR-HA Tg splenocytes were first incubated for 48 h with soluble DEF, HA110-120–pulsed APCs, soluble anti-CD3 mAb, or clonotypic anti–TCR-HA mAb. The phosphorylation-mediated activation of STAT4 and STAT6 proteins has been determined in the purified preparation of T cells, shortly after stimulation with IL-12 and IL-4, respectively. IL-12–induced phosphorylation of STAT4 was impaired in T cells treated with DEF but not in cells treated with other TCR ligands. Although not phosphorylated, STAT4 showed unaltered molecular size, indicating the lack of its proteolytic degradation . In contrast, IL-4–induced phosphorylation of STAT6 was not affected in cells treated with any of the TCR ligands, including DEF. These results suggest that DEF-induced negative regulation of the STAT4 pathway of Th1 differentiation may have been potentiated by the STAT6 pathway of Th2 differentiation. A predominant Th1 response in cells treated with HA110-120–pulsed APCs may have been the result of the antagonist effect of STAT4 on the STAT6 pathway rather than a negative regulation of STAT6 pathways, because both STAT4 and STAT6 proteins were phosphorylated in these cells. STAT4 was shown to activate a genetic program able to antagonize the STAT6 pathway of Th2 differentiation, whereas no antagonistic effect of STAT6 on the STAT4 pathway has yet been described 12 . In this study, we provide evidence that a soluble dimeric HA110-120–MHC class II molecule built on a Fcγ2a scaffold (DEF) can induce in vitro and in vivo differentiation of resting and activated peptide-specific T cells toward the Th2 response. Gajewsky and colleagues proposed a model of T cell differentiation in which naive T cells secreting IL-2 upon primary stimulation in the absence of costimulation either undergo Th1 development or develop into Th0 cells secreting both IL-2 and IL-4 33 38 . Further stimulation of Th0 cells in the presence of costimulation induces Th1 differentiation, whereas stimulation in the absence of costimulation induces a defect in IL-2 but not IL-4 production, leading to Th2 differentiation. Similarly, stimulation of naive HA-specific T cells with soluble DEF in the absence of APC-derived costimulatory signals induced IL-2 secretion and further differentiation toward the Th0 phenotype. In contrast, we found that the presence of APC-derived costimulatory signals augmented polarization of Th0 cells toward the Th2 rather than the Th1 phenotype. This may be explained by different stimuli used to prime the naive T cells, i.e., anti-CD3 mAb, which stimulates an alternative pathway of T cell activation, versus DEF, which stimulates the TCR pathway of T cell activation. At the time of this writing, the extent to which costimulation may have contributed during this stage of differentiation is under investigation. We also found that the presence of HA-pulsed APCs enhanced the IL-4 secretion of DEF-induced Th2 cells. This is consistent with the results of McKnight et al., demonstrating that stimulation of Th2 cells with peptide/MHC class II complexes on APCs enhanced IL-4 secretion 39 . The authors showed that IL-4 secretion by the Th2 cells does not require costimulation. It is thus likely that increased IL-4 secretion detected in our system was induced mainly by the interaction of APC-derived HA/MHC complexes with HA-specific Th2 cells, rather than APC-derived costimulatory signals. Goldstein and colleagues showed that the requirement for costimulation in activating naive CD8 T cells by MHC class I/peptide ligands can be circumvented by potent signaling of TCRs 40 41 . Using a costimulation-free system, Boniface et al. demonstrated that the potency of TCR signaling by soluble peptide/MHC class II ligands parallels the increase in valency from two to four, whereas the monovalent forms are inefficient in stimulating T cells 42 . The monomeric peptide/MHC class II molecules failed to activate T cells at 20,000 times higher concentration than the tetramers, despite the fact that TCR occupancy by the monomeric form was significantly higher. The authors suggested that multivalency-mediated TCR cross-linking, rather than occupancy of individually engaged TCRs, may play an essential role in triggering potent TCR signals by peptide/MHC class II complexes. We found that independent of antigen processing but in the presence of APC-derived costimulation, soluble bivalent DEF was 88 times more potent in stimulating T cells than the HA/MHC complexes expressed on APCs. Soluble DEF induced a potent Th2 differentiation over a large range of doses in vitro (5–0.05 μg/ml) as well as in vivo (130 and 390 μg). For the in vivo experiments, MOPC 173 (γ2a) was used as specificity control for DEF bearing the exon encoding Cγ2a. Although MOPC 173 is a reasonable control, the ideal specificity control would be a DEF-like molecule in which an irrelevant peptide such as hen lysozyme 108–116 43 is linked to the β1 exon. Preparation of such a construct is underway in our laboratory, to be used in future experiments aimed at evaluating the effect of DEF in autoimmune diseases. Hamad et al. showed that various doses of a plastic-immobilized dimeric peptide/MHC class II chimera similar to DEF induced a Th1-like response 21 . Data suggest that not only valency but also the physical form of a dimeric HA/MHC ligand may dictate the outcome in Th effector functions. This may account for a different topology of TCR binding. Dimeric TCR ligands are able to cross-link TCR molecules, and presumably their soluble form can cross-link a larger number of TCRs, whereas surface immobilization can limit this process. High numbers of peptide/MHC TCR complexes can aggregate in large clusters by diffusion on the cell surface. Thus, different degrees of TCR clustering as induced by soluble versus immobilized forms of dimeric ligands may differentially signal the development of T cells. The role of quantitative versus qualitative signaling of T cells at various stages of differentiation upon interaction with TCR ligands is controversial. It has been hypothesized that the nature of TCR ligation dictates conformational changes in the cytoplasmatic domains of the CD3/TCR complex 44 , which in turn favors the engagement of different transductional pathways able to direct the development of T cells toward various effector functions 45 . We found that the same HA110-120 CD4 T cell epitope of PR8 influenza virus induced different T cell responses: (a) a mixed Th1/Th2 response upon immunization with live PR8 virus, (b) a predominant Th1 response upon challenge with synthetic peptide in the context of I-E d αβ class II molecules on APCs, and (c) a Th2 response when the peptide was presented in the context of a soluble, dimeric peptide/MHC class II/Fc chimeric molecule. A soluble clonotypic anti–TCR-HA mAb was unable to stimulate the cognate T cells, suggesting that some conformational constraints cannot trigger productive signaling of T cells. DEF-induced Th2 differentiation depended on the interaction of DEF with TCR and CD4 molecules. Viola et al. showed that Ab-mediated blocking of CD4 molecules precluded T cell activation 46 . Consistent with this report, we found that interaction of soluble DEF with CD4 and TCR molecules was significantly inhibited by Ab-mediated blocking of CD4 and TCR molecules. Brown et al. showed that CD4 molecules preferentially potentiate Th2 differentiation 47 . Although CD4 does not significantly stabilize the interaction of multimeric peptides/MHC class II chimeras with TCRs, the CD4 molecules are able to enhance the threshold of T cell activation 21 48 . This is because signaling of the p56 lck tyrosine kinase causes colocalization with CD4, which then rapidly undergoes autophosphorylation and translocates to the ITAM units on the ξ invariant chains of the CD3/TCR complex, where it mediates phosphorylation of ZAP-70 tyrosine kinase 35 . We found that interaction of soluble DEF with TCR and CD4 molecules induced early phosphorylation of both p56 lck and ZAP-70 tyrosine kinases. Both p56 lck and ZAP-70 kinases are critical for triggering downstream events required for productive signaling of T cells. Phosphorylation-mediated activation of STAT4 is a critical event for the development of Th1 cells 12 , and phosphorylation-mediated activation of STAT6 is required for the development of Th2 cells 13 . In contrast to various TCR ligands, such as HA110-120/MHC complexes expressed by APCs, anti-CD3 mAbs, and clonotypic anti-TCR mAbs, soluble DEF preferentially induced Th2 differentiation, which was correlated to negative regulation of the STAT4 protein. STAT4 could not be phosphorylated in T cells treated with DEF, although its structural integrity was unaltered. Lack of STAT4 phosphorylation may account for several mechanisms, including increased activity of protein tyrosine phosphatases, as previously demonstrated by Haspel et al. in the case of STAT1 protein 49 . Phosphatases may rapidly dephosphorylate STAT4 or alternatively may dephosphorylate the docking site of STAT4 on IL-12R generated by the recruitment of JAK and Tyk upon stimulation with IL-12. Docking of STAT4 on the IL-12R chain is essential for its phosphorylation. Other mechanisms that may be accounted for by lack of STAT4 phosphorylation are competition for the STAT4 docking site by small proteins of the CIS/SOCS/JAB/SSSI family 50 , which may have been upregulated upon DEF interaction with T cells, and downregulation or loss of IL-12R as the only docking site for STAT4 protein. Disregulation and immunodeficiency of IL-12R and/or IFN-γ-Rα chain leading to ineffective Th1 responses has been described in individuals with severe mycotic and bacterial infections 51 as well as in various types of carcinomas 52 . Whether DEF-induced negative regulation of STAT4 may account for one of these regulatory mechanisms remains to be investigated. DEF polarization toward a Th2 response was also detected in activated, peptide-specific T cells, in vitro as well as in mice preimmunized with live PR8 virus. Our working hypothesis is that the peptide- or PR8 virus–induced Th1 response was significantly diminished by negative regulation of STAT4, with compensatory augmentation of the STAT6 pathway of Th2 differentiation. The Th2 response induced by DEF was specific for TCR-HA T cells in vitro as well as in vivo. DEF-like molecules may provide a tool to dissect the intimate mechanisms of Th differentiation and perhaps lead to the development of more efficient immunotherapeutics in autoimmune diseases. | Study | biomedical | en | 0.999996 |
10449526 | Antibodies used in these studies include seven anti-CD86 mAbs; IT2.2 and FUN-1 clone (PharMingen), anti–human CD86 (Serotec), anti–human CD86 (B7-F3, gift from P. Linsley, Bristol Myers Squibb, Princeton, NJ), HF2 3D1, HA5 2B7, HA3 1F9 (gifts from V.J. Kuchroo, Brigham and Women's Hospital, Boston, MA), anti–human CD80 (PharMingen), anti–human CD25 (PharMingen, San Diego, CA); anti–human LFA-3 (IE6, gift from P. Hochman, Biogen Inc., Cambridge, MA), anti–human CD40 (220; a gift from D. Hollenbaugh, Bristol Myers Squibb), anti–human OX40 (Ancell Corp.), anti–human intercellular adhesion molecule (ICAM)-1 (RR1/1, a gift from T.A. Springer, Center for Blood Research, Harvard Medical School, Boston, MA), anti–HLA-DR (LB3.1, a gift from A.H. Lichtman, Brigham and Women's Hospital), and negative control mouse IgG (K16/16, a gift of M. Gimbrone, Brigham and Women's Hospital). Human CTLA4 Ig and control fusion protein were gifts from Dr. Peter Linsley (Bristol Myers Squibb). Cytokines used were recombinant human IFN-γ (Genzyme) and TNF-α (a gift from Biogen Inc.). Other reagents used included recombinant soluble CD154 (a gift from D. Hollenbaugh), LPS (Sigma Chemical Co.), and PHA (Sigma Chemical Co). Endothelial cells were isolated from human umbilical cords as previously described 27 and were grown in M199 medium (BioWhittaker) containing 10% FCS (GIBCO BRL), EC growth supplement, 1% penicillin/streptomycin, l -glutamine, and heparin. Cultured cells were harvested in trypsin/ethylenediaminetetraacetic acid (Sigma Chemical Co.) and subcultured for use at passages 2–4. Saphenous vein ECs were a gift from Dr. P. Libby (Brigham and Women's Hospital) 28 . Single donor, dermal, and lung microvascular ECs were purchased from Clonetics. Cell membrane fractions of ECs or CD4 + T cells were prepared as previously described 29 . In brief, untreated or IFN-γ–treated ECs (2–4 × 10 6 ) were harvested by gentle scraping, washed, and resuspended in lysis buffer containing 0.25 M sucrose, 10 mM Tris (pH 7.4), 10 mM NaCl, 0.1 M MgCl 2 , and 1 mM PMSF. The cells were lysed by homogenization and were centrifuged at 1,000 g for 15 min. Supernatants were centrifuged at 100,000 g for a further 30 min. All manipulations were performed at 4°C. Pellets were resuspended in RPMI and added directly to CD4 + T cells (10 6 ) in 96-well plates. For select experiments, cell membranes were prepared from unactivated or mitogen-activated CD4 + T cells (10 7 cells/condition). PBMCs were isolated by Ficoll-Hypaque gradient centrifugation from blood obtained from healthy volunteers. CD4 + T cells were isolated from PBMCs by positive selection using anti-CD4–coated magnetic beads (Dynal Inc.) according to the manufacturer's instructions. Magnetic beads were subsequently removed using Detachabead (Dynal Inc.). In some experiments CD4 + T cells were further purified by negative depletion of CD14 and HLA-DR expressing cells using a CD14-coated microbead column (MiniMACS separation column; Miltenyi Biotec) and panning on anti–HLA-DR (LB3.1) coated plastic culture dish respectively. The purity of the CD4 + T cells using these methods was 98 and 99.7%, respectively. Purity was assessed by double stain FACS ® analysis for CD3 and CD4 cell surface markers. Purified cells were unactivated as assessed by the lack of spontaneous proliferation, IL-2 and IFN-γ production, and CD25 cell surface expression, as previously described 30 . Human monocytes were isolated from platelet pheresis residues by centrifugation on density gradients (LSM; Organon Teknika), followed by counterflow centrifugation elutriation 31 . Monocytes isolated by this technique are >90% pure and are relatively unactivated as determined by minimal alterations in cell surface activation markers. In some experiments, monocytes were isolated from PBMCs by positive selection using CD14-coated microbeads (MiniMACS separation column; Miltenyi Biotec). CD80-, CD86-, and neomycin-transfected Chinese hamster ovary (CHO) cells (a gift from Dr. G. Freeman, Dana Farber Cancer Institute, Boston, MA) were cultured in collagen-coated tissue culture flasks in complete RPMI with 10% FCS. Cells were harvested by trypsinization and fixed in 0.4% PFA before addition to EC–CD4 + T cell cocultures. Primary cultures of ECs (passages 3–4) were treated with IFN-γ (1,000 U/ml) for 72 h to upregulate class II MHC. IFN-γ–treated ECs (5 × 10 4 /well) were then irradiated (1,750 rads) and cocultured with resting CD4 + T cells (5 × 10 5 /well) in 96-well cell culture plates in a final volume of 200 μl. Additional cells or reagents were added as indicated. Coculture supernatants were taken at days 3 and 5 for cytokine analysis by specific ELISA. Proliferation was assessed after 6 d by [ 3 H]thymidine incorporation for the last 18 h of coculture. Cells were harvested by an automated cell harvester and incorporated radioactivity was assessed by a Beckman Betamax counter. In separate experiments, we examined whether ECs modify CD4 + T cells to express functional CD86. CD4 + T cells were cultured on IFN-γ–treated human umbilical vein EC (HUVEC) monolayers for 24–72 h and then reisolated by positive selection using CD4-coated magnetic beads (Dynal Inc.). These cells were termed “EC-modified” CD4 + T cells. EC-modified CD4 + T cells (10 5 ) were irradiated (1,750 rads) and cocultured with resting autologous CD4 + T cells (5 × 10 5 ) in the presence of submitogenic doses of PHA (0.3 μg/ml). Coculture supernatants were taken at 24 h for specific ELISA. Proliferation was assessed after 3 d of coculture as described above. Transmigration assays were performed using a protocol modified, as follows, from one described previously 32 . HUVECs were seeded at 2 × 10 5 cells/cm 2 on collagen-coated, 8-μm-pore size polycarbonate tissue culture Transwell inserts (Costar Corp.) and were cultured for 5 d to attain confluence. Confluency was assessed by exclusion of FITC-labeled dextran in control wells as previously described 32 . HUVECs were pretreated with IFN-γ (1,000 U/ml) for 72 h to upregulate MHC class II. Purified, resting monocytes (5 × 10 6 ) were added onto the transwell and allowed to transmigrate across untreated or IFN-γ–treated ECs. HUVECs were washed thoroughly to remove IFN-γ before addition of monocytes. Transmigrated cells were counted and RNA was isolated from 2 × 10 6 cells 12 h after transmigration. Induction of monocyte CD86 and cytokine mRNA expression was determined by semiquantitative reverse transcriptase (RT)-PCR and RNase protection assay. Total RNA was prepared using the Ultraspec RNA isolation system (Biotecx) according to the manufacturer's instructions, and was quantified by spectrophotometry. cDNA was prepared by reverse transcription of 5 μg of RNA using random hexamer primers (100 ng/μl) and Moloney murine leukemia virus reverse transcriptase (50 μm/μl) (Stratagene) in a 50-μl reaction. 10 μl of cDNA was used for each PCR amplification reaction. PCR was performed with Taq DNA polymerase using the buffer supplied by the manufacturer (Boehringer Mannheim). The PCR primers were: human CD80, sense: 5′-CAT CAC GGA GGG TCT TCT AC-3′ and antisense: 5′-AGG ATC TTG GGA AAC TGT TGT-3′; and human anti-CD86, sense: 5′-AGG ACA AGG GCT TGT ATC AA-3′ and antisense: 5′-ATT GCT CGT AAC ATC AGG GA-3′. The PCR conditions were 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min for 35 cycles. PCR products were analyzed by ethidium bromide staining in 1.5% agarose gels using standard techniques. RNase protection was performed using the Riboquant™ Multi-Probe RNase Protection assay system (PharMingen). RNA was isolated as described above. 32 P-labeled probes were synthesized from the hCK-2 human cytokine multi-probe template set and were hybridized overnight with RNA samples in hybridization buffer according to the manufacturer's instructions. Samples were digested with RNase and T1 mix in RNase buffer and protected probes were purified and were run on a 5% acrylamide gel in 0.5% TBE buffer. Human control RNA and a dilution of the probe set (serving as size markers) were run in parallel. The gel was absorbed onto filter paper, dried, and exposed onto Kodak photographic paper at −70°C for 24 h. IL-2 was assessed by specific ELISA. Primary and secondary antibodies were purchased from Genzyme and were used according to the recommended protocol. In brief, 96-well flat-bottomed ELISA plates (Falcon; Becton Dickinson Labware) were coated with primary antibody overnight at 4°C. Blocking was then performed with 4% BSA in PBS for 2 h at 37°C and neat coculture supernatants or standards were added to each well in duplicate for 1 h at 37°C. After the incubation, secondary biotinylated anti–IL-2 mAb was added and the ELISA was developed using avidin alkaline phosphatase (Sigma Chemical Co.) and phosphatase substrate (Sigma Chemical Co.). In between each step, the plates were washed in PBS with 0.01% Triton X-100. Plates were read at 405 nm in an E-Max ELISA plate reader (Molecular Devices). Cell suspensions of CD4 + T cells, ECs, or monocytes were analyzed by direct immunofluorescence. In brief, 1–2 × 10 6 cells were incubated with FITC- and/or PE-conjugated mAbs at 4°C for 30 min and were fixed in 1% PFA. Stained cells were then analyzed by FACScan ® (Becton Dickinson). Monocytes were preincubated with buffer containing 20% non-A non-B human serum before flow cytometry to block nonspecific Fc receptor binding and optimize specific binding. Western blot was performed on ∼5 × 10 6 cells per condition. Cells were lysed in PBS containing 1% NP-40, and protease-inhibitor (Boehringer Mannheim) and lysates were separated by standard 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Blots were blocked overnight at 4°C in Tris-buffered saline containing 0.1% Tween 20 and 2% BSA before incubation with optimal concentrations of primary antibody diluted in TBS/0.1% Tween 20 for 12 h at 4°C. After four washes in TBS/0.1% Tween 20, blots were incubated in peroxide-conjugated goat anti–mouse secondary antibody (Jackson ImmunoResearch Labs.) at a 1:2,500 dilution in TBS for 4 h at 4°C. The blots were then washed and developed by chemiluminescence (Amersham Inc.). To investigate a role for CD80 and CD86 in CD4 + T cell–EC interactions, we initially examined the expression of these molecules by resting or activated CD4 + T cells, and ECs alone or after coculture of both cell types . Our findings were that TNF-α, IFN-γ, IL-1, or soluble CD40 ligand (CD40L)–stimulated HUVECs do not express CD80 or CD86 mRNA by RT-PCR; likewise resting CD4 + T cells do not express CD80 and express variable and low levels of CD86 mRNA. In contrast, we found a marked expression of both CD80 and CD86 mRNA in CD4 + T cell–HUVEC cocultures. CD86 mRNA was detected as early as 6 h and was maximal by 72 h of coculture, whereas CD80 mRNA was only expressed in cells harvested at 72 h of coculture . CD86 protein was detected by Western blot in 72-h CD4 + T cell–HUVEC cocultures but not in resting CD4 + T cells or TNF-α, IFN-γ, or soluble CD40 ligand–activated HUVECs . By FACS ® analysis, anti-CD80 mAbs, seven anti-CD86 mAbs, and CTLA4 Ig did not bind to resting or cytokine-activated HUVECs, saphenous vein ECs, dermal microvascular ECs, or lung microvascular ECs; although positive control CD80- and CD86-transfected CHO cells consistently demonstrated high levels of binding . Thus, human ECs express neither CD80 nor CD86, but both these molecules are induced in CD4 + T cell–EC cocultures. We next wished to determine the function of CD86 and CD80 in CD4 + T cell–EC interactions. Purified CD4 + T cells were rested for 48 h and were cocultured with either HUVECs, saphenous vein ECs, dermal microvascular ECs, or lung microvascular ECs, untreated or treated with IFN-γ for 72 h to upregulate class II MHC. Anti-CD86 mAbs, anti-CD80 mAbs, or control isotype antibodies were added to cocultures as indicated by each experiment . We found that anti-CD86 mAbs consistently inhibited IFN-γ–treated HUVEC-induced CD4 + T cell alloproliferation . Maximal inhibition with anti-CD86 mAbs was variable (20–50%) and was less than that observed by blocking CD2–LFA-3 interactions with anti–LFA-3 mAbs. In addition, we found that the combination of anti-CD86 and anti–LFA-3 mAbs provided additive inhibition of CD4 + T cell alloproliferation and IL-2 production (data not shown). The inhibitory effects of anti-CD86 mAbs was dose dependent and was observed when other types of human ECs were cocultured with CD4 + T cells . In contrast, interruption of CD28–CD80 interactions using anti-CD80 mAbs did not inhibit EC-induced CD4 + T cell alloproliferation, most likely because CD80 is not expressed at early time points in the CD4 + T cell–EC coculture . Thus, CD86 but not CD80 is functional in allogeneic EC-induced CD4 + T cell activation. Since we find that human ECs do not express CD86, we next wished to assess CD4 + T cell CD86 expression and function in our model. Previous studies have shown that activated CD4 + T cells may express both CD80 and CD86 33 34 . CD4 + T cells were cultured with IFN-γ–treated HUVECs and CD86 expression determined by double stained FACS ® analysis of cells. As illustrated in Fig. 3 A, we found low levels of CD86 on resting CD4 + T cells, but augmentation of CD4 + T cell CD86 expression after coculture with IFN-γ–treated ECs. Maximal expression of CD4 + T cell CD86 was observed after 72 h of coculture. We also confirmed that CD86 was induced de novo on CD4 + T cells by sorting and culturing CD86 − CD4 + T cells with IFN-γ–treated ECs for 24 h. Consistently, we found that ECs induce de novo expression of CD86 on CD4 + T cells. Furthermore, to determine whether EC-induced CD86 expression by CD4 + T cells was dependent on cell contact, we generated cell membranes from ECs and incubated them with CD4 + T cells. Membrane preparations from IFN-γ–activated ECs but not resting ECs induced CD4 + T cell CD86 mRNA expression by RT-PCR, consistent with the interpretation that induction of CD4 + T cell CD86 is mediated by a cell surface molecule(s) on activated ECs . However, when membranes generated from unactivated or activated CD4 + T cells were incubated with ECs, they failed to induce EC expression of CD86. As a positive control, activated CD4 + T cell membranes enhanced EC E-selectin expression . Having established that ECs induce CD86 expression on CD4 + T cells, we next assessed function. CD4 + T cells were cultured with IFN-γ–treated ECs and were reisolated by positive selection after 24 h of coculture. These EC-modified CD4 + T cells were then irradiated and cocultured either alone or with resting autologous CD4 + T cells in the presence of submitogenic doses of PHA (0.3 μg/ml). Although resting CD4 + T cells alone and EC-modified CD4 + T cells alone failed to proliferate to low dose PHA, coculture of both cell types resulted in enhanced proliferation, which was inhibited by ∼50–80% by anti-CD86 mAbs . This suggests that induced CD86 on EC-modified CD4 + T cells provides an effective costimulatory signal in trans to resting CD4 + T cells. However, the proliferative responses of CD4 + T cells in this trans-costimulation assay were less than those induced by ECs . We note that this is consistent with the ability of ECs to provide additional costimulatory signals such as LFA-3–dependent signals as reported by others 13 35 . To determine the molecular basis for EC modification of CD4 + T cell costimulatory activity, we incubated CD4 + T cells with IFN-γ–treated ECs in the absence or presence of anti–ICAM-1, anti–LFA-3, anti–HLA-DR mAbs, anti-CD40 mAbs, anti-OX40 mAbs, or isotype control antibodies for 24 h. EC-modified CD4 + T cells were then reisolated and irradiated, and were added to resting CD4 + T cells in the presence of submitogenic doses of PHA as described above. We found that anti–LFA-3 and anti–HLA-DR, but not anti-CD40, anti-OX40L, anti–ICAM-1, or control antibody, inhibited the subsequent CD4 + T cell costimulatory effect . Anti-CD40 antibodies also failed to inhibit T cell–T cell trans-costimulation . Thus, induction of functional CD86 on CD4 + T cells is in part dependent on interactions between CD4 + T cells and EC class II MHC and LFA-3. Finally, to confirm functionality of CD86-dependent trans-costimulation for direct allorecognition (when signal one is provided by alloantigen on ECs rather than mitogen), CD4 + T cells were cocultured with IFN-γ–treated ECs in the presence of increasing numbers of CHO cells transfected with CD86. Mock-transfected CHO cells were used as a negative control. CD86-transfected, but not mock-transfected, CHO cells enhanced EC-induced CD4 + T cell alloproliferation and IL-2 production (data not shown) in a dose-dependent manner that is inhibited by anti-CD86 mAbs and CTLA4 Ig . This data confirms that CD4 + T cells can receive CD86 costimulation in trans when signal one is provided by alloantigen on ECs. It has been reported that a subpopulation of peripheral blood CD4 + cells are HLA-DR + and CD3 − myeloid derived dendritic cells 36 . We found that ∼2% of our CD4 + cells were CD3 − , and therefore we wished to confirm that the CD86-dependent trans-costimulation described above is indeed due to CD86 expressed by CD4 + T cells and not to dendritic cell CD86. We further purified our CD4 + cells by negative selection for HLA-DR and CD14 expressing cells (as described in Materials and Methods). The resulting cells were CD4 + CD3 + HLA-DR − T cells and express low levels of CD86. However, consistently after coculture with IFN-γ–treated ECs, these T cells exhibit augmented CD86 expression . Furthermore, these T cells provide effective trans-costimulation to autologous T cells in the presence of low dose mitogen . Trans-costimulation is mediated in part by CD86 (with 25–75% inhibition observed with anti-CD86 mAbs) but also involves other cell surface molecules including LFA-3 . There is no antigen-dependent component to the T cell–T cell proliferative response, since anti–HLA-DR antibody (LB3.1) failed to inhibit proliferation with concentrations of mitogen (PHA > 0.3 μg/ml) used in our model. However, in the absence of mitogen or at low doses of mitogen (PHA < 0.1 μg/ml), anti–HLA-DR partially inhibits proliferative responses. We next wished to determine whether ECs modify the costimulatory and antigen presenting capacity of monocytes. Recent studies suggest that transmigration of monocytes across ECs promotes their differentiation into dendritic cells 37 38 . Indeed, a novel function of ECs may be to enhance the antigen presenting and costimulatory function of infiltrating APCs in the course of alloimmune inflammatory reactions and rejection. Monocytes were isolated by elutriation in order to obtain a relatively unactivated cell population. Cells were then allowed to transmigrate across resting or 72-h IFN-γ–treated confluent EC monolayers in transwells as described in Materials and Methods. After 12 h, cells were harvested from the lower chamber of the transwell and RNA was isolated for analysis by RT-PCR and RNase protection. We found that transmigration of monocytes across IFN-γ–treated ECs, and to a lesser extent resting ECs, resulted in the induction of monocyte CD86 mRNA expression by RT-PCR (data not shown) and enhanced CD86 protein expression by FACS ® analysis . Transmigration across transwell inserts not coated with ECs did not result in induction of CD86 (data not shown). We also found that transmigration of monocytes across IFN-γ–treated ECs resulted in the expression of the cytokine IL-1α as determined by RNase protection assay ; and coincubation of monocytes with IFN-γ–treated ECs resulted in induction of monocyte IL-12 expression . Finally, we compared the ability of resting monocytes with that of EC-modified monocytes to activate allogeneic and autologous CD4 + T cells. After a 72 h coculture with IFN-γ–treated ECs, CD14 + monocytes were reisolated by positive selection and cultured with allogeneic or autologous CD4 + T cells at fixed responder/stimulator ratios. We found that EC-modified monocytes consistently induced greater proliferation of allogeneic CD4 + T cells than did resting monocytes , an effect inhibited by CTLA4 Ig and anti–HLA-DR antibodies . CTLA4 Ig caused a greater percentage of inhibition of CD4 + T cell proliferation induced by EC-modified monocytes, consistent with enhanced expression of CD86 by these cells. Thus, ECs augment the capacity of monocytes to provide costimulatory signals to CD4 + T cells activated by direct allorecognition. Furthermore, EC-modified monocytes induced proliferation of autologous CD4 + T cells. This suggests that ECs may donate alloantigen to monocytes for presentation to autologous CD4 + T cells via the indirect pathway of allorecognition. Microvascular ECs express cell surface molecules that mediate both the recruitment into and the activation of leukocytes within vascularized solid organ transplants. Thus, it is proposed that interactions between CD4 + T cells and microvascular graft ECs are critical for rejection 12 . Antigen-dependent activation of CD4 + T cells is initiated by interactions between the TCR and foreign peptide in association with MHC class II molecules. ECs express MHC class I and class II molecules and provide antigen-dependent signals to T cells in vitro 7 8 9 10 11 12 and in vivo 17 18 . However, the ability of human endothelium to provide effective costimulatory signals for full CD4 + T cell activation is more controversial 12 13 14 15 16 . Surprisingly, only endothelial LFA-3 has been identified to possess costimulatory function with little if any contribution of other human EC cell surface molecules. Endothelial LFA-3 interacts with T cell CD2 and initiates a series of activation responses in CD4 + T cells that result in IL-2, IL-4, and IFN-γ production 10 15 39 . Since naive and previously activated CD4 + T cells are dependent on CD28 signaling for effective activation 22 , we wished to examine in more detail the role of CD28–B7 interactions in EC-induced CD4 + T cell activation. Our results provide insight into how graft ECs may modify infiltrating leukocytes for provision of CD28-mediated costimulation in trans and promote CD4 + T cell activation via direct and indirect allorecognition. In these studies, we confirm that human ECs do not express CD80 or CD86 mRNA, nor protein assessed by RT-PCR, Western blotting, and FACS ® analysis, respectively. However, we do find that CD86 is induced and is functional in CD4 + T cell–EC interactions due to its expression on CD4 + T cells and the ability of these cells to deliver CD86 mediated costimulatory signals in trans. Indeed, we demonstrate induction of expression of CD86 on CD4 + T cells after coculture with ECs. Blockade of CD4 + T cell CD86 using several anti-CD86 mAbs caused a variable but consistent inhibition of CD4 + T cell proliferation and IL-2 production when alloantigen was presented to CD4 + T cells by several different human microvascular ECs. Furthermore, combined blockade of LFA-3 and CD86 results in additive inhibition of CD4 + T cell activation, suggesting that these molecules participate in parallel pathways of CD4 + T cell activation. We interpret these data to suggest that ECs, in addition to providing direct costimulatory signals to CD4 + T cells, predominantly via LFA-3, may promote CD28-dependent trans-costimulation by the induction of CD86 on CD4 + T cells. The ability of ECs to modify leukocytes for provision of trans-costimulatory signals probably provides an additional mechanism whereby ECs regulate inflammatory responses. Although PHA-activated CD4 + T cells have been reported previously to express both CD80 and CD86, the functional importance of CD4 + T cell expression of these molecules is controversial. Azuma et al. have shown that CD4 + T cell clones expressing CD80 are able to stimulate T cell cytokine production and proliferation in a mixed lymphocyte reaction 33 . Furthermore, Jeannin et al. reported that human effector T cells express CD86, and may costimulate naive T cell responses 34 . In contrast, it has been reported that CD4 + T cell CD86 may be nonfunctional due to reduced posttranslational glycosylation 40 . Our studies clearly demonstrate that CD4 + T cell CD86 is functional and may provide CD28-mediated costimulatory signals in trans to autologous T cells when signal one is provided by mitogen or alloantigen on ECs. The high purity of our CD4 + T cell preparations and the high expression of CD86 on CD4 + T cells after coculture with ECs suggests that CD86-mediated costimulation was provided by CD4 + T cells and not low numbers of contaminating CD86 + dendritic cells. To confirm this, we depleted our CD4 + T cell population of HLA-DR– and CD14-expressing cells to eliminate contaminating CD4 + APCs 36 . The resulting highly purified CD4 + T cells proliferated to IFN-γ–treated ECs, expressed enhanced levels of CD86 after 72 h coculture with IFN-γ–treated ECs and provide functional T cell–T cell trans-costimulation. We note that EC stimulation of CD4 + T cells resulted in a discrete population of CD86 expressing CD4 + T cells; which may represent alloactivated CD4 + T cells. Although resting CD4 + T cells may express low levels of CD86, we suggest that this level is insufficient to provide effective costimulation. Indeed, anti-CD86 reagents fail to inhibit CD4 + T cell activation when signal one is provided by mitogen and costimulation is dependent on constitutive cell surface molecules expressed on ECs or resting CD4 + T cells 15 39 . EC induction of CD4 + T cell CD86 is mediated in part by stimulation of the TCR and T cell CD2. Anti–class II MHC mAbs and anti–LFA-3 mAbs inhibit the ability of ECs to modify CD4 + T cells to provide CD86-mediated costimulation. Consistent with these findings, we found by FACS ® analysis that LFA-3 fusion protein and low doses of PHA additively promote CD86 protein expression in purified CD4 + T cells (data not shown). Therefore, EC LFA-3 may provide costimulation to CD4 + T cells via two distinct mechanisms. First, LFA-3 may directly costimulate cytokine production and CD4 + T cell proliferation via interactions in cis 35 ; and second, LFA-3 may promote trans-costimulation via the induction of CD86 on T cells. Although CD40 signals induce CD80 and CD86 expression on B cells 41 , monocytes, and dendritic cells 42 , we find that interruption of CD40L–CD40 interactions does not inhibit EC induction of functional CD4 + T cell CD86. This is consistent with the low levels of CD40 expression on resting and activated CD4 + T cells. Similarly, OX40L-OX40 interactions do not appear to be functional in EC induction of CD86 expression by CD4 + T cells. Consistent with these observations, neither anti-CD40 mAbs nor anti-CD40L mAbs inhibit alloactivation of CD4 + T cells by IFN-γ–treated ECs (data not shown). Our findings that allogeneic ECs can fully activate bulk populations of CD4 + T cells are similar to those reported by other groups 13 15 35 43 . However, they are different from those reported by Marelli-Berg et al., in which ECs were found to have limited costimulatory function 16 . A common finding of all groups is that addition of B7-dependent costimulation in trans reconstitutes the ability of ECs to fully activate T cells, including naive CD4 + T cells 16 21 . We now report that CD28–CD86 interactions are functional in EC-induced CD4 + T cell activation, via induction of functional CD86 on CD4 + T cells. Although B7-mediated costimulation is required for induction of primary immune responses, our studies have not specifically addressed whether ECs can activate naive CD4 + T cell populations. Previous reports have shown that human ECs fail to activate naive (CD45RA) CD4 + T cells 15 16 , which would seem contradictory to our demonstration of functional CD86 on EC-modified CD4 + T cells. It is possible that the levels of CD86 expressed by EC-modified CD4 + T cells is insufficient to activate naive CD4 + T cells. Alternatively, ECs may be unable to induce CD86 expression and thus to promote trans-costimulation in pure populations of naive CD4 + T cells 10 . Endothelial cell modification of leukocytes for subsequent alloantigen-dependent activation may be of great physiologic importance for allograft rejection. We have recently shown that ECs enhance the ability of CD4 + T cells to respond to intragraft cytokines including IL-2, IL-4 and IL-12 via induction of the respective cytokine receptor (39 and our unpublished observations). Therefore, ECs may indirectly influence the differentiation of CD4 + T cells within the graft. In addition, these studies show that ECs may enhance the expression of costimulatory cytokines and cell surface molecules by transmigrating leukocytes and promote alloactivation of T cells within the allograft. Although antigen-specific T cell activation is considered to occur predominantly in primary lymphoid tissue, recent studies have established a requirement for B7 costimulatory signals at sites of inflammation 26 . We suggest that in vivo, EC-modified leukocytes provide B7-dependent costimulation in trans, allowing for local activation of CD4 + T cells via the direct pathway of allorecognition. By enhancing the antigen presenting and costimulatory capacity of transmigrating monocytes, ECs may promote CD4 + T cell activation through the indirect pathway of allorecognition. Indeed, recent studies have demonstrated that ECs may promote monocyte differentiation into dendritic cells, which may subsequently emigrate back to lymph nodes as efficient APCs 37 38 . In support of a role for ECs in promoting indirect allorecognition, Vallee et al. demonstrated that indirect presentation of xenoantigens by human APCs is crucial in the proliferative response of human CD4 + T cells to porcine endothelial cells 44 . We find that EC-modified monocytes promote greater proliferation of CD4 + T cells than do resting monocytes. This data is consistent with the ability of ECs to modify monocytes for enhanced antigen presenting and costimulatory function. Furthermore, the ability of EC-modified monocytes to promote proliferation of autologous CD4 + T cells may suggest that ECs donate alloantigen to monocytes for indirect activation of CD4 + T cells. In summary, we show that CD28–B7 interactions are functional in the antigen-specific alloactivation of CD4 + T cells by ECs. Moreover by inducing CD86 and cytokine expression, human ECs modify the costimulatory capacity of infiltrating CD4 + T cells and monocytes, which may provide costimulation in trans to CD4 + T cells. Our new findings provide evidence for a unique function of the endothelium in allograft rejection in the direct activation of CD4 + T cells. In addition, the modification of monocytes by ECs may provide a mechanism whereby ECs promote indirect allorecognition. | Study | biomedical | en | 0.999995 |
10449527 | Namalwa (American Type Culture Collection) is an EBV + BL line containing one or two copies of the viral genome. This was used as a positive control for the EBV-specific DNA PCR. BJAB is an EBV − B cell lymphoma used as a negative control for all PCR-related experiments. For reverse transcriptase (RT)-PCR of latent membrane protein (LMP)-2a, LMP-1, and EBV nuclear antigen (EBNA)-2, the LCL line IB4 was used as a positive control. Rael, an EBV + BL line, was used as a positive control for EBNA-1(Qp) RT-PCR. All cell lines were maintained in 5% CO 2 with 10% FCS, RPMI 1640 with penicillin and streptomycin. Patients attending the Transplantation Clinic at the New England Medical Center were recruited for blood donation. The patients, including both kidney and liver recipients, were usually 1–2 mo posttransplantation. They all received cyclosporin A or FK506, combined with azathioprine, mycophenolate mofetil, or sirolimus ( Table ). All of the patients received prednisone and were under active immunosuppression, but were otherwise clinically healthy, at the time they were studied and showed no evidence of PTLD. Healthy volunteers were recruited from Tufts University School of Medicine. The patient and control populations were matched for age distribution, sex, and minority representation. PBMCs were isolated and fractionated using the MACS ® (Miltenyi Biotec) cell separation system as described previously 6 . The antibodies and concentrations used were 0.018 μg/ml anti-CD19–biotin, 0.015 μg/ml anti-IgD–biotin (Southern Biotechnology Associates), 0.03 μg/ml anti-CD23–biotin (The Binding Site), and 1 μg/ml each goat anti-IgG, -IgA, and -IgM (Southern Biotechnology Associates). Positively selected fractions were typically >90% pure for the desired marker. Contamination of a specific B cell population with an undesired B cell population was always <5%. Fractionated populations were analyzed on a Becton Dickinson FACScan™ using Lysis II Software. Anti-CD20–FITC (Dako), a pan-B cell marker, and anti-IgD–PE (The Binding Site) were used to assay the purity and depletion of the desired populations. MOPC21 (IgG1 isotype control; Sigma Chemical Co.) and MOPC121 (IgG2b isotype control; Sigma Chemical Co.) were used as negative controls. Isolated populations were distributed to the wells of a V-bottomed microtiter plate (Nunc) at the desired cell number. The cells were pelleted, the cell pellets lysed, and the extract subjected to DNA PCR specific to the W repeat region of the EBV genome exactly as described previously 6 . PCR products were resolved on a 2% Nuseive agarose (FMC Corp.), 1% Seakem LE agarose (FMC Corp.) gel, and Southern blotted to Nytran Plus as described by the manufacturer (Schleicher and Schuell). Specific products were detected using random primed labeled, purified PCR product from Namalwa cells as described previously 6 . Analysis of the cell cycle status of a given population was performed by labeling cells with propidium iodide in the presence of the nonionic detergent Triton X-100 exactly as described previously 9 . The cells were sorted based on DNA content using a Becton Dickinson FACStar Plus™ , into a V-bottomed microtiter plate. DNA PCR was performed as described above. The calculation of expected versus observed numbers of EBV-infected cells in the S-G 2 -M B cell populations has been described previously 9 and was performed as follows. In Table , we used the measured frequency of EBV-infected B cells in the G 0 -G 1 population to estimate the expected frequency of EBV-infected cells in the S-G 2 -M population if all of the infected cells were proliferating and therefore all of the positive cells in the G 0 -G 1 fraction were in G 1 . This number was then compared with the number of signals obtained experimentally. To make the estimate, the following calculation was performed taking patient 2 from Table as an example: 1.07% of the B cell population was in S-G 2 -M. Therefore, for every 10 6 G 0 -G 1 B cells, there are 1.07/99 × 10 6 = 1.08 × 10 4 cells in S-G 2 -M. The frequency of EBV-infected cells was 800/10 6 in the G 0 -G 1 population. In a typical cycling EBV + lymphoblastoid cell line, the percentage of cells in S-G 2 -M is 45 ± 4%. Therefore, if we assume that the EBV-infected cells have a similar cell cycle distribution, then for every 800 EBV + G 0 -G 1 cells there should be 45/55 × 800 = 655 EBV + cells in S-G 2 -M. Therefore, we should find 655 EBV + cells in 1.08 × 10 4 S-G 2 -M cells. In this experiment, we analyzed a total of 6 × 10 2 S-G 2 -M cells. Therefore, we expect 655 × (1.08 × 10 4 )/(6 × 10 2 ) = 36 infected cells in the S-G 2 -M population we tested. We actually obtained two signals. To resolve linear viral genomes from circular viral genomes, the hallmarks of lytic and latent infection, respectively, PCR-modified Gardella gel analysis was used as described previously 5 . In brief, whole cells are lysed in situ in the wells of a 0.75% low melt agarose gel, and the linear and episomal forms of the genome are separated based on their differential mobility in agarose gels: linear genomes migrate faster because they “snake” through the gel. The lanes were sliced into sections, and the agarose from each slice was digested using β-agarase (FMC Corp.). DNA was precipitated, and EBV-specific DNA PCR was performed for each slice as described above. The relative migration points for linear and circular DNA were identified using virions and PBL B cells, respectively, as controls. RNA was isolated from 5 × 10 6 primary cells or 2 × 10 5 culture cells, and cDNA synthesis was performed as described previously 6 . 5 μl of cDNA was then used in PCR for EBNA-1(Qp), EBNA-2, LMP-1, and LMP-2a. This allowed us to test for all four transcripts from one pot of cDNA. EBNA-1(Qp) PCR was carried out using the amplimers described by Schaefer et al. 21 . Reaction concentrations were 50 mM KCl, 20 mM Tris, pH 8.3, 2.5 mM MgCl 2 , 0.2 mM dNTPs, and 20 μM of each primer. Reaction conditions were 15 s at 95°C, 30 s at 62°C, and 30 s at 72°C for 40 cycles, followed by a 5-min 72°C extension step. For EBNA-2 PCR, amplimers described by Chen et al. were used 22 . Reaction concentrations and temperature conditions were the same as for EBNA-1(Qp). LMP-1 PCR amplimers were as described by Chen et al. 22 , and reaction concentrations were 50 mM KCl, 20 mM Tris, pH 8.3, 3.0 mM MgCl 2 , 0.2 mM dNTPs, 20 μM each primer. Reaction conditions were 95°C for 15 s, 65°C for 30 s, and 72°C for 30 s, followed by a 5-min 72°C extension step. LMP-2a PCR amplimers were as described by Tierney et al. 23 , and reaction concentrations were 50 mM KCl, 10 mM Tris, pH 8.3, 2.0 mM MgCl 2 , 0.2 mM dNTPs, and 20 μM of each primer. Temperature conditions were 95°C for 15 s, 55°C for 30 s, and 72°C for 45 s for 40 cycles, followed by a 5-min 72°C extension step. PCR products were electrophoresed on 2% Nuseive agarose (FMC Corp.), 1% Seakem LE agarose (FMC Corp.) gel, and Southern blotted to Nytran Plus as described by the manufacturer (Schleicher and Schuell). Products were detected by probing with the appropriate purified PCR product (from Rael for EBNA-1, and from IB4 for EBNA-2, LMP-1, and LMP-2a) that was random primed labeled (Boehringer Mannheim) as described by the manufacturer. In every case, it was possible to detect the mRNA from a single infected cell (IB4 or Rael) in the presence of 5 × 10 6 uninfected PBL B cells. To identify the type of latently infected cells in the peripheral blood of immunosuppressed patients, we first characterized their cell surface phenotype. PBLs were fractionated using biotinylated antibodies and the MACS ® magnetic bead technique. The antibodies used recognized CD19, a pan-B cell marker; CD23, a marker expressed on naive B cells and strongly expressed on B cell blasts activated by EBV infection; IgD, which is expressed only on naive B cells; and the A, G, and M isotypes of Ig, which are expressed on the surface of memory cells in the absence of IgD. In the first round of experiments, CD19 was used to separate the B and non-B cell populations, and an anti–IgG+IgA+IgM antibody was used to separate cells expressing surface Ig (sIg) from those lacking sIg. The frequency of virus-infected cells in each population was then assayed by limiting dilution DNA PCR. An example of one experiment for each marker is shown in Fig. 1 , and a summary of the quantitation for two patients for each marker is shown in Table . As expected, essentially all of the virus-infected cells were B cells (CD19 + ) expressing sIg. Less than 1% of the infected cells were in the non-B (CD19 − ) sIg − compartment. This number was so small, it could be accounted for by low levels of contamination of the CD19 − cells with B cells. The B cells (CD19 + ) were then further fractionated on the basis of IgD expression to see if there were infected cells in the naive compartment, and CD23 expression to test if the infected cells expressed the lymphoblastoid phenotype. The results of one experiment for each marker are shown in Fig. 1 , and the quantitation for two patients with each marker is summarized in Table . Essentially all of the infected cells reside in the CD23 − and IgD − populations. There was no significant number of infected cells detected in either the CD23 + or IgD + populations. This is not the expected phenotype of a B lymphoblast. Rather, it is the same as the latently infected cells found in the blood of healthy carriers—memory B cells. Therefore, we conclude that the surface phenotype of infected cells in the peripheral blood of immunosuppressed individuals is the same as that found in healthy carriers. The cell surface phenotype of the infected cells in the blood of immunosuppressed individuals, CD19 + sIg + IgD − CD23 − , is the same as that found for healthy carriers, namely resting, memory B cells 6 . To confirm that the infected cells in immunosuppressed patients were indeed resting, purified B cells were permeabilized and stained for DNA content with the dye propidium iodide. The cells were fractionated on the basis of DNA content into G 0 /G 1 and S/G 2 /M populations, and the frequency of virus-infected cells in each population was assessed. The results of three such experiments, on patients with widely ranging frequencies, are summarized in Table . Only a small fraction, ∼1%, of the peripheral blood B cells were in S/G 2 /M. This is probably a result of the immunosuppressive drugs. If the virus-infected cells were in cycle, there should be a large enrichment of virus-positive cells in the S/G 2 /M population. However, EBV was not found in the S/G 2 /M population of B cells from immunosuppressed patients. In fact, so few positive signals were obtained from the S/G 2 /M population that it was impossible to measure a frequency. This was not an artifact of the techniques used, because EBV-infected lymphoblasts could be quantitatively detected in the S/G 2 /M population when spiked into the peripheral B cells before analysis (not shown, and reference 9). The frequencies of infected cells in the G 0 /G 1 populations could be measured and were indistinguishable from the values obtained for the unfractionated cells. These results imply that most or all of the virus-infected cells are in the G 0 /G 1 population. To further quantitate the accuracy of this conclusion, we used the frequency of virus-infected cells, measured in the G 0 /G 1 population, to predict the number of positives we would expect to find in the S/G 2 /M population if the cells had been in cycle. This is the same analysis we have used for healthy donors 9 . As can be seen from Table , the signals obtained represented ∼5% of those expected or ∼10% of the cells in cell cycle (taking into account the cycling cells in G 1 ). Therefore, we estimate that ≥90% of the infected cells are resting. We conclude that the population of EBV-infected cells found in the peripheral blood of immunosuppressed patients has the phenotype of resting, memory B cells. If the phenotype of infected cells in the blood of immunosuppressed individuals is unchanged from that found in healthy carriers, can we be sure that the number of infected cells is changed? Previous studies could not distinguish whether there was truly an increase in the number of infected cells or simply an increase in the fraction of cells replicating the virus. Therefore, we decided to test the relative contributions of latent and lytic genomes to the viral burden. This was done by using a technique, DNA PCR–modified Gardella gels, that can distinguish the linear form of the viral genome, characteristic of cells replicating the virus, from the circular or episomal form characteristic of latent infection. Using this approach, we have shown that infection in the peripheral blood of healthy carriers is tightly latent (7 of 7 tested), with no viral replication detected 5 . An example of such an analysis on a healthy carrier is shown in Fig. 2 A, and the results for virion DNA, which is linear, are shown in Fig. 2 B for comparison. The results for two immunosuppressed patients are shown in Fig. 2C and Fig. D , and a summary of all the patients tested is shown in Table . It is apparent from these studies that in half the patients (nos. 1, 8, 11, and 15), all of the cells are latently infected . However, the other patients (nos. 10, 12, and 17) demonstrated a substantial number of linear genomes in addition to the latent episomal genomes. Comparison of the relative signals in the episomal and linear location indicates that linear genomes were the major contributor to the viral genome burden in these individuals. Our analysis cannot distinguish the fraction of cells that might be replicating the virus. However, comparison with results obtained with cell lines 5 24 suggests that the relative ratio of linear and episomal genomes could be accounted for by a very small (<10%) fraction of the cells replicating the virus at any one time. We do not believe that failure to detect linear genomes in some patients represents a sampling variation or that the appearance of linear genomes is an artifact due to shearing, since when certain patients were retested, the same results were obtained. For example, patient 8 was tested three times over the course of 8 mo under continuous immunosuppression, and never showed detectable levels of linear genomes. The detection of linear genomes was not a function of the number of infected cells present, since the same number of B cells was loaded into the gel for each patient tested, yet there was no correlation between the frequency of infected cells and the presence of linear genomes ( Table ). We conclude from these studies that in approximately half of the patients studied, the viral genome burden can be entirely accounted for by latently infected cells. However, the remaining patients have both latently and lytically infected cells, with linear genomes contributing the largest component of the viral genome burden. Previous studies have claimed that immunosuppression leads to an increase in the number of latently infected cells in the blood. However, these studies have relied on assays that could not distinguish an increase in the infected cell number from an increase in the fraction of cells replicating the virus. We have now shown that the infected cells are not proliferating, and that viral replication is a frequent occurrence in the blood of immunosuppressed patients. Therefore, it was possible that there was no change in the number of infected cells and that the previously described increase in viral burden was entirely due to increased rates of reactivation. We decided to definitively resolve this issue by precisely quantitating the number of virus-infected cells using the limiting dilution DNA PCR assay we have described previously 4 . Because this assay involves the titration and detection of intact infected cells, it provides a measure of the number of virus-infected cells that is independent of the number of viral genomes per cell and therefore will not be artifactually biased by lytically infected cells that have high genome copy numbers. The results from 28 immunosuppressed allograft patients are summarized in Table . As shown previously, the frequency distribution in healthy carriers is skewed; therefore, it is not possible to calculate a meaningful mean value or standard deviation for comparison between populations. However, the data show a normal distribution when the ln frequencies are derived ; this allows the calculation of mean and standard deviation for the ln frequencies. The mean frequency of the ln values for healthy donors is 3.96. Calculating the anti-ln, this is 50 infected cells per 10 7 B cells, in agreement with our previously published estimates on a smaller subset of healthy carriers 4 . When this analysis was applied to the data set from immunosuppressed patients, it was apparent that there are two discrete groups . The first, represented by patients 1–19, also demonstrated a normal distribution of the ln frequencies, with a standard deviation indistinguishable from that of the healthy carriers (0.95 vs. 1.02). The mean frequency of the ln values was 7.36 or, from the anti-ln, 1,600 infected cells per 10 7 B cells, representing a 30-fold increase over the healthy carriers. The fraction of B cells in these patients varied from normal to ∼10% of normal, but in no case could the increase in the frequency of infected cells be accounted for by selective loss of uninfected cells. Therefore, we may conclude that there was also an increase in the absolute numbers of virus-infected cells. The relative ratio of naive to memory B cells, as judged by sIgD expression, was not different from the control population . To confirm that there was an increase in infected cells due to immunosuppressive drugs, the frequency of virus-infected cells was tested in several patients before and after immunosuppression. In every case, the frequencies before immunosuppression were within the range observed for the control population, and after immunosuppression the frequencies were higher than the highest value seen with the controls (not shown). The second group is represented by patients 20–28. Although too small to draw precise conclusions, this group had a mean frequency of virus-infected cells (36 vs. 50) similar to the healthy controls. The simplest interpretation is that this group of patients did not respond to the immunosuppressive drugs; however, they did not show elevated rates of rejection. Confirmation that this was a discrete population came from the observation that they exhibited a large increase in the numbers of uninfected naive B cells , which was never seen in the high frequency group. This increase counterbalanced a modest increase in infected memory cells, resulting in no overall change in the frequency of virus-infected B cells. We have attempted to correlate the status of the two groups of patients with medications, type and origin of the transplanted organ, diagnosis at the time of transplant, sex, ethnicity, and occurrence of rejection episodes. The only factor that correlated was the use of mycophenolate mofetil with the occurrence of high frequencies of virus-infected cells (see Table ). No factor could be associated specifically with the low frequency patients. We conclude that there is an increase in the frequency of infected B cells in the blood of only two thirds of patients receiving immunosuppressive drugs at the New England Medical Center. RT-PCR analysis of virally infected cells from the peripheral blood of healthy carriers suggests that the only viral latent genes detected are LMP-2a and possibly EBNA-1 7 9 22 23 , although the latter result is controversial. LMP-2a contains the same signaling immunoreceptor tyrosine-based activation motif (ITAM) found in the B cell receptor 25 . Because of this, we have suggested that LMP-2a could play a role in the long-term maintenance of the infected memory cells by supplying a surrogate B cell receptor survival signal 6 . Immunosuppressed individuals also carry the virus in the resting memory population, but at a higher frequency. Therefore, we expected to detect LMP-2a with ease in these individuals. However, as shown in Table , LMP-2a was only found in one of the four patients tested, and this was the patient with the highest frequency of virus-infected cells. The results for two patients are shown in Lanes A and C of Fig. 4 , and the results for all four patients are summarized in Table . Lane A shows the result from a patient whose cDNA was tested from a pool of peripheral blood B cells estimated to contain ∼20 infected cells. No LMP-2a message was detected. Lane C is from a patient whose B cell sample was estimated to contain ∼1,700 infected cells. In this case, the signal obtained had an intensity lying between that obtained with one and five cells from the EBV-immortalized cell line IB4 (lanes B or D vs. E). The cell line IB4 was used as a positive control because, in studies not shown, the RT-PCR signal obtained for LMP-2a was similar for IB4 and a number of other in vitro–immortalized lymphoblastoid lines. Note that in control studies, performed for every RT-PCR, the LMP-2a message can be readily detected in a single cell from the IB4 cell line in the presence of 5 × 10 6 uninfected PBL B cells (lane E). The negative PCR signals obtained were not due to an artifact or inhibitory contaminants in the samples, because we could readily detect five IB4 cells when they were mixed with the patient cells before analysis (see lane B, for example). cDNA from the LMP-2a − patients was also tested for expression of the EBNA-2 gene, which is required for expression of the full panoply of latent genes found in the lymphoblastoid type of latent infection, and EBNA-1(Qp), which is derived from a unique promoter that allows the expression of EBNA-1 only in Burkitt lymphoma cells. Again, no signal was obtained, although the techniques readily detected a single spiked EBV-infected cell, IB4 for EBNA-2 and Rael for EBNA-1(Qp), in the presence of 5 ×10 6 uninfected PBL B cells. These results suggest that the vast majority of infected cells in the blood of immunosuppressed individuals may be transcriptionally silent for the known latent genes. The main conclusion of this paper is that latently infected resting, memory B cells expand in the peripheral blood of immunosuppressed individuals. This surprising result means that there is no expansion of EBV-infected proliferating lymphoblasts in the blood, as had been thought previously. Most previous studies on the increased viral burden in the immunosuppressed have focused on the small subpopulation of individuals that have PTLD, whose systems are perturbed not only by immunosuppression but also by the presence of malignancy. This is the first study to characterize the phenotype of cells that expand in the periphery of patients that are immunosuppressed but otherwise clinically healthy. In fact, this is the first study to demonstrate quantitatively that the number of latently infected cells increases in the peripheral blood of most, but not all, patients after immunosuppression and that the cells are all B lymphocytes. The limitations of the previous studies are best exemplified by our demonstration that in approximately half of the patients studied, viral replication contributes a larger portion of the viral genome burden than does the increase in latently infected cells . Our results cannot exclude the possibility that small (<10%) numbers of proliferating infected lymphoblasts are present in the peripheral circulation. We have attempted to confirm the presence of these cells by testing enriched CD23 + B cells for expression of EBNA-2 and LMP-1, latent genes characteristically expressed in the lymphoblastoid form of latency. However, we have only detected expression of these genes in one out of eight patients tested. Therefore, it seems likely that in most patients the small numbers of infected cells detected by DNA PCR in the CD23 + and cycling populations are due to low levels of cross-contamination. We have shown previously that EBV-infected cells in the peripheral blood of healthy carriers are restricted to the memory compartment. This cannot be because infected naive cells are preferentially lysed by CTLs, because we have now shown that a similar restriction applies even after immunosuppression. Therefore, some other mechanism must account for the specific association of EBV with memory cells in the peripheral blood. In addition, the number and frequency of latently infected cells increase in the periphery of immunosuppressed individuals, yet the cells themselves are resting. Therefore, the production of latently infected memory cells must occur elsewhere. The explanation we favor is based on the idea that EBV uses the normal pathways of B cell activation and differentiation to establish and maintain a latent infection. In this model, EBV-infected lymphoblasts are generated from newly infected B cells in secondary lymphoid tissue such as the tonsil. We propose that these latently infected blasts behave like normal B blasts and remain in the lymph nodes, where they follow the normal pathways of B cell activation and differentiation to gain access to the long-lived memory compartment, which we have proposed as the site of long-term persistence. Upon immunosuppression, there is more infectious virus produced in the lymphoid tissue, and therefore more cells are infected and become proliferating lymphoblasts. These blasts expand due to the lack of a cellular immune response, and eventually leave the lymph nodes as increased numbers of differentiated memory B cells. If our hypothesis is correct, then EBV-associated hyperproliferation due to an impaired CTL response should also be limited to the lymph nodes. This is what is observed with PTLDs, the majority of which are limited to the lymph nodes and disappear when immunosuppression is lifted 26 . Escape from the lymph nodes into the peripheral circulation would be atypical behavior for a B blast, and would be expected to be associated with the acquisition of genetic defects. Consistent with this, extranodal PTLDs are less common, associated with genetic anomalies, more malignant, and less likely to respond to reductions in immunosuppression 26 . A second, but not mutually exclusive, explanation for the results in this paper is that the latently infected resting memory B cells are themselves under immunosurveillance, for example due to expression of LMP-2a 27 . An impeded CTL response would allow higher numbers of these cells to be tolerated. The RT-PCR analysis presented here argues against this possibility. Although we can detect LMP-2a message in mixtures where one IB4 cell is diluted with 5 × 10 6 uninfected PBL B cells, we were unable to detect it in as many as 1,000 infected cells from immunosuppressed patients. This suggests either that the transcript copy number in the resting memory cells is extremely low or that the major population that expands in the immunosuppressed is transcriptionally silent for LMP-2a. We have also failed to detect two other markers of latent gene expression, EBNA-2 and EBNA-1(Qp). EBNA-2 is essential for the expression of all the latent genes expressed in the lymphoblastoid form of latency. Its absence was expected, since EBNA-2 activity would be inconsistent with the resting state of the cells. These results suggest that the major infected population in the blood of immunosuppressed patients may not express the known latent proteins and cannot therefore be detected by immunosurveillance. The one major difference in the behavior of the virus between healthy carriers and immunosuppressed patients is the presence of viral replication in the peripheral blood of some patients. We have not tested our patients for lytic gene expression; however, Prang et al. 28 reported finding immediate early and early but not late transcripts of the lytic cycle in the peripheral blood of healthy carriers. This is consistent with our finding that viral genome replication is not detectable. They also demonstrated strong CTL responses to the immediate early proteins 29 and proposed that cells in the periphery occasionally enter the lytic cycle spontaneously, but are eliminated by CTLs before they produce infectious virus. A direct prediction of this idea is that upon immunosuppression a small fraction of the infected cells in the peripheral blood will spontaneously replicate the virus, and indeed this is what we have found. In conclusion, we have shown that immunosuppression does not lead to the appearance of latently infected blasts in the blood. Furthermore, our experiments lend further support to the idea that EBV has evolved to efficiently exploit the normal biology of B lymphocytes to establish and maintain latency in the memory compartment. | Study | biomedical | en | 0.999995 |
10449528 | A18 TCR-transgenic Rag1 −/− C5 −/− mice on an A/J background 5 (TCR specific for epitope 106–121 of mouse complement C5 presented by H2-E k ) were crossed with the BDC2.5 strain 7 recognizing an unidentified peptide from islet cells presented with H-2A g7 , which we received from Drs. Diane Mathis and Christophe Benoist. These mice are Rag +/+ C5 −/− and have been backcrossed onto a NOD background for 16 generations. Both parental strains were heterozygous for TCR expression so that the resulting F1 mice expressed either the A18 receptor alone (F1 A18 + ), the BDC2.5 receptor alone (F1 BDC + ), or both receptors (F1 dual TCR). Lymph node cell suspensions were stained with PE-conjugated anti-CD4 (PharMingen), FITC-conjugated anti-Vβ8.3 13 specific for the A18 TCR β chain, and biotinylated anti-Vβ4 (PharMingen) specific for the BDC2.5 TCR β chain, followed by streptavidin Red 670 (GIBCO BRL). Cells were stimulated in round-bottomed 96-well plates (2 × 10 5 cells/well) with dendritic cells from bone marrow cultures with GM-CSF (2 × 10 4 /well). Culture medium was IMDM (Sigma Chemical Co.) supplemented with 5% FCS, 5 × 10 −5 M 2-ME, 2 × 10 −3 M l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. After culture for 48 h, aliquots of supernatants were removed and tested for IL-2 content in serial dilutions on IL-2–dependent CTLL cells. Bone marrow–derived dendritic cells were generated as previously described 14 15 with some modifications 16 . In brief, 5 × 10 6 bone marrow cells were cultured in petri dishes (9-cm diameter; Nunc, Inc.) in 10 ml culture medium containing 10% supernatant of Ag8653 myeloma cells transfected with murine GM-CSF cDNA (≈ 25 U/ml). On day 4 of culture, nonadherent cells, mostly granulocytes, were removed. Loosely adherent cells were transferred onto a second dish on day 6 of culture. From day 6 to 10, these transferred cells were used as a source of dendritic cells. Islet antigen for stimulation of BDC2.5 was prepared by subcellular fractionation of beta-tumor cells as previously described 17 . Spleen cells from dual or single TCR–expressing F1 mice were depleted of B cells with sheep anti–mouse IgG–coupled Dynabeads (Dynal, Inc.) and cultured at 2 × 10 5 /ml with 2 × 10 4 /ml dendritic cells in 25-ml culture flasks (Falcon Labware). 48 h later, cells were collected, washed, and injected intravenously into host mice. Cell suspensions from pancreatic lymph nodes (2 × 10 5 /well) were prepared by digestion with a cocktail of 1.6 mg/ml Collagenase (Worthington CLS4) and 0.1% DNase (Sigma Chemical Co.) for 30 min at 37°C. 2 × 10 5 lymph node cells per well were then cultured with different doses of C5 protein, and supernatants were removed after 48 h (for IL-2 and IL-10 measurements) or 72 h for measurement of IFN-γ production. IL-2 production was determined by a bioassay with the IL-2–dependent CTLL line, whereas IL-10 and IFN-γ production was measured in a sandwich ELISA using pairs of antibodies for each cytokine (PharMingen cytokine kits). Diabetes was assessed by weekly measurements of venous blood glucose concentration using BM-Test 1-44 strips and Reflolux S glucometer (Boehringer Mannheim). Animals were considered diabetic after at least two consecutive measurements >12 mM. Onset of diabetes was then dated from the first of the sequential (glucose or diabetic) measurements. After sustained hyperglycemia, mice were killed to prevent prolonged discomfort. The BDC2.5 TCR-transgenic strain recognizes an unidentified peptide from islet cells presented by H-2A g7 , whereas the A18 TCR-transgenic strain recognizes a peptide from the mouse complement component C5. Both strains are C5 −/− and all mice are Rag + , as the BDC2.5 strain has not been crossed onto a Rag −/− background. BDC2.5 and A18 mice, heterozygous for expression of their transgenic TCRs, were crossed to obtain F1 offspring transgenic for the A18 TCR (F1 A18 + ), the BDC2.5 TCR (F1 BDC + ), or both (F1 dual TCR). T cells from all F1 mice were analyzed for expression of Vβ4 and Vβ8.3 (the BDC and A18 TCR-β chains respectively). As shown in the Fig. 1 histogram overlays gated for CD4 T cells, expression of the BDC Vβ4 chain is only slightly lower in dual TCR F1 mice compared with F1 mice expressing the parental BDC2.5 TCR alone, whereas expression of the A18 Vβ8.3 chain is 10-fold lower in dual TCR F1 mice compared with F1 mice transgenic for the A18 TCR only. As there are no clonotype-specific antibodies available for the BDC2.5 or A18 TCRs, the constitutive expression of both receptors cannot formally be verified by FACS™ analysis. We therefore compared T cell responses from BDC2.5 and A18 mice as well as the F1 strain expressing both TCRs after stimulation with either C5 protein or a preparation of islet cell membranes that contained the as yet unidentified antigen recognized by the BDC2.5 TCR. As shown in Fig. 1 B, T cells from dual TCR–expressing mice can respond to both islet cell antigen and C5, indicating that both transgenic TCRs on their surfaces are functional. The spontaneous incidence of diabetes in BDC2.5 mice and F1 mice with A18 was determined in a cohort of mice left untreated for 9 mo. Whereas the parental BDC2.5 strain had a very low incidence of spontaneous diabetes, the dual TCR–expressing F1 mice became diabetic from 3–4 mo of age. The incidence of diabetes was higher in F1 mice transgenic for both the BDC2.5 and A18 receptors compared with F1 mice expressing the BDC2.5 receptor alone. F1 mice transgenic for only the A18 receptor never developed diabetes . A standard protocol for testing the diabetogenic potential of T cells is transfer of activated T cells into neonatal recipients. Spleen cells from BDC2.5 × A18 F1 mice transgenic for either both TCRs or only the A18 TCR were activated in vitro with H-2E–expressing A/J dendritic cells and C5 protein and injected into F1 A18 + neonates 48 h later; these adoptive host mice were transgenic for only the A18 TCR and therefore never developed spontaneous diabetes themselves. Neonatal mice injected with C5-activated T cells from dual TCR–expressing mice developed fulminant diabetes within 1 wk after transfer . In contrast, injection of C5-activated F1 A18 + cells, which express only the C5-specific TCR, did not result in diabetes. Similarly, the injection of C5-activated dual TCR T cells resulted in rapid onset of diabetes upon transfer into adult F1 A18 + mice. These data indicate that activation of the second—in this case C5-specific—TCR could indeed activate the diabetogenic potential of dual TCR–expressing T cells. Given that C5 activation in vitro was able to induce the diabetogenic potential of dual TCR–expressing T cells, we proceeded to test this phenomenon in vivo. F1 dual TCR mice were immunized between 6–8 wk of age with C5 protein or C5 peptide either in CFA or with PBS. Control mice received CFA or PBS alone. In contrast to the results we obtained after in vitro activation of T cells, immunization with C5 protein or peptide did not result in onset of diabetes. Other immunization protocols used were subcutaneous injection with Escherichia coli expressing a C5 fusion protein with maltose binding protein, which has previously been shown to be a powerful immunogen; scarification of ear skin with a DNA construct encoding C5; and intravenous injection of dendritic cells pulsed with C5 peptide. We did not observe induction of diabetes under any of these protocols (data not shown). On the contrary, it emerged that mice immunized with various forms of C5 antigen were protected from spontaneous onset of diabetes. Fig. 4 summarizes data from a cohort of mice immunized with either C5 protein or C5 peptide in CFA (mice pooled) compared with control mice that received PBS or CFA alone, showing blood glucose levels for both groups at different ages. Only three mice in the immunized group became diabetic, whereas the majority of the control mice were diabetic by 4 mo of age. Cells from lymph nodes draining the pancreas were analyzed for cytokine secretion after in vitro restimulation with C5. It was evident that cells from immunized F1 dual TCR mice produced less IL-2 and IFN-γ than those of control F1 dual TCR mice . There was no difference in IL-10 production, and IL-4 secretion (data not shown) was not detectable. In this paper, we tested the hypothesis that T cells expressing two TCRs might be involved in triggering the onset of autoimmune disease. We pursued this hypothesis, which was first proposed by Padovan et al. 3 , because of our finding that dual TCR T cells that had escaped from thymic deletion due to low expression of the self-specific TCR could be activated for autoreactive effector function by triggering through the second, nonself-specific TCR 6 . There was no in vivo correlate for the autoreactivity demonstrated in vitro, and we attributed this to the presence of systemic and high levels of self-antigen in the periphery for this model. The choice of the BDC2.5 TCR-transgenic strain for testing the hypothesis in vivo was prompted by the description that this diabetogenic TCR is not subjected to negative selection in the thymus, the autoantigen, albeit not identified on the molecular level, is restricted to pancreas islet cells, autoimmune disease has a delayed onset in these mice, and the introduction of H-2E molecules is not protective as in the nontransgenic NOD strain 8 . T cells in the periphery of genetically susceptible individuals carrying receptors specific for autoantigen expressed in the pancreas normally should not cause any harm given the recirculation characteristics of naive T cells, which exclude their access to peripheral tissues 18 . Therefore, the crucial question is how potentially diabetogenic T cells get activated to allow them to enter the pancreas and cause destruction. Several possibilities exist. For instance, dendritic cells might carry antigens from the pancreas to draining lymph nodes. This scenario took place in a transgenic model in which ovalbumin, exclusively expressed by islet cells, was cross-presented by bone marrow–derived APCs, resulting in activation of ovalbumin-specific transgenic CD8 T cells in lymph nodes draining the pancreas but not in other lymphoid sites 19 . Similarly, activated T cells from BDC2.5 mice were found only in the islets and draining lymph nodes, suggesting transport of islet antigens to this site 20 . However, dendritic cells do not constitutively engage in this form of presentation, termed cross-presentation, but appear to require the donor cell to undergo apoptotic death 21 . It is debatable, therefore, whether antigens expressed by cells in the pancreas (unless they are secreted) are constitutively processed by dendritic cells and transported to lymph nodes, although it may be possible that individuals genetically susceptible to diabetes have a higher baseline rate of apoptosis in the pancreas, which might support such a mechanism. An alternative explanation for how diabetogenic T cells may be activated takes into account that the onset of many autoimmune diseases is correlated with microbial infections, proposing either molecular mimicry, i.e., cross-reactive recognition of peptides shared by pathogens and auto-antigens 12 22 23 or bystander activation by inflammatory cytokines released in the course of immune responses to pathogens 24 25 26 27 . Activation of a second, e.g., pathogen-specific TCR on potentially autoreactive T cells could be another way of involving pathogens in the induction of disease. The results we obtained with mice expressing a diabetogenic TCR from the BDC2.5 TCR-transgenic strain together with the C5-specific TCR A18 indicate that activation of dual TCR cells by stimulation with C5 in vitro and transfer into neonatal or adult BDC2.5 × A18 F1 mice indeed results in rapid development of diabetes. However, immunization in vivo did not give the same results. On the contrary, we observed that immunized mice were protected from the spontaneous onset of diabetes, which occurs with high frequency in this strain combination. This suggests that T cells within the immunized mice exerted a regulatory influence on otherwise diabetogenic T cells newly emerging from the thymus. Although the underlying mechanisms of protection and regulation remain elusive, there are a number of points worth considering. First, the expression of the C5 TCR, as indicated by staining with anti-Vβ8.3 antibody, was drastically reduced in dual TCR F1 mice compared with F1 mice carrying only the A18 transgene. Because we do not have clonotypic or Vα-specific antibodies for either receptor, we cannot formally exclude the possibility that this is due to preferential pairing of the BDC Vβ chain with the A18 Vα chain. However, these cells have reasonable reactivity to C5 stimulation, indicating that they must express the correct TCR. In several other TCR combinations, many of which were on a Rag −/− background, the presence of a second TCR resulted in reduction of the levels of the C5 TCR, even if there was no negative selection pressure from the presence of C5 6 . The only exception was a dual TCR combination with the H-Y specific, H-2E k –restricted A1 TCR 28 in which both receptors were expressed at equivalent levels (our unpublished data); this combination was also the only one in which both TCRs were expressed in the same construct under control of the human CD2 promoter. We assume that the overall level of TCRs on T cells is adjusted during thymic development, but it is not clear what factors are dictating the relative TCR levels. Positive selection in the thymus and subsequent survival signals in the periphery may provide signals that allow high surface expression of a TCR. It is interesting to note that dual TCR expression by thymocytes was prominent in immature subpopulations but much rarer in mature single positive thymocytes 29 ; however, the latter frequently expressed a second TCR intracellularly 30 . Irrespective of low C5 TCR expression, in vitro stimulation with C5 efficiently activated dual TCR T cells from the BDC2.5 × A18 F1 mice. However, in vitro activation, compared with immunization in vivo, is likely to be artificially optimized. Supplementation with highly efficient dendritic cells as APCs as well as optimal contact in close proximity to APC and antigen might allow strong stimulation even if the expression of TCR is reduced, whereas it may be difficult to achieve in vivo. T cells may be driven into different response modes depending on receptor levels and stimulus strength 31 . In several experimental models 32 33 34 , a phenotype of apparent ‘anergy’ is coupled with regulatory activity. Although we have no direct evidence for such a phenomenon in our system, the finding that protection from diabetes takes place despite the presence of a thymus that would continue to export new T cells suggests that an active mechanism is operative. Thus, although in principle the starting hypothesis is not incorrect, it seems that the physiological behavior of dual TCR T cells cannot be predicted in simple terms. Although it has been argued that dual TCR–expressing T cells are immunologically less effective than single TCR cells 35 , this does not seem to preclude functional activity. For instance, T cells expressing low levels of autoreactive receptors together with unidentified additional receptors were capable of initiating autoimmune responses in vivo 36 , whereas using the BDC2.5 strain on different MHC backgrounds, Lühder et al. 37 describe a protective effect of MHC class II molecules that exerts itself through selection of T cells with additional TCRs. In our experimental system, BDC2.5 mice crossed to A18 were not protected from diabetes by the presence of MHC alleles of the A/J strain; in fact, the F1 combination had a far higher incidence of diabetes than the parental BDC2.5 strain. Instead, protection was induced by in vivo stimulation of the C5-specific TCR. It is unclear at the time of this writing what the underlying mechanisms for protection are. IL-10 has been invoked as a ‘suppressive’ cytokine 38 , but in our study we could not detect any significant differences in IL-10 production between cells from immunized and protected dual TCR mice, whereas there was a significant reduction in IL-2 and IFN-γ secretion in the former group. NOD mice can be protected from diabetes by many types of immunostimulation, including nonspecific (e.g., CFA) stimulation 39 40 41 , and perturbations of the cytokine milieu have been suggested as the underlying cause for this effect. It is difficult to rule out subtle changes in the internal cytokine milieu that are not detected in the in vitro assays, but CFA, which protects nontransgenic NOD mice, had no such effect in the dual TCR-transgenic mice . Although the frequency of T cells with two functionally relevant TCRs may be low 42 43 under physiological conditions, it does exist 44 . T cells expressing additional TCRs are not obligatory for development of autoimmune disease, but as our and other data show, they may contribute to it, either by exacerbating or downmodulating the onset of autoimmune destruction 36 37 . Differential expression levels of TCR provide a way for modulation of antigenic signals 45 , and evidence is accumulating that suboptimal signals drive cells into alternative response modes rather than just leaving them unresponsive, so that they may be able to regulate the degree and mode of activation of other T cells. | Study | biomedical | en | 0.999998 |
10449529 | Human CRP was purified from human pleural fluids exactly as previously described 12 . The purity of the preparation was determined by 12% SDS-PAGE. No contaminating proteins were detected on overloaded gels. The monoclonal anti-CRP antibody, 2C10, was the gift of Dr. Larry Potempa (ImmTech, Evanston, IL) and was used as a hybridoma tissue culture supernatant. FITC-conjugated F(ab′) 2 goat anti–mouse IgG (FITC-GAM) was obtained from Caltag Labs. or from ICN. Affinity-purified F(ab′) 2 PE-GAM was obtained from Caltag Labs. Aggregated IgG (aggIgG) was prepared from human IgG (Sigma Chemical Co.) by incubation at 63°C for 30 min at 10–12 mg/ml. AT10 (an anti-CD32 mAb) and FITC-AT10 were purchased from Serotec. FITC-FLI8.26 (an anti-CD32 mAb) was purchased from PharMingen. This antibody was absorbed with mock-transfected COS-7 cells for 30 min on ice to remove nonspecific binding. PE-C1KM5 (an anti-CD32 mAb) was purchased from Caltag Labs. Anti-CD64 mAb 197 and anti-CD32 mAb IV.3 were purchased from Medarex. Protein A–Sepharose CL6B and Affigel 15 were purchased from Sigma Chemical Co. and Bio-Rad, respectively. Bt 2 cAMP was purchased from Sigma Chemical Co. The human erythroleukemia cell line K-562, the human monocytic cell lines THP-1 and U-937, and COS-7 cells were obtained from the American Type Culture Collection. All cell lines were maintained in RPMI with 10% FCS (Hyclone) and 50 μg/ml gentamycin, except for COS-7 cells, which were maintained in DMEM with 10% FCS and 50 μg/ml gentamycin. Cells were counted and resuspended at 0.3 × 10 6 cells/ml in medium containing 1 mM Bt 2 cAMP, and incubated for 60 h before analysis. The human FcγRIIA cDNA clone in the pcDSRα296 transient transfection vector 13 was obtained from Dr. Kevin Moore (DNAX Research Institute, Palo Alto, CA). Cells were transfected using the GenePORTER™ transfection reagent from Gene Therapy Systems Inc. Control cells received GenePORTER™ reagent only. 6-well tissue culture plates were seeded with 2.5 × 10 5 cells/well and cultured for 18–24 h until the cells were 70–80% confluent. Transfection was performed according to the manufacturer's protocol, except cells were centrifuged to increase transfection efficiency. Plates were centrifuged at 300 g for 10 min at 21°C and then incubated at 37°C for 5 h. Cells were cultured overnight in DMEM with 10% FCS. 18 h after transfection, cells were detached using PBS containing 5 mM EDTA and replated on 100-mm tissue culture plates (two 100-mm tissue culture plates for cells from each 6-well plate) and cultured in DMEM. Flow cytometric assays were performed 66–78 h after transfection. The percentage of transfected cells was 75–85% as determined using either FITC-FLI8.26 or PE-C1KM5. Cells were washed twice in ice-cold PBS containing 0.05% azide and 0.1% globulin-free BSA (PAB). Between 5 × 10 5 and 10 6 cells per tube were washed with 1 ml of ice-cold PAB. Cells were pelleted at 180 g for 5 min at 4°C and resuspended in PAB with CRP at the concentrations indicated in a total volume of 80 μl. Cells were incubated for 1 h in the presence of CRP, then washed twice with 1 ml of PAB. Cells were then incubated for 30 min at 4°C with mAb 2C10 culture supernatant. Cells were washed twice with 1 ml PAB and then incubated with PE-GAM (1 μg/10 6 cells) or FITC-GAM (a 1:100 final dilution) for 30 min. After this last incubation, cells were washed twice with 1 ml PAB as above and then resuspended in 0.4–0.5 ml PAB for analysis by flow cytometry. FcγRI levels were determined using mAb 32.2 and PE-GAM. Levels of FcγRII were determined by binding of FITC-AT10. The percentage of dead cells was determined using 7-Aminoactinomycin D (Molecular Probes). To test inhibition by aggIgG, cells were incubated with aggIgG at an increasing concentration for 30 min before the addition of CRP. There was no significant change in the fluorescence of the secondary antibodies in the presence of aggIgG. Cells were analyzed using a Becton Dickinson FACSCalibur ® flow cytometer equipped with CellQuest software (Becton Dickinson). The population analyzed was gated by forward and side scatter to exclude dead cells. A minimum of 30,000 cells was collected. For all measurements of CRP binding, the binding of 2C10 and the anti–mouse secondary antibody were subtracted. Results are presented as the geometric mean channel fluorescence (gMCF). THP-1 cells were treated with 50 μg/ml pronase (Calbiochem) for 30 min at 37°C, washed, and radiolabeled with 125 I using lactoperoxidase 14 . Membrane extracts were prepared at 5 × 10 7 cells/ml using 1% NP-40 in PBS with 1 mM AEBSF, 1 μM pepstatin A, 10 μM E-64, 10 μM bestatin, 100 μM leupeptin, and 2 μg/ml aprotinin (lysis buffer). CRP beads were prepared by coupling 18 mg CRP/ml Affigel 15 according to the manufacturer's protocol. Antibody beads were prepared by incubation of 5 μg anti-CD32 mAb (IV.3 or AT10) or anti-CD64 mAb 197 with 15 μl protein A–Sepharose. 2.5 × 10 6 cell equivalents were incubated with CRP beads, anti-CD32 Sepharose, or anti-CD64 Sepharose in lysis buffer for 4 h at 4°C. The beads were then washed four times with lysis buffer and once with 0.1% NP-40 in the same buffer. The samples were boiled for 3 min in Laemmli's sample buffer and run on 4–20% gradient SDS-PAGE gels (Novex). The gels were dried and autoradiography was performed using a Storm imaging system (Molecular Dynamics). The binding kinetics of CRP to cells were analyzed using GraphPad PRISM™ software (GraphPad Software, Inc.). ImageQuant software (Molecular Dynamics) was used for quantitation of radioactive bands. Experiments were repeated at least twice. Transfection of COS-7 cells with pcDSRα296 containing the cDNA for CD32 resulted in 70–85% of cells expressing the CD32 marker. When these cells were incubated with CRP, a dose-dependent and saturable binding of CRP was seen . The binding of CRP was fitted to a single site model using GraphPad Prism™ software. Saturation was predicted at 100 μg/ml with an apparent equilibrium binding constant ( K D ) of 6.6 × 10 −8 M (7.6 μg/ml). This binding curve is similar to the binding of CRP to K-562 cells, which express FcγRII as the only FcR . The apparent K D is 9.7 × 10 −8 M (11.2 μg/ml) with saturation at ∼100 μg/ml. Background binding to mock-transfected cells was not above baseline binding of secondary antibody. The apparent affinity of CRP for transfected and K-562 cells was significantly higher than the reported values for IgG binding to FcγRII ( K D > 10 −7 M) 15 and similar to the previously reported figure for CRP binding to K-562 cells (3.8 × 10 −8 M) 6 . In separate experiments using 125 I-labeled aggIgG, an apparent K D of 23 μg/ml was measured (data not shown). Binding of CRP to transfected cells was also examined by two-color analysis to confirm that CRP bound to cells expressing CD32 . Single color staining with anti-CD32 or CRP showed staining of 72% (with a background binding of 6% to mock-transfected) and 63% (with a background binding of <1% of mock-transfected) of transfected cells, respectively. In the two-color analysis, CRP binding to CD32-transfected COS cells directly correlated with anti-CD32 binding with 52% of the cells stained for both markers . To determine if CRP was binding to the same site on Fc γ RII as IgG, the effect of aggIgG on CRP binding was examined. As shown in Fig. 3 , aggIgG inhibited CRP binding to K-562 cells in a dose-dependent manner with 50% inhibition at ∼80 μg/ml of aggIgG. This finding is consistent with previous reports by us 10 11 and others 5 6 7 that aggIgG effectively blocks CRP binding to its receptor. A nearly identical inhibition profile was obtained using aggIgG to inhibit CRP binding to CD32-transfected COS cells . For both cell types, aggIgG did not affect the background binding of the secondary antibodies measured in the absence of CRP. Agents that affect the differentiation state may alter the expression of FcR on monocytic cell lines. The U-937 cell line expresses FcγRI and FcγRII. Exposure of U-937 cells to Bt 2 cAMP has been reported to decrease the expression of FcγRI while increasing the expression of FcγRII 17 . We tested the effect of exposure of U-937 cells to Bt 2 cAMP on the amount of binding of CRP. Since both FcγRI and FcγRII bind CRP, a change in the level of CRP binding might suggest that one or the other receptor is more important in CRP binding. As shown in Fig. 4 , Bt 2 cAMP decreased the expression of FcγRI and increased the expression of FcγRII as previously reported. Associated with this increase in FcγRII, a marked increase in CRP binding was seen . Thus, the binding of CRP to FcγRII may be quantitatively more important than the binding to FcγRI. In an attempt to identify proteins reacting with CRP on FcR-bearing cells, receptor precipitation studies were performed. THP-1 cells express both FcγRI and FcγRII. Attempts to immunoprecipitate surface-labeled proteins with immobilized CRP showed weak and nonspecific bands. However, it had previously been determined that the interaction of IgG with FcγRII could be enhanced by pronase treatment of cells 18 . We recently determined that pronase treatment of FcγRII-bearing cells also markedly increases the binding of CRP (Stein, M.-P., J.C. Edberg, R.P. Kimberly, E.K. Mangan, D. Bharadwaj, C. Mold, and T.W. Du Clos, manuscript submitted for publication). When pronase-treated cells were radiolabeled and precipitated with CRP, distinct binding of two major bands was seen. These bands with M r of 70 and 42 kD corresponded to the bands precipitated by mAb to CD64 and CD32 . The binding of CRP to FcγRII was demonstrated in cells transfected with the gene for human FcγRIIA. Binding was dose dependent and resembled the binding of CRP to the erythroleukemia cell line, K-562, which expresses FcγRII as the only FcR. Because accessory molecules are not necessary for the expression of FcγRII, it is probable that CRP binds directly to FcγRII and not to associated molecules. This finding was also confirmed by the complete inhibition of CRP binding to K-562 and CD32-transfected COS cells by aggIgG. The binding and inhibition curves for the two cell types were similar. These studies suggest that not only is CRP capable of binding to FcγRII, there is reason to believe it is the only receptor on K-562 cells, which express only the low affinity IgG receptor. Precipitation of surface-labeled proteins from the monocytic cell line THP-1 with immobilized CRP demonstrated bands with the M r of FcγRI (70 kD) and FcγRII (42 kD). The 42 kD band could be precleared from the lysate by adsorption with immobilized anti-CD32 mAb, confirming its identity as FcγRII (data not shown). Our previous studies demonstrated that CRP also binds to the high affinity receptor for IgG, FcγRI 10 11 . These studies revealed that mononuclear cells were capable of binding CRP through FcγRI, but FcγRI could not account for all of the binding. Mononuclear cells and monocytic cell lines express FcγRII as well as FcγRI. Our current findings demonstrate the precipitation of both FcγRI and FcγRII from the THP-1 cell line, after pronase activation of FcγRII. However, treatment of U-937 cells with Bt 2 -cAMP, which increased FcγRII expression, also increased CRP binding, whereas treatment of U-937 cells with rIFN-γ to increase FcγRI did not increase CRP binding (our unpublished results). In our previous studies, binding to U-937 cells was completely inhibited by aggIgG but only 20% inhibited by monomeric IgG 10 . These findings indicate that CRP binding to FcγRII accounts for most of the binding to these monocytic cell lines. Taken together, our results support the hypothesis that CRP binding to phagocytic cells is mediated by FcRs. This is further supported by studies in a mouse model in which knockout mice lacking all three FcRs fail to bind CRP (Stein, M.-P., C. Mold, and T.W. Du Clos, manuscript in preparation). These findings thus suggest that there is no unique receptor for CRP, as was postulated previously, and that the major receptor for CRP on monocytic cells is FcγRIIa. In conclusion, the acute phase protein CRP, an ancient mediator of innate immunity, binds to FcγRII, considered a receptor for the acquired immune response, with an affinity comparable to that of IgG. It is attractive to speculate that during the acute phase response, high levels of circulating CRP may influence signaling by IgG complexes through FcγRII. It remains to be determined whether CRP will have a positive or negative influence. Future studies will examine the direct effect of CRP on receptor activation and the effect of CRP on IgG-mediated signaling. | Study | biomedical | en | 0.999998 |
10459005 | Agonists for a diverse group of G protein–coupled receptor (GPCR) 1 agonists (purogenic, muscarnic acetylcholine, angiotensin, lysophosphatidic acid [LPA], thrombin, endothelin, adrenergic, and bombesin) have been demonstrated to bring about increased levels of phosphotyrosine on the EGF receptor . Since GPCRs can induce cell proliferation in certain circumstances , these observations suggest that GPCR-dependent mitogenic activity involves receptor networking that couples GPCRs to a growth factor receptor tyrosine kinase. The important issue is whether the EGF receptor is a necessary component for GPCR mitogenic signaling. Numerous reports have now demonstrated that either over-expression of a dominant-negative EGF receptor or the presence of a specific pharmacologic inhibitor of EGF receptor tyrosine kinase activity significantly uncouples GPCR-driven mitogenic responses . In these reports, a variety of biological endpoints (activation of MAP kinase, tyrosine phosphorylation of known substrates, gene expression, stress fiber formation, DNA synthesis) have been measured in both transfected and untransfected cell lines as well as primary cells. In a different approach, Cunnick et al. 1998 demonstrated that B82L cells (an EGF receptor negative cell line) did not produce a mitogenic response to LPA unless exogenous EGF receptors were expressed. When kinase-negative EGF receptors were expressed in these cells, LPA did not produce a mitogenic response. Hence, the capacity of GPCRs to transduce a mitogenic response requires an EGF receptor and its tyrosine kinase activity. The consistency of these reports reinforces the overall conclusion and its biological significance. Clearly, EGF receptor transactivation may be only one of several independent pathways emanating from GPCRs and inputs from other pathways may also be essential for mitogenic signaling. Bradykinin stimulation of protein kinase C seems to be such a required pathway functioning in parallel to EGF receptor–dependent signaling . In nearly all examples cited, heterologous modulation of the EGF receptor occurs too quickly to invoke a mechanism involving the induced synthesis of EGF-like ligands and in some cases this possibility has been directly tested and ruled out. Less well explored, however, is the possibility that heterologous stimuli might stimulate the proteolytic release of cell surface EGF-like growth factor precursors to soluble and diffusible receptor agonists. Since GPCR activation produces a small amount of EGF receptor tyrosine phosphorylation compared with a saturating dose of EGF, only a low level of growth factor would have to be produced. However, in view of other data cited below and in the absence of direct data, this mechanism seems unlikely at present. The mechanism by which GPCRs actually bring about tyrosine phosphorylation of the EGF receptor is centered on the mediation of the non-receptor tyrosine kinase c-Src, which is reported to be coupled to nearly all GPCRs that lead to EGF receptor phosphorylation . Overexpression of either a dominant-negative Src construct or Csk, a regulatory kinase that inhibits Src function, decreases EGF receptor tyrosine phosphorylation provoked by activation of LPA or α2 adrenergic receptors . The mediator role of Src may, in fact, be direct in that Src is able to associate with and phosphorylate the EGF receptor in vivo and in vitro . This mechanism would predict the existence of Src–EGF receptor complexes provoked by activation of GPCR. The evidence for this is shown by the demonstrations that angiotensin II or LPA rapidly increase the amount of Src coprecipitated with EGF receptors. The EGF receptor autophosphorylates at five known tyrosine residues after the addition of EGF. Src-induced EGF receptor phosphorylation has been mapped to most of these sites as well as novel sites. Of particular interest is phosphorylation of Tyr 845 in the EGF receptor, which has been mapped for both in vivo and in vitro Src phosphorylation and which is not a known autophosphorylation site . This residue is highly conserved in tyrosine kinases and in many kinases has a regulatory role. In the EGF receptor, Tyr 845 is predicted to be in the activation loop of the tyrosine kinase domain and, therefore, could function as an activation trigger to increase activity of the kinase domain. There are several indications that the kinase catalytic activity of the EGF receptor is necessary to mediate GPCR-dependent mitogenic responses. First, chemical inhibitors reasonably specific for the EGF receptor tyrosine kinase and competitive with ATP, which suggests an active site mechanism, block GPCR induction of mitogenic responses. Second, a point mutant, kinase-negative EGF receptor does not support GPCR mitogenic signaling . Therefore, it seems unlikely that the receptor is simply phosphorylated by Src and acting as an inert docking site for SH2-containing signal transducers. An outstanding issue is whether GPCRs and/or Src induce dimerization of EGF receptors, a hallmark of EGF-dependent receptor activation. There is but one report which describes carbachol-induced EGF receptor dimerization. Mutagenesis of Tyr 845 in the EGF receptor does not attenuate EGF-dependent receptor autophosphorylation or signaling, but does prevent DNA synthesis induction by the GPCR agonist LPA . However, reports of this mutant include an inconsistency. In the latter report this mutant also attenuated EGF-induced DNA synthesis, while in the former report it did not. Recently, cells genetically deficient for multiple Src family kinase have been described . It will be informative to test the Src requirement for GPCR coupling to the EGF receptor in those cells. That Src may act upstream of the EGF receptor is complicated by the fact that EGF often produces Src activation. Hence, depending on the agonist Src may be upstream and/or downstream of the EGF receptor. The means by which GPCRs activate Src is not understood. However, there is sufficient data to support two alternative potential mechanisms. Many GPCRs that lead to EGF receptor phosphorylation are coupled to G proteins that activate phospholipase C activity provoking Ca 2+ mobilization. Ca 2+ mobilization can lead to the activation of the cytoplasmic tyrosine kinase Pyk2. There is evidence that in certain cell types after GPCR activation, Pyk2 can associate with Src and that this association may lead to Src activation, through a mechanism that is unclear . This putative pathway proceeds as follows: agonist → GPCR → heterotrimeric G protein → PLC → IP 3 → Ca 2+ → Pyk2 → Src → EGF receptor. However, this pathway cannot explain all GPCR activation of Src, as not all agonists that activate PLC activity bring about Src activation. While dominant-negative forms of Pyk2 have been shown to block GPCR activation of MAP kinases, these assays have not included tyrosine phosphorylation of the EGF receptor. Also, the expression of Pyk2 is not ubiquitous and the above scenario may not be applicable in all cells. Recently a second mechanism has received experimental support . In this scheme, developed with β 2 adrenergic receptors, receptor desensitization is coupled to Src activation. After its activation cycle, the β-adrenergic receptor is phosphorylated by specific serine/threonine protein kinases termed βark kinases. These receptor phosphorylation sites then bring about association of the adaptin-type molecule β-arrestin, which recruits the receptor into coated pits. Evidence is presented that Src is recruited to the adrenergic receptor–β-arrestin complex by interacting with β-arrestin and is activated by this interaction. Such a pathway would proceed as follows: agonist → GPCR → heterotrimeric G protein → βark phosphorylation of GPCR → arrestin: GPCR complex → GPCR-arrestin-Src complex → EGF receptor. This large family of receptors is coupled to JAK family non-receptor tyrosine kinases. Recently it has been reported that activation of the growth hormone or prolactin receptors leads to Jak2-dependent tyrosine phosphorylation of the EGF receptor . In cells expressing the EGF receptor, growth hormone was able to promote GRB-2 association with the EGF receptor, MAP kinase activation, and c- fos induction. These growth hormone–dependent events were also produced with a kinase-negative EGF receptor indicating that only the adaptor-docking function of the EGF receptor was essential and not receptor kinase activity. This is in contrast to the previously described EGF receptor kinase activity requirement for GPCR-induced mitogenesis. The mechanism by which growth hormone stimulates tyrosine phosphorylation of EGF receptor was shown to include JAK-2 activation and the formation of growth hormone receptor–EGF receptor heterodimers. Src participation was ruled out. In vitro experiments indicated that JAK-2 may directly phosphorylate the EGF receptor at sites that include Tyr 1086, a known GRB-2–association site. While there is considerable experimental evidence that activation of integrin receptors and cell adhesion in general can modulate EGF responses and postreceptor signaling events, the possibility that integrins influence EGF receptor function, per se, is suggested by two reports. In one study beads coated with ligands that induce integrin aggregation and activation were added to cells and observed to induce the clustering of EGF receptors around the beads . This suggested the coaggregation of integrin and EGF receptors. Other growth factor receptors (platelet-derived growth factor, fibroblast growth factor) were also coclustered with the beads. While the EGF receptors that clustered around these beads were not tyrosine phosphorylated, the growth factor–dependent activation of the EGF receptor was enhanced by the beads. This could be explained either by exclusion of phosphatases from the clusters or by the fact that preclustering increased cross-phosphorylation of EGF receptors after ligand addition. Beads with ligands that induced only integrin receptor aggregation and not activation did not produce EGF receptor clustering around the beads. Subsequently, a second report presented similar observations from a system in which tenascin C, a collagen-binding glycoprotein that also binds to and activates integrin receptors, was added to smooth muscle cells. However, in this cell system cross-linking of integrin receptors, without activation, was sufficient to induce EGF receptor clustering. In a more recent study using fibroblasts and endothelial cells, cell plating on a fibronectin matrix was shown to produce rapid and transient tyrosine phosphorylation of the EGF receptor . Similar results were obtained when the cells were plated on a matrix coated with integrin receptor antibodies that cluster but do not activate integrin receptors. Cell adhesion mediated by poly- l -lysine did not activate EGF receptors in this system. Mechanistically, it was shown that the β1 integrin receptor subunit coprecipitated EGF receptors in a manner that depended on cell adhesion, i.e., coprecipitation did not occur in cells kept in suspension before lysis. Also, the capacity of fibronectin to increase EGF receptor tyrosine phosphorylation was abrogated by the EGF receptor selective inhibitor AG1478 and did not occur in cells expressing a kinase-negative receptor mutant. These results imply that receptor phosphorylation is a consequence of fibronectin-enhanced activity of the receptor tyrosine kinase. Also, this study used both the AG1478 inhibitor and a dominant-negative EGF construct to show that fibronectin activation of MAP kinase requires EGF receptor function. Similar reagents also were used to demonstrate that EGF receptor function was necessary to protect cells from apoptosis when plated on fibronectin in the absence of growth factors. Further evidence with selective chemical inhibitors indicate the role of the EGF receptor in mediating this resistance to apoptosis was, in fact, not due to MAP kinase activation but rather seem dependent on phosphatidylinositol 3-kinase activity. It should be pointed out that integrin-dependent tyrosine phosphorylation of PDGF receptors has been reported and the means by which integrins communicate with receptors tyrosine kinases may lead to the activation of multiple growth factor receptors, depending on the cells employed. Finally, the collagen-binding proteoglycan decorin has been reported to produce in A-431 cells tyrosine phosphorylation of the EGF receptor and to mobilize Ca 2+ and activate MAP kinase in a manner dependent on the tyrosine kinase activity of the EGF receptor . Decorin is not known to interact with integrin or other cell adhesion receptors, but a recent report suggest that it may, in fact, interact directly with the EGF receptor as a low-affinity agonist . In many cells and, in particular, cells of the nervous system, electrical activity initiates intracellular signaling pathways and the generation of cellular responses, such as secretion. In PC-12 cells the application of KCl leads to altered electrical potential across the plasma membrane and activation of the Ras/MAP kinase pathway. The initiating event seems to be an influx of extracellular Ca 2+ elicited by the activation of voltage-sensitive Ca 2+ channels. The artificial influx of Ca 2+ by ionophore treatment can mimic these responses. In these cells, increased levels of intracellular Ca 2+ result in enhanced levels of EGF, but not insulin or nerve growth factor, receptor tyrosine phosphorylation . Transient expression of dominant-negative EGF receptors or application of the selective EGF kinase inhibitor AG1478 prevents the capacity of KCl or ionomycin to increase EGF receptor tyrosine phosphorylation and activate MAP kinases in PC12 cells . Since the EGF receptor is widely expressed in cells of the nervous system, these results may suggest it has role in either the nonmitogenic signaling events present in these specialized cells or in preventing programmed cell death. The means by which intracellular Ca 2+ levels may provoke EGF receptor tyrosine phosphorylation are thought to revolve about the Pyk2 and Src families of tyrosine kinases, which are both known to be activated by membrane depolarization . This pathway (Ca 2+ → Pyk → Src → EGF receptor) is analogous to that described previously for GCPRs. Interestingly, calmodulin-dependent protein kinase has been implicated in EGF receptor tyrosine phosphorylation after membrane depolarization by KCl, but not by GPCRs . The application of certain exogenous stimuli, both physical and chemical, initiates signal transduction pathways in cells that are part of stress responses. Typically, such stimuli activate members of the MAP kinase family and provoke the expressions of genes. The following stress stimuli have been shown to increase the level of tyrosine phosphate on the EGF receptor; arsenite , sulfhydryl reagents , UV radiation , gamma irradiation , hyperosmotic conditions , oxidants , and heat shock . The tyrosine phosphorylation of other receptor tyrosine kinases is also affected and the influence on the EGF receptor can not be characterized as specific. However, in a number of instances the capacity of these stressors to activate MAP kinases and provoke the expression of certain genes is blocked by selective chemical inhibitors of EGF receptor tyrosine kinase activity and/or by the expression of dominant-negative EGF receptors. These results imply that EGF receptor involvement is a necessary element for the initiation of signaling in response to such stimuli. Perhaps the most investigated stress stimulus, as it relates to the EGF receptor, is UV radiation. UVA, UVB, and UVC have all been shown to very rapidly, within seconds, promote enhanced tyrosine phosphorylation of the EGF receptor. Also, UV induces the phosphotyrosine-dependent association of signaling molecules, such as GRB-2, with the receptor and induces the tyrosine phosphorylation of EGF receptor substrates, such as Shc and PLC-γ1 . That functional receptors are produced by UV exposure is also indicated by studies demonstrating the formation of receptor dimers, receptor aggregation, and receptor internalization . Hence, by many parameters, UV seems to provoke receptor activation that mimics addition of a direct ligand. It is clear, however, that UV does not provoke receptor activation by a means that involves autocrine production of EGF-family agonist. Less clear, though unlikely, is the possibility that stressors promote the proteolytic release of an EGF family molecule from cell surface precursors. However, as UV activates v-erbB, an oncogenic isoform of the chicken EGF-receptor that lacks a ligand-binding domain, the possible role of ligand involvement would seem unlikely . That it is the kinase activity of the EGF receptor that promotes receptor autophosphorylation is indicated by the fact that selective kinase inhibitors, such as AG1478 and tyrphostin 23 block UV-induced EGF receptor phosphorylation. Similarly, expression of a dominant-negative EGF receptor prevents UV-induced receptor phosphorylation presumably by the formation of dimers incapable of cross-phosphorylation . Finally, in cells expressing a kinase-negative EGF receptor, UV exposure did not increase receptor tyrosine phosphorylation . Hence, the evidence suggests that UV stress activates the kinase domain and that EGF receptor phosphorylation is not primarily a consequence of phosphorylation by other kinases. There is one reported exception in cells expressing kinase-negative EGF receptors and wild-type ErbB-2 . UV does activate ErbB-2 and ErbB-2, which heterodimerizes with EGF receptors, is able to cross-phosphorylate the EGF receptor. While UV does seem to mimic growth factor activation of the receptor, no phosphotyrosine maps have been published for UV-activated EGF receptors. Therefore, it is not clear that UV and growth factors result in identical receptor activation. UV treatment of cells activates a plethora of signaling pathways. Experiments using chemical inhibitors of the EGF receptor or expression of dominant-negative receptor mutants show that the EGF receptor mediates, at least in part, several UV-induced signaling events, including activation of erk1 and 2, production of prostaglandins and leukotrienes, and the expression of several genes including c- fos and erg -1 . Less clear is the mechanism by which UV actually activates the EGF receptor. The prevailing evidence would suggest that the activation is indirect through inactivation of phosphotyrosine phosphatase activity. UV is known to produce reactive oxygen species in cells, perhaps through the generation of H 2 O 2 . Interestingly, EGF also promotes H 2 O 2 formation in cells . Several studies have shown that preincubation with antioxidants, such as N -acetylcysteine, prevent UV but not EGF-induced receptor autophosphorylation . Reducing agents also protect against UV-induced receptor activation. As the catalytic sites of phosphotyrosine phosphatase have a highly conserved sulfhydryl group as an essential element, this is the likely target for oxidation-induced by UV. There is experimental evidence at the biochemical level which shows that a phosphatase-containing membrane preparation can dephosphorylate a second membrane preparation that contains activated EGF receptor . The rate of receptor dephosphorylation in this system is markedly decreased by UV treatment of the phosphatase preparation before its addition to EGF receptors. This result implies that basal EGF receptor kinase activity is quite significant, which is true of purified receptor preparations. It is interesting that various stimuli provoke EGF receptor tyrosine phosphorylation by two distinct means. While physical and chemical stressors inactivate downstream phosphotyrosine phosphatases, heterologous receptors and membrane depolarization bring about a similar result by activating upstream tyrosine kinases. It is possible, however, that this division into separate mechanisms is not absolute and some level of contribution by kinases and phosphatases exists with each stimulus, as recently described for carbachol regulation of the Kv1.2 potassium channel . | Study | biomedical | en | 0.999998 |
10459006 | The first step in most cells undergoing apoptosis is to partially detach from the ECM and “round up.” The process is most dramatic in cells that are spread out, with firm matrix attachments and stress fibers, such as fibroblasts, epithelial, and endothelial cells . The process is least pronounced in cells with weak ECM attachments, such as lymphocytes. The release stage has been partially elucidated in endothelial cells, wherein strong peripheral–lateral FA complexes disassemble with reconcentration of focal adhesion complexes ventrally underneath the newly rounded cell body . Concomitantly, actin rearranges into a peripheral ring in preparation for blebbing. Inhibiting actin polymerization with low concentrations of cytochalasin D maintains the spread state in the face of an apoptotic stimulus, suggesting that actin rearrangement is critical . Anoikis (apoptosis due to ECM detachment) represents direct entry into the Release stage. In anoikic cells, FA kinase (FAK) signaling is disrupted , leading to changes in a variety of key signal transduction pathways . Intracellularly, FAK is cleaved, as are three other FA structural proteins (α-actinin, talin, and p130-CAS) that link actin to focal adhesions . Paxillin, another structural FA protein, is dephosphorylated and dissociates from the FA . hsp27, which mediates actin reorganization, is critical for actin rearrangement in apoptosis of endothelial cells . Gelsolin is also implicated in this phase based on studies with gelsolin −/− cells showing significant delay in onset of blebbing, though blebbing eventually occurs . Microtubule (MT) disassembly occurs early in the execution phase and may be necessary for cells to round up . Besides crippling intracellular transport, disassembly of MTs alters cellular compartments and releases a number of regulatory proteins that are normally bound to MTs . Caspases are implicated in this phase, as many cell types do not begin morphological manifestations of the execution phase if caspases are inhibited (however, this may occur predominately in systems where signal transduction of the apoptotic stimuli requires upstream caspases). Caspases cleave FA proteins including FAK and p130CAS . However, many cell types retract and/or begin blebbing despite caspase inhibition . Calpains, which cleave α-actinin, fodrin, and talin (structural proteins linking actin and the plasma membrane) are also implicated in release . p38MAP kinase signaling activates hsp27 and actin reorganization . In anoikis, MEKK-1, an upstream regulator of p38MAP kinase, is activated by caspases and may in turn play a role in caspase-7 activation ; this potential positive feedback loop may explain why entry into the execution phase is apparently irreversible. The p21-activated kinase 2 (Pak2), which is activated by the small GTPases, Rac and Cdc42, and is known to reorganize the actin cytoskeleton, is cleaved into an active form by caspases . In nondying cells, a different family member, Pak1, causes stress fiber disassembly and retraction by phosphorylating myosin light chain kinase (MLCK), and decreasing myosin activation . Thus, Pak1 could be important for stress fiber disassembly and, indirectly, actin reorganization (see also below). After the cell rounding up that occurs during release, the model we propose involves myosin II activation centripetally contracting the cortical actin ring. At the same time, membrane-actin linkages weaken focally, resulting in bleb extrusion in areas of weakness . A counterforce (perhaps myosin I or VI) retracts the blebs and the cycle repeats. Actin and myosin may concentrate at the base of blebs, and a thin rim of membrane-associated actin lines the blebs. Blebbing does not occur in some cells lacking caspase 3 ; cells appear to release but do not bleb (Pittman, R., unpublished observations), suggesting a cellular checkpoint exists between release and blebbing. Myosin II, or conventional myosin, seems to provide the force for dynamic membrane blebbing in both apoptotic and nonapoptotic systems . Inhibition of myosin motor activity or the myosin activators MLCK or RhoA stops bleb formation in apoptotic cells . A role for nonconventional myosins is based on the fact that the general myosin motor inhibitor butanedione monoxime (BDM) inhibits bleb retraction and based on the role nonconventional myosins like myosin I and VI have in regulating protrusive membrane structures in general . Disruption of the actin cytoskeleton with cytochalasin D decreases membrane blebbing , implying that actin polymerization and/or polymerized actin are needed for force generation. The links these proteins provide between the actin cytoskeleton and the plasma membrane may be broken focally, allowing blebs to protrude at foci where the plasma membrane is no longer anchored to the cytoskeleton. Fodrin (nonerythrocyte spectrin) has been implicated in blebbing numerous times, as it is readily cleaved in multiple places by caspases and calpains . The ezrin, moesin, radixin family of actin membrane–linking proteins is also dephosphorylated and dissociates from the membrane during the execution phase , and ezrin can be cleaved by calpains . Although caspases cleave cytoskeletal components like fodrin and, therefore, have been implicated in this stage, many cells bleb for days with caspases apparently inhibited . MLCK, which activates nonmuscle myosin II by phosphorylating the regulatory light chain, is necessary for initiation and propagation of blebbing . RhoA has also been shown to be important for blebbing , probably by activating Rho kinase, which phosphorylates and inhibits myosin phosphatase . Large amounts of ATP are required for blebbing (to maintain myosin contractility), so cellular energy generation most likely is not compromised . All cells eventually stop blebbing. Under normal conditions, this usually happens with striking regularity after about an hour. Cessation of blebbing is followed by fragmentation into small apoptotic bodies or condensation into a small ball with actin and MTs largely disassembled or degraded . The Condensation Stage may simply represent the end result of blebbing, when contraction occurs strongly enough to pinch off apoptotic bodies. However, caspase inhibition can stop apoptotic body formation without stopping blebbing . Hence, apoptotic body formation, although a direct offshoot of blebbing, must represent a distinct stage of the execution phase, immediately downstream of blebbing. There may be a cellular checkpoint between blebbing and condensation, or perhaps cells can condense only after enough cytoskeleton has been dismantled/reorganized to allow cytoplasmic dissolution. Little is known about specific, regulatable aspects of this final active stage of apoptosis; however, F-actin seems to be required for apoptotic body formation . Caspases almost certainly play a role, as inhibition of caspases leads to either no morphologic changes during apoptosis or leads to cells trapped in a blebbing state, unable to condense . Perhaps the role of caspases in cell shrinkage events is merely to limit blebbing and induce condensation. This might happen by caspases slowly degrading proteins necessary for blebbing (e.g., actin). Other proteases might also be important (e.g., serine proteases and proteasomal proteases). The p21-activated kinase Pak1 appears critical for apoptotic body formation . In some cells, activation of the protein cross-linking enzyme, transglutaminase, has been implicated in cytoplasmic packaging during condensation . Apoptosis is known to be a broadly conserved means of eliminating cells without damaging neighboring cells, without spreading pathogenic DNA, and without inciting an immune response. It is during the execution phase that these evolutionary directives are carried out. However, it is not yet understood exactly which events in the execution phase correlate with the evolutionarily driven functions of apoptosis. For example, although blebbing is an almost universal feature of apoptosis, it is not clear why cells bleb. Perhaps, cytoplasmic blebs represent a mechanostructural communication to neighboring cells to begin the process of phagocytosis, or maybe membrane blebbing functions within the dying cell to deplete ATP, to mix compartments as part of cellular packaging, or as a prerequisite for apoptotic body formation. In any case, the release, retraction, and condensation stages culminate with the creation of a smaller cell or cell fragments for facilitated phagocytic clearance. For cells that maintain strong cell–cell contacts (such as epithelial or endothelial cells), the cytoplasmic execution phase machinery may be very important for another reason. The contraction of an apoptotic cell may be an altruistic means of preserving an intact monolayer, as the dying cell expends its energy contracting neighboring cells to cover the potential gap that would be created by the dying cell . In epithelial monolayers the execution phase of a cell results in stretching of neighboring cells toward the dying cell . Stress fibers form in the neighbors, suggesting that they are being pulled rather than locomoting themselves. The dying cell likely generates force by the same mechanism underlying membrane blebbing (actin-myosin contractions); therefore, inhibiting blebbing and/or condensation phases should decrease barrier function in epithelia. In conclusion, from the standpoint of the organism, the most important aspect of apoptosis is that death occurs without the release of potentially pathogenic or harmful macromolecules and without inflammation or injury to neighboring cells. A primary role of the execution phase is to insure that the dying cell is packaged to avoid these potentially devastating sequelae. The past two years have been marked by considerable increase in basic information on mechanisms underlying the extranuclear execution phase. While it is still too early to generate a precise model or scheme of this critical aspect of apoptosis, it is possible to organize most of the results as they pertain to three distinct stages: release, blebbing, and condensation. It is becoming clear that these stages represent specific, inhibitable underlying processes with evolutionarily important roles. Given the recent flurry of information, it appears that research on the extranuclear execution phase is now primed to advance our fundamental understanding of apoptosis into uncharted areas. Besides the obvious impact of this area of work for understanding the apoptotic process, an additional benefit of investigation into cytoplasmic execution should be a better understanding of the cytoskeleton in general with potential novel roles for microtubules, actin, and myosin. | Study | biomedical | en | 0.999998 |
10459007 | CHOC 400 is a CHO cell derivative in which a 243-kb segment of DNA containing the DHFR gene has been amplified ∼500-fold by stepwise selection in methotrexate . CHOC 400 cells were grown as monolayer cultures in DME (GIBCO) supplemented with nonessential amino acids and 5% FBS (GIBCO) at 37°C in a 5% CO 2 atmosphere. Homogeneous populations of cells blocked in metaphase (≥95%) were obtained by mechanical shake off after 4–5 h incubation with nocodazole (Calbiochem-Novabiochem) at 50 ng/ml as described . G1-phase populations were prepared by washing mitotic cells with warm medium and plating in free medium (cells were collected at 2 and 6 h to obtain pre-ODP or post-ODP cells, respectively). G1/S-phase cells were prepared by releasing mitotic cells in free medium for 2–6 h, after which aphidicolin (Calbiochem-Novabiochem) was added at 10 μg/ml and the cells were incubated for another 8–12 h. Cells were released from the aphidicolin block by washing with warm PBS and subsequently incubating for 5 min at 37°C in free medium; under these conditions, ∼70–95% of the cells entered S-phase. CHOC 400 S-phase cell populations were prepared by further incubation in free medium for 3 h (early S-phase) or 6–8 h (mid/late S-phase). CHOC 400 cells, synchronized at the G1/S border as described above, were pulse-labeled with 10 μM 5-chloro-2′-deoxyuridine (CldU; Sigma Chemical Co.) for 5–20 min (designated earliest-replicating sequences). The CldU-medium was removed, cells were washed with warm PBS, chased with 200 μM thymidine (Sigma Chemical Co.), and then regrown in fresh medium. CHOC 400 cells in late S-phase (typically 6–8 h after release from aphidicolin block) were pulse-labeled with 10 μM 5-iodo-2′-deoxyuridine (IdU; Sigma Chemical Co.) for 10 min (designated late-replicating sequences), the IdU-medium was removed, the cells were washed with warm PBS and regrown in fresh medium. The differential staining of DNA sites, substituted with CldU or IdU, was performed according to the protocol described in , with some modifications. In experiments where only CldU- or IdU-substituted DNA was detected, cells were washed with PBS, fixed with cold 70% ethanol and stored at 4°C for an indefinite period of time. Immediately before immunostaining, cells were incubated for 30 min at room temperature in 1.5 N HCl, then washed and incubated with the primary antibody. Sites of CldU incorporation were detected using rat anti-bromodeoxyuridine (BrdU) antibody (no. MAS250b; Harlan-Sera Lab) and FITC-conjugated donkey anti–rat IgG (no. 712-095-153; Jackson ImmunoResearch Laboratories). Sites of IdU incorporation were detected using mouse anti-BrdU antibody and FITC-conjugated donkey anti–mouse IgG (no. 715-095-151; Jackson ImmunoResearch Laboratories). Antibody concentrations were adjusted specifically for every experiment. Incubations with antibodies were carried out in a humidified chamber for 0.5–1 h at room temperature, or for longer time periods at 4°C. Multiple controls, using nonlabeled DNA or omitting the respective primary antibodies, were performed to ensure that there was absolutely no cross-reactivity in double-staining experiments. For immunostaining of protein antigens, CHOC 400 cells grown on coverslips were washed with cold PBS and cold cytoskeleton buffer (CSK: 10 mM Hepes-KOH, pH 7.4, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl 2 ) and then extracted for 2 min on ice with 0.5% Triton X-100 (Triton; Sigma Chemical Co.) in CSK buffer supplemented with 1 mM PMSF (Boehringer Mannheim), 1 μg/ml each pepstatin, chymostatin, leupeptin, and aprotinin (Sigma Chemical Co.), 50 mM sodium fluoride and 0.1 mM sodium vanadate. The cells were fixed for 20 min at room temperature with 4% formaldehyde in PBS, washed with PBS, treated for 5 min with 0.5% NP-40 (Boehringer Mannheim) in PBS, and stored in PBS at 4°C. The following primary antibodies were used for detection of protein antigens: (a) affinity-purified rabbit polyclonal anti-human Mcm2 antibody ; (b) rabbit polyclonal anti-human RPA antibody ; and (c) mouse monoclonal anti-human PCNA antibody (PC10 mAb, no. sc-56; Santa Cruz Biotechnology). The secondary antibodies used were Texas Red (TxRed)-conjugated donkey anti–rabbit IgG (no. 711-075-152; Jackson ImmunoResearch Laboratories), FITC-conjugated donkey anti–rabbit IgG (no. 711-095-152; Jackson ImmunoResearch Laboratories) and TxRed-conjugated donkey anti–mouse IgG (no. 715-075-151; Jackson ImmunoResearch Laboratories). The order of addition of the primary antibodies was found to be essential in the Mcm2+PCNA staining experiment in Fig. 4 A. Incubating the cells simultaneously with the monoclonal anti-PCNA and the polyclonal anti-Mcm2 antibodies resulted in normal staining for Mcm2, but complete lack of or very faint staining for PCNA. Hence, the cells were first incubated with the anti-PCNA, followed by the anti-Mcm2 antibodies. This effect was not observed in the RPA+PCNA staining experiment in Fig. 4 B, in which the cells were incubated simultaneously with the monoclonal anti-PCNA and the polyclonal anti-RPA antibodies. All washes after antibody incubations were done with 0.5% Tween-20 (Sigma Chemical Co.) in PBS (PBS/Tween) at room temperature. In double-staining experiments where proteins were colocalized with sites of CldU- or IdU-substituted DNA, the cells were fixed according to the protocol described for the respective protein and immunolabeling of the protein antigen preceded detection of the halogenated nucleotides. The primary and secondary antibodies specific for RPA, Mcm2, or PCNA were fixed in place with 4% formaldehyde for 20 min at room temperature, the cells were treated for 5 min with 0.5% NP-40 in PBS, DNA was depurinated with HCl and the coverslips were subsequently washed with PBS/Tween and incubated with the anti-CldU or anti-IdU antibodies as described above. Coverslips were mounted in Vectashield (Vector Laboratories). Conventional epifluorescence microscopy was performed with a Nikon Labophot-2 microscope equipped with a Nikon PlanApo 100× 1.4 NA oil-immersion objective, a dual FITC/Rhodamine (Merge images) and single FITC and TxRed fluorescence filters. Photographs were taken on Kodak Ektachrome P1600 films, scanned with a Nikon Coolscan device and assembled in a Power MacIntosh and Apple G3 computers using Adobe Photoshop 5.0.2 and Claris Draw 1.0v4 software. In Fig. 2 C and 4, dual-color confocal laser scanning microscopy was performed with a MRC 1024 ES system (Bio-Rad Laboratories) equipped with a Nikon Eclipse E600 microscope. A Nikon PlanApo 60× 1.4 NA oil-immersion objective was used, and a Krypton/Argon laser to excite FITC and TxRed (at 488 and 568 nm, respectively). Optical sections of 512 pixels × 512 pixels × 8 bits/pixel were collected through the nuclei at 0.5-μm intervals (with Kalman averaging of 6 images). The fluorescence signals from the two fluorochromes were recorded sequentially. Images were processed using LaserSharp software and assembled in an Apple G3 computer using Adobe Photoshop 5.0.2 and Claris Draw 1.0v4 software. CHOC 400 cells were trypsinized, washed with cold PBS and CSK buffer, and then resuspended at 2.5 × 10 7 cells/ml in CSK buffer containing 0.5% Triton, 1 mM PMSF, 1 μg/ml each pepstatin, chymostatin, leupeptin, and aprotinin, 50 mM sodium fluoride and 0.1 mM sodium vanadate. Cell extraction was carried out for 5 min on ice. Identical results were obtained with different types and concentrations of nonionic detergent (0.05–0.5% Triton or NP-40) and different extraction times (1–10 min). Cell lysates were separated into a soluble fraction and a nuclear pellet by centrifugation for 3 min at 1,500 g at 4°C. The pelleted nuclei were washed once with CSK buffer and resuspended in CSK at 2.5 × 10 7 nuclei/ml. To analyze the amount of total nuclear protein, intact nuclei were prepared by cell permeabilization with digitonin as described , with some modifications. CHOC 400 cells (5 × 10 6 cells/ml) were incubated for 5 min on ice in transport buffer (TB) containing 70–80 μg/ml digitonin, 1 mM PMSF, 1 μg/ml each pepstatin, chymostatin, leupeptin, and aprotinin, 50 mM sodium fluoride, and 0.1 mM sodium vanadate. Cytosolic proteins were removed by immediate centrifugation (without addition of BSA stop-solution) at 1,500 g for 2 min at 4°C. The intactness of nuclei was verified before they were used further in the experiment. Digitonin-permeabilized nuclei were prepared by raising the concentration of digitonin to 250 μg/ml . Nuclear pellets were washed once with cold TB and resuspended in TB at 2.5 × 10 7 nuclei/ml. Proteins were separated by electrophoresis in SDS–polyacrylamide gels as described and electroblotted to nylon membranes (Immobilon, Millipore) using a semi-dry system (Bio-Rad Transblot SD). The membranes were blocked for 1 h in 4% nonfat dry milk in TBS-T buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.05% Tween-20) and probed with the respective primary antibodies, followed by horseradish peroxidase-conjugated goat anti–rabbit or anti–mouse IgG. Antibody binding was detected by enhanced chemiluminescence system (ECL; Amersham). Specificity of initiation in the DHFR locus in pre- and post-ODP CHOC 400 nuclei was determined by the early labeled fragment hybridization (ELFH) assay as described . In brief, ∼5 × 10 5 intact nuclei were incubated for 90 min in a Xenopus egg extract supplemented with 100 μg/ml aphidicolin. Nuclei were then washed free of aphidicolin and the earliest-replicating nascent DNA chains were labeled briefly with α-[ 32 P]dATP (New England Nuclear). 17 unique probes distributed over a 120-kb region that includes the DHFR ori-β were immobilized on nylon filters (Hybond N+; Amersham) and hybridized to the 32 P-labeled early replication intermediates. Relative cpm were obtained by PhosphorImaging analysis (Molecular Dynamics) and adjusted for differences in probe size, deoxyadenine content, and hybridization efficiency by normalizing to the corresponding values for parallel hybridizations with labeled replication intermediates from exponentially growing cells. In principle, a pre-RC protein could either associate with all replicons during G1-phase or it might associate only with the very first replicons preparing to initiate S-phase. We wanted to design a protocol that could distinguish these two possibilities. In mammalian cells, several replication proteins (DNA polymerase α, cyclinA/cdk2, DNA methyltransferase, DNA ligase I, and PCNA) have been shown to be present at sites of DNA synthesis by simultaneous indirect immunofluorescent labeling of protein antigens and BrdU-substituted nascent DNA . However, by design this approach cannot detect the assembly of pre-RCs because it requires that DNA synthesis be already ongoing in order to label sites on DNA. We reasoned that if we could tag early- and/or late-replicating DNA sequences in one cell cycle, then we could follow the association of various proteins with the prelabeled DNA sites during G1-phase of the subsequent cell cycle. To accomplish this, we took advantage of the ability of specific antibodies to distinguish segments of DNA substituted with iodinated or chlorinated nucleotide analogues to develop a method of differentially tagging the earliest- and the later-replicating DNA . CHOC 400 cells were synchronized at the G1/S border by mitotic selection and subsequent incubation in the presence of aphidicolin. Aphidicolin inhibits the processive elongation of nascent DNA strands but does not prevent the initiation of replication and the formation of short (100–500 bp) primers . Thus, cells accumulate with replication forks arrested close to their sites of initiation. These cells were then released into S-phase by removal of aphidicolin and the very earliest-replicating chromosomal domains were briefly labeled with CldU, followed by a chase period of several hours. Late-replicating DNA was then labeled with IdU, the cells were subsequently synchronized in metaphase by mitotic shake off and released into G1-phase. It has been shown that the distribution of replication sites in the nucleus follows a defined spatio-temporal program during S-phase, which is typical for each cell type . Examples of these patterns in CHOC 400 cells (a derivative of CHO cells in which the DHFR locus has been amplified 500-fold) are shown in Fig. 1 . Each of the fluorescent foci consists of a cluster of replicons that are synchronously replicated within the span of ∼60 min . Early replication patterns (visualized as a few tens to a few hundred small fluorescent foci scattered throughout the nuclear interior) persist for the first 5–6 h of a 10–12-h S-phase and consist of multiple sets of replicon clusters. With the protocol shown in Fig. 1 , only the very earliest subset of these replicon clusters is labeled with CldU. Since cells do not traverse S-phase in perfect synchrony, the late S-phase IdU–label highlights all three late spatio-temporal replication patterns, providing a convenient means to visualize the entire spectrum of late-replicating domains within the same cell preparations. We have demonstrated that each of these spatio-temporal patterns of labeled replicon clusters persists throughout interphase and is reproduced in the subsequent cell cycle within 2 h after metaphase. Furthermore, we have shown that the earliest subset of clusters tagged with CldU are reproducibly activated at the onset of subsequent S-phases (Dimitrova, D.S., and D.M. Gilbert, manuscript submitted for publication). Based on this evidence, we conclude that it is feasible to compare the assembly of pre-RCs onto the earliest- vs. later-replicating chromatin by following the behavior of the CldU-tagged and IdU-tagged DNA sites during G1-phase of the subsequent cell cycle. We examined the localization of chromatin-bound Mcm2 relative to earliest- and late-replicating chromatin in CHOC 400 cell populations prelabeled as described above and released in the subsequent cell cycle for different periods of time. Cells were first extracted with Triton X-100 to remove soluble nuclear proteins, then fixed and stained with a polyclonal antibody specific for Mcm2 and a monoclonal antibody specific for either CldU or IdU. We observed that Mcm2 began to associate with both early- and late-replicating chromatin within Triton-extracted nuclei as soon as the daughter nuclei were formed at the end of mitosis. There was no qualitative difference in the Mcm2 staining pattern between pre-ODP and post-ODP nuclei . To verify that these cells were in fact in the pre-ODP and post-ODP stages of G1-phase, intact nuclei prepared from aliquots of the same cell cultures were introduced into Xenopus egg extract and the sites of in vitro initiation of replication at the DHFR locus were mapped . These results confirmed that neither the complicated synchronization procedure, nor the tagging of DNA sequences with halogenated nucleotides exerted any deleterious effects on the progression of cells through G1-phase. We conclude that Mcm2 association with chromatin is predominantly upstream of the DHFR ODP and that no qualitative changes in Mcm2 distribution take place at the ODP. Nearly uniform association of Mcm2 with chromatin continued throughout G1-phase and persisted in aphidicolin-arrested (G1/S) cells. Remarkably, within a few minutes after the release of cells into S-phase, when CldU-tagged sequences are actively replicating, a significant amount of Mcm2 was absent from these earliest-replicating clusters. Since early foci take an average of 60 min to complete replication , Mcm2 is either released from active replicons or the epitope is rapidly masked. Mcm2 proteins were clearly still present on the remaining chromatin, as manifested by the intense staining of the nuclear interior. These results are most consistent with the rapid release of Mcm2 proteins from the very first replicons to fire and the persistence of Mcm2 on the remaining sets of replicon clusters, including the remaining early-firing replicons. By 3 h into S-phase, when more than half of the early replicating foci have completed replication (Dimitrova, D.S., and D.M. Gilbert, manuscript submitted for publication), detectable Mcm2 was cleared from a large fraction of the early-replicating chromatin and the intensity of the immunofluorescent signal in the nuclear interior had significantly decreased. However, Mcm2 proteins remained associated with late-replicating chromatin throughout early S-phase and were gradually released from these domains at times corresponding to their replication times. The data described above were obtained with a conventional epifluorescence microscope equipped with a high numerical aperture lens to restrict the image collected to a relatively thin focal plane. To further substantiate that the colocalization of Mcm2 with earliest- and late-replicating chromatin was not due to overlap of independent signals from different focal planes, confocal images were collected using z-sections of 0.5 μm. This was particularly important to confirm colocalization of Mcm2 with the earliest-replicating sequences (which are distributed throughout the interior of the nucleus) and the separation of Mcm2 from those sequences shortly after initiation of replication. Results confirmed that detectable Mcm2 is released from chromatin domains shortly after they initiate DNA replication. As a control, aliquots of these same cells were used to colocalize the earliest-replicating DNA domains with PCNA, which functions at the elongation stage of DNA replication and is clearly not a component of the pre-RC. PCNA is present in the nucleus throughout the cell cycle but associates with chromatin only during S-phase, localizing to sites of DNA synthesis that appear as punctate foci within the nucleus . Since both PCNA and IdU are detected with mouse monoclonal antibodies, we could not visualize them simultaneously. Instead, we prepared parallel cultures of prelabeled cells, singly pulse-labeled with CldU late in S-phase. Consistent with previous reports , indirect immunofluorescent staining showed that, during G1-phase, PCNA was completely removed from CHOC 400 nuclei by extraction with Triton . PCNA assembled into detergent-resistant replication granules at the onset of S-phase , colocalizing to a significant extent with the earliest-replicating chromatin. As cells progressed further into S-phase, the percentage of PCNA foci that colocalized with earliest-replicating chromatin was reduced and PCNA was redistributed to later-firing subsets of euchromatic replicons. However, in contrast to Mcm2 proteins, PCNA was completely excluded from heterochromatin for the first 5–6 h of S-phase . PCNA associated transiently with late-replicating domains exclusively at their respective scheduled replication times . The data in Fig. 2 indicate that Mcm2 is released from the earliest-firing replicons before the completion of their replication. This result appears to be in contradiction to experiments in S . cerevisiae that have revealed the presence of Mcm proteins at active replication forks . The fact that PCNA colocalizes with the CldU-labeled sequences suggests that, if Mcm2 was also a replication fork protein, then we should also have observed a significant degree of colocalization of Mcm2 with the CldU-labeled sequences. Nonetheless, to directly examine the presence of Mcm2 at replication forks, we stained aliquots of the same cells shown in Fig. 2 C simultaneously for PCNA and Mcm2 and analyzed them by confocal microscopy . No colocalization of PCNA and Mcm2 was observed at any time during S-phase. We considered the possibility that the monoclonal anti-PCNA and polyclonal rabbit anti-Mcm 2 antibodies might somehow mask each other and prevent the detection of colocalizing signals at replication forks. Hence, we performed control experiments double-staining aliquots of these same samples with monoclonal anti-PCNA and polyclonal rabbit anti-RPA antibodies (RPA is also present at the replication fork, see below). As expected, a high degree of colocalization was observed between PCNA and RPA throughout S-phase . We conclude that there is no immunologically detectable Mcm2 protein at mammalian replication forks. Although there was no qualitative change in the binding of Mcm to chromatin during G1-phase, we noticed that the intensity of anti-Mcm2 staining was generally brighter in later stages of G1-phase. In particular, confocal analysis indicated that the colocalization of Mcm2 signal (amount of yellow color) with both early- and late-replicating domains increased from early G1 to late G1-phase. To obtain a more quantitative estimate of the amount of Mcm2 bound to chromatin at different times during G1-phase, immunoblotting experiments were performed with either whole cell extracts, intact nuclei, permeabilized nuclei or detergent-extracted nuclei. Results revealed that the total amount of Mcm2 per cell did not vary significantly throughout the cell cycle and remained exclusively nuclear during interphase. However, the fraction bound to chromatin was regulated during the cell cycle, consistent with previously reported results with HeLa cells . Triton extraction of chromatin was slightly more effective at removing soluble Mcm2 than digitonin permeabilization of nuclei. Varying the concentration of Triton (from 0.05 to 0.5%) or the length of exposure to Triton (1–10 min) did not remove more Mcm2, suggesting that this fraction is tightly associated with chromatin. Consistent with results obtained in HeLa cells , Chinese hamster Mcm2 displays an apparent molecular mass of ∼120 kD and can be resolved as a doublet in the detergent-soluble fraction and as a predominantly single band of lower mobility in the chromatin-bound fraction . Previous studies of the cell cycle localization of Mcm were not able to evaluate the association of Mcm proteins with chromatin during late mitosis and early G1-phase. These experiments were either done with poorly synchronized cells or failed to extract soluble Mcm proteins from the nucleus. Careful inspection of the time between metaphase and early G1-phase revealed that Mcm2 first bound to chromatin within 40–60 min after release from nocodazole , coincident with the formation of an intact nuclear envelope in telophase. The percentage of cells in different stages of mitosis was determined by staining aliquots of these cells with DAPI and microscopic observation of mitotic figures . The kinetics of formation of an intact nuclear envelope could be further inferred from the retention of Mcm2 in digitonin-permeabilized cells . 20 min after release from nocodazole block, only metaphase and anaphase cells were observed, and no cells had completed the formation of an intact nuclear envelope. Immunofluorescent staining of Mcm2 proteins in Triton-extracted metaphase and anaphase cells did not detect any Mcm2 bound to chromosomes at these stages of mitosis . The appearance of chromatin-bound Mcm2 at 40 min after release from nocodazole block was coincident with the formation of an intact nuclear envelope in a fraction of the cells and the appearance of telophase cells in the cell population. Immunofluorescent staining revealed that Mcm2 was undetectable in Triton-treated early-telophase nuclei and weakly detectable in the late-telophase nuclei . We conclude that Mcm2 proteins start to associate with chromatin as soon as an intact nucleus is formed. Also evident from the immunoblots displayed in Fig. 5 B is an increase in the amount of chromatin-bound Mcm2 throughout G1-phase, peaking at the start of S-phase. To determine what percentage of the total Mcm2 was bound to chromatin, serial dilutions of the supernatant, obtained after centrifugation of Triton-washed nuclei, were subjected to immunoblotting in parallel with samples of the pelleted chromatin (not shown). Results revealed that, 90 min after metaphase (the pre-ODP stage of G1-phase), only 20% of the total Mcm protein was bound to chromatin. This amount increased to 30% at 6 h after metaphase (post-ODP), reached a peak of 45% at the onset of S-phase , and then gradually decreased during S-phase progression. Thus, the association of Mcm2 with chromatin does not occur as a single defined step at the end of mitosis; instead, there is a continuous loading of Mcm2 onto chromatin throughout G1-phase. In parallel control experiments, the same protein preparations were subjected to immunoblotting with the antibody against PCNA, which was detected as a single band with an apparent molecular mass of 35 kD . As expected, PCNA was detected in the nucleus throughout the cell cycle. It was absent from insoluble nuclear structures during G1-phase, first associated with nuclear components at the G1/S-phase transition and a fraction remained nuclear bound throughout S-phase . Whereas the maximal Mcm2 bound was observed at the G1/S boundary within aphidicolin-blocked cells, the maximal amount of PCNA bound was observed after release from the aphidicolin block, in early S-phase. Previous studies of RPA in mammalian cells have detected discrete nuclear RPA sites in non-S-phase HeLa cells or presented direct evidence for a diffuse nuclear distribution of RPA during G1-phase in human cells . These results suggested that mammalian RPA may form pre-RCs similar to those observed in Xenopus egg extract and encouraged us to determine exactly when they form in relation to Mcm binding and the ODP. Since the optimal technical protocol for immunostaining of RPA (Dimitrova, D.S., and D.M. Gilbert, manuscript in preparation) was the same as for Mcm2, we were able to use the same populations of synchronized cells shown in Fig. 2A and Fig. B , allowing a direct comparison of the cell cycle behavior of these two proteins. Simultaneous staining of 2 h or 6 h G1-phase nuclei with the anti-RPA antibody and either anti-CldU, or anti-IdU antibody, revealed that RPA was completely absent from G1-phase chromatin . RPA bound to chromatin at the onset of S-phase , when it formed discrete foci that colocalized with sites of early-, but not late-replicating chromatin. Late in S-phase , RPA continued to exhibit punctate distribution but at that time it associated exclusively with late-replicating chromatin domains. Contrary to previous reports , RPA foci were not detected in G2-phase nuclei (not shown). To confirm that there was not a chromatin-bound fraction of RPA in G1-phase nuclei that is not detectable by immunofluorescence, we performed immunoblotting experiments with aliquots of the same protein extracts from synchronized cells shown in Fig. 5 . Using the same polyclonal antibody used in Fig. 6 , Chinese hamster proteins of 14- (not visible), 30-, and 70-kD apparent molecular masses were detected . Results with synchronized cells revealed that, as with PCNA, no RPA was detected associated with chromatin during G1-phase. However, detergent-resistant RPA was readily detected in S-phase cells. The experiments described above demonstrate that RPA is present in a soluble nucleoplasmic form during G1-phase and binds tightly to the earliest-replicating DNA sites at the onset of S-phase. However, these experiments do not distinguish whether RPA binds nuclear components at some detectable time after the ODP but before initiation of replication. To address this, we prepared mitotic CHOC 400 cells and released them into fresh medium without aphidicolin. At 7, 8, or 9 h after release, just when the fastest cells in the population start to enter S-phase, we pulse-labeled aliquots of the cells for 1 min with CldU, then fixed and double-stained with the anti-RPA and anti-CldU antibodies . We reasoned that, with very brief CldU-labeling time, if RPA associated with chromatin several minutes or more before the onset of DNA synthesis, we should be able to detect cells that stained positively for RPA, but not for CldU. Counting the number of CldU-positive cells indicated that the percentage of S-phase cells was 9% at 7 h, 20% at 8 h, and 31% at 9 h. These numbers are consistent with results obtained by applying longer CldU pulses. As expected, in double-labeled cells the RPA and CldU sites colocalized, verifying that a 1-min CldU pulse was sufficient to identify nuclei that synthesized DNA. At each of these time points, ∼5% of cells exhibited positive staining only for RPA, but not for CldU. No RPA-negative, CldU-positive cells were found at any time point. Similar results were obtained by staining aliquots of the same cells with antibodies specific for PCNA and CldU . Thus, we conclude that RPA functions as a protein of the replication machinery assembling into multiprotein nuclear complexes at the G1/S-phase transition, at or very shortly before initiation of DNA synthesis. The order of assembly of replication proteins onto metazoan replication origins is poorly understood due to the paucity of experimental approaches applicable to these systems. In this report, we employ a newly developed indirect immunofluorescence approach to compare the cell cycle–regulated association of early- and late-replicating chromosomal domains with Mcm2 and RPA proteins, two essential replication factors that have been proposed to be a part of mammalian prereplication complexes. We demonstrate that, in CHO fibroblasts, Mcm2 associates with both early- and late-replicating chromatin as soon as nuclear envelopes are assembled in telophase. Subsequently, additional Mcm2 is loaded onto chromatin throughout G1-phase and is maximal at the G1/S border. Detectable Mcm2 is then displaced from replicons shortly after their initiation. By contrast, we found no evidence for stable association of RPA with chromatin during G1-phase. These properties are consistent with Mcm proteins, but not RPA, being a part of mammalian early G1-phase pre-RCs. Previous studies in human, mouse, and Xenopus cells have concluded that members of the Mcm family of proteins are present in the nucleus throughout interphase but associate with chromatin only during part of the cell cycle, beginning at some undefined point in G1-phase . Synchrony methods employed in these prior studies were not sufficient to determine whether Mcm proteins were binding during early or late G1-phase and were not able to relate chromatin association of Mcm proteins to specific G1-phase hallmarks, such as the ODP . When synchronized in metaphase with a brief nocodazole block, followed by mechanical shake-off of mitotic cells, CHOC 400 cells proceed into G1-phase within 60–90 min in a highly synchronous fashion. This allowed us to look more precisely at the timing of the Mcm-chromatin interactions during the mammalian G1-phase. We show here that ∼20% of hamster Mcm2 associates with chromatin as cells exit mitosis, several hours before specification of the DHFR replication origin at the ODP. In fact, initial binding of Mcm2 coincides with the assembly of a nuclear envelope, which would allow active transport to concentrate Mcm proteins in the nuclear compartment. Significantly, our novel indirect immunofluorescence approach for visualization of proteins and tagged DNA sites allowed us to demonstrate for the first time that Mcm2 proteins bind simultaneously to both early- and late-replicating chromatin regions at the very beginning of G1-phase. Quantitative immunoblotting analysis showed that an additional 25% of Mcm2 binds chromatin gradually and cumulatively throughout G1-phase until, at the G1/S border, ∼45% of Mcm2 is bound, with the remainder present in the soluble nucleosolic fraction. A similar increase in the amount of chromatin-bound Mcm proteins during G1-phase has been documented in S . cerevisiae . The observation that the initial binding of hamster Mcm2 to chromatin occurs in late telophase is similar to the situation in yeast and Xenopus egg extracts and implicates this protein as a pre-RC component. The fact that this event is upstream of the ODP suggests that the formation of Mcm-containing pre-RCs is not sufficient for the specification of mammalian replication origins. This view is also supported by studies in Xenopus egg extracts, where ORC-Cdc6-Mcm–dependent replication occurs without the use of specific origins . However, since we found that additional Mcm is continuously loaded onto hamster chromatin throughout G1-phase, it is still formally possible that a critical threshold of loaded Mcm in some way focuses initiation to specific sites. Alternatively, the specification of origins could be a gradual process, with different origins specified at different times in G1-phase corresponding to the binding of Mcm proteins. To date, the ODP has been determined only for the DHFR origin. In eukaryotic nuclei, the activation of replication origins occurs according to a strictly regulated temporal program . Although the mechanism that establishes this program has not been elucidated, it has been suggested that replication timing might be influenced by the amount or kinetics of Mcm loading onto early- vs. late-replicating chromatin . The technique described in this report allowed us to distinguish the association of proteins with early- and late-replicating chromosomal domains during G1-phase. We consistently observed equivalent association of Mcm2 with early- and late-replicating chromatin. Hence, the establishment of a chromosomal domain as early- or late-replicating does not appear to involve quantitative differences in the association of Mcm2 proteins. In S . cerevisiae chromatin immunoprecipitation experiments have produced direct evidence for the binding of members of the Mcm protein family (Mcm4 and Mcm7) to yeast chromosomal replication origins . One of these studies further showed that shortly after origin firing Mcm4 and Mcm7 proteins dissociate from origins and move with replication forks, along with DNA polymerase ∈ . This observation raised an apparent paradox regarding the behavior of Mcm proteins , since previous reports (and our own unpublished data) have shown that, in Xenopus egg extracts and in cultured mammalian cells, members of the Mcm family do not colocalize with sites of newly synthesized DNA . However, these results are difficult to interpret because proteins are localized to their positions at the moment of fixation, whereas sites of newly synthesized DNA were labeled before fixation. In the approach described in this report, proteins can be localized to sites that are actively engaged in replication. Mcm2 was still associated with the earliest-firing replicon clusters after initiation (at the aphidicolin-arrested step) but was cleared from those earliest replicating clusters within 5 min after DNA synthesis was allowed to proceed. At this time, these CldU-tagged sequences were actively being replicated and colocalized with the replication fork proteins PCNA and RPA. Since these earliest replicons take ∼60 min to complete replication , we are forced to conclude that all detectable (by immunofluorescence) mammalian Mcm2 is cleared from replicons shortly after initiation and does not associate with replication forks thereafter. Furthermore, direct inspection of Mcm2 and PCNA revealed a complete lack of colocalization . The possibility remains that other members of the Mcm complex, such as Mcm4 , behave differently than Mcm2. However, Tanaka et al. 1997 did not observe association of Mcm4 with S . cerevisiae replication forks using the same technique as . It is also possible that there is a distinct minor population of Mcm proteins that escape immunodetection but are present at replication forks. This would imply the existence of three populations of Mcm proteins: a soluble form (at least half), a chromatin-bound immunodetectable form that is cleared upon initiation, and a replication fork-associated form. While Mcm2-binding to chromatin before the ODP implicates it as a component of the CHO pre-RCs, the behavior of RPA is most consistent with its involvement in the initiation and elongation of nascent DNA strands. RPA is a well-characterized complex of three polypeptides that is a key participant in the initiation step of DNA replication. Several groups have shown that, in Xenopus egg extracts, RPA associates with sperm chromatin before the start of DNA synthesis. We have applied the same immunofluorescent technique employed to examine the association of Mcm2 with chromatin and failed to detect any stable interaction between RPA and chromatin during G1-phase in CHOC 400 cells. Hamster RPA assembled into distinct nuclear granules within a few minutes before DNA synthesis and could be detected in association with the earliest-firing replicon clusters at the onset of S-phase, similar to PCNA. This coincides with the initial appearance of replicative megacomplexes, termed replication factories, which have been observed to assemble at the G1/S transition in human fibroblasts and contain both PCNA and DNA polymerase α. Our results are also consistent with recent chromatin immunoprecipitation studies in S . cerevisiae that did not detect any association of RPA with yeast chromatin until the onset of S-phase . We suggest that previous data on G1- or G2-phase nuclear association of RPA proteins derive from the presence of a soluble, detergent-extractable form of RPA (Dimitrova, D.S., and D.M. Gilbert, manuscript in preparation) whose functional significance for DNA replication remains unclear. The approach described in this report also allowed us to demonstrate that RPA does not associate with late-replicating chromosomal regions until the late stages of S-phase, at the time when these sequences engage in replication. This behavior parallels that of PCNA and supports a role for RPA in the initiation and elongation steps of replication, not the formation of pre-RCs. Most of our knowledge of the role of RPA in replication has resulted from in vitro studies of SV-40 replication or Xenopus sperm chromatin replicating in Xenopus egg extracts. In the SV-40 system, RPA has been shown to be required for the stabilization of unwound origin DNA during the initiation step . In Xenopus egg extracts, RPA is essential for replication and forms punctate foci on sperm chromatin that resemble sites of DNA synthesis . The assembly of RPA foci onto chromatin precedes DNA unwinding and the initiation of replication and is independent of cdk2 activity in the extracts, implying that RPA may play a role at an earlier, preinitiation stage of chromosome replication . Subsequently, it was shown that the formation of prereplicative RPA foci on sperm chromatin is dependent on the presence of another protein, FFA-1, later shown to be the Xenopus homologue of a human DNA helicase defective in individuals with Werner syndrome . RPA-containing pre-RCs do not colocalize with Xenopus Mcm proteins and are assembled in extracts that have been immunodepleted of Xenopus ORC and Cdc6 proteins and cannot form Mcm-containing pre-RCs . Based on these findings, it has been concluded that two separate and independent assembly pathways are essential for the initiation of replication in eukaryotic cells. After these studies, it has been assumed that similar prereplicative RPA foci exist in mammalian cells. However, we could find no evidence for their existence and recent studies in S. cerevisiae found no evidence for RPA association with yeast chromatin in G1-phase . In fact, it still remains to be demonstrated that FFA-1/RPA foci formed in Xenopus egg extracts are involved in DNA replication. Oddly, RPA foci were found to persist on sperm chromatin even after completion of DNA replication . Most importantly, while it has been shown that RPA foci do not form in FFA-1–depleted extracts, the critical experiment to determine whether FFA-1–depleted extracts could support sperm DNA replication was not performed . FFA-1 is a homologue of Werner's helicase which, like RPA, and possibly together with RPA, is involved in DNA repair and recombination . Hence, it remains possible that FFA-1–mediated formation of RPA foci is unrelated to DNA replication and that the role of RPA in DNA replication is restricted to its role as defined in the SV-40 in vitro studies. | Study | biomedical | en | 0.999997 |
10459008 | General chemicals were from Merck or Sigma Chemical Co. α[ 32 P]GTP (3,000 Ci/mmol), [ 35 S]methionine, and the ECL system were purchased from Nycomed Amersham, Inc. SR was purified by immunoaffinity chromatography . Antibodies were raised in rabbits to a peptide corresponding to residues 137–150 of human SRα, coupled to keyhole limpet haemocyanin with sulphosuccinimidyl 4-( n -maleimidomethyl)cyclohexane-1-carboxylate (Sulpho-SMCC; Pierce Chemical Co.). The antibodies were affinity-purified against the immobilized peptide (Sulpholink gel; Pierce Chemical Co.) and then immobilized on protein A–Sepharose (Pharmacia Biotech, Inc.) with dimethylsubermidate. The affinity column was then used to purify SR from a digitonin extract of dog pancreas rough microsomes, essentially as described by Görlich and Rapoport 1993 . The yield of SR was ∼1 mg from 30,000 equivalence of rough microsomes. Sec61p complex was purified from a ribosome-associated membrane protein fraction by ion-exchange chromatography according to Görlich and Rapoport 1993 . TRAM protein was purified as described . Protein purity was assessed by 10–15% SDS PAGE and silver staining . Detergent exchange of translocon components from digitonin to deoxyBigCHAP, followed by reconstitution into proteoliposomes was performed as described . SRP was purified from a high salt extract of canine rough microsomes by gel filtration (Sephadex G-150), followed by ion-exchange chromatography (DEAE–Sepharose) according to Martoglio et al. 1998 . SRP was then further purified by sucrose density centrifugation . RNC complexes bearing preprolactin 86mer nascent chains (PPL86) were synthesized in the wheat germ lysate translation system . Translation was allowed to proceed for 10 min at 25°C in the presence of unlabeled amino acids. Further initiation of synthesis was blocked by the addition of 7 methyl guanosine-5′-monophosphate ( 7 me-GMP) to 2 mM. After a further incubation at 25°C for 10 min, the RNCs were stabilized by the addition of 2 mM cycloheximide. Translation reactions were adjusted to 500 mM KOAc and then the RNCs isolated by centrifugation through a sucrose cushion (1 M sucrose, 25 mM Hepes-KOH, pH 7.8, 500 mM KOAc, 5 mM Mg(OAc) 2 , 1 mM cycloheximide, and 1 mM DTT) for 1 h at 400,000 g at 4°C. The RNCs were then resuspended in half the original volume of the translation reaction in HMC buffer (25 mM Hepes-KOH, pH 7.8, 5 mM Mg(OAc) 2 , 1 mM cycloheximide) with 500 mM KOAc and treated with 10 mM NEM for 10 min at 25°C before addition of 20 mM DTT. The RNCs were then reisolated by centrifugation as before. RNCs were finally resuspended in HMDC buffer (HMC with 1 mM DTT) and 150 mM KOAc at a concentration of 56 OD 260 units/ml. SR, purified and reconstituted into liposomes , was mixed at 25 nM in the presence or absence of purified RNCs and/or purified SRP (5.6 OD 260 /ml) with 0.5 μM α[ 32 P]GTP (3,000 Ci/mmol) or concentrations as indicated in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc) 2 , 2 mM cycloheximide. After incubation for 20 min on ice and 5 min at 25°C, the 10 μl reactions were transferred onto a silanized glass plate precooled on ice-cold metal blocks and irradiated with UV light at 4,000 W/cm 2 in a stratalinker™ for 5 min to cross-link the radiolabeled GTP to the proteins . The solutions were then transferred to an Eppendorf tube and proteins precipitated with an equal volume of 20% TCA in the presence of 0.15% deoxycholic acid and 10 mM GTP. The pellet was washed with 10% TCA and with 80% acetone to remove uncross-linked radiolabeled nucleotides, and analyzed by 12.5% SDS-PAGE , followed by PhosphorImaging. Quantification was done using the PhosphorImager (Fuji). Curves connecting data points and the apparent dissociation constants (K d ) were calculated using the nonlinear regression program GraphPad Prism™ (GraphPad Software Inc.). SR, purified and reconstituted into liposomes , was mixed at 25–50 nM in the presence or absence of purified RNCs (8.4 OD 260 /ml) and/or purified SRP (25–50 nM) with 0.5 μM α[ 32 P]GTP (3,000 Ci/mmol) in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc) 2 , and 2 mM cycloheximide at 25°C for the indicated time points. Aliquots of the samples were spotted onto polyethyleneimine cellulose thin-layer plates; α[ 32 P]GDP was resolved from α[ 32 P]GTP using 0.75 M KH 2 PO 4 , pH 3.3, as solvent. Radioactive TLC spots were quantitated using a PhosphorImager. The percentage of GTP hydrolysis was calculated from the amount of α[ 32 P]GDP divided by the sum of the amounts of α[ 32 P]GTP and α[ 32 P]GDP. Liposomes lacking or containing 50 nM SR or trypsinized SR were incubated in 50 mM Tris-OAc, pH 7.8, 150 mM KOAc, 2 mM DTT, 5 mM Mg(OAc) 2 , 2 mM cycloheximide with purified RNCs (2,8 OD 260 /ml) containing [ 35 S]methionine-labeled PPL86 and 0.5 mM GMPPNP or GDP in the presence or absence of 50 nM SRP. Wheat germ cytosol (1.8 μl), which was previously depleted of ribosomes by pelleting the ribosomes at 400,000 g for 20 min, was subsequently added to 5 μl reactions to block unspecific binding of ribosomes to liposomes lacking SR. After 30 min incubation at 25°C, 60 μl of ice-cold sucrose buffer containing 2.2 M sucrose, 10 mM Tris-OAc, pH 7.8, 500 mM KOAc, 2 mM DTT, and 5 mM Mg(OAC) 2 was added, thoroughly mixed, and layered under a 100 μl 1.8 M sucrose cushion containing CR buffer (50 mM Tris-OAc, pH 7.8, 500 mM KOAc, 2 mM DTT, 5 mM Mg(OAc) 2 , 2 mM cycloheximide, 0.5 mM guanine nucleotides) in a TLA 100 tube that was preincubated with a 10 mg/ml BSA/PBS solution. A 0.25 M sucrose cushion in CR buffer (40 μl) was laid over the 1.8 M sucrose cushion. After centrifugation at 400,000 g for 60 min, the amount of labeled PPL86 in the top and bottom fractions was determined by scintillation counting. The amount of labeled PPL86 that was recovered in the top fraction in the absence of liposomes was taken as background and was subtracted. To test GTP binding to SR subunits, we used UV light-mediated cross-linking of α[ 32 P]GTP to purified SR reconstituted into liposomes (SR liposomes). This approach allows analysis of GTP binding to each SR subunit . Fig. 1 A shows the purified SR analyzed by SDS-PAGE and silver staining. α[ 32 P]GTP cross-linked to SR liposomes is revealed after SDS-PAGE followed by PhosphorImaging, and shows labeling of both SRα (70 kD) and SRβ (30 kD). An unidentified protein of ∼50 kD was also found in various amounts, as has been reported previously . To determine the apparent affinity of SRα for GTP, we added increasing concentrations of unlabeled GTP to the cross-linking reactions with α[ 32 P]GTP. Proteins were analyzed by SDS-PAGE, the amount of label in SRα was quantified after PhosphorImaging, and was plotted against the concentration of added GTP. From these data, an apparent K d of 14 μM was calculated . To determine the apparent affinity of SRβ for GTP, we used increasing amounts of α[ 32 P]GTP in the cross-linking reactions. The amount of labeled SRβ was plotted against the concentration of α[ 32 P]GTP . We determined an apparent K d of 20 nM GTP for SRβ. Thus, the affinity of SRβ for GTP is ∼700-fold higher than the affinity of SRα for GTP. To test whether translocon components affect α[ 32 P]GTP cross-linking to the SR subunits, we used SR liposomes containing, in addition, the Sec61p complex and the TRAM protein. Fig. 2 shows the purified proteins analyzed by SDS-PAGE and silver staining, and the proteins cross-linked to α[ 32 P]GTP after SDS-PAGE and PhosphorImaging. As can be seen in Fig. 2 , cross-linking of α[ 32 P]GTP to SRα and SRβ is not changed by the inclusion of SR/Sec61p/TRAM liposomes to the assay . Furthermore, we found that the K d of SRβ and SRα for GTP remained unchanged in the presence of SR/Sec61p/TRAM liposomes, as compared with SR liposomes (data not shown). Thus, we conclude that neither the TRAM protein, nor the Sec61p, influence α[ 32 P]GTP cross-linking to the SRα or SRβ. To test whether a component of the targeting complex affects GTP binding to SR, we added purified SRP and/or purified RNCs bearing PPL nascent chains of 86 amino acids to SR liposomes. As shown in Fig. 3 A (lanes 1 and 2), the presence of SRP did not affect α[ 32 P]GTP cross-linking to either SRα or SRβ. When RNCs were added, α[ 32 P]GTP cross-linking to SRα remained the same, however, α[ 32 P]GTP cross-linking to SRβ was strongly reduced . This indicates that the RNC interacts with SR and selectively reduces α[ 32 P]GTP cross-linking to SRβ. Reduction in α[ 32 P]GTP cross-linking to SRβ was also seen when SRP was added in addition to RNCs . In this case, cross-linking of α[ 32 P]GTP to SRP54 was found to be increased in the presence of RNC, as shown previously . The apparent affinity of SRβ for GTP in the presence of RNCs was determined as described. We found that the apparent K d was 1 μM, ∼50-fold higher than that observed in the absence of RNCs . Cross-linking of α[ 32 P]GDP to SRβ was also found to be reduced by the interaction with RNC (data not shown). This indicates that GTP, as well as GDP, binding to SRβ is decreased by RNC. We next asked whether the decrease in α[ 32 P]GTP cross-linking to SRβ is caused by a direct interaction between RNCs and SRβ, or if it requires the presence of both SR subunits. To test the latter possibility, we removed SRα by mild trypsin digestion . Fig. 4 A shows that SRα was largely removed by treatment with 2 ng/ml trypsin and high salt, whereas SRβ resisted proteolysis and remained bound to the membranes. Liposomes containing the β-subunit of SR (SRΔα liposomes) were tested in the α[ 32 P]GTP cross-linking assay. Strong α[ 32 P]GTP cross-linking to SRβ was seen, while α[ 32 P]GTP cross-linking to SRα was greatly reduced . The presence of SRP did not affect α[ 32 P]GTP cross-linking to SRβ . When SRΔα liposomes were combined with RNCs, α[ 32 P]GTP cross-linking to SRβ was reduced similarly, as was seen with SR liposomes . SRP had no effect on the RNC-mediated reduction of α[ 32 P]GTP cross-linking to SRβ . This indicates that RNCs directly interact with SRβ, resulting in reduced α[ 32 P]GTP cross-linking. We next investigated the effect of the RNC–SR interaction on GTP hydrolysis. SR liposomes were incubated with RNC and/or SRP and GTP hydrolysis determined by chromatographic analysis of α[ 32 P]GDP generated in the assay. No significant GTP hydrolysis was observed with SR liposomes or RNC alone . However, when SR liposomes were combined with RNCs an increase of GTP hydrolysis was observed . This confirms that RNC interacts with SR and indicates that RNC stimulates GTP hydrolysis by SR. As shown previously, a large additional stimulation of GTP hydrolysis is observed when SRP is also added . This stimulation of GTP hydrolysis by SRP was about eight times that observed in the presence of RNCs and SR alone . This was calculated from the initial slope of the GTP hydrolysis curves shown in Fig. 5 A. SRP alone or in combination with SR or RNC showed only background level of GTP hydrolysis. To investigate the RNC-stimulated GTP hydrolysis of SR in more detail, we used increasing concentrations of RNCs in the assay. When SR liposomes were tested alone, or in the presence of SRP, we found that the amount of GTP hydrolyzed at a given time point was saturable . This indicates a specific interaction between RNC and SR. To identify the subunit of the SR that hydrolyzes GTP in the presence of RNC, we used the SRΔα liposomes containing only SRβ. SRΔα liposomes alone, or combined with SRP, showed only background levels of GTP hydrolysis as observed with SR liposomes . When SRΔα liposomes were combined with RNC, the stimulation of GTP hydrolysis was similar to that observed with SR liposomes , suggesting that the RNC directly interacts with SRβ and stimulates its GTP hydrolysis. Addition of SRP and RNC to SRΔα liposomes did not significantly enhance GTP hydrolysis above the level seen with RNC alone . A significant further stimulation of GTP hydrolysis is, as expected, observed with SR liposomes in the presence of RNC and SRP . This also has been observed previously, and reflects the reciprocal GTPase stimulation of SRP54 and SRα . This indicates that RNCs stimulate the GTPase activity of SRβ, independent of the presence of SRα. The observation that RNC reduces α[ 32 P]GTP cross-linking to SRβ and stimulates GTP hydrolysis by SRβ indicates that the RNC contacts SRβ. To test this directly, we allowed interaction of RNC to SR liposomes and then floated SR liposomes with bound RNC to the top of a sucrose gradient to separate them from unbound RNCs. To test for a GTP dependence of this binding, we performed the assays in the presence of either GDP or the nonhydrolyzable GTP analogue GMPPNP. SR liposomes were incubated with purified RNCs bearing 35 S-labeled PPL86. To reduce unspecific binding to the lipids, we included wheat germ cytosol from which endogenous ribosomes had been removed. SR liposomes were then floated and the amount of nascent chains ( 35 S-labeled PPL86) associated with the floated SR liposomes and in the pellet was determined. 35 S-labeled PPL86 nascent chains were not found associated with liposomes lacking SR . In the presence of GDP, only a small amount of 35 S-labeled PPL86 nascent chains were found associated with SR-liposomes . In contrast, with GMPPNP, a significantly increased amount of nascent chains was recovered with the floated SR liposomes . This suggests that RNCs bind to the SR liposomes in a GTP-dependent manner. To test the effect of SRP on RNC interaction with SR liposomes, we included SRP in the assay system. We found that, even in the presence of GDP, a further increase in RNC binding to SR liposomes compared with the absence of SRP . But, in the presence of GMPPNP, a substantially higher amount of RNCs was found associated with SR liposomes . Taken together, this suggests two GTP-dependent interactions, one between the ribosome and SR and the other between SRP and SR. To test whether the RNC binds to SRβ in the absence of SRα, we used SRΔα liposomes in the floatation assay. As was seen with SR liposomes, a significantly higher amount of RNC floated with SRΔα liposomes in the presence of GMPPNP, as compared with GDP . These data suggest that the RNC directly interacts with SRβ in a GTP-dependent manner. The addition of SRP in the assay led to an increased binding of RNC to SRΔα liposomes in the presence of GDP, as was seen with SR liposomes. This might point to a GTP-independent interaction between SRP and SRβ. In the presence of GMPPNP, a further increase in binding was observed, but much less than observed with SR liposomes . This indicates that the binding of SRP to SRα is drastically reduced, whereas the binding between SRβ and RNC is not affected . The functions of GTPases are regulated by guanine–nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which mediate GTP binding and stimulation of GTP hydrolysis, respectively. Here, we show that the ribosome interacts with SRβ in its GTP-bound state, functions as a GAP for SRβ, and reduces the affinity of SRβ for guanine nucleotides. Previously, it has been shown that the ribosome functions as a GEF for SRP54 by increasing its affinity for GTP. Thus, the ribosome regulates the GTPases of the SRP/SR targeting system at two stages, first after signal sequence recognition by SRP54 and then at the ER membrane when it contacts SRβ. To identify components that regulate the SRβ GTPase, we have used liposomes containing purified SR alone or together with translocon components, namely Sec61p complex, TRAM protein, and components of the targeting complex, namely RNC and SRP. Consistent with previous observations, we found that SRα alone has a very low affinity for GTP. The apparent K d of SRα for GTP was ∼14 μM . This is in good agreement to the K d of 10 μM, which has been previously reported . However, we found a considerably higher affinity of GTP for SRβ alone or SRβ in association with SRα (K d = 20 nM) than previously reported by Miller . In the experiments shown here, SR reconstituted into liposomes was used, whereas previously, detergent solubilized SR was used. It is therefore conceivable that GTP binding to SRβ is reduced in the presence of detergents. However, in all studies, SRβ has been found to have a higher affinity for GTP than SRα. Including components of the translocation machinery into the proteoliposomes with SR did not affect GTP binding to SRα nor to SRβ, suggesting that these components do not directly regulate the GTPases of SR. In contrast, RNCs were found to drastically reduce GTP binding to SRβ. In addition, they specifically stimulate GTP hydrolysis by SRβ. We suggest that the RNC induces a conformational change of the GTPase domain of SRβ that leads to both an increased GTP hydrolysis and a reduced guanine nucleotide binding. As free SRβ binds GTP with high affinity, interaction of SRβ with the ribosome first induces hydrolysis of bound GTP, and the resulting GDP is then bound with low affinity. The low GDP affinity might increase the dissociation of the bound GDP, creating an empty state of the GTPase domain. SRP alone or in combination with RNC showed no effect on GTP binding and hydrolysis by SRβ, indicating that it functionally interacts only with SRα. When SRβ is associated with SRα, the presence of SRP leads to the observed burst in GTP hydrolysis via the reciprocal stimulation of GTP hydrolysis by SRP54 and SRα, which was previously shown . The difference in regulation of SRβ and SRP54/SRα is in agreement with the difference in the primary GTPase domain structure of these molecules. The GTPase domains of SRP54 and SRα are related and contain an insertion box that stabilizes the nucleotide-free form of the proteins, resulting in the low affinity for GTP . In contrast, the GTPase domain of SRβ is structurally distinct and falls into its own subfamily of GTPases . Ribosomes can bind to ER membranes independently of a nascent chain or SRP . This suggests that there are ribosome receptor proteins at the ER membrane. Several ribosome receptors have been identified. Based on ribosome binding assays , ribosome receptors of 34 kD and 180 kD have been identified. However, both were shown not to be essential for the translocation of proteins across the ER membrane . Therefore, they may play a role in modulating ribosome-binding to the ER membrane or become engaged at times when ribosomes are not active in translation or translocation . Studies on ribosome binding during ongoing translocation using SR/Sec61p liposomes have revealed that the Sec61p complex of the translocon forms the translocation channel and directly binds to the ribosome . Binding of ribosomes to the Sec61p complex also has been visualized by EM and revealed contacts to the large ribosomal subunit, suggesting that the nascent chain is directly transferred from the exit site on the ribosome into the protein conducting channel of the translocon . The interaction between ribosomes and SRβ described here is unlikely to directly contribute to binding of ribosomes to the ER membrane. For this, the high affinity binding between the Sec61p complex and the ribosome is probably sufficient . Recent data with the yeast SRβ showed that a functional GTPase domain of SRβ, but not its membrane-spanning region, is required for efficient translocation . This is consistent with a regulatory role of SRβ, rather than a role in binding RNC/SRP to the ER membrane. How is the GTPase cycle of SRβ related to the function of the other two translocation GTPases, SRP54 and SRα? The first step in targeting nascent secretory and membrane proteins to the ER membrane is the interaction of SRP with the signal sequence exposed on a ribosome . The additional interaction of SRP54 with the ribosome leads to GTP binding and an activated RNC–SRP–GTP targeting complex . Binding of the targeting complex to the ER may proceed in distinct steps and may involve, besides the core components of the Sec61p complex, many accessory factors . For simplicity, we consider here only the minimal translocation machinery, the SR and the Sec61p complex of the translocon. Because of the high affinity between ribosomes and the Sec61p complex , we envisage that the first contact between the RNC–SRP–GTP with the ER membrane is the interaction between the ribosome and the Sec61p complex . This interaction is transient, as it can be competed by 80S ribosomes . The ribosome of a membrane-bound RNC–SRP–GTP complex may then recruit SR by interacting with SRβ–GTP . This would allow SRα to scan the ribosome for the presence of SRP and trigger the release of the signal sequence from SRP54 when GTP has been bound . The dual contacts between RNC/SRP and the membrane, via an interaction between the ribosome and SRβ, and SRP and SRα may ensure that only the combination of ribosomes and SRP make a functional targeting complex. | Other | biomedical | en | 0.999996 |
10459009 | All fly strains were grown and collected according to standard conditions . The original screen alleles wrt ER1 , wrt AM4 , and wrt AS1 , as described in Verheyen et al. 1996 were progeny of the w 1118 stock exposed to either EMS (AM4 and ER1) or X-irradiation (AS1). The wrt MEF allele was also derived from the w 1118 stock, but as a spontaneous mutation found in homozygosing an unrelated transgene on 2R. From the Bloomington Stock Center, P2352 [l(2)08232] was determined to be a warthog allele as it failed to complement the bristle phenotype of the original screen alleles. To create excision alleles of the P2352 allele, 50 P2352/CyO; D2-3 Sb/ + males were independently crossed to Adv/CyO; ry − virgins. Up to four ry − Sb + male progeny carrying the altered P2352 chromosome were retained from each pair mating and were used for complementation testing against the starting P2352 stock as well as the original screen alleles. If differing phenotypes were found to exist for any of the four male progeny, one representative of each was kept. The progeny could be grouped into three different phenotypic classes. Two of the classes constituted new warthog alleles: 14 were homozygous lethal, lethal with the P2352 allele, and failed to complement the bristle phenotype of the wrt screen alleles (D1A, D5C, D6E, D9C, D12C, D15A, D17B, D22A, D23D, D24A, D28E, D29D, D37A, D39B, and D40B), whereas 9 alleles were homozygous viable with the wrt bristle phenotype and failed to complement the bristle phenotype of wrt AM4 , wrt ER1 , wrt AS1 , and wrt MEF , but did complement the lethality of P2352 (D2B, D7C, D9A, D9D, D10A, D14E, D23A, D35A, and D40C). 13 revertants constituted the third class of excision alleles; they were homozygous viable without any bristle aberrations and complemented the wrt screen alleles as well as the P2352 allele (D1B, D2D, D3A, D4A, D5A, D6B, D11A, D13E, D17E, D27A, D28B, D30D, and D31B). Molecular analysis showed this last class of alleles to be precise excisions of the P2352 insertion, whereas the former two classes were imprecise excisions with or without duplications and deletions. For clonal analysis, the wrt P2352 , wrt ER1 , wrt AS1 , wrt D17B , and wrt D23D mutations were recombined onto chromosome 40-2pM ( w; P[mini-w + ; hs-pM]21C, 36F, P[ry + ; hs-neo; FRT]40A). Methods described in Rooke et al. 1996 were followed using the flipase stocks 40y+F ( y , w, phs-FLP; P[ry +;y+]25F, P[ry+; hs-neo; FRT]40A) and 40-2pM F ( w phs-FLP; P [ mini-w + ; hs-pM]21C, 36F, P[ry + ; hs-neo; FRT]40A). Germline mosaics were obtained from phs-FLP/ +;ovo D -FRT/P2352-FRT females. The Berkeley Drosophila Genome Project (BDGP) had sequenced 300 bp of genomic DNA neighboring the P insertion site of l(2)08323. To determine which P1 in the 33C-D region contained this sequence, primers were designed from either end of the corresponding STS 0355. These primers [C: ctt ctc gct ccg ctc cgc tct cac c and D: gat tcc cgct ctg gtc aca cac aac] were used in a PCR reaction with various BDGP P1 clones assigned to this chromosomal region. With P1 DS00299 as a template, a fragment of the correct size and restriction site pattern was produced, indicating it contained the genomic DNA neighboring the P insertion. The DNA from this PCR reaction was used as a probe to start a genomic walk along the P1 DS00299. Subclones extending 15 kb to the left and 10 kb to the right of the initial fragment were obtained, sequenced, and then analyzed with DNAStar software (DNASTAR Inc.). The BLASTX program was used for homology comparison. For cDNAs within the region, both a 0–12-h embryonic library and an imaginal disc library (gift of T. Xu and G. Rubin) were screened with each of the genomic subclones obtained in the walk. Sequencing was performed by the W.M. Keck Foundation Biotechnology Resource Lab at Yale University. Primers were synthesized by the Oligoz-R-Us at Yale University. Whole genomic Southerns were prepared with DNA from all warthog alleles digested with EcoRI or BamHI. Each of the P1 subclones from the genomic walk was used for hybridization. For a subset of mutants, additional Southerns were prepared using the following restriction enzymes: EcoRV, SacII, PstI, ClaI, or a combination of these. From these studies, the alleles D17B, D39C, and D40B were found to have retained the 3′ end of the PZ insert, whereas the alleles D12C and D23E retained the 5′ end. Alleles D6E, D24A, and D29D had small insertions of genomic or transposon DNA, whereas the D23D allele had a 1.8-kb genomic deletion. For the original screen mutants, no disruption in the restriction pattern was found by whole genomic Southerns, therefore, the particular mutation was determined by sequencing subcloned PCR fragments. DNA from wrt AM4 / wrt AM4 , wrt MEF /wrt MEF , wrt AS1 /Df 3344 , wrt ER1 /Df 3344 , and w 1118 was used as templates for a PCR reaction with primers CS-T7-2 (ccc cat tat aaa cag tga gg) and CS-T3-7 (cgt gtc aat gag tta gca ttc gc) that encompass the open reading frame (ORF) of Drab6. The single band obtained from gel purification was subcloned into the pGEM-T Easy vector (Stratagene) and sequenced. More than four independent PCR reactions were performed for each mutant and for w 1118 as a control. To determine the exact extent of the genomic deletion of D23D, PCR was also performed using DNA from D23D/CyO flies with the primers CS-T3-8 [gga atc att gaa cac aga ctg gc] and 5′-1-s[cct gct ggt tag ccg ata tcc] or CS-T3-9 [ggg ata gtc atg cga aca gag gtg cgc] and 5′-1-s[cct gct ggt tag ccg ata tcc]. For each reaction, two bands of the expected sizes were obtained, a 3.3-kb fragment derived from the balancer chromosome and a 1.5-kb fragment derived from the mutant D23D chromosome. Sequencing was performed on the 1.5-kb fragment. P element–mediated germline transformation was performed as per Go et al. 1998 using w 1118 as the parental strain for all germline transformations. Genomic rescue fragments were constructed in the pCaSpeR4 vector. For the medrab6 rescue construct, a 4.5-kb EcoRI-EcoRI fragment (named 9B4) from D9.2 of M. Noll was adjoined to a 2.2-kb EcoRI-XhOI fragment (named RX11) from the P1 subclones. Sequencing of this construct showed it contained the entire ORF for the rab6-like gene and Phae1 but a 0.5-kb deletion removed the last half of Phae2 . All other genomic rescue constructs contained DNA only from the P1 DS 00299. The 9-6 construct contained the entire ORF of the rab6-like gene. The X-X5 construct truncated the rab6-like ORF three amino acids before the STOP codon so any translated protein would not contain the isoprenylation site CAC. The SacII-RI construct truncated the rab6-like gene two thirds into the ORF and the RX11 construct contained no sequence of the rab6-like gene. Transgenic flies of the different cDNA constructs were created using the phsCaSpeR vector. For wild-type warthog , the PCR product of w 1118 flies that was used as a control for sequencing of the screen alleles was subcloned into the pGEM-T Easy vector (Stratagene), and then transferred to the phsCaSpeR vector. This DNA contained the complete ORF of Drab6 as well as 110-bp upstream and 310-bp downstream of the coding region. To overexpress the R62C mutant form of Drab6, subcloned DNA from a PCR reaction of the AM4 allele was inserted into the phsCaSpeR vector. To generate the Q71L mutant form of Drab6, the subcloned w 1118 PCR product above was used as a template for site-directed mutagenesis (Stratagene). The following primers were used to induce the point mutation that resulted in Q71 being converted to a leucine: Q71L-S (g gat acg gcg gga CTC gag cga ttc cgc) and Q71L-AS (gcg gaa tcg ctc GAG tcc cgc cgt atc c). Our lab had previously performed a genetic screen to isolate new genes that altered Notch signaling . One complementation group, named warthog ( wrt ), enhanced the Notch eye phenotype although it did not visibly affect eye development outside of this interaction . It was also noted to have a recessive bristle phenotype independent of its interaction with the aberrant Notch signaling in the eye . In wild-type flies, bristles are part of mechanosensory organs and develop shortly after puparium formation as the trichogen, or shaft cell, sends a cytoplasmic extension from the epidermis into the overlying cuticulin . At the center of this extension is a longitudinal core of microtubules. Around the circumference and positioned near the plasma membrane are regularly spaced bundles of actin filaments. These filaments are hexagonally packed and run parallel to the microtubule core. As development proceeds, continued growth of the shaft occurs in two directions. One is elongation at the distal tip, whereas the second is throughout the width of the shaft as regions of the cytoplasm protrude from between the actin fibers to produce the characteristic ridges seen in a cross-section of the bristle . The five warthog alleles ( wrt AM4 , wrt ER1 , wrt AS1 , wrt BU1 , and wrt BN7 ) recovered in the screen had considerably shortened bristles as homozygotes or transheterozygotes . This defect was present only for macrochaete of the ocelli, notum, and scutellum, whereas the bristles of the eye, wing, and leg appeared normal. Scanning electron micrographs of warthog bristles showed, in addition to the aberrant length, that the morphology of wrt bristles was altered. The wrt bristles did not have finely tapered ends nor did they show the regularly spaced ridges from the membranous protrusions. Instead, the tips were mangled and the surface was either smooth or had very mild and disorganized ruffling. The wrt locus had been mapped to the 32F1-3;33F1-2 region of the second chromosome . Of the P alleles in this region, only P2352 [l(2)08323] failed to complement the bristle phenotype of the original screen alleles. Sequencing of the subcloned DNA in a 25-kb genomic walk surrounding the P insertion site of P2352 revealed five ORFs . Each of these ORFs was homologous to a known gene in other organisms. The most distal within the genomic walk was named Patsas , a novel gene with at least six tandem ankyrin-like repeats with homology to mouse and human p19 protein, to the Drosophila expressed sequenced tags GH10910.5prime and GH15747.5prime, and the human proteins Q92556 and BAA7679.1. The next two ORFs had homology to threonyl-tRNA synthetase and rab6 proteins in other organisms, respectively. The P insertion site of allele P2352 was ∼30 bp upstream of the starting methionine of Drab6. 800-bp proximal to this P insertion site were two tandem ORFs, named Phaedra1 ( Phae1 ) and Phaedra2 ( Phae2 ) , both of which were homologous to serine proteases of the kallikrein family, including Drosophila trypsin proteins, mammalian NGF-g subunit, and EGF-binding protein type 1. To determine which potential transcript corresponded to the wrt gene, rescue constructs containing different portions of the subcloned DNA were generated . Only fragments that contained the complete ORF of the rab6-like gene were capable of rescuing the bristle phenotype of wrt AM4 , wrt MEF , wrt ER1 , and wrt AS1 (data not shown). The warthog cDNA was obtained by screening two independent cDNA libraries with a genomic subclone that straddled the P insertion site sequence. A 2.1-kb cDNA was recovered from an embryonic library and a 1.9-kb cDNA from an imaginal disc library. Sequencing showed both cDNAs to be from the same transcript with varying amounts of the 5′ untranslated region. Comparison to genomic DNA showed no introns but revealed several base pair polymorphisms that did not alter the amino acid sequence. Translation of the sequence showed this transcript to have 89% identity to human rab6, 72% to the yeast rhy1 protein, and has subsequently been cloned as Drab6 . Additionally, two putative C . elegans proteins were also found to be homologous (84% and 75%) . Transgenes of this cDNA were also capable of rescuing the wrt bristle phenotype indicating the wrt gene encodes the Drosophila homologue of rab6. The molecular lesions in the warthog locus recovered from the Notch screen were determined. As none of the alleles (eAS1, eER1, and eAM4) showed molecular aberrations on whole genomic Southern blots, PCR products of the coding region were sequenced. This data showed they each harbored the same point mutation; at base pair 313 from the starting methionine, a C was converted to a T that resulted in an amino acid change of arginine 62 to a cysteine (R62C). The location of this point mutation resides one amino acid from the second conserved GTP-binding domain . It is surprising that each of the alleles contained the same mutation as they were from independent mutagenesis crosses, and it is unlikely that the same point mutation would be created multiple times from both EMS and X-ray mutagenesis. It is more probable that the mutation existed in the genetic background of our starting stock w 1118 and was recovered repeatedly given the sensitivity of its interaction with the Notch phenotype used in the screen. Consistent with this interpretation, a spontaneous allele ( wrt MEF ), also derived from the w 1118 stock, was found to contain the same R62C point mutation. The original alleles recovered were homozygous viable with short bristles, but the P2352 allele used to clone the warthog gene, produced shorter macrochaete in trans to the R62C alleles and was homozygous lethal. This variability of phenotype was also seen in excision alleles generated from the P2352 allele. Upon mobilization of the P insert, two classes of new wrt mutants were recovered. Nine were phenotypically similar to the original screen alleles; they were homozygous viable with the bristle defect and they complemented the P2352 lethality giving transheterozygotic progeny with the wrt bristle phenotype. Sixteen of the excision alleles were phenotypically similar to the parental P2352 allele; they were homozygous lethal but viable with the bristle defect in trans to the original screen alleles. The phenotypic pattern of these alleles suggests the R62C mutation may be a less severe loss-of-function allele, whereas the P2352 allele and several of its excision progeny are more severe loss-of-function alleles. Various genomic transformants were tested for the ability to rescue the lethality of the parental stock and representative excision alleles (D1A, D5C, and D6C). As with the bristle phenotype, only genomic constructs with the complete ORF of Drab6 rescued the lethality. Different lines of the phs Drab6 cDNA transformants were also tested and all were capable of rescuing the lethality. These studies show that both the lethality and the bristle phenotype were due to perturbations in the same gene, Drab6. The original screen alleles (AS1, ER1, and AM4) and the spontaneous allele (MEF) constitute hypomorphic warthog alleles, whereas the lethal alleles are more severe alleles of wrt . Unexpectedly, a subset of the Drab6 cDNA transformant lines rescued the lethality to produce flies with bristle defects more subtle than the original wrt alleles. As these same transformant lines were capable of rescuing the bristle defect of the screen alleles with the R62C point mutation, this indicates that bristle development is more sensitive to the quantity or timing of Drab6 expression or function than is lethality. To determine the molecular lesions associated with the different excision alleles, whole genomic Southerns were prepared and probed with each subclone of the genomic walk. The allele wrt P2352 showed a single insertion between Drab6 and Phaedra1 . Restriction digests showed the expected pattern for the PZ construct, with the 3′ end of the insert lying closest to the warthog locus . Generated excision lines from this P allele that were homozygous viable without any bristle defect were all precise excisions, showing a return of the 2.6-kb BamHI fragment to the size of wild-type flies. The severe loss-of-function excision alleles (homozygous lethal and failed to complement the bristle defect of the original alleles) all contained molecular aberrations centering around the P insertion site of wrt P2352 . Most were imprecise excisions with deletions of only one end of the PZ insert as well as some flanking genomic DNA. Of interest was the allele D23D that showed complete excision of the PZ insert as well as a 1.8-kb genomic deletion in the region of the warthog gene . To determine the extent of this deletion, DNA from these flies was used as a template in PCR reactions with four different sets of primers known to extend beyond the deletion. Sequencing of the PCR products showed the deletion extended from 72-bp upstream of Phae1 to 365-bp downstream of the STOP codon of the wrt gene. Therefore, this allele constituted a genomic null of warthog . Since the deletion extended into a potential upstream regulating region of Phae1 , genetic testing with the different rescue constructs was performed. Flies that were homozygotic for the D23D chromosome were viable and phenotypically wild-type if they also carried the wrt cDNA transgene or a genomic fragment with only the wrt ORF. Therefore, the wrt null did not produce any phenotypic defects other than those of the wrt deletion. To establish the time period of Drab6 expression critical for viability, homozygotic P2352/P2352 , or D23D/D23D were monitored at different stages of development. Eggs with these homozygotic genotypes would proceed through embryogenesis to the larval stage, but would not continue to develop into pupae. Therefore, the more severe alleles of warthog were larval lethal. To determine if the lack of embryonic lethality was due to a maternal contribution of wrt, the FLP-FRT system was used to generate females with wrt − /wrt − germlines. All progeny with the genotype P2352/P2352 developed past embryogenesis, showing that a maternal contribution is not responsible for survival of wrt P2352 through embryonic development. To study the effect of the more severe disruptions in Drab6 function during later stages of development, mosaic clones were induced using the FLP-FRT system . Within heterozygotic flies , patches of homozygotic tissue were generated by heat shock regimes throughout development. As with the original screen mutants, no defects of eye, wing, or leg development were noted. The defects on macrochaete were more severe and more variable than that seen with the wrt R62C mutants . Contrary to the R62C alleles, this mutation also affected the smaller bristles, called microchaete, on the head and thorax (data not shown). The defects seen in these smaller shafts mirrored those seen in the macrochaete of Drab6-R62C mutants; distal tip growth was stunted and the circumferential ridges produced from cytoplasmic protrusions were nearly absent. Surprisingly, the clonal analysis also showed that wrt P2352 was nonautonomous . Whereas portions of the mosaic clones contained mutant bristles, phenotypically wild-type bristles were also present in patches of P2352/P2352 tissue, indicating that wrt protein is not required within the cell producing the shaft of the bristle. The function of rab proteins in mammalian systems has been elicited by studying the effects of overexpression of wild-type and mutant forms of these proteins. The best characterized forms are those modeled after ras mutations and are known to alter the ability of rabs to cycle between the GDP- and the GTP-bound states. The state of continued GTP binding has been produced by altering the Q of the second conserved GTP-binding domain to a leucine . This abolishes intrinsic GTPase activity and decreases GAP-stimulated hydrolysis as well . To study the effects of this mutation in the whole organism, we constructed the similar mutation in warthog (Drab6-Q71L). cDNAs of wild-type Drab6, the R62C mutation, and the Q72L mutation were placed under the control of the heat shock promoter to drive expression at different stages of development. Whereas overexpression of the wild-type form and the R62C mutation produced no visible phenotype in the background of wrt + /wrt + , the Q71L mutation altered the direction of bristle growth at any point along the bristle shaft . Overexpression of this GTP-bound mutant produced smoothly curving bristles or bristles with sharp changes in the orientation of growth, followed by continued growth in two opposite directions. Normal morphology appeared distal to the alteration, presumably because of the return of normal Drab6 function after the pulsed overexpression of Drab6 Q71L passed. Aberrations in the circumferential ridges was also seen, indicating that the membranous protrusions from between the actin bundles was also disrupted. Interestingly, basal expression of the Q71L mutant cDNA without heat shock, was capable of rescuing the bristle phenotype of the R62C alleles, indicating that even small amounts of the Q71L form of Drab6 can rescue the phenotypic effects of the loss-of-function R62C mutation. The Notch signal transduction pathway is used in many species to modulate the ability of precursor cells to respond to developmental cues. This signal is activated by the binding of the ligand Delta to its receptor Notch to activate downstream proteins. However, the selection of which cells undergo this activation is influenced by the amount of the Notch receptor at the cell surface; Notch is one of only a handful of genes to produce a visible phenotype with either an extra copy of the gene or in missing one copy. In a search for genes that modified an activated Notch phenotype, a novel bristle mutant named warthog was found. Cloning of the gene revealed it encoded the Drosophila homologue of rab6, or Drab6 . Mammalian and yeast forms of this gene are involved in Golgi trafficking . In mammalian tissue culture cells , a mutation (Q72L) in rab6 that impairs GTP hydrolysis, leads to a morphological disruption of Golgi structures and a decrease of marker proteins in the late Golgi network. Conversely, a mutation resulting in a GDP-bound form of rab6 (T27N) shows more prominent Golgi structures and an accumulation of marker proteins in the late Golgi network. Both of these rab6 mutations led to a kinetic inhibition of proteins presented to the cell surface; in pulse–chase experiments, cells that overexpress wild-type or either mutant form of rab6 (Q72L or T27N) eventually secrete the same quantity of extracellular proteins as controls, but the rate of release is markedly decreased. From these tissue culture experiments, mutations in Drab6 would be expected to delay the surface presentation of the Notch receptor. Given that the amount of Notch present on the cell surface is critical for the adoption of different cellular identities, such a delay in transportation of the Notch receptor to the plasma membrane would alter Notch signaling. The phenotypic interaction of the wrt screen alleles was consistent with a decrease in the amount of N available for signaling on the cell surface . Another explanation for the modification of Notch signaling by wrt is suggested by a paper from McConlogue et al. 1996 . They showed that rab6 specifically functioned at the critical junction of sorting between the amyloidogenic and nonamyloidogenic pathways for the β-amyloid precursor protein. This role of rab6 in the proper sorting of molecules into different compartments within or from the TGN may account for the interaction between Notch and warthog . Notch undergoes proteolytic cleavage by a furin-like convertase within the TGN to produce a heterodimeric receptor at the cell surface . If rab6 determines which Golgi and post-Golgi enzymes transported proteins encounter, then alterations in warthog function could potentially lead to a missorting of Notch into a transport pathway where the receptor is not cleaved properly. Having found genomic mutations in Drab6, we were able to study its effect on the development of a multicellular organism. In contrast to the single-cell organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe , the rab6 homologue was an essential gene in Drosophila . To determine how Drab6 affects Drosophila development beyond the larval stage, three different types of mutants were analyzed. The first was the arginine to cysteine point mutation (R62C) recovered from the screen. The second type was the more severe alleles, which included the P2352 insertion and several of its excision progeny, most notably the genomic null D23D. The third was an engineered mutation, Q71L, which by analogy to mutations in other ras-like proteins, confers a GTP-bound state to Drab6. Overexpression of this protein in the background of wild-type Drab6 was analyzed. Each of these mutants affected bristle morphology in a manner similar to other bristle mutants known to affect the structural integrity of cytoskeletal components . That Drab6 mutations altered cytoskeletal elements would be consistent with recent results involving rab-interacting proteins. Rabphilin, the effector protein of rab3, promotes the actin bundling activity of α-actinin and this activity is blocked by rab3-GTP . Also, a rab6-interacting protein, rabkinesin-6, was shown to bind microtubules and has ATPase activity similar to the plus end motors to which it is homologous; rab6-GTP was postulated to regulate the association and dissociation of rabkinesin-6 to microtubules . However, for warthog , no additive or synergistic interactions were seen with many mutations known to affect bristle structure (e.g., Sb , sn 3 , f 36a , Pr 1 , ss , Bsb , Bsb Pr , and Pr Dr ; data not shown). More importantly, the nonautonomous phenotype seen in the severe warthog mutants implies the Drosophila homologue of rab6 modifies the surface presentation of other proteins. Nonautonomous phenotypes are typically seen with secreted or transmembrane proteins that signal to neighboring cells. This effect is consistent with results from yeast and mammalian tissue culture experiments that establish the role of rab6 in the proper secretion of other proteins . Mutations that have previously been studied for rab6 are those engineered based on the GTP- and GDP-bound forms of ras-like molecules. From our screen , a novel mutation that resulted in the conversion of an arginine to a cysteine at amino acid 62 (R62C) was obtained. From biochemical and crystallographic data of other GTPases, the R62C mutation is expected to lie next to a defined GTP-binding domain (DX 2 G) where the invariant aspartate binds the catalytic Mg 2+ through an intervening water molecule . However, in vitro studies revealed R62C mutant protein was capable of binding and hydrolyzing GTP (data not shown), suggesting that this point mutation affects Drab6 function through another mechanism, perhaps by altering it interaction with regulatory proteins. This hypomorphic mutation altered rab6 function differently than the Q71L mutation, which resides next to the same GTP-binding domain. Overexpression Q71L Drab6 disrupted the orientation of bristle growth, whereas overexpression of R62C Drab6 in a wild-type background elicited no effects. Q71L Drab6 was also capable of rescuing the bristle defect of the R62C mutation. Therefore, studying the R62C mutation may reveal new information of Drab6 function. Perhaps the most interesting aspect of this phenotypic analysis is the limited requirement of a rab6 homologue throughout development. While an essential gene, Drab6 mutations did not affect the development of the eye, wing, and leg, nor the bristle structures within these tissues. This paucity of developmental phenotypes mirrors yeast studies that show null mutations in Ypt6 or rhy1 are not lethal, implying transport redundancy exists as proteins travel to the cell membrane . This redundancy could be the result of more than one rab6 protein, which is supported by the discovery of two putative rab6 homologues in C . elegans . Alternatively, it may be a functional redundancy where parallel but independent trafficking pathways through the Golgi/TGN can compensate for alterations in one another. Recent studies in mammalian systems support the existence of these independent trafficking pathways. The study of McConlongue et al. showed the secreted protein, β-APP, was processed in a different compartment if rab6 was mutated and a study of cell surface antigen presentation also showed alterations in rab6 affected one transport pathway but not another. The bristle phenotype of the warthog mutants, however, reveals there is a limitation to which an organism can compensate for mutations in Drab6, even if redundant or independent pathways exist for transport through the Golgi. This limitation may also be seen only after prolonged Drab6 dysfunction. Shetty et al. 1998 showed that overexpression of Drab6 Q71L in a subset of cells within the eye led to degeneration after two weeks. Having phenotypes associated with this limitation in redundancy through the Golgi/TGN will provide a novel means to dissect Golgi transport mechanisms. Identifying proteins that modify the wrt bristle phenotype will allow an ordered dissection of the protein cascade required for rab6 function. These mutants may also lead to a better understanding of how the cell regulates trafficking of signaling receptors such as Notch. Capitalizing on the interaction between wrt and Notch in sensitized backgrounds, genetic screens may help identify the proteins required for surface presentation of a functional Notch receptor. | Study | biomedical | en | 0.999999 |
10459010 | The wild-type S. cerevisiae strain used was X2180-1A (MATα SUC2 mal gal2 CUP1 ). 10 liter fermentation cultures of wild-type cells were grown at 24°C in liquid YPD medium (1% Bacto yeast extract, 2% Bacto peptone [Difco Laboratories Inc.], 2% glucose). Peroxisomes were isolated from the wild-type strain D273-10B cultivated under inducing conditions (0.3% Bacto yeast extract, 0.5% Bacto peptone, 0.5% KH 2 PO 4 , pH 6.0, 0.1% oleic acid [Merck], 0.2% Tween 80 [Merck]). The elo3 Δ mutant strain EMA40 (MATa ura3 trp1 leu2 elo3::URA3 ), together with the corresponding wild-type strain EMY30 (MATα ura3 trp1 leu2 ), were obtained from G. Loison and cultivated in liquid YPD medium at 30°C before isolation of the plasma membrane. For the isolation of mitochondria, microsomes, nuclei, peroxisomes, vacuoles, and lipid particles, late exponential phase cultures were harvested by centrifugation and converted to spheroplasts essentially as described by Daum et al. 1982 . Mitochondrial subfractions enriched in markers of the outer membrane, the inner membrane, and the contact sites were isolated after swelling and shrinking of intact mitochondria as described in Pon et al. 1989 . Microsomal membranes were obtained from the postmitochondrial supernatant by differential centrifugation at 40,000 and 100,000 g ; the 100,000 g supernatant being the soluble cytosolic fraction. Nuclei were enriched by sucrose density gradient centrifugation of the postmitochondrial supernatant as described . Vacuoles were isolated as described by Uchida et al. 1988 , with a modification that yields the lipid particle fraction . Peroxisomes were isolated as previously described . Plasma membranes were isolated following the procedure described by Serrano 1988 which relies on the disruption of intact cells by glass beads. Golgi membranes were isolated from early exponential phase wild-type cells as described by Lupashin et al. 1996 . Deuterium oxide, sucrose, and ATP were purchased from Sigma Chemical Co. Protein was quantified by the method of Lowry et al. 1951 using BSA as standard. Before quantification, proteins were precipitated with 10% TCA and solubilized in 0.1% SDS, 0.1 M NaOH. Proteins were separated by SDS-PAGE and transferred to Hybond-C nitrocellulose filters (Nycomed Amersham Inc.). Relative enrichment and degree of contaminations of subcellular fractions were determined by immunoblotting. Antigens were detected by antibodies against the respective protein followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescent signal detection using SuperSignal™ (Pierce Chemical Co.). Signal intensity was quantified by densitometric analysis using the wand tool present in NIH Image 1.61 (http://rsb.info.nih.gov/nih-image/download.html). Rabbit polyclonal antisera against the following proteins were employed: Kar2p/BiP (1:5,000; M. Rose, Princeton University, Princeton, NJ); Kre2p, ; Gas1p ; carboxypeptidase Y (CPY; 1:500; R. Schekman, University of California, Berkeley, CA); Erg6p ; porin and MDH (both 1:1,000; G. Schatz, Biocenter Basel, Basel, Switzerland); Aac1p (1:1,000; W. Neupert, University of Munich, Munich, Germany); Pox1p (1: 1,000; W.-H. Kunau, University of Bochum, Bochum, Germany). The mouse mAb against the soluble cytosolic 3-phosphoglycerate kinase PGK (mAb 22C5-D8) was purchased from Molecular Probes and used at 2 μg/ml. Lipid extracts of subcellular membrane fractions were dried under a stream of nitrogen and redissolved in a small volume (20–200 μl) of methanol/chloroform (2:1) containing either 10 mM ammoniumacetate (added from a 100 mM stock solution in methanol) for positive ion analyses or no additive for negative ion measurements. Total phospholipid content of samples was measured by the method of Rouser et al. 1970 . For the quantification of free ergosterol, lipid extracts of subcellular membrane fractions were dried in a vacuum concentrator and sulfated before mass spectrometry as described . Mass spectrometric analyses were performed with a triple quadrupole instrument model QUATTRO II (Micromass) equipped with a nano-electrospray source operating at a typical flow rate of 20–50 nl/min. The instrument was used either in the single-stage MS mode for the detection of total negative and positive ions or in tandem MS mode for product ion, precursor ion, or neutral loss scan analysis, as previously described . When used in single stage MS mode, relative peak intensity of different phospholipid classes may depend on the lipid concentration of the sample analyzed. PA, for example, was found to strongly quench the ionization of other lipids in samples with a high lipid concentration. The relative intensity of each (M − H) − and (M + H) + ion was determined from phospholipid class specific scan analyses as described . To identify the fatty acid composition of each molecular species, product ion scan analyses of the different molecular species and parent scan analyses for all major fatty acids were performed. Before nano-ESI-MS/MS analysis, the lipid extracts were centrifuged in a benchtop centrifuge for 5 min, and then a 3–10 μl aliquot was transferred into the electrospray capillary (type D; Micromass). The spray was started by applying 400–700 V to the capillary. For each spectrum, 20–100 repetitive scans of 10-s duration were averaged. All tandem MS experiments were performed with argon as collision gas at a nominal pressure of 2 mTorr. Collision energy settings employed were as follows: +par 184, 30 eV; +nl 141 and +nl 185, 28 eV; −par 241, 45 eV; −nl 87, 28 eV; −par 195, 50 eV; product ion scans, 30–50 eV; fatty acid specific scans, 40–50 eV. For ultrastructural analysis of subcellular membranes by EM, freshly prepared membrane fractions were fixed in 1% glutaraldehyde in 0.06 M phosphate buffer, pH 7.2, containing 1 mM CaCl 2 and 0.6 M mannitol for 45 min at room temperature. Membranes were then washed three times for 20 min each in phosphate buffer, postfixed in 1% OsO 4 for 45 min at room temperature, washed twice for 10 min each in phosphate buffer, dehydrated in a graded series of ethanol (50–100%, with en bloc staining in 1% uranylacetate in 70% ethanol overnight), and embedded in Spurr's resin. 70 nm ultra-thin sections were stained with lead citrate and viewed with a Philips CM 10 electron microscope. The aim of the present work was to determine qualitative differences between the lipid molecular species composition of distinct organellar membranes. To minimize alterations in the lipid composition due to culture variations, as many membranes as possible were isolated from a single batch of cells. The fractionation strategy designed to simultaneously isolate nine different organellar membranes is outlined in Fig. 1 . A 10 liter fermentation culture of wild-type cells was grown to late exponential phase, harvested, and one fifth of the total cell mass was used for the isolation of the plasma membrane fraction, which required disintegration of intact cells by glass beads . The remainder of the culture was converted to spheroplasts, split into three aliquots, each of which was then specifically processed for the isolation of particular fractions, i.e., nuclei , vacuoles and lipid particles , and mitochondria, which were further subfractionated into inner and outer mitochondrial membranes and contact sites . The postmitochondrial supernatant was subfractionated into heavy (40,000 g ) and light (100,000 g ) microsomes, and the soluble cytosolic fraction . Since Golgi membranes could not be isolated in sufficient yield and quality from a late exponential phase culture, an early exponential phase culture of the same wild-type strain was processed in parallel for the isolation of this organelle . The isolation of peroxisomes from yeast requires prior induction of the organelle by oleic acid. Peroxisomes were thus isolated from an independent culture cultivated under inducing conditions . Biochemical and morphological criteria were subsequently employed to assess the quality of the isolated membranes. Relative enrichment and degree of cross-contamination of the fractions was judged by immunoblot analysis with antibodies against the following marker proteins: Kar2p/BiP, a HSP70 family member and soluble lumenal ER protein, served as marker for the ER ; α1,2-mannosyltransferase , was used as marker for the Golgi membrane; Gas1p, a glycosylphosphatidylinositol (GPI)-anchored membrane protein , was the marker for the plasma membrane; CPY served as a lumenal marker for the vacuole; 3-phosphoglycerate kinase (PGK) was used as marker for soluble cytosolic protein; Δ24-sterol C-methyltransferase (Erg6p) served as marker protein for the lipid particle fraction ; Acyl-CoA oxidase (Pox1p) was a marker for peroxisomes ; porin was used as marker for the outer mitochondrial membrane ; ADP/ATP carrier protein (Aac1p) marked the inner mitochondrial membrane; and malate dehydrogenase (MDH) was used as marker of the mitochondrial matrix. Immunoblot analyses of all the subcellular fractions probed with these antibodies are shown in Fig. 2 and a quantification of the results is given in Table . As shown in Fig. 2 and Table , the plasma membrane was the subcellular fraction with the highest enrichment for its marker protein. The GPI-anchored Gas1p was enriched 185.5-fold. The isolated nuclei were 3.7-fold enriched for Kar2p. Enrichment factors of 10–15 for ER proteins in isolated yeast nuclear fractions have been reported for cells harvested at an early logarithmic phase . The lower enrichment seen in our preparations likely is due to the late growth phase at which these cells were harvested. Kar2p was detected in various fractions, which is in line with the contamination of most organelles by the poorly defined microsomes. The Golgi membrane was 23.5-fold enriched for the medial-Golgi apparatus marker Kre2p, which was also detected in the heavy microsome fraction (2.4-fold). Based on enzyme activity, this particular Golgi membrane fraction has been reported to be 205-fold enriched for the cis-Golgi membrane localized GDPase . Since early and late Golgi compartments differ in density , the Golgi fraction analyzed in this study is more representative of an early Golgi compartment when compared with the Kex2p enriched late Golgi compartment whose lipid composition has previously been analyzed . The lipid particle fraction was highly enriched for its marker protein Erg6p, but also contained Kar2p, consistent with the proposed relationship of this compartment with the ER membrane . The vacuolar fraction was 15.2-fold enriched in the lumenal vacuolar CPY. Mitochondria were enriched for porin, a major protein of the outer mitochondrial membrane , and for the ADP/ATP carrier protein, Aac1p, of the inner mitochondrial membrane. Both markers were further enriched in their corresponding subfractions. The marker protein for the mitochondrial matrix, MDH, was enriched in the mitochondrial fraction, but was also present in the inner mitochondrial membrane fraction, as has been observed previously . The dense microsomal fraction (40,000 g ) was 7.0-fold enriched for Kar2p. None of the marker proteins were selectively enriched in the light microsomal fraction (100,000 g ). The soluble cytosolic fraction was 5.2-fold enriched for phosphoglycerate kinase and did not show any enrichment for other marker proteins tested. Peroxisomes were enriched ∼30-fold for Pox1p relative to the corresponding homogenate (not shown), with no major cross-contaminations detectable. To further assess the purity and homogeneity of the isolated fractions, membranes were fixed and examined by ultra-thin section EM. The results of this analysis are shown in Fig. 3 . A distinct membrane/vesicle morphology for each of the isolated fractions was readily visible and the fractions appeared homogenous for the type of membrane isolated. The 40,000 g microsomal fraction contained the most morphologically inhomogeneous materials, such as big and small vesicles next to stacked sheets of membranes. Lipid bilayer thickness has been discussed as a possible sorting determinant for integral membrane proteins . We thus measured membrane thickness of the different fractions on high magnification prints of electron micrographs. The values obtained from this analysis are listed in Table . The average thickness of the membranes was 7.1 ± 0.4 nm. The plasma membrane was significantly thicker (9.2 ± 0.4 nm), and the lipid particle membrane was significantly thinner (4.5 ± 0.4 nm), as expected for a lipid monolayer that delineates the lipid particles . Lipids from the various subcellular membrane fractions were extracted, their phosphate content was determined, and set in relation to the protein content of the respective fraction ( Table ). The ergosterol content of the membranes was determined by quantitative nano-ESI-MS/MS analysis of lipid extracts containing a defined amount of [ 13 C]cholesterol as internal standard, added to the fractions before lipid extraction . Ratios of phospholipid to ergosterol content of the subcellular fractions are also listed in Table . These analyses complement previously published data , and show that the yeast plasma membrane has a phospholipid to ergosterol ratio of 2.2, which is comparable to a value of 1.9–2.9 reported for higher eukaryotic cells . Limiting amount of material did not allow quantification of the phospholipid content of the Golgi, nuclei, and mitochondrial fractions, which are therefore not represented in Table . Lipid extracts of the subcellular membrane fractions were then analyzed by nano-ESI-MS/MS. A comparison of negative ion mode spectra of the different membranes is shown in Fig. 4 . For peak assignment, each major ion was subjected to product ion scan analyses (data not shown). Interpretation of the spectra was facilitated by the relative simple fatty acid composition of S . cerevisiae phospholipids. As can be seen from the overview shown in Fig. 4 , the plasma membrane differed most from all other fractions. Free ceramide (Cer-C, containing phytosphingosine with a α-hydroxylated C26 fatty acid) was readily detectable in this membrane fraction, whereas the mature sphingolipids (M(IP) 2 C, IPC-C, and IPC-D) were also detected in the heavy microsome fraction . The m/z values of these sphingolipids were identical to those reported previously . To determine the relative abundance of each molecular species within its lipid class, scans specific for single phospholipid classes were recorded for every membrane fraction . Specifically, the molecular species profile of PS was calculated from positive mode ion scans specific for the neutral loss of m/z 185 (specific for serine phosphate; Table and Table ). The molecular species profile of PE was calculated from positive mode ion scans specific for the neutral loss of m/z 141 (specific for ethanolamine phosphate; Table ). The molecular species profile of PC was calculated from positive ion precursor scans for m/z 184 (specific for choline phosphate; Table ). The molecular species profile of PI was calculated from negative ion precursor scans for m/z 241 (specific for the dehydration product of inositol phosphate; Table ). Since peroxisomes were isolated from cells cultivated in the presence of oleic acid (C18:1), the lipid molecular species profile of this membrane was shifted dramatically towards oleic acid-containing species . For the comparison of the molecular species profiles of the different membranes that follow, peroxisomes were thus not further taken into account. Moreover, molecular species containing double desaturated acyl chains were observed in peroxisomal phospholipids ( Table , VI–VIII). These are due to contaminations of the oleic acid preparation used to supplement the culture media by C18:2, as revealed by GC-MS analysis (data not shown). A molecular species profile, calculated by averaging the values of ten different membranes, not including peroxisomes, is listed in Table and Table Table Table . In the right-most column of these Tables, the percentage of saturated acyl chains in the respective phospholipid class is given for each of the subcellular membranes. Averaged over all membrane fractions, PS 34:1 (i.e., composed of oleic acid [C18:1] and palmitic acid [C16:0]) constituted the main species of PS with 40.9%. This was followed by PS 34:2 (i.e., composed of oleic acid [C18:1] and palmitoleic acid [C16:1]) with 30.6%, PS 32:1 (15.7%), and PS 32:2 (10.2%). In the plasma membrane, PS 34:1 was the most prominent species (64.1%). Surprisingly, this rise in PS 34:1 at the plasma membrane was compensated by a greatly diminished PS 32:2 level (1.9%). A similar, albeit less pronounced, reduction was observed for PS 34:2 (16.8%), suggesting that PS species containing two unsaturated fatty acids are replaced by and/or remodeled to species containing one saturated fatty acid at the plasma membrane. Interestingly, in the lipid particle monolayer membrane, a PS profile contrasting that of the plasma membrane was observed. Diunsaturated fatty acid-substituted PS species, i.e., PS 34:2 (40.0%) and PS 32:2 (25.0%), were enriched at the expense of saturated fatty acid-containing species. The Golgi membrane, on the other hand, was greatly enriched in the short chain lipid PS 28:0, i.e., containing C14:0/C14:0 (10.6%). Yeast has a plasma membrane phospholipid asymmetry typical of eukaryotic cells, with PS mainly, if not exclusively, in the inner leaflet, and sterols and sphingolipids in the outer leaflet . The yeast plasma membrane has been estimated to contain most of the PS (90%), PE (80–90%), and PI (85%) in the inner leaflet and the sphingolipids (30% of total phospholipids) on the outer leaflet . The acyl chain composition of the sphingolipids in yeast is unusual in that they contain the saturated very long chain C26:0 fatty acid . The observation that monounsaturated PS species were greatly enriched at the expense of diunsaturated species suggested that the C26:0 containing sphingolipids may affect the acyl chain composition of lipids in the inner leaflet of the plasma membrane. To test this hypothesis, the PS profile of plasma membrane isolated from a yeast mutant, elo3 Δ , that is defective in the final step of elongating the C24 to the C26 fatty acid, and hence, does not contain any C26 fatty acid, was analyzed. Compared with the corresponding wild-type strain, in the plasma membrane from the elo3 Δ mutant, PS 34:2 is the most abundant species (40.3%), followed by PS 32:2 (24.6%), PS 32:1 (15.3%), and PS 34:1 . This dramatic shift towards diunsaturated fatty acid-containing species in cells that synthesize C24-substituted sphingolipids thus suggests that the precise acyl chain length of very long chain fatty acid-substituted sphingolipids affects the acyl chain composition of neighboring glycerophospholipids. Averaged over all membranes, the most abundant PE species was PE 34:2 (36.4%), followed by PE 32:2 (25.3%), PE 34:1 (15.5%), and PE 32:1 (14.8%). The tendency towards enrichment of saturated species (PE 32:1 and PE 34:1) at the expense of diunsaturated species (PE 32:2 and PE 34:2) at the plasma membrane, noted for PS, was also observed for PE (see Table ). Lipid particles were again enriched in the diunsaturated PE species, PE 32:2, as was the case for PS. The molecular species profile of PC did not display significant variation between the different membranes ( Table ). Overall, PC 32:2 (40.0%) was the most abundant species, followed by the 34:2 (25.4%), 32:1 (14.9%), and 34:1 (9.3%) species. The molecular species profile of PI was generally more heterogeneous than that of the other glycerophospholipids. This was mainly due to the fact that disaturated species of PI, but not of PS, PE, or PC, were present in all of the membranes (see Table ). The most abundant PI species was PI 34:1 (34.0%), followed by PI 32:1 (21.8%), PI 32:0 (7.5%), PI 34:0 (7.3%), and PI 34:2 (7.0%). PI thus contained a significantly higher proportion of saturated acyl chains (53.1%) than PS (29.4%), PE (16.2%), and PC (15.4%). The unusual short-chain substituted PI 28:0 was enriched at the plasma membrane (8.1%, compared with an average of 3.4%). The mitochondrial contact sites, on the other hand, were enriched in a second unusual short-chain containing PI, namely PI 30:0 (9.3%; see Table ). This enrichment of PI 30:0 in contact sites appeared to be at the expense of PI 34:1, which was reduced to 16.2%, compared with an average of 34.0%. PI 34:1, on the other hand, was enriched in the Golgi membrane (45.1%), but reduced at the plasma membrane (29.4%). PI 34:2 and PI 32:0 were both enriched in the lipid particle membrane. In general, the four main glycerophospholipid classes, PS, PE, PC, and PI, each were comprised of two main species, which together made up 56–72% of the respective lipid class, and two (or four in the case of PI) minor species that contributed 24–30% to the total. The two main PS species, PS 34:2 and PS 34:1, represented 71.5%, and the two minor species, PS 32:1 and PS 32:2, represented 25.9% of all PS. Similarly, the two major PE species, PE 34:2 and PE 32:2, comprised 61.7%, whereas the two minor species, PE 34:1 and PE 32:1, comprised 30.3% of PE. The PC species were dominated by PC 32:2 and PC 34:2, which together comprised 65.4% and were complemented by the two minor species, PC 32:1 and PC 34:1, which contributed 24.2%. PI contained two major species, PI 34:1 and PI 32:1 (55.8%), and four minor species, PI 32:0, PI 34:0, PI 34:2, and PI 36:1, which together made up 28.7%. Remarkably, not all possible combinations of the four major fatty acids were found esterified to the different glycerophospholipid classes. Disaturated fatty acid-containing species (32:0 and 34:0) were only observed for PI . Nano-ESI-MS/MS analysis, together with improved procedures for the isolation of individual subcellular membranes from yeast, S. cerevisiae , enabled us to perform what is, to our knowledge, the first analysis of the lipid molecular species composition of a comprehensive set of organellar membranes from a eukaryotic cell. The ESI-MS/MS analyses employed in this study were performed with intact phospholipid molecules, thus permitting direct structural assignments that avoid indirect inferences of identities of glycerophospholipid species inherent in HPLC- and GC/MS-based methods. Scan modes specific for the different phospholipid classes allowed us to resolve the complex mixture of lipid molecular species into readily assignable molecular species profiles for each of the different membranes. This analysis revealed a number of remarkable differences in the lipid molecular species composition of yeast subcellular membranes. The molecular species composition of the plasma membrane deviated most from that of all other membranes. Even though this was unexpected, it is not surprising, given that the lipid class composition of the plasma membrane with its high enrichment in sterols and sphingolipids, is much different from that of all the other membranes. It also contained large amounts of free ceramide-C, not readily detected in any other membrane. Furthermore, the molecular species profile of PS and PE at the plasma membrane was shifted dramatically towards species containing one saturated acyl chain (34:1) at the expense of diunsaturated species (32:2 and 34:2). A comparable enrichment of saturated PS species also has been reported for the plasma membrane of mammalian cells . This enrichment of saturated species of PS and PE is particularly noteworthy in light of the fact that PS and PE constitute, 33.6% and 20.3%, respectively, the majority of the glycerophospholipids in the plasma membrane of yeast . Moreover, in contrast to all other glycerophospholipids, PS preferentially incorporates radiolabeled palmitic acid into position sn-1 , suggesting that remodeling of the acyl chain composition of this lipid follows a unique pathway . As inferred from the established lipid asymmetry at the plasma membrane of eukaryotic cells, these PS species appear to be concentrated in the inner leaflet of the membrane and thus may interact with the saturated C26 very long acyl chain of sphingolipids from the opposing membrane leaflet. An effect of the C26-substituted sphingolipids on the molecular species composition of the plasma membrane was evident from the dramatic alteration of the PS profile observed in elo3 Δ mutant cells. The degree of acyl chain saturation of plasma membrane lipids is, due to the higher packing density of saturated acyl chains, regarded as a critical parameter for the generation of detergent insoluble membrane domains . The plasma membrane is the terminal compartment of lipids whose transport and/or maturation depends on a functional secretory pathway, i.e., the sphingolipids , PS, and ergosterol (Pichler, H., and G. Daum, unpublished observation). The question that arises is how this peculiar lipid composition of the plasma membrane is established and maintained. Three alternative, but not necessarily exclusive, hypotheses may be considered: (i) lipid selection and/or remodeling in the Golgi apparatus, i.e., by synthesis of specific species of PE and PC via the Kennedy pathway, results in the formation of secretory vesicles that already have an established lipid asymmetry and a molecular species composition typical of the plasma membrane; (ii) the acyl chain composition of lipids delivered to the plasma membrane is subject to extensive remodeling within their target membrane, i.e., unsaturated fatty acids on PS and PE are replaced by saturated fatty acids, resulting in the in situ generation of the molecular species profile of the plasma membrane; and (iii) the molecular species composition of the plasma membrane is established by selective removal of species that do not fit the particular requirements of that membrane, i.e., by selective phospholipase-mediated degradation of diunsaturated fatty acid-substituted species, or by concentrating such species in endocytotic vesicles and delivery to the vacuole. The question whether the plasma membrane lipid composition is defined in the exocytic or the endocytic pathway may be answered by analyzing the molecular species profile of the appropriate vesicles . Our previous observation that sphingolipids, ergosterol, and PS are enriched in the late (Kex2p-enriched) Golgi compartment (∼11% PS) and in late secretory vesicles (∼13% PS) suggests a significant contribution of the Golgi/secretory pathway in establishing the lipid composition of the yeast plasma membrane . In lipid particles, a phospholipid monolayer surrounds a hydrophobic core of triacylglycerols and steryl esters . Surprisingly, this membrane was particularly rich in diunsaturated fatty acid-substituted species of all major phospholipid classes, PS, PE, PC, and PI (see Table , VI–VIII). The lipid particle membrane is the subcellular membrane with the highest content of PI and is furthermore rich in PC (36.4%) and PE (20.0%), but contains little PS . The enrichment of lipid species with bulky head groups (PI and PC) and acyl chain substituents (diunsaturated species), suggests that during the biogenesis of this membrane type selected lipid species get incorporated into or are retained within the prospective lipid particle membrane. Intermediates in the de novo synthesis of PC via methylation of PE, i.e., monomethyl-PE (MMPE) and dimethyl-PE (DMPE), were detected in the Golgi membrane. No such methylation intermediates were detected in any of the other subcellular membranes . The de novo synthesis of PC occurs by sequential three-step methylation of PE first to MMPE, then to DMPE, and finally to PC. This reaction sequence is generally thought to occur in the ER and is catalyzed by two methyltranferases in yeast, Cho2p/Pem1p and Opi3p/Pem2p, with overlapping substrate specificity . Cho2p catalyzes the first step, methylation of PE to MMPE, and Opi3p catalyzes the subsequent two steps from MMPE to PC . The fact that methylation intermediates were observed in the Golgi fraction rather than in the ER led us to consider whether methylation may be, in part, a Golgi apparatus-localized process. Inspection of the two protein sequences revealed the presence of a COOH-terminal ER retention signal in Opi3p (KAKKNM), but no such signal could be found in Cho2p . The subcellular localization of Cho2p thus needs to be reinvestigated in more detail. Alternatively, since the Golgi membrane was isolated from exponentially growing cells, the presence of methylation intermediates in this fraction may be due to export of not yet fully matured phospholipids from the ER of rapidly dividing cells. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g., PS, PE and PC, and PS and PI), noted already in our earlier study of the acyl chain composition of total phospholipids , were also observed in this study, suggesting that pathways exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. Consistent with the results obtained by Wagner and Paltauf 1994 , we find that the percentage of saturated fatty acids is low in PC (15.4%) and PE (16.2%). PS (29.4%), the direct precursor to PE via the de novo pathway, has a significantly higher content of saturated fatty acids. Although both PS and PI are derived from cytidine diphosphate-diacylglycerol (CDP-DAG), the fatty acid distribution between these two phospholipid classes is remarkably different, with PI having the highest percentage of saturated fatty acids . These species differences may be explained by different substrate specificity (species preferences) of enzymes catalyzing individual steps in the synthesis of the major lipid classes, or by distinct subcellular localization of specific substrates for the synthesis of the different lipid classes. For example, the enzymes that catalyze the final step in the synthesis of PI and PS (PI synthase and PS synthase, respectively) may possess different preferences for distinct molecular species of their common substrate, CDP-DAG, hence yielding products with different acyl chain compositions. Alternatively, these two enzymes may be localized in different subdomains of the ER and therefore may have access only to local pools of CDP-DAG molecular species. Apart from such species differences established by de novo synthesis of the various lipid classes , species differences may also be established later, by the salvage/Kennedy pathway for PE and PC synthesis from local pools of DAG species produced in the Golgi apparatus . Furthermore, pathways may exist to generate specific molecular species by remodeling the acyl chain composition of existing lipids ; different molecular species may have different rates of turnover , or may be transported with different efficiency . Minor lipid classes, such as plasmalogens and cardiolipin, and unusual molecular species of the major lipid classes were not analyzed in detail in this study. Molecular species of cardiolipin from yeast previously have been characterized and shown to be of mainly tetramonounsaturated type . Low levels of plasmalogens, mainly of PE, were detected in most of the subcellular fractions analyzed and were also observed in lipid extracts from cells cultivated on synthetic minimal media, suggesting that S . cerevisiae has the enzymatic repertoire to synthesize these lipids de novo (data not shown). The dynamic nature of the lipid molecular species composition of the different subcellular membranes has not been addressed in this study. Data reported here thus may merely serve as a point of reference for future more detailed and focused analyses of lipid molecular species profiles of, for example, the plasma membrane of various mutant cells or of vesicles of different origin. Moreover, comparison of molecular species profiles from a variety of growth conditions should provide a minimum set of lipid molecular species whose presence in a given membrane compartment is required for normal vegetative growth and another set composed of species that are synthesized only in response to specific environmental stimuli or during more specialized processes. The data presented here thus provide basic information integral to the interpretation of such future experiments. | Study | biomedical | en | 0.999997 |
10459011 | Clathrin was purified from bovine brain–coated vesicles as described . d -myo-Inositol hexakiphosphate (IP 6 ) was obtained from Calbiochem. [ 3 H]IP 6 was from DuPont NEN, 35 S-Translabel was from ICN. l -1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was from Worthington Biochemical, Inc. Restriction and modification enzymes were purchased from Boehringer Mannheim. S-Sepharose and Sepharose 2B were from Sigma Chemical Co. TnT rabbit reticulocyte transcription-translation system was from Promega. All other chemicals were reagent grade or better and were purchased from Sigma Chemical Co. or Fisher. Deletion and site-directed mutations in the α5-80 insert in the plasmid pMAL αA5-80 were performed using a combination of subcloning procedures and polymerase chain reaction. Details of these procedures are available upon request. The fusion proteins were expressed and purified as described previously . Purified maltose-binding protein chimera were dialyzed into 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 2 mM CaCl 2 , and digested with the Factor Xa (1 mg enzyme/200 mg fusion protein) for 24–36 h at 4°C. The digestion mixture was then incubated with amylose beads to remove MBP and undigested fusion protein. The supernatant was then applied to an S-Sepharose column (Pharmacia) and the column was washed with 20 mM sodium phosphate, pH 7.3, 200 mM NaF. The bound peptide was eluted with 20 mM sodium phosphate and 1 M NaF. The eluate was dialyzed into 10 mM sodium phosphate, pH 7.3, and 100 mM NaF, and used for circular dichroism spectroscopy. Purity of the peptide was checked by SDS-electrophoresis on an 8–25% gradient gel using a PhastSystem (Pharmacia). The binding of [ 3 H]IP 6 to recombinant fusion proteins was determined by a polyethylene glycol precipitation procedure as described previously . Circular dichroism (CD) spectroscopy was performed on an Aviv-62DS instrument at ambient temperature under nitrogen atmosphere with peptides at 0.1–0.4 mg/ml in 10 mM sodium phosphate, 100 mM NaF. The CD spectra were analyzed using secondary structure prediction software based on the method described by Andrade et al. 1993 . Wild-type and mutant α A subunits were expressed in vitro using a TnT rabbit reticulocyte lysate transcription-translation system. First, plasmid pSP65α A was modified to optimize expression by removing most of the 5′ untranslated region beyond 10 bp upstream of the initiation codon of α A cDNA. Analysis showed that the resulting plasmid pSP65α A 2 gave 5–10-fold higher expression than pSP65α A . Mutant α A cDNA fragments were cloned into the pSP65α A 2 by a series of subcloning procedures, the details of which are available on request. The resulting plasmid pSP65 α A KKK-Q and the plasmid pSP65α A 2 were used for in vitro expression of mutant and wild-type α A . Transcription-translation reactions were performed according to the manufacturer's recommendations in the presence of 35 S-Translabel. After incubation for 2 h at 30°C, translation reactions were centrifuged at 100,000 rpm for 20 min at 4°C in a TLA100 rotor (Beckman). Limited tryptic proteolysis and clathrin cage binding experiments with in vitro–translated wild-type and mutant AP-2α polypeptides were performed essentially as described previously . Plasmid containing the αγα construct (derived from bovine AP-2α C ) in pBluescript SK− was digested with EcoRI, treated with Klenow fragment and dNTP, and subsequently digested with SacI. Excised fragments were ligated into the plasmid pSP65 that had been digested with SacI and SmaI, resulting in the plasmid pSP65-dN-αγα. This plasmid was cut with SacI and SalI, and the excised fragment was ligated together with the linker connecting NdeI and SacI sites into the plasmids pSP65α A 2 and pSP65 α A KKK-Q from which NdeI-SalI fragments were excised. The resultant plasmids pSP65-WTαγα and pSP65-M18αγα contained wild-type and mutant αγα chimeric inserts. These plasmids were consecutively treated with SalI, blunted with Klenow fragment and dNTP, and then digested with EcoRI. The excised inserts were ligated into eukaryotic expression vector pcDNA3 that had been cut with EcoRI and EcoRV, resulting in plasmid constructs pcDNA3αγαWT and pcDNA3αγαKKK-Q. For transient expression, BALB/c-3T3 cells were grown in T-75 flasks in a humidified atmosphere with 5% CO 2 in DME supplemented with 5% fetal bovine serum, 5% calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were grown to 60–70% confluence and transfected with expression constructs using Lipofectamine reagent. In brief, 15 μg of pcDNA3αγαWT or pcDNA3αγαKKK-Q were incubated with 80 μl of Lipofectamine in 5 ml of DMEM for 30 min at room temperature. After the incubation 5 ml of DME was added and the mixture was transferred to a flask with DMEM-rinsed BALB/c-3T3 cells. After 4 h of incubation the mixture was substituted with complete media and the cells were incubated for 15–18 h. Cells were then trypsinized and plated on 12-mm round glass coverslips in a 24-well plate. Immunofluorescence analysis was performed 48 h after transfection. MOP8 cells transiently transfected for 48 h with the wild-type αγα or KKK/Q mutant αγα constructs (3 T-75 flasks) were washed with PBS and scraped into 5 mM Tris-HCl, pH 7.0, supplemented with protease inhibitors. After homogenization, 1 M Tris-HCl, pH 7.0, was added to the broken cells to a final concentration of 0.5 M, the suspension was incubated for 30 min on ice and centrifuged at 100,000 rpm for 30 min in TLA100 rotor (Beckman). The supernatant was diluted to yield a concentration of 125 mM Tris-HCl and supplemented with protease inhibitors and Triton X-100 (0.05%). The AP-2 αγα protein was immunoprecipitated with the antibody 100/3 and resolved by SDS-PAGE. Western blot analysis was performed using antibodies R2 and 100/3, specific for the β- and γ-adaptins, respectively, as well as affinity-purified antibodies against μ2 and σ2 . Immunofluorescence analysis was performed as described previously . In brief, cells were washed, fixed, permeabilized, and exposed to mouse monoclonal antibody 100/3 (50 μg/ml) for detection of αγα polypeptide and rabbit Ab31 (1:150) for detection of endogenous AP-2 . We have found that Ab31, though it was made against a fragment containing both the hinge and ear sequences of rat brain α C , reacts only with α hinge and not with the ear domain (data not shown). Treatment of the α subunit with trypsin is known to generate two major fragments corresponding to the core and the intact hinge + ear domains, while elastase cleaves the ear fragment leaving intact core + hinge . On immunoblotting, Ab31 recognized the intact hinge + ear domain generated by trypsin, but failed to recognize the separate ear domain produced by elastase. Antibody 100/2, specific for the α ear, recognized both the hinge + ear fragment generated by trypsin and the ear domain generated by elastase. This allowed us to use Ab 31 to detect endogenous α but not αγα, which lacks the hinge region of α. Clathrin was detected using rabbit polyclonal antibody 27004 (1:150 dilution) . Primary antibodies were detected with fluorescein-conjugated donkey anti–mouse IgG or rhodamine-lissamine–conjugated donkey anti–rabbit IgG secondary antibodies (Jackson ImmunoResearch). Confocal microscopy was performed on a Bio-Rad MRC-600 laser scanning confocal microscope, using a Zeiss Plan-Apo 63× 1.40 NA oil immersion objective. Images were collected sequentially in the photon counting mode using single line excitation. In our previous study, using photoaffinity labeling and bacterially expressed fusion proteins, we localized the high affinity PPI binding site on the clathrin adaptor protein AP-2 to the region between residues 5 and 80 at the NH 2 -terminal of the α subunit. To determine whether PPI binding could be localized to a shorter sequence within this region, we produced several maltose-binding protein (MBP) fusion proteins containing smaller fragments . Among fusion proteins containing either residues 5–21, 21–80, 5–49, or 50–80, only those containing residues 21–80 retained specific IP 6 binding, with affinity similar to that of the full fragment 5–80 ; the other fusion proteins did not display any detectable binding. This suggested that the PPI binding site in the AP-2 α subunit may not be represented by a short stretch of residues, but that a relatively large portion of the sequence between amino acids 21–80 may be required to form a discrete domain with proper tertiary structure. The AP-2 α sequence between residues 5 and 80 is a fairly basic region with several clusters of cationic residues. As it is likely that positive charges are involved in the interaction with the negatively charged phosphate groups of PPIs, we investigated more closely the role of these basic residues in IP 6 binding. Accordingly, we produced a series of fusion proteins of MBP with the AP-2 α5-80 fragment (denoted MBP-α5-80) in which each basic amino acid (10 lysines and 4 arginines) was changed to a glutaminyl residue. Glutamine was chosen because it contains a substantial side chain, similar to lysyl and arginyl residues, but is uncharged. Each of these fusion proteins was purified by affinity chromatography and tested for IP 6 binding. We found that residues scattered throughout the α 5–80 region affected IP 6 binding, though to differing extents . The mutations could be divided into several groups in terms of their effects on IP 6 binding: no reduction in IP 6 binding activity (R21); slight (∼20%) reduction (K24, K26, R41, K43, K48); substantial (∼40%) reduction (K31, R32, K35, K45, K61); and large (>60%) inhibition (K55, K56, K57). To investigate further the role of lysyl residues 55–57 whose alteration to glutamines had the most pronounced effect on IP 6 binding, we generated an additional mutant in which residue K56 was changed to glutamic acid with reversal of charge. The IP 6 binding ability of this mutant was even more greatly diminished, to ∼30% of the wild-type protein, compared with substitution with glutamine. When all three lysyl residues were changed to glutamines in a single mutant, denoted KKK/Q, the IP 6 binding ability was decreased to <10% of the wild-type protein. This mutant, essentially devoid of IP 6 binding, was characterized further using biophysical methods and functional assays described below to determine whether these residues are directly involved in PPI binding, or whether the decrease in PPI binding is the result of gross conformational change in the structure of the protein. CD spectroscopy is a very useful method for the rapid determination of secondary structures of peptides and proteins. We employed CD spectroscopy to characterize the secondary structure of the AP-2 PPI binding domain and to monitor any changes resulting from the mutations introduced in its sequence. The AP-2 α5-80 fragments of the wild-type and KKK/Q mutant fusion proteins were cleaved with Factor Xa and purified from MBP by consecutive affinity and ion-exchange chromatography steps as described in Materials and Methods. The purification procedure resulted in peptide preparations that were uniformly >95% homogeneous (data not shown). A CD spectrum of the isolated wild-type AP-2 α5-80 fragment is shown in Fig. 3 a. The positive absorption peak at 195 nm and two negative peaks at 207 and 222 nm indicate that the conformation of the α5-80 peptide has substantial α-helical content. Secondary structure calculated by the method of Andrade et al. 1993 yielded estimates of ∼37% α-helix and 26% β sheet. Also shown in Fig. 3 a is the CD spectra of the wild-type AP-2 α5-80 fragment in 50% trifluoroethanol, known to induce an α-helical conformation in oligopeptides . Under these conditions, the spectrum of the wild-type fragment exhibited much more pronounced maximum and minima, corresponding to almost 100% α-helicity. The CD spectrum of the mutant AP-2 α5-80-KKK/Q fragment is presented in Fig. 3 b, along with the spectrum of the wild-type peptide. The mutant α5-80-KKK/Q has secondary structure content practically identical to that of the wild-type protein. The CD spectrum of the purified peptide derived from the charge inversion mutant AP-2 α5-80-K56E was also indistinguishable from that of the wild-type (data not shown). These results argue that the decrease in PPI binding observed with these mutants is not the result of gross conformational changes induced by the amino acid substitutions, but rather results from disruption of direct interactions between the ligand and basic residues on the protein. We tried to determine whether ligand binding induces any conformational change in the PPI binding domain α5-80. Unfortunately, in the presence of IP 6 at concentrations as low as 1 μM the peptide aggregated. This problem could not be overcome by the addition of salt and/or nonionic detergents. To further evaluate the effects of the KKK/Q mutation on the overall properties of the AP-2α subunit, we performed structural and functional assays on full-length wild-type and mutant AP-2 α polypeptides generated in a rabbit reticulocyte in vitro translation system. We have previously shown that the in vitro translated AP-2 α polypeptide is folded similarly to that in the native AP-2 complex isolated from bovine brain . For example, limited tryptic proteolysis of in vitro–translated AP-2α generates fragments of ∼55–66 kD and 40 kD, corresponding to the NH 2 -terminal core and COOH-terminal appendage domain generated on similar treatment of the native bovine brain AP-2 . Similar limited proteolysis of in vitro translated mutant KKK/Q α A polypeptide produced a pattern with the characteristic core and appendage domains, virtually identical to that of the wild-type polypeptide . This result demonstrates that alteration of the K(55-57) residues in the NH 2 -terminal region of the AP α polypeptide does not cause gross misfolding of the entire subunit on synthesis. The isolated AP-2 α subunit generated by in vitro translation has also been shown to bind specifically to clathrin . This provided a useful assay to ask whether lysyl residues 55–57 were directly involved in clathrin binding, or whether their substitution with glutamines altered structural properties of the isolated AP-2 α polypeptide required for this interaction. We found that binding of the in vitro–translated mutant KKK/Q α polypeptide to clathrin cages was indistinguishable from that of the wild-type polypeptide . Collectively, these results indicate that the mutant KKK/Q AP-2 α polypeptide retained the native tertiary structure and function of the wild-type protein, but that it is essentially devoid of PPI binding ability. To investigate the functional role of the AP-2 PPI binding site in intact cells, we employed an αγα construct described previously and kindly provided by Dr. M.S. Robinson (University of Cambridge). The αγα construct encodes mouse α C polypeptide in which the hinge region between the core and appendage domains, corresponding to α C residues 620–700, has been substituted with the hinge region of the bovine Golgi-specific AP-1 γ subunit. This enabled us to specifically localize the expressed αγα polypeptides in transiently transfected mouse fibroblasts using a γ-specific monoclonal antibody (mAb 100/3) which does not recognize the endogenous (mouse) protein . Additionally, the endogenous AP-2 α polypeptide could also be uniquely localized using Ab31, a rabbit polyclonal anti-α antibody kindly provided by Dr. A. Sorkin (University of Colorado). We found that though Ab31 was produced by inoculation with a fragment consisting of the hinge and appendage domains of the rat brain α C subunit , it reacts only with the α hinge region and not with the α appendage (see Materials and Methods). Thus, the endogenous and the transiently expressed exogenous α polypeptides could be detected independently, providing important tools for the study of mutant α subunits. First, we asked whether wild-type and KKK/Q mutant AP-2 αγα polypeptides expressed after transfection are incorporated into AP-2 adaptor complexes in intact cells. Lysates of mock, wild-type αγα, and KKK/Q mutant αγα–transfected MOP8 mouse fibroblasts were challenged with monoclonal antibody 100/3, and the resultant immunoprecipitates were fractionated by SDS-PAGE and analyzed by immunoblotting with antibodies to the other AP-2 subunits. As shown in Fig. 5 , reactivity with the anti-γ 100/3 antibody was detected only in immunoprecipitates from cells transfected with the wild-type or mutant αγα constructs, consistent with the inability of this antibody to recognize the endogenous mouse AP-2 γ polypeptide. On immunoblotting with antibodies to the β, μ2, or σ2 subunits of AP-2, no signal was detected in the immunoprecipitates from mock-transfected cell lysates demonstrating that recovery of the endogenous AP-2 subunits were dependent on their incorporation into complexes containing exogenous αγα polypeptide. However, anti-γ immunoprecipitates of cells transfected with either the wild-type or the KKK/Q αγα constructs contained the endogenous β2, μ2, and σ2 subunits in similar amounts. These findings confirm the results of Page and Robinson 1995 in indicating that the wild-type αγα polypeptide becomes incorporated into AP-2 complexes, which we denote AP-2 WT . Furthermore, the results presented here demonstrate that the mutant KKK/Q αγα polypeptide behaves indistinguishably from the wild-type, associating with the other AP-2 subunits and forming complexes (which we denote AP-2 PPI −) in the transiently transfected cells. To investigate the cellular phenotype resulting from knockout of the PPI binding site of AP-2, we analyzed transfected BALB/c-3T3 cells by confocal fluorescence microscopy. Though the transfection efficiency of BALB/c-3T3 cells was lower than that of MOP-8 cells in our hands, the former were chosen for this experiment because their morphology after fixation is much more amenable to immunofluorescence analysis of plasma membrane coated pits. Cells transfected with wild-type (AP-2 WT ) or mutant KKK/Q (AP-2 PPI −) αγα constructs were double-labeled with mouse monoclonal antibody 100/3 to reveal the localization of the exogenous αγα product, and with either rabbit Ab31 or 27004 to localize endogenous α-adaptin or clathrin, respectively. Fig. 6 a shows the localization of the AP-2 WT αγα product at several different expression levels in transiently transfected cells. The vast majority of the expressed AP-2 WT protein (upper panels) had a punctate distribution in the plane of the plasma membrane, with very little diffuse signal detectable. Comparison with the distribution of endogenous AP-2 α (lower panels) indicated almost complete colocalization . The images also show that the presence of the γ hinge did not misdirect the protein to the Golgi region. Consistent with this finding, the AP-2 WT distribution was also largely coincident with the localization of plasma membrane coated pits stained with anti-clathrin antibody, but did not colocalize with anti-clathrin staining in the trans-Golgi network (data not shown). Similar observations were made by Robinson 1993 on her initial use of the αγα construct for expression in Rat1 cells. At low expression levels, AP-2 WT had no detectable effect on the distribution of the endogenous α adaptin . Interestingly, in cells with higher levels of expression there is an apparent dominant-negative effect in that the level of endogenous α adaptin in clathrin-coated pits is decreased compared with untransfected cells in the same field . Only at unphysiologically elevated levels of expression is there any evidence for significant accumulation of soluble AP-2 WT protein (data not shown), and there is no detectable effect on the normal distribution of clathrin at the plasma membrane or in the Golgi region. The localization of the mutant AP-2 PPI − protein at several different levels of expression are shown in Fig. 6 b and 7. The distribution of the mutant protein differed radically from that of the wild-type protein. Generally, most of the AP-2 PPI − localization was diffuse and at any level of expression, no significant amount of the mutant polypeptide could be detected in clathrin-coated pits at the plasma membrane. In some cells a small amount of finely punctate signal was detectable, most of which was intracellular. With few exceptions this signal did not coincide with that of endogenous AP-2 α , nor did it colocalize with either early or recycling endosomes (labeled with endocytosed fluorescent transferrin), or with the late endosome/lysosomal compartment (labeled with endocytosed fluorescent dextran) (data not shown). Interestingly, with increasing expression levels of the mutant AP-2 PPI − protein, the proper localization of AP-2 to discrete plasma membrane sites was diminished . Similarly, at low levels of mutant AP-2 PPI − expression, the localization of clathrin to plasma membrane was not noticeably affected . However, clathrin localization was clearly abnormal at higher levels of mutant expression with a reduced number of plasma membrane coated pits present in comparison to adjacent, nonexpressing cells . Interestingly, the clathrin signal in the Golgi region also seemed to be affected by elevated levels of AP-2 PPI − expression, consistent with continuity between the plasma membrane and Golgi pools of clathrin. Finally, we evaluated the internalization of the fluorescently tagged transferrin by cells expressing αγα constructs. In cells expressing low levels of either the AP-2 WT or AP-2 PPI − mutant proteins, internalization of transferrin was indistinguishable from that in neighboring cells that were not expressing either product (data not shown), consistent with the absence of an effect on coated pit distribution in these cells. Interestingly, transferrin internalization was greatly diminished in cells expressing moderate levels of the mutant AP-2 PPI − protein, consistent with the disruption of clathrin-coated pits in that population. In contrast, AP-2 WT did not detectably affect transferrin uptake until very high levels of expression were attained. In this study, we have sought to determine the importance of the high affinity PPI binding site located in the NH 2 -terminal region of the AP-2α subunit in the process of receptor-mediated endocytosis. In previous reports we identified the polypeptide region involved in binding and provided in vitro evidence that PI-3,4,5-P 3 , a product of phosphatidylinositol 3-kinase, is the ligand of highest known affinity for AP-2 in assembled coat structures . Though these results suggested that PIP 3 is a physiologically relevant ligand for AP-2, in the absence of definitive data from intact cells this conjecture remains uncertain, as does the precise identity of other ligand(s) that may interact with AP-2. Accordingly, we undertook a complementary approach to study the physiological function of the AP-2 PPI binding site. We sought to identify and alter amino acid residues critical for PPI binding to AP-2, and to evaluate the effects of expression of this mutant AP-2 in intact cells. The results of these efforts lead to the conclusion that an active PPI site is indeed required for AP-2 function in receptor-mediated endocytosis. In some PPI-binding proteins short peptides (8–20 residues) have been found to be sufficient for high affinity binding of inositol phosphates or phosphoinositides. Examples include certain actin-associated proteins such as gelsolin and profilin and some C2 domain–containing proteins such as synaptotagmin . Pleckstrin homology (PH) domains, which are found in a number of proteins involved in signal transduction and are believed to function in membrane recruitment and regulation of enzymatic activity , provide a contrasting pattern. In these proteins essentially the entire ∼100 amino acid module is necessary for high affinity ligand binding. The mutagenesis analysis reported here suggests that the latter characterization is more applicable to the PPI binding site in AP-2 α. It has a highly organized secondary structure and seems to require a ≥60 residue region for full binding activity, from which we infer that this portion of the α structure comprises a distinct structural and functional domain. Positively charged amino acids throughout the region contribute to the binding interaction , with two clusters of basic residues toward each end (a lysine triad at 55–57 and K31/R32/K35) appearing to be most important. In parallel with the PH domains whose tertiary structure in complex with ligands has been determined , the PPI binding region in AP-2 may be a large positively charged surface with some residues in direct contact with the bound ligand, while others may be responsible for the initial electrostatic recruitment of the PPI to the binding pocket or the formation of the charged surface. It is increasingly appreciated that protein domains may have remarkably similar three-dimensional structure but share very limited sequence homology. Consequently, the relationship of the AP-2 binding site to other PPI binding domains with which it does not share detectable sequence identity will probably only be answered after determination of its tertiary structure. This region of the AP-2 α sequence, and the basic residues in particular, are virtually entirely conserved in both Drosophila and C . elegans homologues of mammalian α A . Furthermore, although the overall identity of two recently identified yeast α homologues with the mammalian protein in this region is 30–40%, most of the basic residues required for inositide binding in the mammalian protein, in particular the lysine triad, are also conserved . This extends the inference of a functional PPI binding domain to these lower eukaryotes. Interestingly, the mammalian AP-1 γ, AP-3 δ, and recently identified ∈ subunit of a novel AP-4 complex show distinct but considerably less conservation of several of these basic residues : to the best of our knowledge the PPI binding properties of these proteins have not been reported. Finally, the COPI coatomer and AP180 also bind PPIs but have no discernible sequence similarity to AP-2 α. Collectively, these observations suggest that PPI binding by coat subunits involved in membrane transport is a ubiquitous phenomenon, and that the nature of specific residues in this binding domain may impart inositide binding specificity. There is increasing evidence for an essential role of phosphoinositides in transport vesicle function at different locations in mammalian cells. Phosphoinositides, particularly PIP 2 , formed secondarily to ARF activation of phospholipase D, have been implicated in the recruitment of COPI coat proteins onto the membranes of the Golgi stacks . The specific interaction between these acidic phospholipids and coatomer, which has been shown to bind PPIs and particularly PIP 3 with high affinity , could contribute to recruitment of the coatomer to a specific membrane location, though this is controversial . With regard to AP-2, broken cell assays have shown that PIP 2 sequestration, accomplished either pharmacologically with neomycin or biochemically using the PH domain of PLCδ, had an inhibitory effect on AP-2 recruitment to the plasma membrane, indirectly implicating phosphoinositides in AP-2 targeting . Our in vitro binding data indicated that the (assembled) coat form of AP-2 shows the highest affinity for phosphoinositides, as compared with inositol phosphates, and that the converse is true for the soluble (disassembled) AP-2 protein . These observations suggest that the presence of phosphoinositides will drive the AP-2 molecule toward its higher affinity, assembled form. This conjecture is supported by the observation reported here that AP-2 lacking a functional PPI site is not incorporated into coated pits. Further, it has been reported that the receptor cytoplasmic tail interaction of AP-2 with bound phosphoinositide is comparable to that with AP-2 in an assembled coat structure, and that both are of higher affinity than that with free AP-2 , again suggesting that inositide binding drives AP-2 toward an assembled conformation thereby promoting its ability to interact with clathrin. The results reported here provide direct support for the notion that PPIs play a physiologically important role in membrane recruitment of AP-2. The mutant AP-2 PPI− assayed in vitro is almost totally defective in PPI binding, but otherwise indistinguishable from the wild-type protein by multiple structural and functional criteria. However, in intact cells AP-2 PPI− is almost completely defective in incorporation into plasma membrane clathrin-coated pits. Unlike the wild-type protein, it tends to have a diffuse distribution throughout the cell . At high expression levels, a small amount of punctate signal is also detectable which may reflect the inability of AP-2 PPI − to bind PPIs and resist self-association . These observations are generally consistent with earlier results of Page and Robinson . Using α/γ chimeras, their results indicated that the plasma membrane/Golgi targeting signals are localized primarily between residues 130 and 330–350 in the α and γ sequences, respectively. Interestingly, chimeric proteins in which 132, or even 36, residues from the NH 2 -terminal region of the AP-2α subunit had been replaced by corresponding γ sequences gave substantial diffuse signal and considerably reduced, though still detectable, recruitment to plasma membrane coated pits . This may reflect cooperation of the plasma membrane targeting signal, localized by these workers to the distal α sequence, with the action of a hypothetical PPI binding domain in the NH 2 -terminal region of the γ sequence . According to this reasoning, the AP-2 PPI− is not detectably recruited to coated pits despite presence of a plasma membrane targeting signal because it lacks PPI binding. Interestingly, AP-2 PPI − also acts as a dominant-negative inhibitor of coated pit formation. This suggests that excess inactive AP-2 PPI− complexes effectively sequester the other AP-2 subunits and/or occupy the limited sites that must be available for coat formation , indicating in either case that the binding to AP-2 of PIP 3 or another specific PPI ligand in the membrane is important early during the receptor mediated endocytosis process, i.e., at the stage of clathrin-coated pit formation. We have recently demonstrated that clathrin-coated pits form at specific and defined sites on the plasma membrane, and that a cytoskeletal framework in tight association with the membrane likely plays a major organizational role in this process . Together, these results suggest a model in which coat formation is initiated and anchored by interactions oriented both outward toward the plasma membrane and inward toward a neighboring skeletal structure. This general model is supported by our recent demonstration that PPIs are also involved in the ligand-dependent internalization of another class of receptors, the G-protein–coupled receptors. Nonvisual arrestins, which have been shown to act as adaptors in the internalization of β 2 -adrenergic receptors , bind PPIs with high affinity. We found that soluble PPIs and phosphoinositides differentially modulate arrestin interaction with clathrin and receptor. Furthermore, as in the case of the AP-2 adaptor reported here, a functional PPI binding site is critical to the ligand-dependent recruitment of the receptor-arrestin complex to clathrin-coated pits . Together, these findings point to common themes of phosphoinositide action in membrane trafficking events: they may serve either as recruitment signals for coat components and/or to modulate the interaction of coat components with receptor complexes. The presence in clathrin-coated pits and vesicles of synaptojanin , a phosphoinositide 5-phosphatase, suggests that adaptor functions may be regulated by a complex interplay of different enzymes involved in site-specific phosphoinositide metabolism. Additional enzymes involved in adaptor/coat regulation, and the factors, which modulate their activity, are yet to be discovered. | Other | biomedical | en | 0.999996 |
10459012 | Full-length recombinant VAMP-7 protein lacking its COOH-terminal hydrophobic domain was used to immunize both a rabbit and a mouse. Affinity-purified polyclonal antibodies against VAMP-7 were then prepared from rabbit antisera as described previously . mAbs against VAMP-7 were obtained from the corresponding hybridoma cell lines produced by fusion of NS-1 mouse myeloma cells with spleen cells from an immunized BALB/c mouse as described previously . The mAbs were isotyped using an ELISA system (Boehringer Mannheim). Ascites fluid was prepared by Joseph Beirao (Josman Laboratories, Napa Valley, CA). The mAb epitopes were mapped by Western blotting against three glutathione S -transferase (GST)–fusion protein constructs consisting of various portions of the cytoplasmic domain of VAMP-7: 1–23 amino acids (aa), 1–123 aa, or 1–182 aa. Rat anti–lysosome-associated membrane protein 1 (LAMP-1) mAbs were purchased from PharMingen. Mouse anti-TfR mAbs were purchased from Zymed Laboratories, Inc. Mouse anti-p115 antibodies have been described previously . Texas red–labeled anti–rabbit IgG, FITC-labeled anti–mouse IgG, and FITC-labeled anti–rat IgG antibodies were obtained from Jackson ImmunoResearch Laboratories. Rabbit antibody lym3 against the cytosolic tail of lgp120 was kindly provided by Dr. I. Mellman (Yale University, New Haven, CT). Rabbit antibody MSCI against the cytosolic part of the cation-dependent mannose 6-phosphate receptor (CD-MPR) was a gift from Dr. A. Hille (Georg-August University, Gottingen, Germany). In some single-labeled sections, a swine anti–rabbit IgG (SWAR) antibody was used (Nordic Immunological Laboratories) after the incubation step with VAMP-7 antibody to enhance the density of immunostaining. Western blotting was performed using ECL (Amersham Pharmacia Biotech). Indirect immunofluorescence localization was performed on NIH-3T3 cells as described previously . Antisera were used at the following dilutions: anti–VAMP-7 polyclonal antibody at 2 μg/ml; anti–LAMP-1 mAb at 0.05 μg/ml; anti-TfR mAb at 1 μg/ml; and Texas red–labeled anti–rabbit IgG, FITC-labeled anti–mouse IgG, and FITC-labeled anti–rat IgG antibodies at 7.5 μg/ml. PC12 cells were prepared for ultrathin cryosections, and (double)-immunogold labeling according to the protein A–gold method was carried out as described previously . For a quantitative analysis of the subcellular distribution of VAMP-7, well-preserved areas of two different grids single-immunogold labeled for VAMP-7 were selected, and at a magnification of 25,000 in the electron microscope, all gold particles were counted and subscribed to the compartment over which they were located. The definition of the distinct compartments was based on their morphology. The Golgi complex was recognized by its characteristic appearance of stacked cisternae, and the array of tubulovesicular membranes that were located at the trans -side of the Golgi was considered the TGN. EEs were defined as elongated, sometimes curved, electron-lucent vacuoles with few internal vesicles. LEs were defined as globular shaped compartments with numerous internal vesicles. Connected to or in close association with these endosomal vacuoles, many tubulovesicular membrane profiles were present that we indicated as endosome-associated vesicles. Electron-dense compartments without or with degraded internal membranes were categorized as lysosomes. These definitions are based on the description of the endocytic compartments in a variety of cell types . In PC12 cells, the compartments that by these definitions were defined as LEs and lysosomes stained positive for the LE/lysosomal marker protein lgp120 , whereas TfR localized to the compartments defined as EEs (data not shown). Finally, when the endocytic tracer BSA-gold was added to PC12 cells, it appeared in EEs, LEs, and lysosomes after 1-, 10-, and 60-min uptakes, respectively (Klumperman, J., manuscript in preparation). Membrane extraction studies of VAMP-7 were carried out on NIH-3T3 cells as described previously . To measure EGF and Tf trafficking, we adopted with slight modifications the streptolysin-O (SLO)-permeabilized cell system . In brief, HeLa cells were loaded with either 125 I-EGF or 125 I-Tf at 18°C for 1 h and then extensively washed to remove unbound EGF/Tf. Cells were then permeabilized with SLO as described previously . Where indicated, cells were incubated at 37°C for varying periods of time. To remove cytosol, cells were incubated for 1 h on ice with KTM buffer (115 mM potassium acetate, 25 mM Hepes, pH 7.4, 2.5 mM magnesium acetate, and 1 mg/ml BSA) and then washed extensively with ice-cold KTM buffer. Under these conditions, ∼95% of the cytosol can be removed from the permeabilized cells . Permeabilized cells were then resuspended in 0.5 ml of KTM buffer with or without 3 mg/ml rat brain cytosol. In all cases, KTM buffer was supplemented with 0.5 mM ATP and an ATP regeneration system (80 mM creatine phosphate, 9 U/ml creatine kinase). EGF/Tf trafficking was induced by incubating samples at 37°C for specified amounts of time. Where indicated, permeabilized HeLa cells were preincubated for 1 h with 100 μg/ml IgG, affinity-purified rabbit polyclonal anti–VAMP-7 antibodies, mouse monoclonal 22G2 and 1D9 anti–VAMP-7 antibodies, or anti–syntaxin 6 antibodies. Where indicated, affinity-purified rabbit polyclonal anti–VAMP-7 antibody was preincubated with 150 μg/ml GST–VAMP-7 or GST. After incubation at 37°C, KTM buffer was removed and cells were solubilized with 0.5 ml KTM buffer containing 2% Triton X-100 to determine the amount of EGF/Tf remaining in the cell. The removed buffer represents released EGF/Tf. To determine the amount of degraded EGF, the removed buffer was extracted with 10% TCA and the precipitate containing recycled intact EGF was sedimented by centrifugation at 10,000 g for 15 min. Remaining soluble 125 I represents degraded EGF . Similar TCA extraction of the buffer containing released 125 I-Tf resulted in the complete sedimentation of all 125 I (data not shown), in agreement with the data that Tf does not get targeted to the lysosomes for degradation. The amounts of 125 I-EGF degraded and 125 I-Tf recycled were determined by scintillation counting and expressed as the percentage of total EGF/Tf (the sum of released and intracellular Tf/EGF for each sample). The rat brain cytosol for EGF degradation and Tf recycling assays was prepared as described previously . In brief, fresh rat brains were homogenized in 25 mM Hepes, pH 7.4, containing 115 mM potassium acetate, 2.5 mM magnesium acetate, 0.1 mM EGTA, 2 mM DTT, 4 μg/ml aprotinin, and 0.8 μg/ml pepstatin. This homogenate was then centrifuged at 10,000 g for 20 min, followed by 100,000 g for 45 min. Cytosol was then flash-frozen in liquid nitrogen and stored at −80°C. The protein concentration was determined by the Bradford assay according to the manufacturer's instructions (BioRad). Where indicated, cytosol was pretreated with 0.2 mM NEM for 30 min on ice. To further investigate the function of VAMP-7, we generated one rabbit polyclonal and five mouse mAbs using the full-length VAMP-7 protein lacking its COOH-terminal hydrophobic region as the immunogen. All six antibodies recognized a single band of the expected molecular mass, 25 kD, on Western blots of rat kidney postnuclear supernatant (PNS) . To define more precisely the region of VAMP-7 recognized by the mAbs, each antibody was tested for binding to three different GST–fusion protein constructs of VAMP-7 . mAbs 8B7 and 23B8 bind to aa 23–123. 1D9, 22G2, and 24C3 bind in the region encompassed by aa 123–182, the approximate region of the helical domain important in forming the core fusion complex. The rabbit polyclonal antibody used in these studies recognizes epitopes in both these domains. A previously reported Northern blot analysis indicates that VAMP-7 mRNA is present in brain, kidney, spleen, thymus, and liver while no RNA was detected in heart . To analyze the protein expression pattern of VAMP-7, affinity-purified polyclonal antibodies were used to detect VAMP-7 protein in several tissues. In agreement with the Northern blot results, VAMP-7 protein was expressed in all tissues tested except heart and muscle . In liver, in addition to the 25-kD band, an additional immunoreactive species of ∼27 kD was detected. This band could represent either a posttranslational modification, an alternatively spliced isoform, or a cross-reactive protein distinct from VAMP-7 that is present only in liver. We used several cell lines for further investigation of VAMP-7 function and localization, including PC12, NIH-3T3, and HeLa cells. The antibodies detect a single band of 25 kD in these cell lines as exemplified by the pattern seen in PC12 cells . Based on the broad mRNA and protein distribution of VAMP-7, it appears that VAMP-7 is involved in a trafficking pathway that is common to most cell types. VAMP-7 contains a hydrophobic COOH-terminal sequence thought to be a membrane anchor . To determine if VAMP-7 behaves as a membrane protein, NIH-3T3 cells were homogenized and the PNS was fractionated into cytosolic and membrane fractions. The membranes were then extracted under various conditions and fractionated into supernatant and pellet fractions. Even when extracted with 1.5 M NaCl or pH 11.0, VAMP-7 was recovered in the pellet fraction. Only when the membranes were solubilized with 2% Triton X-100 did the protein redistribute to the supernatant, consistent with VAMP-7 being an integral membrane protein . Understanding the localization of a SNARE within a cell is critical information for understanding the membrane trafficking step mediated by that protein. We reported previously that epitope-tagged, full-length VAMP-7 displayed a perinuclear punctate staining pattern, and showed significant overlap with the late endosomal/lysosomal protein lgp120 . To ascertain the localization of endogenous VAMP-7, we stained NIH-3T3 cells with anti–VAMP-7 mAb 1D9, anti–VAMP-7 mAb 24C3, and anti–VAMP-7 affinity-purified polyclonal antibodies. In double labeling experiments, each mAb exhibited a staining pattern identical to that of the polyclonal antibody, and addition of VAMP-7 cytoplasmic domain blocked the immunostaining. These control experiments demonstrate that the localization is due to specific recognition of VAMP-7 (data not shown). To begin a more detailed study of the intracellular localization of endogenous VAMP-7, we compared its immunostaining pattern with those of well-characterized markers of Golgi, endosomal, and lysosomal compartments. VAMP-7 showed little if any colocalization with TfR, a marker of EEs, and p115, a marker of the Golgi . However, endogenous VAMP-7, like the transfected protein, is localized in a pattern that overlapped considerably with LAMP-1, a marker of LEs and lysosomes . In NIH-3T3 cells, EEs, LEs, and lysosomes are all clustered around the Golgi stacks making it difficult, at the level of light microscopy, to resolve these organelles in this region of the cell. However, it is possible to differentiate these membrane compartments in the periphery of the cells where the organelles are more dispersed. In the cell periphery, the precise colocalization of VAMP-7 and LAMP-1 in many puncta becomes most evident. The intensity of VAMP-7 and LAMP-1 staining for the individual puncta varies: some show more intense VAMP-7 immunoreactivity, whereas others show more intense LAMP-1 immunoreactivity. The effect of membrane trafficking perturbants such as nocodozole and brefeldin A (BFA) on vesicle trafficking proteins can unveil features of their native localization and cycling patterns. The microtubule-depolymerizing agent, nocodozole, has been shown to cause vesiculation and dispersal of both Golgi cisternae and early endosomal compartments . Treatment of NIH-3T3 cells with 10 μM nocodozole did cause TfR-positive EEs to scatter throughout the cytoplasm . However, both LAMP-1–positive and VAMP-7–positive organelles were relatively unaffected by nocodozole treatment . BFA treatment results in a block in anterograde ER-to-Golgi traffic, and due to the fact that the drug does not disrupt retrograde trafficking, Golgi proteins redistribute to the ER . Additionally, in certain cells, BFA treatment results in the collapse of the TGN and certain classes of endosomes onto the microtubule-organizing center . Again, BFA seems to have little effect on the distribution of organelles containing either VAMP-7 or LAMP-1 . In contrast, TfR staining collapses into the microtubule-organizing center . These results are consistent with the localization of VAMP-7 on LEs and/or lysosomes. To gain a more precise understanding of the subcellular distribution of VAMP-7, ultrathin cryosections were prepared from PC12 cells and immunogold labeled with affinity-purified polyclonal anti–VAMP-7 antibody. The amount of labeling on various membranes was quantified by defining the distribution of 372 gold particles. A discussion of the criteria used for morphological definition of the membrane compartments is provided in Materials and Methods. Approximately 8% of the labeling was found to be within the Golgi stacks, whereas 30% was found within the TGN region . The plasma membrane was virtually devoid of label. Previously, we localized two other SNARE proteins, syntaxin 6 and VAMP-4, in the TGN region of PC12 cells, and found that these SNAREs were incorporated into clathrin-coated vesicles and immature secretory granules . Unlike these SNAREs, VAMP-7 did not significantly label clathrin-coated vesicles or immature secretory granules in the TGN. Within the endosomal compartments, 5% of the VAMP-7–representing gold particles were found on EEs, 22% on LEs, and 30% on LE-associated vesicles. 5% of the gold particles were found on lysosomes . On LEs, VAMP-7 colocalized with lgp120 . 200 endocytic compartments were categorized into EEs, LEs, or lysosomes to quantify the number of these organelles in PC12 cells. According to the criteria we used to define these organelles (see Materials and Methods), 19% of the structures were EEs, 25% LEs, and 57% lysosomes. Thus, we conclude that VAMP-7 is enriched in the LE compartment. We further studied the sublocalization of VAMP-7 within the endosomes. Within EEs (89 particles), VAMP-7 was found at the limiting membrane of the vacuole (39%) and on associated vesicles (60%), whereas there was virtually no labeling of internal vesicles (1%). Within the LE compartment (195 particles), 44% of the gold particles were found on the vacuole membrane, 41% on associated vesicles, and 15% on internal vesicles. Vesicles that label for VAMP-7 do not generally contain the MPR, suggesting VAMP-7 functions in a trafficking pathway distinct from TGN-to-endosome transport . In addition, the vesicles that contain VAMP-7 were generally large and less electron-dense than the typical MPR-carrying vesicles . The light and EM immunohistochemical studies suggest that VAMP-7 plays a role in transport within the endosomal pathway, perhaps from EEs to LEs or from LEs to the lysosome. Although we are not able to precisely define the compartment labeled within the TGN, the morphological data are also consistent with a role in TGN trafficking. To investigate the role of VAMP-7 in Tf recycling and EGF breakdown, we use a modified SLO-permeabilized HeLa cell system . HeLa cells are incubated for 1 h at 18°C with either 125 I-EGF or 125 I-Tf and then washed to remove the unbound ligand. In intact cells, recycling of TfR to the plasma membrane results in the release of Tf, a process that occurs over a period of ∼80 min . Similarly, ∼10% of the EGF is rapidly released from cells and recovered as intact protein. The level of released, intact EGF plateaus at ∼20 min. Degraded EGF, defined as EGF that is not precipitated by TCA, begins to increase after ∼40 min and continues out to 120 min when we ended the measurements. We further studied the release of intact and degraded EGF in SLO-permeabilized cells . Release of the two forms of EGF occurs similarly in SLO-permeabilized cells as in intact cells, increasing to 45% of the total EGF released in degraded form after 180 min . The remainder of the EGF is still present within the cell, suggesting that longer chase periods would be required for its breakdown. The cellular processes required for recycling of Tf and breakdown of EGF are retained when cells are permeabilized with SLO. We assayed effects of various antibodies on the trafficking of the two ligands 2 h after incubation at 37°C. In the absence of cytosol or in the presence of NEM, EGF degradation is largely blocked . Furthermore, addition of IgG or anti–syntaxin 6 mAb to the permeabilized cells has no effect on EGF breakdown. However, when anti–VAMP-7 mAbs (1D9 or 24C3) are added to the permeabilized cells, a significant reduction in EGF breakdown is observed. While the reduction is only ∼25%, it is statistically significant, reproducible, and not observed with control antibodies . Addition of anti–VAMP-7 polyclonal antibodies to EGF-loaded, permeabilized cells resulted in a similar inhibition of EGF breakdown. To further control for the specificity of the antibody inhibition, we demonstrated that addition of VAMP-7 protein, but not GST, to the antibody reversed the inhibition of EGF degradation . The inhibition of EGF breakdown is dependent on the concentration of anti–VAMP-7 antibody used in the assay, further confirming the specificity of the inhibition . Since lysosomal degradation of EGF is likely to depend on several trafficking steps, we wondered if it would be possible to chase the EGF past the point at which the anti–VAMP-7 antibodies block. After EGF loading at 18°C, we incubated the cells for 0, 10, 20, or 40 min before permeabilization. Increasing the incubation time resulted in a progressive decrease in the level of EGF breakdown . These data suggest that we are able to chase the EGF into a compartment, past the EE, which no longer depends on VAMP-7 for further trafficking to the lysosome. Finally, we further investigated the effect of anti–VAMP-7 antibodies on the recycling of Tf; however, we did not observe any effects on this process . We interpret these data as demonstrating that antibody binding to VAMP-7 inhibits interactions with other proteins. Since inhibition of these protein interactions blocks EGF breakdown, we suggest that VAMP-7 is necessary for a vesicular trafficking step needed for EGF breakdown. The formation of helical bundles of SNARE proteins from opposite membranes likely drives intracellular membrane fusion. The discovery of syntaxin 1A, a SNARE largely localized to the plasma membrane, and Sed5p (syntaxin 5 in mammalian cells) , a syntaxin isoform largely localized to the cis -Golgi complex and ER-Golgi intermediate compartment, led to the idea that intracellular membrane trafficking and membrane fusion will be mediated by sets of SNARE proteins. Furthermore, if SNARE proteins pair specifically, part of the specificity of membrane trafficking may be mediated by the formation of specific sets of core fusion complexes. These ideas have become known as the SNARE hypothesis . For this hypothesis to be correct, several criteria must be met. First, there need to be enough SNARE proteins so that specific SNAREs, or at least a specific combination of SNAREs, could direct each vesicular trafficking step. In mammalian cells, there are in fact many different SNARE proteins, and they do have specific patterns of localization within cells. For example, syntaxin 5, rsec22b, rbet1, and membrin are important in antero- and/or retrograde trafficking between the ER and the cis -Golgi apparatus . In contrast, syntaxin 1A, VAMP-1, SNAP-25, and their closely related homologues are required for exocytosis . In this report we define a function for VAMP-7. The protein is localized to LEs, where our functional data suggest that it mediates trafficking to the lysosomes. The histochemical data are supported by both polyclonal and multiple mAbs, which strengthens the contention that the immunolocalization is specific. However, the EGF breakdown monitored in our assay is only partially inhibited by antibodies against VAMP-7. Complete inhibition of a transport step has never been observed in these types of assays. The partial inhibition may be explained in several ways. The antibodies may have access to only some of the active VAMP-7, or the antibody affinity may not be sufficient to block all SNARE interactions effectively. The antibodies used for the transport inhibition studies recognize the coil domain of VAMP-7 involved in SNARE complex formation. Although this is a domain of critical functional importance, and therefore an excellent site for function-blocking antibodies, the high stability of SNARE complexes may be such that the antibody can be displaced from VAMP-7 during complex formation. Alternatively, only a portion of the EGF may travel to a degradative compartment via a VAMP-7–mediated process. Since VAMP-7 is not present on vesicles that contain the MPR, we conclude that this SNARE is not likely to be involved in trafficking of enzymes to the lysosome. VAMP-7 that is present on the internal vesicles of LEs is likely to be degraded. This may partially explain why only low levels of VAMP-7 are found at steady state in lysosomes. In addition, we assign a significant amount of the VAMP-7 immunoreactivity to the TGN although we have not defined the subregion of the TGN that is immunoreactive for VAMP-7. The morphological definition of the TGN may include vesicles or tubules that are more appropriately considered part of the endosomal system. Since nocodozole and BFA do not affect the localization of VAMP-7, VAMP-7–positive organelles in the TGN region are more likely to be related to endosomal compartments. Consistent with the TGN localization, some VAMP-7 may be retrieved from LEs to participate in another round of membrane transport. Our data do not rule out the possibility that VAMP-7 also has a role in a distinct TGN transport process and is not used for a single trafficking step. For example, previous studies of VAMP-7 suggest a role in transport to the apical membranes in MDCK cells . In general, the morphological and functional data we present support the idea that different trafficking steps are mediated by distinct SNARE proteins, consistent with the SNARE hypothesis. A second criteria necessary for the SNARE hypothesis to be correct is that the SNAREs pair specifically. The full spectrum of SNAREs important in trafficking through the endosomal system to the lysosomes, including those that might pair with VAMP-7, is not yet fully defined. Recent experiments have shown that many combinations of SNAREs pair with VAMP-7 to form stable complexes in vitro . Syntaxin 1A/SNAP-25, syntaxin 4/SNAP-23, and syntaxin 13/SNAP-29 all form SDS-resistant complexes with VAMP-7; the midpoint temperatures ( T m ) of the unfolding transitions for these complexes are 92°C, 88°C, and 85°C, respectively . However, relatively small differences in the thermal stabilities of SNARE complexes can result in dramatic differences in the abilities of the complexes to mediate membrane fusion. Therefore, the simple formation of a complex cannot be taken as an indication of a functional complex, nor can one easily draw conclusions regarding specificity based on complex formation in vitro. Given these caveats, it is the case that syntaxin 1A and SNAP-25 form the most stable complex with VAMP-7 in studies so far. The 92°C unfolding transition for this complex is more stable than that for the unfolding transition of syntaxin 1A/SNAP-25 with their known SNARE partner, VAMP-2 ( T m , 90°C) . Therefore, it is unlikely that information for the specificity of membrane trafficking is contained within the core complex-forming helical domain of VAMP-7. If the core complex formation is not the specificity-determining event, then why have such a large number of SNAREs, particularly within the endosomal pathways? Many SNAREs, including VAMP-7, are considerably larger than the 70 aa required for fusion complex formation. In addition, these non–core complex–forming regions are more highly divergent in aa sequence between the SNAREs expressed within a species than those sequences involved in SNARE complex formation. For example, the cytoplasmic domain of VAMP-7 is 180 aa, and the function of the NH 2 -terminal 120 residues of the protein is not known. Perhaps non–core complex–forming regions of SNAREs are important for interactions with proteins that regulate aspects of vesicle targeting specificity. These interactions are likely to involve interactions with Rab proteins and their effectors, and perhaps members of the sec1p family of syntaxin-binding proteins. The mechanisms whereby SNAREs are localized to distinct compartments are an additional critical issue for understanding the organization of membrane compartments. The aa sequence motifs that direct SNARE localization are not well understood. It is also not clear for most SNAREs if particular coat or adaptor proteins are important in localizing the proteins to specific vesicles or matching the SNAREs to particular sets of cargo proteins. It is intriguing that VAMP-7 contains a potential adaptor protein binding motif, D/EXXXLL (aa 162–167), within the SNARE coil domain. Perhaps each of the SNAREs will contain specific sequences that direct binding to particular adaptor proteins. The small number of aa that are so far defined to be important for adaptor binding interactions makes it difficult to understand their significance simply by inspection of the aa sequences of the SNAREs. Further experiments are needed to clarify these issues. Only when we have a complete understanding of the localizations and protein interactions of all of the membrane fusion proteins of the VAMP, syntaxin, and SNAP-25 families will we be in a position to fully consider their roles in specificity. In addition, a full definition of the complexity of SNARE protein function and organization will lead to a more precise definition of complex membrane trafficking pathways within cells. | Study | biomedical | en | 0.999997 |
10459013 | The S . pombe strains used in this study are listed in Table . All strains are isogenic to 972 . Fission yeast media, growth conditions, and manipulations were carried out as described previously . Except where noted, cells were grown in YE medium. Unless otherwise indicated, all experiments involving temperature-sensitive strains were done at a permissive temperature of 25°C and a restrictive temperature of 36°C. Standard genetic and recombinant DNA methods were used except where noted. S . pombe transformations were carried out using either a lithium acetate method or electroporation . DNA was prepared from bacteria and isolated from agarose gels using Qiagen kits, and from yeast cells as described by Hoffman and Winston 1987 . DNA sequencing was performed at the University of Massachusetts Medical School's Nucleic Acid Facility. Oligonucleotide primers were obtained from Integrated DNA Technologies or from Operon Inc. A sid2 -null mutant strain was constructed by direct chromosomal integration into a diploid strain of a fragment generated by PCR using plasmid pFA6a-kanMX6 as template, as described . The two primers had 75-bp tails corresponding to the regions immediately 5′ to the sid2 start codon and immediately 3′ of the sid2 stop codon. The PCR fragment was gel purified and transformed into the diploid strain (YDM105 crossed with YDM108) using the lithium acetate method. Transformants were selected on YE G418 plates. PCR analysis of DNA prepared from individual transformants identified a strain bearing the sid2 deletion (YDM468). The sid2 deletion strain was sporulated and tetrads were dissected, which resulted in only two viable G418-sensitive progeny per tetrad. The sid2 deletion strain was transformed with a sid2– green fluorescent protein (GFP) construct (described below) by electroporation. A haploid-null strain (YDM470) bearing an episomal copy of sid2- GFP was obtained by plating a spore preparation from YDM468 expressing sid2- GFP, and selecting for G418-resistant and Ura+ haploid colonies. This strain was grown under conditions that did not select for the plasmid, allowing the plasmid to be lost and the null phenotype examined. Cells that had lost the sid2- GFP plasmid had no GFP signal (see below) and became long and multinucleate (data not shown). Costaining with antibodies against tubulin, as well as Cdc4p and Arp3p, which stain the medial ring and actin patches, respectively, also showed no readily apparent abnormalities in these structures (data not shown). Thus, the sid2 -null phenotype was indistinguishable from that of the temperature-sensitive allele of sid2, sid2-250 . A strain expressing a Sid2p-GFP COOH-terminal fusion was prepared by first amplifying the last 800 bp of the sid2 gene by PCR. The 5′ primer contained an Xba1 site followed by an in frame stop codon, and the 3′ primer contained an Nde1 and Kpn1 site. The PCR product was directionally cloned into the integrating vector pJK210 , using the Xba1 and Kpn1 sites from the primers. The GFP (S65T) DNA fragment was cloned in frame at the 3′ end of the sid2 gene as an NdeI fragment to yield plasmid pDM238. The final construct was linearized in the sid2 coding sequence using EcoR1 and transformed into yeast (YDM105). Note that the integration of this plasmid results in the full-length gene fused to the GFP tag, as well as an untagged COOH-terminal fragment of the sid2 gene which is not expressed due to the in frame stop codon at the 5′ end of this fragment. Integrants were selected on ura(−) plates, and correct integration was confirmed by PCR of integrant genomic DNA. The final strains (YDM415/YDM416) were then crossed to various mutant strains, producing the strains listed in Table (YDM420–YDM442, YDM541, YDM614). Sid2p was tagged at its COOH terminus with 13 tandem copies of the myc epitope (13Myc) by direct chromosomal integration into strain YDM105 of a fragment generated by PCR using plasmid pFA6a-13Myc-kanMX6 as template . The two PCR primers had 20 bp homologous to the DNA template followed by 75-bp tails corresponding to the regions 5′ and 3′ of the sid2 stop codon. The resulting strain (YDM497 or YDM514) was checked for correct integration by PCR. This strain was then crossed with various mutant strains, producing strains listed in Table (YDM500-515, YDM545). All sid2 epitope–tagged strains were indistinguishable from wild-type with respect to growth rates and cellular morphology. A number of different plasmids were constructed for expressing sid2 or tagged versions of sid2 in S . pombe cells. Plasmid pDM264 allows for expression of sid2 from its own promoter with a COOH-terminal GFP fusion in the vector pUR19 . Plasmid pDM264 was constructed by starting with the sid2 genomic clone, pDM99, in the vector pUR19, and then replacing the COOH-terminal fragment of sid2 between the Nhe1 site in the sid2 coding region, and the Kpn1 site in the 3′ flanking region, with the Nhe1 to Kpn1 fragment of plasmid pDM238 (see above), which contains the COOH terminus of sid2 fused with GFP. Several other plasmid vectors were constructed for expression of sid2 or a kinase-dead version of sid2 (see below), either alone or as NH 2 -terminal GFP or triple hemagglutinin (HA) epitope fusions from the thiamine-repressible promoter in vector pRep42 . The sid2 gene or a kinase-dead version, sid2-K238R (see below), were amplified by PCR using oligos that added Ase1 and EcoICR1 sites at the NH 2 and COOH termini, respectively. These fragments were then cloned into the Nde1/Sma1 cut vectors pRep42 , pRep42GFP, and pRep42HA . Constructs bearing the nmt1 promoter were regulated by the addition of thiamine to a final concentration of 2 μM. Site-directed mutagenesis was employed to create a point mutation in the sid2 gene at a site that would alter the proposed ATP-binding domain of the kinase (K238 to R). A sid2 genomic clone in the plasmid pUR19 was mutated by a PCR-based Quikchange mutagenesis system (Stratagene), resulting in the plasmid pDM343. The presence of the K238R mutation was confirmed by DNA sequencing. Indirect immunofluorescence staining was performed as described previously . The tubulin antibody TAT-1 was a generous gift from K. Gull (University of Manchester, Manchester, UK). Cdc4p and Arp3 antibodies from our lab stock were used at a 1:100 dilution. HA antibodies (BABCO) were diluted 1:200. Primary antibodies were detected with anti–rabbit or anti–mouse Texas red or FITC-IgG (Molecular Probes). DNA was visualized with DAPI (Sigma Chemical Co.) at 20 μg/ml. Images were captured using a Nikon Eclipse E600 microscope with a cooled CCD camera (Dage 300; MVI) and IPlab Spectrum software (Signal Analytics Corp.). Immunoelectron microscopy was done as described in Ding et al. 1997 with antibodies against GFP (a generous gift of P. Silver, Harvard Medical School, Boston, MA) or the Myc epitope (a generous gift of H. McDonald, Vanderbilt University, Nashville, TN). Protein lysates were prepared from 1.0–2.5 × 10 9 cells, which were collected by centrifugation and frozen on dry ice. All subsequent manipulations were carried out at 4°C or on ice. Cells in NP-40 buffer (1% NP-40, 150 mM NaCl, 2 mM EDTA, 6 mM NA 2 HPO 4 , 4 mM NaH 2 PO 4 , 0.15 μg/ml PMSF, 5 μg/ml each aprotinin, leupeptin, and pepstatin) were lysed by vortexing vigorously with glass beads (Sigma Chemical Co.). Protein lysates were prepared as described in Moreno et al. 1991 . Relative protein concentrations were determined using a Coomassie assay (BioRad). Immunoprecipitations were carried out by adding to the NP-40 cell lysates either 1 μl of anti-myc 9E mouse monoclonal IgG (M. Jacobs, Tufts University, Boston, MA) or 1 μl anti-HA mouse monoclonal IgG (H. McDonald, Vanderbilt University) as appropriate, followed by incubation for 1 h on ice. Immune complexes were purified by adding 25 μl of a 1:1 slurry of protein G–Sepharose beads (Amersham Pharmacia Biotech), incubating for 30 min on a rocker at 4°C, and pelleting bound material by centrifugation in a microfuge for 1 min. Beads were washed three times with 1 ml NP-40 buffer. For detection of Myc or GFP epitope–tagged Sid2p in total protein lysates, NP-40 cell lysates (described above) were separated by SDS-PAGE (7%), and transferred to Immobilon P nylon (Millipore Corp.) using a semidry blotting apparatus (Owl Scientific). Blots were probed with the anti-myc IgG or anti-GFP IgG (Clontech) at a 1:1,000 dilution, and developed using an alkaline phosphatase chemiluminescent system (BioRad). Immune complex bead preparations were washed once in 1 ml of kinase assay buffer (25 mM MOPS, pH 7.2, 60 mM β-glycerol phosphate, 15 mM MgCl 2 , 15 mM p -nitrophenylphosphate, 1 mM DTT, 0.1 mM sodium vanadate, 1% NP-40, 50 μM ATP (unlabeled), 5 μg/ml each aprotinin, leupeptin, and pepstatin). Washed immunoprecipitates were incubated at 30°C for 30 min in 20 μl kinase assay buffer with 10 μg myelin basic protein (MBP) or histone (Sigma Chemical Co.), and 2 μl of a 1:10 dilution of 10 mCi/ml (3,000 Ci/mmole) [ 32 P]ATP . Reactions were stopped with the addition of SDS-PAGE sample buffer and half of each sample was resolved on 15% polyacrylamide gels. Gels were dried and imaged on a PhosphorImager (Molecular Dynamics). The other half of each sample was probed for Sid2p-13Myc or HA-Sid2p by Western blotting, and used to normalize Sid2p kinase activity. For each experiment, a cell lysate from cells that do not contain Sid2p-13Myc or HA-Sid2p was used as a control, and the amount of MBP phosphorylation observed was used to determine the amount of background for each experiment. For the cell cycle profile of kinase activity, strain YDM500 ( cdc25-22 , sid2- 13Myc) was grown to log phase at 25°C, shifted to 36°C for 3 h, then shifted back to 25°C. Cells were collected every 15 min for 4–5 h. At each time point, a 50 ml culture of cells was pelleted and frozen in a dry ice/ethanol bath, a sample of the cells was fixed in −20°C methanol and stained with DAPI, and a sample of the cells was examined by phase–contrast microscopy for the appearance of a septum. Once all time points were collected, cell pellets were assayed for in vitro kinase activity as described above. To better understand how the Sid2p kinase controls initiation of medial ring constriction and septation, we sought to determine the localization of the protein in S . pombe cells. A strain was constructed that expressed a Sid2p-GFP fusion from the normal chromosomal locus (see Materials and Methods). In interphase cells, a single spot of GFP fluorescence was observed at the periphery of the nucleus, whereas two spots of GFP fluorescence were seen in mitotic cells . At the end of anaphase, Sid2p appeared as a band in the medial region as well as two nuclear-associated dots . This band of Sid2p colocalized with the medial ring during initiation of ring constriction . As the ring constricted and the primary septum formed, Sid2p localized along either side of the developing septum . Following septum formation, but before cell separation, Sid2p disappeared from the middle of the cell . Real-time examination of living cells stained with calcofluor showed that Sid2p localized to the medial region just before the appearance of septal material (Sparks, C., S. Wheatley, and Y.L. Wang, unpublished observations). The nuclear-associated spot-like localization of Sid2p suggested that Sid2p was a component of the SPB. To confirm SPB localization, we fixed and stained the Sid2p-GFP–expressing cells with anti-tubulin antibody. Sid2p was localized at the ends of the mitotic spindle in cells undergoing mitosis, consistent with its localization to the SPB . To unequivocally establish that Sid2p was a component of the SPB, as well as to assess if Sid2p localized to the nuclear or cytoplasmic faces of the SPB, Sid2p localization was examined using strains expressing Sid2p-GFP by immunoelectron microscopy. As shown in Fig. 1 D, the SPB appears as an electron-dense disk-shaped structure at the edge of the nucleus by electron microscopy. Using GFP-specific antibodies, we found that Sid2p localized to the outer cytoplasmic face of the SPB . Serial sections through a representative SPB body are shown. The distribution pattern of Sid2p at the SPB was the same in both interphase and mitotic cells (data not shown). Essentially identical results were obtained following electron microscopy when Sid2p-13Myc cells were stained with Myc-specific mAbs (data not shown). Thus, Sid2p is a bona fide component of the SPB and is associated with the cytoplasmic face of the SPB. Sid2p was not detected at the division site in septating cells using either Myc- or GFP-specific antibodies. This may be due to inaccessibility of Sid2p to antibodies when it is at the division site, since we were also unable to observe Sid2p staining at the division site by conventional immunofluorescence using either Myc- or GFP-specific antibodies (data not shown). To determine if localization of Sid2p to the middle of the cell required the medial ring, we examined its localization in two different types of medial ring mutants. In cdc3 mutants, medial rings are not formed and septal material is deposited in irregular patches on the cell cortex. We found that in cdc3 mutants, Sid2p never formed the medial band, but faint patches of Sid2-GFP were occasionally observed at the cell cortex . Another type of medial ring mutant examined was a cdc15 mutant. In these cells, an actin-containing medial ring will form in the middle of the cell but these rings lack the Cdc15p component . Interestingly, we could not detect any Sid2p at the medial cortex in cdc15-140 mutants . In both cases >200 cells were examined. These observations indicate that Sid2p requires the actin ring for its localization to the medial cortex, and that there may be an interaction between Cdc15p and Sid2p at the medial ring. Next, we asked whether microtubules were required for the redistribution of the Sid2p kinase from the SPB to the division site. We began by examining the distribution of Sid2p-GFP in the cold-sensitive tubulin mutant strain nda3-KM311 . After 4 h at the restrictive temperature of 19°C, 70% of cells had arrested in mitosis with condensed chromosomes, and no cells had septa . Also, no intact microtubules were detected in these cells by indirect immunofluorescence microscopy . We found that Sid2p remained at the SPB in these cells . Thus, the microtubule cytoskeleton is not required to maintain the localization of Sid2p to the SPB. At the block-point, nda3 -KM311 mutants undergo mitotic arrest with fully formed medial rings . Sid2p failed to accumulate at the medial rings in cold-arrested nda3 - KM311 cells, suggesting that an intact microtubule cytoskeleton, perhaps in the form of the postanaphase array, was required for accumulation of Sid2p at the division site. There are several possible explanations for why Sid2p failed to accumulate at the medial rings formed in the tubulin mutant cells. Sid2p could be transported from the SPB to the cleavage site down cytoplasmic microtubules, or alternatively, the medial ring of microtubules may be required to maintain Sid2p at the division site. Another distinct possibility is that these cells have arrested in metaphase, before the end of anaphase when activation of the pathway to signal the initiation of cytokinesis is presumably turned on. Of course, it is also possible that Sid2p localization to the cell middle requires both activation of the signaling pathway and the presence of intact microtubules. To test between these possibilities, we examined the Sid2p distribution in a strain carrying a deletion of the spindle checkpoint gene, mad2 , in addition to the nda3 tubulin mutation. These cells do not arrest in metaphase, but instead continue to cycle without any microtubules present, eventually undergoing cytokinesis and cleaving through unsegregated chromosomes . As expected, we found that unlike the nda3 mutant alone, these cells did not remain blocked in mitosis. Out of >200 cells counted, only 7% had condensed chromosomes, 30% of the population had septum staining, and like the nda3 mutant cells, these cells had no intact microtubules by immunofluorescence after 4 h at 19°C . Upon initial examination of these cells, it appeared that although 30% of the cells displayed septum staining, Sid2p was not localized to the medial region of these cells. However, careful scrutiny of these cells showed that a very faint Sid2p signal could be observed in the medial region of 5% of the septated cells. The faint Sid2p medial signal was always observed in cells that had not completed septum formation , although not all septating cells had an obvious Sid2p signal in the medial region . It is not surprising that Sid2p was not observed in the medial region in the majority of the cells that displayed septum staining, since most of these cells had completed septum formation, and in wild-type cells, Sid2p normally disappears from the medial region at that time . Quantitation of the fluorescence intensity of the medial staining in 20 wild-type and 20 of the nda3 mad2 cells in which a medial Sid2p-GFP signal could be visualized showed that the Sid2p-GFP signal was ∼10-fold less intense in the nda3 mad2 cells than that observed in wild-type cells. The simplest explanation for these results is that normal Sid2p localization to the medial ring requires both anaphase-induced activation of the signaling pathway and the presence of intact microtubules. In the nda3 mad2 mutant cells, enough activated Sid2p may be able to diffuse to the medial ring, causing septation to initiate, but Sid2p is unable to accumulate to the levels seen in wild-type cells in the absence of microtubules. It has been proposed that the Sid groups of proteins (Cdc7p, Cdc11p, Cdc14p, Sid1p, Sid2p, Spg1p, and Sid4p) function as a part of a novel signal transduction cascade . Thus, in the following sections we describe several lines of experimentation designed to test if Sid2p functions in a signaling cascade, and if so, where it functions with respect to the other Sid proteins. As one way to assess the order of function for these genes, we asked if any of the other sid genes were required for the distinctive distribution pattern of Sid2p. We found that Sid2p-GFP could not localize to both the SPB and the medial region in sid4 , cdc7 , and cdc11 mutants . In sid1 , spg1 , and cdc14 mutants, Sid2p-GFP localized to the SPB but not to the medial region . In all cases, the Sid2p localization pattern was examined in at least 200 cells. These results are consistent with the possibility that sid2 functions downstream of all of the other known genes in this signaling cascade. We also checked Sid2p-GFP protein levels in the mutant strains in which the Sid2p-GFP signal was lost from the SPB at the restrictive temperature, to test whether Sid2p was being degraded or simply displaced in these mutant cells. We found that the Sid2p protein levels were similar to wild-type cell levels and, therefore, the Sid2p appears to be blocked from localizing properly in the mutant backgrounds. Thus, the localization of Sid2p to the division site requires the function of all of the Sid proteins. In addition, Cdc7p, Cdc11p, and Sid4p are also required for its localization to the SPB. A previous study has shown that Cdc7p functions downstream of Spg1p . The Sid2p localization experiments described above suggest that Sid2p may function downstream of both Spg1p and Cdc7p. To test this hypothesis in another way, we asked whether Cdc7p depends on Sid2p for its proper distribution pattern. We examined the localization of an HA epitope–tagged Cdc7p (a gift from V. Simanis, ISREL, Epalinges, Switzerland) in a sid2-250 mutant. Cells grown at the permissive temperature, or incubated at the restrictive temperature for 3 h, were fixed and stained with HA antibodies. At permissive temperature, 28% (61/219) of cells examined displayed Cdc7p staining at the SPB. At the restrictive temperature, 53% (156/294) of cells showed localization of Cdc7p to one or more SPBs . The increased percentage of cells with Cdc7p SPB staining at the restrictive temperature reflects the fact that these cells had become multinucleate. Therefore, Cdc7p does not require the function of Sid2p to localize properly, suggesting that Cdc7p is upstream of Sid2p in this cascade. Cells that overexpress spg1 undergo multiple rounds of septation but never complete cell separation . If Sid2p functions downstream of Cdc7p, then it should be downstream of Spg1p as well. To test this, we asked if the sid2-250 phenotype was epistatic to the Spg1p overproduction phenotype. We examined the consequences of overexpressing the spg1 gene using the thiamine-repressible nmt1 promoter in the sid2-250 mutant. Overexpression of spg1 at the permissive temperature caused cells to form multiple septa as reported previously in wild-type cells . Of 100 cells counted, 98 had 2 or more septa. However, at restrictive temperature, out of 100 cells counted, none of the cells formed septa, and these cells instead became long and multinucleate like sid2-250 mutants , demonstrating that the sid2-250 phenotype is epistatic to the spg1 overexpression phenotype. Together, these experiments are consistent with Sid2p functioning downstream of Cdc7p and Spg1p in the pathway to initiate actin ring constriction and septation. Sequence analysis of the sid2 gene predicts that the protein functions as a protein kinase . To test if Sid2p had kinase activity in vitro, we used a strain which expressed a 13Myc epitope tag inserted in frame at the 3′ end of the chromosomal sid2 gene (see Materials and Methods). Sid2p immune complexes were prepared from these cells using anti-myc antibodies and assayed for kinase activity using MBP as an artificial substrate. Sid2p was found to efficiently phosphorylate MBP . Appreciable nonspecific kinase activity in cells not expressing Sid2p-13Myc was not detected . We tested the ability of the Sid2p to autophosphorylate by eliminating the substrate in the assay, however no autophosphorylation was detected. To ensure that the kinase activity that we were measuring was due to Sid2p and not an associated kinase, we created an episomally expressed HA-tagged kinase-dead version of the protein by introducing a point mutation in the proposed ATP binding site of the kinase that changed lysine 238 to arginine (K238R) (see below). Both HA-Sid2p and HA-Sid2-K238R were expressed in wild-type cells and their kinase activity was measured. This showed that although HA-Sid2p did display kinase activity, the kinase-dead version HA-Sid2-K238R did not . We also wanted to test the importance of Sid2p kinase activity for the function of the protein in vivo. The K238R mutation was introduced into a plasmid carrying a sid2 genomic clone, or because we suspected that overexpression of the sid2-K238R allele might be toxic, we expressed sid2-K238R in the vector pREP42 under the control of an attenuated version of the thiamine-repressible nmt1 promoter either with or without a GFP or HA tag (see Materials and Methods). The GFP tag allowed us to determine if Sid2p kinase activity was required for proper localization. The sid2-K238R gene expressed off its own promoter was presumably lethal to cells, since no colonies formed after transformation of this plasmid into wild-type cells. The pRep42 sid2-K238R constructs were not lethal when weakly expressed under thiamine repression in wild-type yeast cells. However, when induced, the cells grew very slowly and appeared to be quite sick (data not shown). We then transformed each of these plasmids into the sid2-250 strain to ask if the kinase-dead version of sid2 could rescue the sid2 mutant phenotype at 36°C. Each strain was grown on selectable plates at 25°C, then shifted to 36°C in the presence of thiamine . Only the wild-type version of the sid2 gene could rescue the sid2-250 strain at 36°C. This showed that under these conditions, sufficient GFP-Sid2p (or Sid2p, not shown) was expressed to rescue the sid2-250 mutant. However, the Sid2p-K238R protein was not capable of rescue under the same circumstances. Removing thiamine and thus allowing for the overexpression of the kinase-dead Sid2p results in a lethal phenotype at the permissive temperature (data not shown). Overexpression of wild-type Sid2p had no effect on cell growth or morphology in both wild-type and sid2-250 mutant cells. Both the wild-type and kinase-dead GFP-Sid2p were expressed in wild-type cells and localized properly to the SPBs and the cleavage furrow (data not shown), indicating that Sid2p kinase activity is not required for proper localization of the protein. Next, we were interested in determining if Sid2p kinase activity was cell cycle regulated. To test this, we prepared a strain that expressed Sid2p-13Myc in a cdc25-22 background. When shifted to 36°C, cdc25-22 mutant cells arrest at the G2/M transition. Upon release to the permissive temperature, these cells proceed synchronously through the cell cycle. Thus, the cdc25-22 mutation was used to generate populations of synchronous cells for analysis of Sid2p kinase activity throughout the cell cycle. Cells were collected every 15 min after release from a 36°C block, and Sid2p kinase activity was measured at each time point . The percentages of cells undergoing anaphase and septation were also determined at each time point . Interestingly, the Sid2p kinase activity peaked during actin ring constriction and septation at the end of anaphase . Low levels of kinase activity were also observed earlier in mitosis. The significance of this earlier activity is not clear. However, the majority of the Sid2p kinase activity peaks when Sid2p is accumulating at the medial ring during ring constriction and septation. Having demonstrated that the Sid2p localization was dependent on the activity of the other gene products that function in septation and medial ring constriction , we now wanted to see if these same gene products were also required for Sid2p kinase activity. Sid2p kinase activity was measured using strains expressing Sid2p-13Myc in each of the septation initiation mutant backgrounds, which had been grown at permissive or restrictive temperatures. In all of the mutant backgrounds, Sid2p kinase activity was greatly diminished at restrictive temperature . The reduced kinase activity of Sid2p in these mutant strains does not appear to be due to a nonspecific cell cycle block, since nuclear division cycles continue in these mutants and they display a mitotic index similar to that of wild-type cells . Thus, the Sid2p kinase appears to require the function of the other septation initiation gene products to be active. This would support our other results which place Sid2p at the end of the signaling cascade that regulates medial ring constriction and septation. It could also be proposed that the sid group of genes is only required for Sid2p to localize to the medial ring, and then the protein becomes activated at the ring. To test this, we also examined Sid2p kinase activity in the medial ring mutant cdc15 , which is also required for medial Sid2p localization. Sid2p activity was not lost at the restrictive temperature in the cdc15 mutant, suggesting that Sid2p does not have to localize to the medial ring to become activated. To maintain genomic stability and proper ploidy, it is crucial that cell division occurs at the end of anaphase after chromosome segregation. Genetic analysis in S . pombe has identified a group of interacting genes, which includes cdc7 , cdc11 , cdc14 , sid1 , sid2 , sid4 , and spg1 , required to initiate constriction of the medial ring and septation at the end of anaphase. It was shown that the Spg1p GTPase and the Cdc7p kinase both reside at the SPBs , perhaps to be able to sense that anaphase was complete and then to pass on a signal to initiate cell division. Here we show that Sid2p may function downstream of Spg1p and Cdc7p to pass the signal on to the medial cortex that it is time to divide. It had been shown previously that Spg1p and Cdc7p localize to the SPBs but not the cell division site , raising the question of how the signal to divide becomes transmitted from the poles to the division site. Here we show that Sid2p localizes to both the SPBs and to the cell division site at the time of cell division, suggesting that Sid2p may transmit the signal to divide from the poles to the division site. We also identified a number of proteins required for proper localization of Sid2p to both the SPB and the division site. It appears that Sid2p is an actual component of the SPB since it does not require microtubules for localization to the SPB. However, localization of Sid2p to the SPB does require Cdc7p, Cdc11p, and Sid4p. One explanation of these results could be that there is a protein complex between these proteins at the SPB, and if it is disrupted, other components do not localize properly. However, initial coimmunoprecipitation experiments, using mild conditions (1% NP-40 lysis buffer), looking at endogenous protein–protein interactions failed to detect any complex formation between Sid2p and either Cdc7p or Sid4p (Sparks, C., and D. McCollum, unpublished observations), although it is possible that the methods used to prepare the cell lysates did not solubilize or preserve protein complexes at the SPB. A complex between Sid2p and Cdc7p also seems unlikely, since Cdc7p only resides at the SPB during mitosis, whereas Sid2p is always at the SPB. Furthermore, in an spg1-B8 mutant, Sid2p does localize to the SPBs even though Cdc7p does not , suggesting that Cdc7p may function in the cytoplasm to promote Sid2p localization to the SPB. These results are somewhat surprising and we do not have a simple explanation for them at this time. Further experiments showed that Sid2p requires microtubules for it to efficiently accumulate at the division site. The fact that the nda3 mad2 cells are able to septate despite poorly localizing Sid2p to the division site poses somewhat of a paradox. Although Sid2p accumulates inefficiently at the division site in the absence of microtubules, the fact that some Sid2p can still localize to the division site suggests that binding interactions between freely diffusing Sid2p and medial ring components are sufficient to localize enough Sid2p to the medial ring to trigger septation. Since Sid2p presumably functions in a catalytic manner, it may not be necessary have large amounts of Sid2p at the division site to initiate septation. Similarly, it has been observed that in budding yeast, myosin rings can form in the absence of actin; however, ring formation is much less efficient, suggesting that transport of myosin, facilitated by F-actin, is important for accumulation of myosin at the medial ring, but in the absence of F-actin, myosin can accumulate by diffusion mechanisms . Although cells can clearly septate without the efficient microtubule-mediated accumulation of Sid2p at the division site, this mechanism may be important in normal cells to ensure that cytokinesis initiates precisely at the end of anaphase. It is worth noting that precisely at the time when Sid2p begins to appear at the division site, the spindle is breaking down, the postanaphase array of microtubules is forming, and a medial band of microtubules is present . At this time, microtubules can be observed running from the cell division site to the SPBs. It has been proposed that these microtubules function to position the nucleus in the middle of the new cell away from the division site to ensure that the nucleus does not get cut by the septum . Given the timing of the appearance of these microtubules, it is tempting to speculate that Sid2p moves along these microtubules from the SPB to the division site. This would serve as an efficient mechanism to couple proper positioning of the nucleus with initiation of cell division. The localization of Sid2p to the outer cytoplasmic face of the SPB places it in a position from which it could move down astral microtubules emanating from the pole. However, it is also possible that the medial band of microtubules is important for Sid2p localization, and at this point we cannot say how microtubules function to help localize Sid2p to the division site. The fact that one of the putative upstream activators of Sid2p, Cdc7p, only localizes to one of the SPBs instead of two, like Sid2p, suggests that if Sid2p moves along microtubules from the SPB to the division site, it may only come from one of the two poles. However, we have not been able to detect any obvious diminution of Sid2p signal from one of the poles at this time. It is possible that this is due to insufficient sensitivity of our instruments or Sid2p could be rapidly replenished at the pole from cytoplasmic pools of the protein. Although it appears from these studies that Sid2p requires microtubules and the presence of a medial ring for it to localize to the division site, it will be important in future studies to address the target(s) and/or binding partners of Sid2p at the cleavage site. Although Sid2p initially appears to localize to the medial ring, once the septum has begun to form Sid2p also localizes on either side of the developing septum. Thus, Sid2p may have targets involved in both medial ring constriction and in forming the septum. One candidate binding partner for Sid2p is Cdc15p. cdc15 mutants are capable of forming medial rings that contain actin and Cdc4p , but not Cdc15p . However, Sid2p cannot localize to the medial rings that form in a cdc15 mutant. Thus, Sid2p requires Cdc15p or a Cdc15p-associated protein as a docking protein at the medial ring. The inability of cdc15 mutants, unlike mutants in other ring components, to form any deposits of septum material may be due to its inability to recruit Sid2p to the ring. The sequence of sid2 predicted that the protein may function as a kinase , and in this study we demonstrate that Sid2p does in fact possess in vitro kinase activity. Sid2p kinase activity is essential for the function of the protein, since a kinase-dead version of Sid2p does not display in vitro kinase activity and will not rescue the sid2-250 mutant. In fact, stronger expression of the Sid2p kinase-dead mutant has a dominant negative phenotype similar to the sid2 loss of function phenotype in either wild-type or sid2-250 cells, indicating that Sid2p-K238R may be titrating out essential regulatory factors or substrates. Sid2p kinase activity peaks precisely when the medial ring is constricting and septum is deposited. There also appears to be some Sid2p kinase activity early in mitosis, before the anaphase peak. The presence of this activity could suggest a function for Sid2p earlier in mitosis, although the phenotype of the sid2 mutant gives no indication of this. Since total Sid2p protein levels were examined and found not to change appreciably throughout the cell cycle (data not shown), the peak in Sid2p kinase activity is presumably regulated posttranslationally. It will be important to determine in future studies if Sid2p kinase activity is regulated by phosphorylation and/or binding to regulatory subunits. Interestingly, the kinase appears to be most active during the time when it transiently accumulates at the cleavage site, suggesting that it may function to activate, by phosphorylation, the machinery at the cleavage site involved in initiating medial ring constriction and septum deposition. It will be important to determine if a single regulatory event triggers both activation of the kinase and its relocalization. It is clear that Sid2p's own kinase activity does not play a role in directing it to the cleavage site, since kinase-dead versions of Sid2p were still capable of localizing to the cleavage site. Sid2p kinase activity is diminished at the restrictive temperature in all of the mutants required for medial ring constriction and septation, suggesting that Sid2p may function at the end of the signaling cascade. A variety of other data are consistent with this hypothesis. Sid2p-GFP does not localize to both the SPB and the medial ring in sid4 , cdc7 , and cdc11 mutants, and it localizes to the SPB but not the medial ring in sid1 , spg1 , and cdc14 mutants. Thus, Sid2p does not localize properly in mutants in any of the other components of this pathway. Interestingly, although Sid2p does not localize to the medial region in the medial ring mutant cdc15 , its kinase activity is unaffected, showing that localization of Sid2p to the medial ring is not necessary for the kinase to become activated. Also, Cdc7p localization is unaffected in a sid2-250 mutant, and the sid2-250 phenotype is epistatic to the Spg1p overproduction phenotype, which further supports Sid2p functioning downstream of spg1 . Thus, loss of any of the putative upstream signaling molecules shuts off the cascade, leaving Sid2p in an inactive state and unable to distribute properly and presumably phosphorylate its target substrates. Many recent studies have begun to identify the SPB as the site of localization of many molecules that regulate cytokinesis. In S . pombe , Plo1p , Cdc7p, Spg1p , and Sid2p (this study) all localize to the SPB and are required for initiation of medial ring constriction and septation. The reason for their localization to this site is unclear. It is possible that by virtue of their localization at the SPBs they are in a position to receive a signal that anaphase is completed and cell division can be initiated. Two proteins that are required for formation of the medial ring, the IQGAP protein Rng2p and Plo1p kinase , have been shown to localize to both the spindle poles and the medial ring. This theme may not be unique to fission yeast. In animal cells, a number of proteins localize to the centrosome early in mitosis and then move to the central spindle and cleavage furrow. At least two of these, the Polo and AIM-1 kinases, have been demonstrated to be required for cytokinesis . In Saccharomyces cerevisiae , there exists a set of genes similar to the sid group of genes in S . pombe . The S . cerevisiae genes cdc5 , cdc15 , tem1 , and dbf2 are homologous to the S . pombe genes plo1 , cdc7 , spg1 , and sid2 , respectively. Mutations in the S . cerevisiae genes cause a block at the end of anaphase with an intact spindle and a failure in cytokinesis. Interestingly, like Sid2p, Dbf2p protein kinase activity peaks at the end of anaphase . Recent work has suggested that the primary function of these genes may, like in S . pombe , be in cytokinesis . Like its counterpart, Plo1p, Cdc5p localizes to the SPB . The other S . cerevisiae genes in this pathway have not been localized. Recently, a protein called Mob1 was identified which interacted with the Sid2p homologue, Dbf2 . Using database searches of genome sequencing projects, we noticed that homologues of Mob1 exist in both S . pombe and humans, suggesting that this signaling pathway may be conserved in higher eukaryotes as well. In this study we have identified the Sid2p kinase as a component of a novel signaling cascade residing at the SPB that may transmit a signal that anaphase is complete from the SPB to the cell division site, thereby causing the cell to divide. From our studies it appears that Sid2p may function downstream of the other known components of the signaling pathway. It will be important in future studies to determine how and where the other known components of this pathway function in the cascade. Although the medial ring forms early in mitosis, it does not initiate constriction until the end of anaphase after chromosome segregation. The events that take place at the ring at the end of anaphase to cause the ring to begin to constrict and the septum to form are not known. Sid2p is the first protein required for initiation of ring constriction and septation to be localized to the medial ring. The identification of Sid2p should facilitate the use of biochemical and genetic approaches to identify targets of Sid2p involved in medial ring constriction and cell division. | Study | biomedical | en | 0.999996 |
10459014 | Human Cdc20 and Mad2 cDNA were prepared by reverse transcriptase–PCR. Two human expressed sequence tags homologous to Cdh1/Hct1 were used as probes for the screening of full-length human Cdh1 cDNA in a λgt10 human erythroleukemia K562 cDNA library. The T7- and His 6 -tagged Cdc20, Cdh1, and Mad2 in pET-23d were expressed in NovaBlue (DE3) in the presence of 0.5 mM isopropyl-β- d -thiogalactopyranoside at 25°C for 20 h. The cell lysates were solubilized (10 mM Tris, pH 8.0, 0.1 M NaH 2 PO 4 , and 6 M guanidine isothiocyanate), bound to His-Bind resins, washed (10 mM Tris, pH 8.0, 0.1 M NaH 2 PO 4 , and 8 M urea) five times, eluted (10 mM Tris, pH 4.5, 0.1 M NaH 2 PO 4 , and 8 M urea), and dialyzed. Rabbit antisera against tagged Cdc20 and Cdh1 were prepared and each specific antibody was purified by antigen affinity chromatography. The purified Cdc20, Cdh1, or Mad2 (1 μg) was incubated with 10 μCi of γ-[ 32 P]ATP (3,000 Ci/mmol) and human Cdc2-GST-Cyclin B prepared by baculovirus (0.1 μg) or Plk (0.1 μg) in 50 μl of a kinase buffer (MPF: 10 mM Tris, pH 7.4, 10 mM MgCl 2 , 0.1 mM EGTA, and 0.05% β-mercaptoethanol; Plk: 20 mM Tris, pH 7.4, 10 mM MgCl 2 , 25 mM NaCl, 0.2 mg/ml BSA, and 0.05% β-mercaptoethanol), at 37°C for 30 min. The labeled proteins were immunoprecipitated with anti-T7 mAb (Novagen), washed, and resolved in 7% SDS-PAGE. The immunoprecipitates of anti–Cyclin B1-specific antibody in nocodazole-treated K562 cell extracts were incubated with 10 μCi of γ-[ 32 P]ATP in 20 μl of MPF kinase buffer at 30°C for 1 h. 1 μl of 20% SDS was added, mixed, and the sample was centrifuged. The sample was diluted with 500 μl of the kinase buffer and the labeled Cdc20 and Cdh1 were immunoprecipitated with anti–Cdc20- or anti–Cdh1-specific antibody, respectively. The samples were resolved in 7% SDS-PAGE. Mouse NIH3T3 cells were maintained in DME supplemented with 10% FCS. Cells were synchronized at the G1/S boundary by double blocking with 1 μg/ml aphidicolin and harvested 2 h after drug release. APC in the S phase was prepared by Resource Q chromatography and immunoprecipitation with anti-Cdc27 antibody (a gift of Drs. P. Hieter and A. Page, University of British Columbia, Vancouver, Canada) as described . Immunoprecipitates were washed three times with a buffer containing 500 mM KCl to remove MPF and Plk, and then washed with the buffer for kinase or ubiquitination reaction. Cdc20 and Cdh1 (1 μg) were phosphorylated by incubating with 0.1 μg of Cdc2-GST-Cyclin B and 0.1 mM ATP in 10 μl of MPF kinase buffer at 37°C for 30 min. The phosphorylated Cdc20 and Cdh1 were bound to His-Bind resin, washed with the kinase buffer containing 0.5 M KCl three times to remove MPF, and washed with the ubiquitination buffer. In some cases, the purified APC was phosphorylated by PLK phosphorylated by MPF (pPlk) and/or PKA in 50 μl of Plk kinase buffer containing 1 mM ATP, 0.1 μg of human Cdc2-GST-Cyclin B prepared by baculovirus and 0.1 μg of Plk, or in PKA buffer (10 mM Tris, pH 7.4, 10 mM MgCl 2 , and 0.1 mM EGTA) with 0.5 μg of bovine PKA catalytic subunit, and washed twice with 5 mM Tris, pH 7.6, and 0.5 mM MgCl 2 . The phosphorylated or untreated APC was incubated with 2 μg of GST-Cut2 (a gift of Dr. M. Yanagida, Kyoto University, Kyoto, Japan) or 2 μg of human Cdc2-GST-Cyclin B (a gift of Dr. N. Watanabe, RIKEN, Japan) in the presence of various amounts (0–1 μg) of phosphorylated or unphosphorylated forms of Cdc20 and Cdh1 in 30 μl of 5 mM Tris, pH 7.6, 0.5 mM MgCl 2 , 2 mM ATP, 2 mM DTT, 2 mM creatine phosphate, 1 μg/ml creatine phosphokinase, 0.2 mg/ml bovine ubiquitin (Sigma Chemical Co.), 40 μg/ml mouse recombinant E1 and 50 μg/ml human recombinant hE2-C , and then incubated at 25°C for 30 min or the indicated time. The samples were applied to 7% SDS-PAGE, and the ubiquitinated GST-Cyclin B or GST-Cut2 was detected by immunoblotting with anti–Cyclin B1 antibody and anti–GST antibody, respectively, and visualized by the enhanced chemiluminescence reaction. The purified APC in the S phase, which was immunoprecipitated with anti–Cdc27 antibody, was incubated with or without pPlk or PKA as described. The pretreated APC was incubated with T7-tagged human Cdc20, pCdc20, Cdh1, or Cdh1-phosphorylated MPF (pCdh1) in the ubiquitination buffer at 25°C for 30 min, washed with the buffer five times, and the proteins bound to the APC were resolved by 7% SDS-PAGE. The bound proteins were detected by immunoblotting with anti-T7 antibody. The purified S phase APC was incubated with Cdc2-GST-Cyclin B and γ-[ 32 P]ATP in the presence of Cdc20 or Cdh1 in MPF kinase buffer at 37°C for 30 min. The 32 P-labeled proteins were immunoprecipitated by anti–Cdc27 antibody and were resolved in 7% SDS-PAGE. The T7-tagged human Cdc20, Cdh1, and Mad2 were prepared in Escherichia coli , and their possible kinases, human Cdc2-GST-Cyclin B (MPF) prepared by baculovirus and His-tagged Plk produced in E . coli , were homogeneously purified as shown in Fig. 1 A. The in vitro phosphorylation was performed by incubating these recombinant Cdc20, Cdh1, and Mad2 with recombinant MPF or Plk in the presence of γ-[ 32 P]ATP. Both Cdc20 and Cdh1 could be phosphorylated by MPF but not by Plk in vitro. Mad2 was not phosphorylated by these kinases (data not shown). Next, we performed in vitro phosphorylation of APC by MPF in the presence or absence of recombinant purified human Suc1/Cks1 (CksHs-1 or CksHs-2). There exist two human Suc1/Cks1 homologues, CksHs-1 and CksHs-2. As shown in Fig. 1 C, none of the APC core subunits could be phosphorylated by MPF alone . In the presence of CksHs-1 or CksHs-2 , however, MPF could clearly phosphorylate APC1/Tsg24 and APC3/Cdc27 but not APC6/Cdc16, which is consistent with the results of Patra and Dunphy 1998 . The MPF-activated Plk could phosphorylate APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and an unidentified protein of 85 kD as we previously showed . In the presence of all three components (MPF, CksHs-1 or CksHs-2, and Plk), these APC core subunits were most efficiently phosphorylated . These results indicate that none of the APC core subunits can be phosphorylated by MPF alone, but in the presence of Suc1/Cks1, two APC core subunits, APC1/Tsg24 and APC3/Cdc27, can be phosphorylated by MPF. In vitro reconstituted ubiquitination assay was performed to assess the substrate- and time-specific activation or inactivation of mammalian APC, and the effects of phosphorylation of the regulatory factors as well as APC on the ubiquitination activity were determined. Fig. 2A and Fig. B , show that the purified APC in the S phase had no activity to ubiquitinate GST-Cut2 or GST-Cyclin B , even in the presence of Cdc20 . Since we found that Cdc20 could be phosphorylated by MPF, we next examined the effect of Cdc20 phosphorylated by MPF (pCdc20) on APC activity. The Cdc20 was phosphorylated by MPF, bound to His-Bind resin, and washed with 0.5 M KCl to remove MPF. It was confirmed by the immunoblot and MPF assay that the pCdc20 prepared in this way was free of MPF (data not shown). Interestingly, when the APC was incubated with pCdc20, both GST-Cut2 and GST-Cyclin B were ubiquitinated in a dose-dependent manner . Furthermore, Mad2 inhibited these ubiquitination activities in a dose-dependent manner . These results indicate that pCdc20 but not Cdc20 activates ubiquitination of both Cut2/Pds1 and Cyclin B, and the pCdc20-dependent APC activation can be suppressed by Mad2. No GST-Cut2 was ubiquitinated even in the presence of a large excess of Cdh1 or Cdh1 phosphorylated by MPF (pCdh1) . The pCdh1 was prepared by the same procedure as pCdc20, and it contained no MPF activity. The ubiquitination of GST-Cyclin B was activated by Cdh1 in a dose-dependent manner , and the activation was not blocked by the addition of a large excess of Mad2 . In contrast, even sufficiently large amounts of pCdh1 could not activate the ubiquitination of GST-Cyclin B . These results indicate that APC in the presence of Cdh1 but not pCdh1, effectively and specifically ubiquitinates Cyclin B (but not Cut2/Pds1) at least in vitro, and that Mad2 suppresses only pCdc20-dependent APC activation. Next, we examined the effect of phosphorylation of APC itself on APC activity in the presence of the activated form of the regulatory factors, pCdc20 and Cdh1. As previously reported, the pPlk-activated APC (pAPC) ubiquitinated GST-Cyclin B , but its activation was suppressed by phosphorylation with PKA . However, pAPC could not ubiquitinate GST-Cut2 . Furthermore, if the APC or pAPC was once phosphorylated by PKA, the GST-Cyclin B could not be ubiquitinated even in the presence of the active regulatory factors, pCdc20 or Cdh1 . These results indicate that the ubiquitination of Cyclin B is suppressed by PKA as well as by Mad2, and that pPlk specifically stimulates the ubiquitination of Cyclin B but not Cut2/Pds1. Time course experiments of Cyclin B ubiquitination with pAPC demonstrated that the kinetics of the ubiquitination reaction became much faster in the presence of the active regulatory factors, pCdc20 or Cdh1 , i.e., a shorter incubation time (10 min) was enough to reach the saturation level of GST-Cyclin B ubiquitination , indicating that pCdc20 or Cdh1 acts synergistically with pPlk on Cyclin B ubiquitination. It was also found that Mad2 does not inhibit pAPC activity ; thus, Mad2 inhibits only pCdc20-dependent APC activation, whereas PKA suppresses both pCdc20- and pPlk-dependent APC activation. The binding assay of the regulatory factors to APC demonstrated that both Cdc20 and pCdc20 constitutively bound to APC and to pAPC but not to APC phosphorylated by PKA (pAPC(PKA)) . These results indicate that pCdc20-induced APC activation is dependent on the phosphorylation of Cdc20 with MPF rather than on the binding preference of pCdc20 to APC. Furthermore, the binding of Cdc20 and pCdc20 to APC or to pAPC was not affected by the addition of Mad2 , suggesting that Mad2 inhibition of pCdc20-induced APC activation is not due to the binding inhibition of pCdc20 to APC, but due to the direct functional inhibition of pCdc20 by Mad2. In contrast, Cdh1 could bind to either APC or pAPC but not to pAPC(PKA) , whereas pCdh1 did not bind to any forms of APC . This indicates that Cdh1-induced APC activation totally depends on the binding of Cdh1 to APC or pAPC. These results suggest that Cdh1 (but not pCdh1) specifically binds to and activates APC, and that the PKA-induced inhibition of APC activity is due to the inhibition of the binding of these active regulatory factors to APC. To confirm these binding data, the purified S phase APC was incubated with Cdc2-GST-Cyclin B and γ-[ 32 P]ATP in the presence of Cdc20 or Cdh1, and the 32 P-labeled proteins were immunoprecipitated by anti–Cdc27 antibody. The results showed that MPF directly phosphorylated Cdc20 but none of the components of the purified APC , and that pCdc20 actually bound to APC but pCdh1 could not bind to APC . These results also confirmed that there is no need to consider the effect of phosphorylation of APC by MPF on ubiquitination activity. To demonstrate that MPF phosphorylates Cdc20 and Cdh1 during mitosis in vivo, the immunoprecipitates of anti–Cyclin B-specific antibody from mitotic K562 cell extracts were incubated with γ-[ 32 P]ATP in MPF kinase buffer. The sample was washed with 1% SDS, diluted 25-fold with the kinase buffer, and the labeled Cdc20 and Cdh1 were immunoprecipitated with anti–Cdc20- or anti–Cdh1-specific antibody, respectively. The 32 P-labeled Cdc20 and Cdh1 can be clearly seen . Fig. 7 , lane 1, shows the total 32 P-labeled proteins without immunoprecipitation by anti–Cdc20 or anti–Cdh1 antibody, and the most prominent band corresponded to the 32 P-labeled Cyclin B as shown. The preimmune antibody could immunoprecipitate none of these proteins . The phosphorylation could not be observed in cell extracts prepared from cells in S phase (data not shown). These results strongly suggest that MPF phosphorylates Cdc20 and Cdh1 during mitosis in vivo, while it cannot be completely ruled out the possibility that another kinase coimmunoprecipitated with anti–Cyclin B antibody phosphorylates these proteins. Next, we examined in vivo phosphorylation of Cdc20 and Cdh1 during mitosis and the interaction of these phosphorylated factors with APC in vivo. K562 cells were labeled with [ 32 P]orthophosphate during mitosis in vivo and the APC was purified by immunoprecipitation with anti–Cdc27 antibody. The 32 P-labeled Cdc20 could be immunoprecipitated with anti–Cdc20-specific antibody from this purified APC and from the total cell lysates , indicating that Cdc20 can indeed be phosphorylated and binds to APC during mitosis in vivo. In contrast, the 32 P-labeled Cdh1 could not be immunoprecipitated with its specific antibody from the purified APC, whereas the 32 P-labeled Cdh1 could be immunoprecipitated from the total cell lysates , demonstrating that Cdh1 can actually be phosphorylated but pCdh1 cannot interact with APC during mitosis in vivo. These results perfectly agree with the findings shown in the in vitro reconstituted system . Taken together with the findings described above and other observations recently reported, the scheme of regulation of APC activity is depicted in Fig. 9 . We showed in this paper that phosphorylation of Cdc20 is required for Cdc20-dependent APC activation at least in vitro . It has been reported that Cdc20 is expressed during G2 phase and mitosis and binds to APC in early mitotic stages . Whereas, Fang et al. 1998a described that the in vitro translated Cdc20, which might be phosphorylated during preparation, activated APC in vitro and that the phosphatase treatment of Cdc20 had no effect on APC activity, it was recently demonstrated that Cdc20 is clearly phosphorylated during mitosis in HeLa cells , and that Cdc20 is phosphorylated by MPF in Xenopus embryos . Actually, the possible Cdc2 phosphorylation site is conserved in budding yeast Cdc20, fission yeast Slp1, and mammalian Cdc20. Therefore, it is most likely that Cdc20 is indeed phosphorylated during mitosis in vivo. Taken together, we concluded that Cdc20 activates APC when it is phosphorylated by MPF. The activation of APC can be blocked by the binding of Mad2 to pCdc20 . Therefore, at least two events may be required to activate APC at metaphase–anaphase transition: release of Mad2 from pCdc20 after spindle assembly checkpoint is released, and phosphorylation of Cdc20 bound to APC by MPF or binding of pCdc20 to APC. Furthermore, we found that at least in vitro APC activation can be suppressed by PKA , which phosphorylates two APC subunits, APC1 and APC3 . Thus, dephosphorylation of PKA phosphorylation sites on APC by a specific phosphatase yet unidentified, which may be PP1 as previously suggested , might also be required for the onset of anaphase. Mad2 cannot inhibit Cdh1-induced APC activation , and pAPC can ubiquitinate Cyclin B even in the presence of Mad2 . Thus Mad2 inhibits APC activation only through Cdc20. Further, the binding of Cdc20 or pCdc20 to APC or pAPC was not affected by Mad2 , indicating that Mad2 inhibits the function of pCdc20 but not the binding of pCdc20 to APC. Very recently, it was reported that whereas Mad1, Mad2, Mad3/Bub1, and Bub3 suppress Cdc20-dependent APC activation, Bub2 localized in the spindle pole body regulates Cdh1-dependent APC activation . Further detailed functional analyses of the Mad family and Bub family are required to clarify the molecular mechanisms of spindle assembly checkpoint and of APC regulation. It was clearly shown that pAPC can ubiquitinate Cyclin B but not Cut2 in vitro, but it remains unclear whether free pAPC actually exists during mitosis in vivo. It is most likely that the majority of pAPC forms a complex with either pCdc20 or Cdh1 that ubiquitinates Cyclin B in vivo. Furthermore, phosphorylation of APC by pPlk may not be essential for APC activation at the metaphase–anaphase transition, but our results suggest that it is required for complete Cyclin B ubiquitination in later stages of mitosis. We showed here that pCdc20 and Cdh1, but neither Cdc20 nor pCdh1 activates APC in vitro, and the following switching mechanism from pCdc20 to Cdh1 could be speculated. When the cells enter anaphase after Cut2/Pds1 is entirely degraded, the pAPC–pCdc20 complex steadily ubiquitinates Cyclin B and, consequently, MPF activity decreases by the end of mitosis. The pCdc20 can be dephosphorylated by a specific phosphatase and released from pAPC or it may be degraded as previously suggested . When MPF activity is high, Cdh1 is phosphorylated and remains inactive, but when Cdc14 is activated, Cdh1 is dephosphorylated by Cdc14 as suggested and binds to and activates APC. Consequently, the pAPC–pCdc20 complex is replaced by the pAPC–Cdh1 (or APC–Cdh1) complex in late mitosis. The pAPC–Cdh1 or APC–Cdh1 complex further ubiquitinates Cyclin B in late mitosis and G1 phase. Thus, the switch mechanism from pCdc20 to Cdh1 may be dependent upon the MPF activity during mitosis, and the MPF activity itself is controlled by the pCdc20- and Cdh1-dependent APC activity. Cdc20 has been reported to activate ubiquitination of the factors regulating sister chromatid separation and Cdh1 promotes ubiquitination of mitotic cyclins . However, it was recently found that in Xenopus early embryos, Cdh1 is not expressed , which is consistent with the observation that Cdh1 is not expressed before stage 13 in Drosophila embryos . It was also described that Cdc20 regulates ubiquitination of both Cut2/Pds1 and Cyclin B in the early embryonic cell cycle . Very recently, Clute and Pines 1999 reported that ubiquitin-dependent proteolysis of Cyclin B begins at metaphase–anaphase transition in HeLa cells, which is consistent with the results obtained with clam embryo . These findings are consistent with our observation that pCdc20 can activate ubiquitination not only of Cut2/Pds1, but also of Cyclin B. Thus, pCdc20 alone may be enough for cells to go through mitosis without Cdh1. However, in the somatic cell cycle, Cdh1 in addition to pCdc20, may be required for effective and complete ubiquitination of Cyclin B in later stages of mitosis and G1 phase. We demonstrated in this paper that Cdh1 can be phosphorylated by MPF , and Cdh1 but not pCdh1 binds to and activates APC only after dephosphorylation . These in vitro results are consistent with in vivo data in budding yeast that the dephosphorylated form of Cdh1/Hct1 activates APC . It was recently described that Cdh1 is constantly expressed throughout the cell cycle , binds to APC in late mitosis and G1 phase , and is phosphorylated during mitosis in HeLa cells . It was also reported that Cdh1/Hct1 binding to APC is regulated by cyclin-dependent kinases . Very recently, it was demonstrated that Cdc14 dephosphorylates Cdh1/Hct1 and inactivates APC . Further, it was found that the activation of Cdc14 is regulated by Bub2/Byr4 and RENT complex , although it remains unresolved whether the regulation by RENT complex works in the mammalian system. All these results in vivo agree well with our results in vitro. In our scheme, it is possible that Cdh1–APC complex activity is maintained until late G1 phase, while it might be diminished by the phosphorylation of Cdh1 with Cdk2, Cdk4, or another unidentified specific kinase that is active at G1/S transition . It has been reported that the low level of Cyclin B is translated even in G1 phase . In the G1 phase, APC–Cdh1 complex may, thus, effectively ubiquitinate this newly translated Cyclin B to avoid activation of MPF. The possible involvement of G1/S Cdks or other specific kinases in Cdh1-dependent APC inactivation must be further studied. Patra and Dunphy 1998 recently reported that Cdc2-Cyclin B phosphorylates APC3/Cdc27 and APC1/BIME of Xenopus APC in the presence of the Xenopus Suc1/Cks1 protein, Xe-p9. We confirmed that in the presence of human Suc1/Cks1 (CksHs-1 or CksHs-2), MPF could phosphorylate APC1/Tsg24 and APC3/Cdc27 but not APC6/Cdc16, whereas none of the APC core subunits could be phosphorylated by MPF alone . The MPF-activated Plk could phosphorylate APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and one more additional unidentified protein as we previously showed . It has been reported that in Xenopus the phosphorylated APC components during mitosis are at least APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and APC8/Cdc23 . Therefore, the kinase that phosphorylates Cdc23/APC8 and may affect APC activity has not yet been identified. Furthermore, Patra and Dunphy 1998 have not demonstrated that phosphorylation with Cdk1-Suc1/Cks1 actually activates ubiquitination of cyclin B. Very recently, however, Shteinberg and Hershko 1999 demonstrated that in early embryonic cell cycle Cdk1 activated Xenopus APC in the presence of Suc1/Cks1. Further, Shteinberg et al. 1999 described that phosphorylation of APC by MPF is required for its stimulation by Cdc20. In contrast, Kaiser et al. 1999 very recently reported that Cks1 neither bound to nor activated APC, but that it did regulate proteasome activity in yeast. We also found that the APC was not activated only by phosphorylation with MPF. These data support the notion that MPF can phosphorylate APC in the presence of Suc1/Cks1, but itself may not be enough to completely activate APC, and that other kinases like Plk and/or factors such as Cdc20 and Cdh1 are required for full APC activation. Our findings in vitro and in vivo are consistent with the other in vivo observations , and strongly support the notion that pCdc20 but not Cdc20 activates APC, and that Cdh1 but not pCdh1 binds to and activates APC. Therefore, phosphorylation and dephosphorylation of APC regulatory factors by MPF are critical for their binding to APC and/or APC activation. The MPF activity itself is regulated by the pCdc20- and Cdh1-dependent APC activity, and this feedback control precisely regulates APC activity. Taken together, the APC activity is regulated by the phosphorylation and dephosphorylation of APC and of the regulatory factors, Cdc20 and Cdh1, by MPF, Plk, PKA, and PP1, as well as by the binding of positive and negative regulatory factors, Cdc20, Cdh1, and Mad2, to APC. These elaborate regulatory mechanisms might control the precise progression of mitosis. | Study | biomedical | en | 0.999995 |
10459015 | 35 kb of genomic DNA in the region of the Dhc1 gene was recovered from a large insert, wild-type (21gr) Chlamydomonas library. The position of the Dhc1 transcription unit within this region was determined by probing Northern blots of wild-type RNA with selected subclones, and the Dhc1 transcription unit was thereby narrowed down to ∼22 kb of genomic DNA . Sequence information was obtained from both strands of subclones A–D, and G using a series of nested deletions (Erase-a-Base System; Promega Corp.) and Sequenase 2.0 (Amersham Life Science, Inc.) following the manufacturer's instructions. Subclones E and F were sequenced by the DNA Sequencing Facility (Iowa State University) on an ABI Prism sequencer (Perkin Elmer Corp.). The sequence data were assembled using the GCG software package versions 8 and 9 (Genetics Computer Group). Potential open reading frames were identified using the GCG program CodonPreference and a codon usage table compiled from the coding regions of 73 different Chlamydomonas nuclear sequences . Potential splice donor and acceptor sequences within the open reading frames were identified based on splice junction consensus sequences found in Chlamydomonas nuclear genes . In five regions of the Dhc1 gene, the presence of multiple potential splice donor or acceptor sequences did not allow a confident prediction of the putative exons. In those cases, the splice junctions were determined directly by sequence analysis of reverse transcriptase–PCR (RT-PCR) products generated from the Dhc1 transcript . Total RNA was isolated from wild-type cells 45 min after deflagellation, and then 5 μg of total RNA was reverse transcribed using either a random primer or a sequence-specific reverse primer and the Superscript Preamplification System (GIBCO BRL) according to manufacturer's instructions. 5 μl of the resulting 25-μl cDNA product was used in a 100-μl PCR reaction with sequence specific primers. PCR reactions were performed using 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl 2 , 2 mM deoxynucleotide triphosphates, 0.2 mM of each primer, and 2.5 U Taq polymerase (Life Technologies, Inc.). Some reactions also contained 3% DMSO. The PCR reactions were first denatured at 94°C for 3 min, followed by 30 cycles of 58°C for 1 min, 72°C for 3 min, and 94°C for 1 min, and then completed with a final cycle of 58°C for 1 min and 72°C for 5 min. The final reaction products were analyzed on agarose gels, and then purified using Wizard PCR preps (Promega Corp.) for direct sequencing with sequence-specific primers. The proposed translation start site was determined by the recovery of an RT-PCR product using a forward primer downstream of the TATA box sequence and a reverse primer in exon 3. The resulting RT-PCR product contained stop codons in all three frames immediately preceding the proposed start codon. The predicted amino acid sequence encoded by the Dhc1 gene was analyzed using the GCG program Motifs. The programs Bestfit, Compare, and Pileup were used to compare the 1α Dhc sequence to Chlamydomonas outer arm Dhc sequences α, β, and γ and the cytoplasmic Dhc from Dictyostelium . Regions with the potential to form α-helical coiled coils were identified using the program COILS, version 2.2 . To identify clones that might contain a full-length Dhc1 gene, we screened two different Chlamydomonas cosmid libraries that were generously provided by S. Purton (University College, London) and D. Weeks (University of Nebraska). Because the Purton library contains the ARG7 gene within the cloning vector, the cosmid clones can be used to directly transform arg7 strains. 10 5 independent clones from each library were screened on Magnagraph (Micron Separations, Inc.) nylon membrane lifts in duplicate with probes from the 5′ and 3′ ends of the Dhc1 gene . Probes used for hybridization were purified in low melting point agarose (GIBCO BRL) and radiolabeled with [ 32 P]dCTP and random hexamer primers using the Prime It II kit (Stratagene). Conditions for prehybridization and hybridization were as described previously . After single colony isolation, cosmid DNA was purified using alkaline lysis procedures and CsCl gradient centrifugation . A truncated version of the Dhc1 gene was constructed by fusing sequences from the 5′ end to sequences from the 3′ end. To recover the 5′ end, a 19-kb SalI fragment was subcloned to form the plasmid pSM8 . pSM8 contains ∼1.7 kb of genomic DNA located 5′ of the coding region, but ends in the middle of the Dhc1 transcription unit. pSM8 was digested with SalI and AscI to release the Dhc1 gene as an 11-kb fragment that is truncated before the region encoding the ATP hydrolytic site (P1). The 3′ end of the Dhc1 gene was subcloned as a 4.3-kb SalI, EcoRI fragment to form the plasmid p14SE, which was digested with SalI and AscI to release the region 5′ of the AscI site. The SalI-AscI fragment from pSM8 was ligated into the digested p14SE subclone to form the construct pD1SA . pD1SA joins sequences from the 5′ end of the Dhc1 gene to the 3′ end at the AscI site. It is predicted to encode the first 1,956 amino acids of the 1α Dhc, and then terminate translation after adding nine novel amino acids (QCHGCGPGV) to the COOH terminus of the polypeptide. A modified pBELO BAC library containing Chlamydomonas genomic DNA was screened with selected subclones to identify large insert BAC clones containing the Dhc1 gene. This library was constructed by N. Haas and P. Lefebvre (University of Minnesota, St. Paul, MN) using genomic DNA from the cell-wall less strain cw92 and is currently available from Genome Systems, Inc. BAC DNA was isolated from positive clones using a modified version of the manufacturer's protocol available from C. Amundsen (University of Minnesota, St. Paul, MN) at the following URL: http://biosci.cbs.umn.edu/~amundsen/chlamy/methods/bac.html. The final pellet of BAC DNA was resuspended in 200 μl of TE and stored at −20°C. To identify clones containing full-length Dhc1 genes, 5 μl of BAC DNA was digested with the restriction enzyme SacI and analyzed on Southern blots using subclones from the 5′ and 3′ ends of the Dhc1 gene. Large-scale preparations of genomic DNA were isolated from wild-type and mutant transformant strains using CsCl gradients as described in Porter et al. 1996 . A smaller scale mini-prep procedure was used to isolate DNA samples from tetrad progeny and some of the transformants. Restriction enzyme digests, agarose gels, isolation of total RNA, and Southern and Northern blots were performed as previously described . The strains used in this study are listed in Table . All cells were maintained as vegetatively growing cultures at 21°C as previously described . The arg2 strains were grown on rich medium that contained reduced ammonium nitrate (one tenth the normal concentration), but was supplemented with l -arginine to 0.6 mg/ml. The pf9-2 arg2 strain was crossed to the outer arm mutant pf28 using standard genetic techniques to obtain the triple mutant pf9-2 pf28 arg2 . The pf9-2 pf28 arg2 strain assembles short, immotile flagella, and requires arginine for growth. After growth in tris-acetate phosphate (TAP) media supplemented with 0.6 mg/ml l -arginine, this strain was cotransformed using the glass bead method with various constructs of the Dhc1 gene (2–4 μg) and the plasmid pARG7.8, which contains a wild-type copy of the ARG7 (argininosuccinate lyase) gene . After transformation, cells were washed and plated on TAP media lacking arginine to select for arg+ transformants. After 10 d of growth, single arg+ colonies were picked into liquid media and tested for rescue of the flagellar assembly and motility defects. Positive transformants were picked into 96-well plates and screened for motility on an inverted microscope (Olympus CK). Wells containing motile cells were streaked for single colonies and rescored on a phase-contrast microscope (Axioskop; Carl Zeiss, Inc.) using a 40× objective and a 10× eyepiece. The phenotypes of motile transformants were further analyzed by measuring forward swimming velocities and beat frequencies as previously described . Transformants were tested for their ability to phototax using two different assays. In the first assay , actively swimming cells were put in a dark box with a 3-cm-wide horizontal slit cut out along the bottom such that only the lower portion of a 10-ml suspension was illuminated. The box was placed ∼35 cm from a fluorescent light source for 40 min. Positively phototactic cells would concentrate in the lower, illuminated portion of the tube, whereas phototaxis defective cells would remain uniformly suspended throughout the tube. In the second assay, motile cells were transferred to a 96-well plate and placed on a dissecting microscope with substage illumination. Thick posterboard was placed under the plate and positioned to cover half of the well. The location of the cells within the well was followed over a 60-min time course. Positively phototactic cells would move quickly to the side of the well exposed to light, whereas cells unable to phototax would remain uniformly distributed throughout the well. To verify that the rescued motility in the transformants was due to expression of the Dhc1 transgene and not a reversion event at the PF9 locus, the motile transformants were backcrossed to a pf28 allele ( oda2 ), which lacks the outer arms but is wild-type at the PF9 locus . Tetrad progeny were recovered following standard genetic methods , and their motility phenotypes were scored using a phase-contrast microscope as described above. Axonemes were prepared from large-scale (5–40 liters) liquid cultures of vegetative cells using procedures described by Witman 1986 and S.M. King et al. 1986 , as modified by Gardner et al. and Myster et al. 1997 . Crude dynein extracts were obtained by brief (∼30 min) high salt extraction of whole axonemes . To isolate I1 complexes, crude dynein extracts were fractionated by sucrose density gradient centrifugation as previously described . Aliquots of each fraction were analyzed by SDS-PAGE and Western blotting. Protein samples from whole axonemes and sucrose gradient fractions were separated on 5% polyacrylamide gels using the Laemmli 1970 buffer system and either stained directly with Coomassie brilliant blue (R250; Sigma Chemical Co.) or transferred to either nitrocellulose (Schleicher and Schull, Keene) or Immobilon-P (Millipore) in 25 mM Tris, 192 mM glycine, and 12.5% methanol at 800 mA for 90 min at 4°C using a Genie electroblotter (Idea Scientific, Co.). Nitrocellulose blots were blocked in 1× PBS (0.58 M Na 2 HPO 4 , 0.017 M NaH 2 PO 4 ·H 2 O, 0.68 M NaCl), 5% normal goat serum (Sigma Chemical Co.), and 0.05% Tween 20 (polyethylenesorbitan monolaurate), whereas the Immobilon-P membrane was blocked in 0.2% I-Block (Tropix) in 1× PBS and 0.5% Tween 20. Four different antibody preparations were used to probe the blots. The 1α Dhc antibody has been previously described in detail and is highly specific for the Dhc1 gene product . This antibody was raised against the peptide sequence DGTCVETPEQRGATD, which corresponds to amino acids 1,059–1,073 of the 1α Dhc polypeptide. The 1α Dhc antibody was affinity-purified on Western blots of dynein extracts, and then used at a 1:10 dilution. The IC140 antibody was provided by P. Yang and W. Sale (Emory University, Atlanta, GA). This antiserum was raised against a fusion protein containing a fragment of the 140-kD intermediate chain of the I1 complex , and it was typically used at a dilution of 1:3,000. The rabbit polyclonal antibody R5205, which was raised against a fusion protein of the human 14-kD dynein LC , was provided by S. King (University of Connecticut). The R5205 antibody cross-reacts with the 14-kD LC (Tctex1) of the I1 complex and was used at a dilution of 1:50. After incubation overnight at 4°C, the blots were washed in 1× PBS and 0.05% Tween 20. Immunoreactivity was detected using an alkaline phosphatase–conjugated secondary antibody, BCIP (5-bromo-4-chloro-3-indolyl phosphate), and NBT (nitro blue tetrazolium) following the manufacturer's instructions (Sigma Chemical Co.). An mAb to tubulin was used at a dilution of 1:1,000, and then detected using an HRP-conjugated secondary antibody, 4-chloro-1-naphthol, and hydrogen peroxide following the manufacturer's protocol (Sigma Chemical Co.). To view the I1 complex in strains with rescued motility, selected transformants were crossed to a pf9-3 strain to recover strains with rescued I1 complexes and the wild-type complement of outer dynein arms. Axonemes were prepared and processed for EM as previously described . Longitudinal images were selected, digitized, and averaged using the methods described in Mastronarde et al. 1992 . Averages of individual axonemes were obtained by analyzing at least six 96-nm radial spoke repeats, and then averages from several axonemes were combined to obtain a grand average for each strain. The methods used to compute differences between two strains are described in detail in Mastronarde et al. 1992 . To identify the 3′ end of the Dhc1 transgene in the G3 transformant (which assembles the shortest 1α Dhc fragment), genomic DNA was isolated from wild-type and G3, digested with the restriction enzymes SacI and KpnI, and analyzed on Southern blots probed with Dhc1 subclones. A polymorphic 7.2-kb SacI-KpnI fragment was identified in G3 using subclone C. This polymorphic fragment was recovered from G3 genomic DNA by constructing a size-selected minilibrary, and then screening the library with subclone C. After single colony purification, the 3′ end of the truncated Dhc1 transgene was sequenced with Dhc1 specific primers to determine the predicted amino acid sequence at the COOH terminus of the 1α Dhc fragment. In previous work, we identified a null mutation in the Dhc1 gene that resulted in the failure to assemble the I1 inner arm complex into the flagellar axoneme . To understand how the Dhc1 gene product might contribute to dynein complex formation, we have now sequenced the entire Dhc1 transcription unit . A map of the deduced gene structure is shown in Fig. 1 b. Sequence analysis of subclone A identified the 3′ end of the neighboring gene (geranyl geranyl pyrophosphate synthase), ∼800 bp of intervening sequence, and a TATA box sequence 144 bp upstream of the proposed translation start site of the Dhc1 gene (see Materials and Methods). All of the 5′ sequence elements required for regulated Dhc1 expression should therefore be contained within an ∼1-kb region. The next 20 kb of the sequence contains the coding region located within 29 exons. The 3′ end of the gene is located in subclone G, which contains the last exon encoding the COOH-terminal 552 amino acids, a stop codon, and a consensus polyadenlyation signal sequence (TGTAA) 471 bp downstream. The predicted amino acid sequence of the encoded 1α Dhc contains 4,625 amino acid residues and corresponds to a polypeptide of 522,806 D . A search for potential nucleotide binding sites within the 1α Dhc sequence identified six consensus or near consensus phosphate-binding (P-loop) motifs with the sequence A/GXXXXGKT/S . Four of the P-loop motifs (P1–P4) are located within the central region of the Dhc, and both spacing and sequences of these P-loops are similar among all Dhc sequences reported thus far . Two additional P-loop motifs were identified in the NH 2 -terminal (Pn) and COOH-terminal (Pc) regions of the 1α Dhc respectively; these appear to be unique to the 1α Dhc . The predicted amino acid sequence of the 1α Dhc was compared with the three Dhc sequences (α, β, and γ) that form the outer dynein arm in Chlamydomonas and the cytoplasmic Dhc from Dictyostelium . In each case, a high degree of sequence similarity was apparent over long stretches of the polypeptide, especially in the central and COOH-terminal thirds of the Dhc (28–38% identity, 58–67% similarity). However, the more variable NH 2 -terminal third of the 1α Dhc also shares significant homology with the β and γ Dhcs of the outer arm . Alignment of the Dhc sequences using the GCG program PILEUP confirmed that the presence of conserved domains within the NH 2 -terminal region, but also revealed several short stretches of unique peptide sequence in the 1α Dhc, including the region previously used to generate a monospecific 1α Dhc antibody . The 1α Dhc sequence was also analyzed using programs that predict secondary structure to identify regions with the potential to form α-helical coiled-coil domains . One region located before P-loop 1 (residues 1,227–1,409) and a second after the P-loop 4 (residues 3,192–3,297, 3,400–3,494, and 3,701–3,789) show the highest probability of forming α-helical coiled coils. The presence of limited coiled-coil domains separating the central portion of the Dhc from the NH 2 -terminal and COOH-terminal regions has been observed in other Dhc sequences . These conserved structural domains are thought to play an important role in protein interactions within the dynein arms. To better understand how the specific domains of the 1α Dhc polypeptide might be involved in the assembly and activity of the I1 inner arm dynein, we decided to analyze constructs of the Dhc1 gene in vivo in a pf9 mutant background. Because of the large size of the Dhc1 gene (∼21 kb), two cosmid libraries and one BAC library were screened with probes representing the 5′ and 3′ ends of the Dhc1 gene to improve the chances of recovering clones that contain the full-length gene . The first cosmid library yielded a single clone, cA1, which was positive with both probes, but upon further analysis proved to be lacking a small portion at the 3′ end of the gene . Screening the second cosmid library resulted in the recovery of a single clone, cW1, which contained the complete Dhc1 transcription unit as well as additional genomic sequences both 5′ and 3′ that might be required for proper expression in vivo . Four larger clones (100–135 kb) containing the Dhc1 transcription unit were recovered from the BAC library; two of these clones were used in subsequent cotransformation experiments . We also constructed a truncated version of the Dhc1 transgene known as pD1SA by fusing an 11-kb region encoding the NH 2 -terminal 1,956 amino acids to a 1-kb region containing the 3′ end of the gene . All of the Dhc1 transgenes were tested for their ability to rescue the pf9 mutant defects in vivo. Mutations at the PF9/IDA1 locus typically result in strains that have a slow, smooth swimming behavior . To increase the sensitivity of the screen for rescue of the pf9 mutant phenotype, we introduced a second motility mutation into the pf9-2 background. pf28 is a mutation in the γ Dhc gene that results in the failure to assemble the outer dynein arms . Cells carrying the pf28 mutation swim with a jerky phenotype that can be easily distinguished from the slow, smooth swimming behavior of the pf9 mutant cells. In addition, pf9-2 pf28 double mutants assemble short, paralyzed flagella and sink in liquid medium . This short, paralyzed flagellar phenotype makes it very straightforward to identify transformants that have rescued the pf9 mutant defects by screening for cells that assemble full-length, motile flagella and swim with a pf28 -like motility phenotype. The pf9-2 pf28 arg2 strain was first cotransformed with the selectable marker pARG7.8 and the cW1 cosmid containing the complete Dhc1 transcription unit. Positive transformants were selected by growth on solid medium lacking arginine, and then single colonies were picked into liquid media and screened for motility. Fig. 5 C illustrates the combined results of several independent cotransformation experiments with the cW1 cosmid. Although arg+ transformants were recovered at expected frequencies , only 11 out of 2,880 transformants screened displayed any motility, for a frequency of rescue of <0.4%. Moreover, the motility of the cW1 rescued strains was not the same as pf28 (see below and Table ). Given these preliminary findings, we decided to test the other Dhc1 transgenes for their ability to rescue the mutant phenotypes. The cosmid cA1 contains an incomplete copy of the Dhc1 transcription unit but also contains the ARG7 gene cloned within the bacterial vector sequences, thereby physically linking the selectable marker and the Dhc1 gene. Therefore, all arg+ transformants might be expected to contain the Dhc1 sequence integrated along with the ARG7 gene. However, the frequency of rescue with the cA1 cosmid was only slightly better than the cW1 cosmid. These observations indicated the Dhc1 transgenes were probably being fragmented during the transformation protocol, but the recovery of motile strains also suggested that a truncated version of the Dhc1 sequence was capable of restoring some function. Previous study of an outer arm mutation, oda4-s7 , had indicated that the NH 2 -terminal third of the β Dhc polypeptide is sufficient for assembly of a dynein complex . To test if this is also true for the 1α Dhc, we cotransformed the pf9 pf28 mutant with the smaller pD1SA construct, which encodes ∼40% of the 1α Dhc sequence. These experiments yielded seven motile strains , which represented only a modest (0.87%) increase in the frequency of rescue, but these rescues confirmed that truncated Dhc1 transgenes could restore partial motility. To see if it was possible to completely rescue the motility defects, we also transformed the pf9 pf28 mutant with two BAC clones that contained the full-length Dhc1 gene located in the middle of ∼100–135-kb genomic inserts . The frequency of rescue (∼0.1%) was still quite low, but the motility phenotypes of the rescued strains were very similar to pf28 (see below). The recovery of motile isolates after cotransformation could also be due to an intragenic reversion event at the PF9 locus during the course of transformation and/or selection. To confirm that the motility of the transformants was due to the successful expression of the Dhc1 transgene and not a reversion event, two of the cW1 transformants (E2 and G4) were crossed with oda2 , another mutant allele at the PF28 / ODA2 locus, and the progeny from 11 complete tetrads were analyzed for each cross. If the rescued motility was due to reversion of the pf9-2 mutation, all the resulting tetrad progeny would be motile and swim with a pf28 / oda2 –like motility phenotype. However, if the rescued motility was due to the presence of the Dhc1 transgene, then the restored motility phenotype would be expected to segregate independently of the pf9-2 mutation, and a class of immotile progeny with the original pf9-2 pf28 genotype should be recovered. Surprisingly, we observed three different motility phenotypes in the tetrad progeny. The first class swam with a motility phenotype that was indistinguishable from either pf28 or oda2 . The second class swam with a jerky motion like the pf28 / oda2 strains but appeared slower. The third class was immotile with short, stumpy flagella. The recovery of aflagellate strains demonstrated that the original pf9-2 mutation was still present in the genetic background of the two transformants and that the rescued motility was due to the presence of the Dhc1 transgene. Although the frequency of rescue was low, it was clear that the rescued motility was due to the presence of the different Dhc1 transgenes, and so the motility phenotypes of the Dhc1 transformants were analyzed in greater detail. More specifically, we measured the flagellar beat frequency, the forward swimming velocity, and the ability to phototax ( Table ). Transformants with complete rescue of the pf9 mutation would be expected to have a swimming phenotype nearly identical to that of pf28 . The flagellar beat frequencies of the Dhc1 transformants were almost identical to the beat frequency of pf28 , but measurements of forward swimming velocities clearly indicated that most of the transformants swam more slowly than pf28 ( Table ). In particular, the swimming velocities of the rescued strains obtained by transformation with the cosmid clones and pD1SA were slower than those obtained by transformation with the BAC clones. These results suggested that there were still some inner arm defects in most of the Dhc1 transformants. We next tested if the Dhc1 transformants had recovered the ability to phototax. King and Dutcher 1997 have previously used a photoaccumulation assay to demonstrate that pf9 mutant cells do not phototax effectively. Using similar conditions, we have found that pf28 cells, which lack outer arms but have the full complement of inner dynein arms, are able to phototax, as assayed by their tendency to become concentrated in the illuminated portion of a tube within 40 min of exposure to a directional light source ( Table ). However, all of the Dhc1 transformants obtained with the cosmid clones remained equally distributed between the illuminated and darkened regions of the tube. To confirm these findings by direct observation of individual cells, the ability to phototax was also monitored in 96-well plates over a 60-min time course (see Materials and Methods). In the absence of outer arms, the Dhc1 transformants obtained with the cosmid clones remained equally distributed in both the illuminated and darkened portions of the microtiter well. Conversely, the majority of pf28 cells became concentrated on the illuminated side of the well within 15 min. These results indicated that this group of Dhc1 transformants does not phototax as effectively as pf28 control cells. To examine the motility of the transformants in the presence of outer arms, two strains, G4 and E2, were crossed to pf9-3 and tetrad products containing the Dhc1 transgene in a wild-type outer arm background (G4+OA and E2+OA) were recovered. The two strains have beat frequencies almost identical to wild type, but their swimming velocities are intermediate in speed between pf9 and wild type ( Table ). Moreover, in the presence of the outer arms, the two strains could photoaccumulate as effectively as wild type. These results suggest that the outer arms can compensate in some way for the phototaxis defects in the Dhc1 transformants. The swimming behavior of the Dhc1 transformants obtained with the cosmid clones demonstrated that the introduction of these Dhc1 clones resulted in only a partial rescue of the pf9 motility defects. Given the large size of the Dhc1 transcription unit (>22 kb), we were initially concerned that these transgenes might not be expressing wild-type levels of the Dhc1 gene product. To address this question, we isolated axonemes from the Dhc1 transformants and analyzed the components of the I1 complex. Previous work has shown that the I1 complex is composed of eight polypeptides, two Dhcs (1α and 1β), three ICs (IC140, IC138, and IC110) , and three LCs (LC8, LC12, and LC14) . The Dhc1 gene encodes the 1α Dhc, which can be identified on Western blots using antibody directed against a peptide epitope in the NH 2 -terminal region . Fig. 6 A shows three Coomassie blue–stained polyacrylamide gels containing whole axonemes isolated from several mutant strains: the 11 motile Dhc1 transformants obtained with the cW1 cosmid, the 4 motile Dhc1 transformants recovered with the cA1 construct, and the 5 transformants recovered with the Dhc1 BAC clones. Fig. 6 B shows the corresponding Western blots probed with the 1α Dhc antibody. The 1α Dhc antibody identified the ∼520 kD 1α Dhc in the pf28 control sample, but polypeptides significantly smaller than the 1α Dhc were identified in all of the motile strains obtained by transformation with the Dhc1 cosmids. In contrast, all of the axoneme samples prepared from rescued strains obtained by transformation with the Dhc1 BAC clones contained full-length 1α Dhc polypeptides. These results indicated that the partial rescue phenotype seen with the Dhc1 cosmid clones was not due to low levels of expression of a full-length 1α Dhc, but instead due to the expression of truncated 1α Dhcs, ranging in size from ∼165 to ∼300 kD. To determine if other I1 subunits were associated with the truncated 1α Dhcs, Western blots of isolated axonemes were probed with an antiserum raised against the 140-kD intermediate chain . This antibody detects the IC140 in wild-type axonemes, but not in I1 mutant axonemes. As shown in Fig. 6 C, the IC140 antibody recognized a single polypeptide of ∼140 kD in pf28 and each rescued transformant, but did not detect the IC140 in any of the pf9 mutant strains. Similar results were seen using the antibody directed against the 14-kD Tctex1 light chain (data not shown). To confirm that the other polypeptide subunits were assembled into an I1 complex, we isolated whole axonemes from large-scale cultures of two Dhc1 transformants, E2 and G4, as well as from control pf28 cells. Partially purified I1 complexes were obtained by high salt extraction of the isolated axonemes followed by sucrose density gradient centrifugation. The resulting fractions were analyzed by both SDS-PAGE and Western blotting. Fig. 7 A shows the 19S region of a sucrose gradient that was loaded with the pf28 dynein extract. The two Dhcs and three ICs of the I1 isoform cosediment as a complex that peaks in fraction number 4. Duplicate samples tested on Western blots probed with the 1α Dhc antibody confirmed the presence of the 1α Dhc in the 19S region (right). Fig. 7 B shows four fractions from the sucrose gradient that was loaded with the G4 dynein extract. The gel on the left reveals that the 1β Dhc and the three intermediate chains of the I1 complex have shifted and now cosediment at ∼16S, peaking in fraction 6. A novel polypeptide of ∼183 kD cosediments in the same region (see asterisks). Western blot analysis with the 1α Dhc antibody identified this novel band as the truncated 1α Dhc . Identical results were observed with dynein extracts isolated from the E2 strain (data not shown). The truncated 1α Dhcs in the Dhc1 transformants therefore form stable complexes with the other polypeptides of the I1 complex, but the resulting mutant complexes sediment more slowly than wild-type complexes. To analyze the structure of the I1 complex in the Dhc1 transformants, we prepared purified axonemes from wild-type and mutant strains for thin section EM. To facilitate the analysis of the images, the transformants G4 and E2 were crossed to a pf9-3 strain to recover the Dhc1 transgene in a wild-type outer arm background (G4+OA and E2+OA, see Materials and Methods). Fig. 8 a shows the grand average of the 96-nm repeat from wild-type axonemes, and Fig. 8 b indicates the corresponding densities. Previous work has shown that the inner dynein arms repeat as a complex group of structures every 96 nm in register with the radial spokes . The I1 complex is a trilobed structure located proximal to the first radial spoke (S1) in each 96-nm repeat . These three lobes are missing in pf9-3 axonemes , which lack the I1 complex . Fig. 8e and Fig. g , show the grand averages of the axonemes from the G4+OA and E2+OA strains, which contain I1 complexes with truncated 1α Dhcs. Lobes 1 and 3 of the I1 complex are present in the axonemes from these samples, but lobe 2 is still missing. Difference plots between these images and wild type confirms that the loss of lobe 2 is the only significant defect in the two Dhc1 transformants . These images demonstrate that the I1 complex is assembled and targeted to the appropriate axoneme location. In addition, these images suggest that the region of the 1α Dhc that is missing in the Dhc1 transformants corresponds to lobe 2 of the I1 structure. Although the initial cotransformation experiments involved the use of a full-length or near full-length Dhc1 cosmid clones, all of the motile transformants recovered with these clones assemble partially functional I1 complexes with truncated 1α Dhcs . To understand how the 1α Dhc fragments were related to the Dhc1 sequence, we analyzed the Dhc1 transcripts from several of the rescued transformants on Northern blots. Total RNA was first isolated from the G4+OA and E2+OA strains, which contain the Dhc1 transgene in the pf9-3 null mutant background. This background facilitated our analysis because the pf9-3 mutation is a large deletion (∼13 kb) in the Dhc1 gene and does not generate an endogenous Dhc1 transcript . Fig. 9 A shows a partial restriction enzyme map of the Dhc1 gene and the subclones that were used as probes to analyze the Dhc1 transcripts. As shown in Fig. 9 B, probe A3′, which spans the Dhc1 transcription start site, identified a single, large (>13 kb) transcript in wild-type RNA. However, in G4+OA and E2+OA, the transcripts recognized by the A3′ probe were significantly smaller than the wild-type Dhc1 transcript, but these smaller transcripts were still upregulated in response to deflagellation (compare lanes 0 and 45). Identical results were observed with the next two subclones, probes B and C. Probe D, which includes the conserved region encoding the primary ATP hydrolytic site, hybridized to the truncated transcripts in E2+OA, but it did not recognize the truncated transcripts in G4+OA. Probes E–G did not hybridize with any transcripts in the transformants . The Dhc1 transcripts in G4 and E2 are therefore truncated from the 3′ end of the Dhc1 gene, and G4 is truncated before E2. To characterize the Dhc1 transcripts present in other transformants, RNA was isolated from three additional strains: G3, G9, and A2. G3 and G9 are the cW1 transformants that assemble the smallest and largest 1α Dhc fragments respectively, whereas A2 is a cA1 transformant that assembles the largest (>300 kD) 1α Dhc fragment obtained thus far . As shown in Fig. 9 C, in each strain, probe C hybridized to a truncated transcript that is significantly smaller than the endogenous Dhc1 transcript derived from the pf9-2 mutant background. However, probe D, which corresponds to the region encoding the ATP hydrolytic site, failed to hybridize with the truncated transcripts present in the G3, A2, and G9 samples. Similar results were seen with probes E and F (data not shown). Therefore, all three strains encode 1α Dhc fragments that are truncated before the proposed motor domain. To identify the sites where the Dhc1 cosmid clones were being modified during transformation, we isolated genomic DNA from the transformants and analyzed the structure of the integrated Dhc1 transgenes on Southern blots. Fig. 10 A shows a blot of SacI digested genomic DNA that was hybridized with a probe for subclone C. As expected, this probe hybridized to the endogenous Dhc1 gene present in the pf9-2 mutant background of the transformants. However, for each transformant, a second polymorphic band could be detected using either probe C or probe D (data not shown). From these and other blots, we concluded that the Dhc1 cosmids were being rearranged during integration into genomic DNA. In addition, the region of the Dhc1 gene encoding the conserved motor domain appeared to be the most common target for disruption during these integration events. Because the G3 transformant assembles the smallest 1α Dhc fragment identified thus far , we recovered the modified Dhc1 transgene using probe C to screen a mini-library made from genomic DNA of the G3 transformant (see Materials and Methods). The Dhc1 transgene was then sequenced with Dhc1 specific primers to identify the junction between the Dhc1 sequence and the site of integration in G3 genomic DNA. The Dhc1 sequence in G3 is fused to an unidentified DNA sequence, and the resulting hybrid gene is predicted to encode up to amino acid residue 1,249 of the 1α Dhc, followed by the addition of 17 novel amino acids before encountering a stop codon . Sequence analysis of an RT-PCR product derived from G3 RNA has confirmed the presence of this hybrid transcript. The polypeptide encoded by the modified transgene would, therefore, correspond to a 1α Dhc fragment of ∼143 kD that is truncated just COOH-terminal to the epitope recognized by the 1α Dhc antibody . The recovery of this fragment in G3 axonemes reveals that the NH 2 -terminal coiled-coil domains of the 1α Dhc are not required for I1 complex assembly. 16 different Dhc genes have been identified in Chlamydomonas : 2 cytoplasmic Dhc sequences , 3 genes that encode the Dhcs of the outer dynein arm , and 11 other Dhc genes whose expression patterns are consistent with gene products that are involved in flagellar function . The analysis of specific Dhc mutations has revealed the role of each outer arm Dhc in flagellar motility , but the specific functions of the multiple inner arm Dhcs are largely unknown. We have previously demonstrated that the Dhc1 gene product plays an essential role in the assembly and function of the I1 inner arm complex . To better understand how this inner arm Dhc contributes to flagellar motility, we have now analyzed the structure and function of the Dhc1 gene and the encoded 1α Dhc in detail. As described in Fig. 1 , we have determined the nucleotide sequence for the complete Dhc1 transcription unit and used this information to obtain the predicted amino acid sequence of the 1α Dhc . To our knowledge, this is the first full-length, inner arm Dhc sequence to be reported in any organism. Comparisons to other full-length Dhc sequences indicate that the 1α Dhc is most similar to the β and γ Dhcs of the outer arm, and that these sequence similarities extend into the NH 2 -terminal region . As the NH 2 -terminal region of the Dhc is thought to be involved in the association with isoform specific IC and LC subunits, these observations suggest that the 1α Dhc and the β and γ Dhc contain conserved sites for the binding of accessory subunits. Indeed, recent studies have revealed that the I1 complex and the outer arm do contain similar IC and LC components, such as the 8-kD LC, the Tctex1 and Tctex2 LCs, and a family of WD-repeat containing ICs . However, a novel feature of the 1α Dhc sequence is the presence of a P-loop motif (Pn) at amino acid residues 960–967 . A weakly conserved P-loop motif has also been identified within the first 200 residues of the β Dhc . Whether the Pn sequence in the 1α Dhc is a bona fide nucleotide binding site is unknown, but the future sequence analyses of 1α Dhc homologues in other organisms should indicate whether the Pn motif is a conserved feature of this class of Dhc. The central and COOH-terminal thirds are the most highly conserved regions of the 1α Dhc, and our transformation experiments are consistent with previous proposals that this region corresponds to the dynein motor domain . The amino acid residues around P1 (the primary ATP hydrolytic site) and P4 are highly conserved with other axonemal Dhcs, whereas the sequences around P2 and P3 are less well conserved . For example, P3 does not strictly conform to the P-loop consensus sequence GXXXXGKS/T , as the glycine in position 6 is substituted by an alanine in the 1α Dhc sequence, but the same amino acid substitution was found in the γ Dhc sequence . The COOH-terminal third of the 1α Dhc also contains a small region ∼340 amino acids downstream from P4 that is predicted to form a limited coiled-coil domain . A similar region in cytoplasmic Dhc sequences has been identified recently as the stalk structure that extends from the globular head domain and forms the microtubule binding site . Mutations in this region in the outer arm β Dhc can have dramatic effects on flagellar motility . Interestingly, a sixth P-loop motif (Pc) has been identified in the 1α Dhc sequence, ∼280 amino acids downstream from the proposed microtubule binding site. The function of this sixth P-loop in the 1α Dhc is unknown, but its position downstream from the microtubule binding site is intriguing. Recent sequence analysis has suggested that all dyneins may contain six ATPase-like repeat regions: the four central P-loops previously identified and two additional, less well conserved repeats after the COOH-terminal coiled-coil domains . Using the Dhc1 sequence information, we recovered two BAC clones and two cosmid clones containing full-length or near full-length Dhc1 genes, and then used these constructs to rescue the pf9 motility defects . 20 independent transformants with rescued motility were recovered. Backcrossing the transformants confirmed that the rescued motility was due to the presence of the Dhc1 transgene and not to a reversion event at the PF9 locus. However, analysis of the Dhc1 transformants produced two unexpected results. First, the frequency of rescue (<1%) was much lower than previously observed with other flagellar genes (5–10%) , and second, the 1α Dhcs were truncated in most of the motile transformants recovered thus far. One reason that the cotransformation frequencies were so low might be due to the large size of the Dhc1 transcription unit, which could make the Dhc1 transgenes more susceptible to damage during the transformation protocol. Exogenous DNA sequences often undergo deletions as they integrate into the Chlamydomonas genome . The recovery of truncated 1α Dhc fragments indicated that the Dhc1 cosmids were being disrupted during the transformation protocol, and both Southern and Northern blot analyses of the rescued transformants have confirmed that deletions from the 3′ end of the Dhc1 cosmids did occur. The presence of additional genomic DNA flanking the Dhc1 transcription unit in the BAC clones may have served to protect the Dhc1 transgenes and thereby permitted the full-length rescues observed with these clones . Another reason for the low frequency of rescue might be the relatively small amount of genomic DNA present on the 5′ end of the Dhc1 gene in certain constructs . If this region was randomly deleted, the resulting cotransformants would not retain the sequences necessary for expression of the Dhc1 transcript and rescue of the mutant phenotype. Recent experiments with constructs encoding the IC140 subunit have indicated that sufficient DNA upstream from the 5′ end of the IC140 gene is essential for efficient rescue of the ida7 mutation . The second unexpected result was the frequency with which we recovered motile transformants expressing only NH 2 -terminal fragments of the 1α Dhc . Northern and Southern blot analyses have shown that the rescued strains retained the 5′ sequence elements required for regulated expression of the Dhc1 transcripts, but several of these strains lacked the 3′ end of the transgene . Therefore, the truncated 1α Dhcs represent those NH 2 -terminal fragments that were competent to assemble with other subunits into the I1 complex . The observation that none of the 1α Dhc fragments is smaller than ∼143 kD may indicate that this is the shortest NH 2 -terminal fragment capable of complex assembly. Studies of outer arm mutants have identified a novel β Dhc mutation with similar properties. The oda4-s7 mutant expresses a 160-kD fragment of the β Dhc that is capable of coassembly with other outer arm subunits at the correct axoneme location . Likewise, low level expression of cytoplasmic Dhc constructs in Dictyostelium indicates that a 158-kD NH 2 -terminal fragment is also capable of complex assembly . Although the NH 2 -terminal third of the 1α Dhc is the most variable region, secondary structure programs have identified a region just before P1 that is predicted to form a limited coiled-coil domain . This domain, which has been identified in nearly all Dhcs sequenced to date , has been proposed as a potential region that might mediate interactions between the Dhcs and their associated ICs and LCs. To determine if this coiled-coil domain is required for assembly of the I1 complex, we recovered the Dhc1 transgene from the G3 transformant, which expresses the shortest 1α Dhc fragment . Sequence analysis of the truncated Dhc1 transgene demonstrated that the 1α Dhc sequence terminates before the region predicted to form the NH 2 -terminal coiled-coil domain . Given that this 1α Dhc fragment still assembles with other I1 components into the flagellar axoneme , other sites within the NH 2 -terminal region must be required for complex formation. We plan to analyze additional Dhc1 constructs to further delineate the domains required for specific subunit interactions and complex assembly. If the Dhc1 transgenes were deleted randomly from the 3′ end, we would expect to recover a broad distribution of Dhc fragments ranging in size from the minimum required to assemble the I1 complex to nearly full-length. Therefore, why are almost all of the 1α Dhc fragments smaller than ∼217 kD ? One possibility may be that larger 1α Dhc fragments are unstable and prevent assembly of the I1 complex into the axoneme. Studies in Dictyostelium have shown that constructs of cytoplasmic Dhc lacking significant portions of the COOH terminus are expressed poorly as compared with other constructs that contain the entire motor domain . Alternatively, the presence of larger 1α Dhc fragments with partial motor domains may inhibit flagellar motility. If so, we would not recover such transformants in our screen, which was based on the rescue of a motility defect. Indeed, Northern blot analysis of the two transformants (A2 and G9) that assemble the largest 1α Dhc fragments demonstrates that the sequences encoding the dynein motor domain are not present in the associated Dhc1 transcripts . The NH 2 -terminal regions of these larger 1α Dhc fragments must, therefore, be fused to other protein sequences. Interestingly, the absence of motile transformants with partial motor domains is consistent with previous reports that nucleotide binding by the cytoplasmic Dhc is inhibited by deletion of the COOH terminus, leading to the formation of rigor complexes . Structural studies of the isolated I1 complex by negative staining or rotary shadowing have shown that it is a two-headed isoform but the physical relationship between the globular heads seen in vitro and the structural domains identified in situ has been unknown. Because some of the Dhc1 transformants assemble I1 complexes that lack central and COOH-terminal regions of the 1α Dhc , we can now identify the position of the 1α motor domain within the I1 structure. EM analysis of axonemes isolated from the E2 and G4 transformants has revealed that lobe 2 of the I1 structure corresponds to the missing 1α motor domain . Lobe 2 is close to the first radial spoke, in a position that may permit direct signaling between the radial spoke and the 1α Dhc motor domain. We predict that the remaining two lobes of the I1 structure represent the positions of the 1β Dhc motor domain and stem region containing the I1 ICs and LCs respectively. We are currently transforming other I1 mutants with the genes for other I1 subunits to identify the polypeptide components that are located within these structural domains . The recovery of transformants missing only the 1α Dhc motor domain but containing the other components of the I1 complex has allowed us to analyze the specific role of the 1α Dhc motor domain in flagellar motility. I1 mutants lacking both the 1α and 1β Dhcs have a slow, smooth swimming phenotype with an altered flagellar waveform . Measurements of swimming velocities reveal that the Dhc1 transformants with truncated 1α Dhcs swim faster than I1 mutants but slower than control strains containing both Dhcs ( Table ). The 1α Dhc motor domain, therefore, contributes directly to force production during motility. The I1 complex is also an essential component of the phototaxis response in Chlamydomonas . Strains that have defects in outer dynein arms, the dynein regulatory complex, or other inner arm isoforms can phototax, but mutants lacking the I1 complex cannot . Analysis of other phototaxis mutants that retain the I1 complex reveals that the phosphorylation state of IC138 is altered . In vitro sliding assays have shown that the phosphorylation state of the IC138 affects microtubule sliding velocities . These results suggest a model in which the phosphorylation state of the IC138 modulates the activity of the I1 Dhc motor domains. To assess the specific role of the 1α Dhc motor domain in phototaxis, we compared the swimming behavior of the Dhc1 transformants to that of control cells in response to a directional light source. pf28 cells were clearly phototactic, but the Dhc1 transformants with truncated 1α Dhcs remained uniformly dispersed during the time course of our assays. These observations indicate that the motor activity of the 1α Dhc contributes to phototaxis, at least in the absence of the outer arms. However, if the outer arms were present, the Dhc1 transformant strains could undergo phototaxis, whereas I1 mutant strains could not ( Table ). This difference in behavior in the presence or absence of outer arms suggests that there are cooperative interactions between the I1 complex and the outer arms during the phototaxis response. Previous studies have demonstrated that differences in the activity of the cis and trans flagellum are the basis of the phototaxis response . This differential activity includes both differences in beat frequency between the two flagella as well as differences in flagellar waveforms . The outer dynein arms are responsible for generating the differences in beat frequency observed between cis and trans flagella, as the cis-trans frequency differential is lost in mutants that lack the outer arm or the outer arm α Dhc . More recent work has demonstrated that this differential beat frequency depends on the presence of the docking structure that facilitates the attachment of the outer arm to its specific binding site on the A-tubule . The differential in beat frequency is not essential for the phototaxis response , as outer arm mutant cells such as pf28 are capable of phototaxis ( Table ). However, it is clear that the cis-trans differences in beat frequency and flagellar waveform must be coupled in some way, because phototaxis mutants such as ptx1 are defective in both . In this context, it appears that regulation of the I1 complex is important for generating the asymmetries in flagellar waveform between the cis and trans flagella that contribute to phototaxis , whereas regulation of the outer arm contributes to the differential in flagellar beat frequency . Phototaxis can occur in the absence of the outer arms, but not the I1 complex . However, if both the outer arms and part of the I1 complex are present (such as in G4+OA or E2+OA), then their combined activity can apparently compensate for the absence of the 1α Dhc motor domain. These observations raise several interesting questions about the mechanism by which changes in the phosphorylation state of IC138 might contribute to phototaxis. For example, where is IC138 located in the axoneme relative to the motor domains of the two I1 Dhcs and the three outer arm Dhcs? Is it in lobe 3 of the I1 structure, which is also in close proximity to at least one outer arm per axoneme repeat? Does IC138 interact directly with either the 1α or 1β Dhc? We are planning to address these questions by analyzing subunit interactions within the I1 complex. Other important questions concern the identity and location of the axonemal kinases and phosphatases that modulate the phosphorylation state of IC138. Work from other laboratories has indicated that many of these regulatory components are tightly bound to the flagellar axoneme , but the specific enzymes that act on either the outer arm or the I1 IC138 in situ have not yet been identified. The future identification and localization of these regulatory components will provide new insights into the pathway that governs the activity of the multiple dynein motors during flagellar motility and phototaxis. | Study | biomedical | en | 0.999999 |
10459016 | A 6.5-kb BamHI fragment was isolated from a λDASHII phage containing 16 kb of the mouse villin gene (kindly furnished by G. Tremp, Rhône Poulenc RORER) and subcloned in pBS/KS+ (Stratagene). The neomycin resistance gene (pMC1neo; Stratagene) was introduced in a unique KpnI site in villin exon 2. This disrupts the open frame reading of the villin gene. The neo cassette was placed in the reverse transcriptional orientation compared with the villin gene. The construct contained 3.5 and 3 kb of homology regions, 5′ and 3′, respectively. In addition, the Herpes simplex virus (HSV) thymidine kinase expression cassette was inserted in the unique ClaI site flanking the 5′ end of the construct. This resulted in the p villin neo targeting construct. The CK35 ES cell line was cultured in DMEM (GIBCO BRL) supplemented by 1 mM Na-Pyruvate, 15% FCS (Techgen International), 1,000 U/ml LIF (ESGRO; GIBCO BRL), and 50 mM β-mercaptoethanol (GIBCO BRL) as described . 2 × 10 7 CK35 ES cells were electroporated with 20 μg of the p villin neo targeting construct linearized in the plasmid backbone (PvuI site). G418 (300 μg/ml) was added 36 h after plating for 12 d. Gancyclovir (2 μM) was added with G418 from day 2 to day 8 of selection. The G418-resistant clones were isolated and their genotype analyzed by Southern blot. Chimeric mice were generated by microinjection of the targeted ES cells into C57Bl6 blastocysts as described . Crosses between chimeric male and C57Bl6/DBA2 females generated heterozygous animals which were then intercrossed to generate homozygous animals. The following experiments were performed in sibling villin-null mice and wild-type animals with the same mixed genetic background. The mice were killed with an intraperitoneal injection of a lethal dose of pentobarbital, or anesthetized when necessary with a 50 μl/10 g body wt of a mixture of 750 μl xylazine (Rompun™ 2%; Bayer), 6 ml Imalgene™ (Rhône Merieux), and 300 μl Flunitrazepan (20 mg in 5 ml 100% ethanol; Sigma Chemical Co.) dissolved in 12 ml of PBS. The abdominal cavity was opened and the blood sample was obtained from aorta in heparinized tubes for further centrifugation and plasma-obtaining or EDTA-containing tubes (Hémo Kit 200; Melet Schoesing Laboratories) for blood cell counting. The intestine was then isolated and its length measured. Schematically, the intestine was divided in three identical parts in length corresponding to the duodenum, jejunum, and ileum. The proximal large intestine was isolated near the caecum and the distal part close to the rectum. The intestinal tube was washed with PBS containing 1 mM CaCl 2 , 1 mM MgCl 2 . Kidney was sampled and frozen in liquid nitrogen. Brush border membranes were prepared from mucosa freshly scraped from the whole small intestine. The mucosa was diluted in 10 vol/mg wt with buffer A (10 mM imidazole, 5 mM EDTA, 1 mM EGTA, pH 7.4, 0.2 mM DTT, 200 μg/ml Pefabloc, 1 μl/ml of a mixture of protease inhibitors: 1 μg/ml antipapaine, 1 μg/ml pepstatine, 15 μg/ml benzamidine) (Sigma Chemical Co.) and stirred at 4°C for 1 h. A mechanical cellular disruption was then obtained with 5 strokes in a Dounce homogeneizer (model S852; Braun). After centrifugation (10 min, 4°C, 1,000 g ), the pellet was washed 3 times with 10 ml buffer A followed by centrifugation. At this step, two procedures were carried out: either the crude membranes were directly used for studying the Ca 2+ effect (see below) or brush border membranes were purified in a sucrose gradient. For the latter procedure, the pellet was resuspended in 10 ml buffer B (75 mM KCl, 5 mM MgCl 2 , 1 mM EGTA, 10 mM imidazole, pH 7.4, and similar protease inhibitors to those in buffer A) and then mixed with a sucrose solution (40% sucrose final concentration in buffer B). This sample was collected in a tube containing the same volume of 65% sucrose (in buffer B), and centrifugation was performed at 4°C, 30 min, 15,000 g . The purified brush border membranes were obtained at the interface of the 40%:65% sucrose gradient. The protein content was determined and normalized with the initial weight of scraped mucosa. In some experiments, brush border crude preparation was incubated for 10 min in a solution containing 10 −5 to 10 −3 M CaCl 2 in imidazole buffer (10 mM, pH 7.4, without EGTA and EDTA, plus protease inhibitors similar to those in buffer A). Microscopic observation was immediately performed using Nomarski optics (DMRD) or transmission electron microscopy after fixation (see below). DNA from embryonic stem (ES) cell pellets or mouse tails was extracted in a lysis buffer (50 mM Tris, pH 7.5, 0.1 M NaCl, 0.5% SDS, 5 mM EDTA, 100 μg/ml proteinase K), and analyzed by Southern blots or PCR. Southern blots were performed using DNA digested with HincII or Sca1 restriction enzymes and analyzed with a 0.4 kb BamHI-HincII fragment , and a 0.5-kb BglII-StuI fragment (data not shown) as 3′ and 5′ external probes, respectively. PCR analysis was performed using DNA in 50 μl for 30 cycles. Each cycle consisted of 60 s at 94°C, 60 s at 67°C, and 90 s at 72°C, using the 0.5 μl of Taq polymerase (Quantum Biotechnologies, Inc.) in a homemade buffer (67 mM Tris HCl, pH 8.8, 16.6 mM [NH 4 ] 2 SO 4 , 0.45% Triton X-100, 0.2 mg/ml gelatin, 2 mM MgCl 2 ). 2 pmol of 5′-GTCAAAGGCTCTCTCAACATCAC-3′ villin sense oligonucleotide (primer 1), 1 pmol of 5′-GACTACATAGCAGTCACCATC-3′ villin antisense oligonucleotide (primer 3), and 1 pmol of 5′-TCTGGATTCATCGACTGTGGC-3′ neo antisense oligonucleotide (primer 2) were used, generating a 0.9-kb fragment for the wild-type allele and a 1.2-kb fragment for the targeted allele . Total RNA was extracted from different tissues using RNA Now kit (Ozyme). For Northern blot analysis, RNA samples (10 μg) were separated on 4.2% formaldehyde-agarose gel and blotted onto nylon membrane (Amersham). The 32 P-labeled probe was synthesized with TransProbe T kit (Pharmacia Biotech). The RNA probe used corresponded to 530 bp of the 3′ end of the smaller human villin mRNA . For reverse transcription PCR analysis, 10 pmol of 5′-TCCAGCCAGCACATTCCTCTTCCC-3′ was hybridized with 2 μg of total RNA at 70°C for 10 min in distilled water. Reverse transcription with 200 U of Moloney murine leukemia virus reverse transcriptase (SuperScript II; Life Technologies, Inc.) was carried out at 37°C for 60 min in a 20 μl solution of 1× First Strand Buffer (Life Technologies, Inc.), 10 mM DTT, 0.5 mM deoxynucleoside triphosphates, and 0.4 U/ml RNasin. 3.5 μl of the resulting cDNA was amplified by PCR reaction in 50 μl for 35 cycles. Each cycle consisted of 60 s at 94°C, 60 s at 55°C, and 60 s at 72°C. 10 pmol of primers, 5′-ATGCCCAAGTCAAAGGCTCTCTCAACATCAC-3′ coding strand and 5′-TGCAACAGTCGCTGGACATCACAGG-3′ noncoding strand, was used, generating a 400-bp product. Proteins were separated by standard gel electrophoresis and then transferred to nitrocellulose membrane (0.2 μm; Schleicher and Schuell). Immunostaining was performed using primary antibodies visualized by peroxidase-conjugated antiimmunoglobulin antibodies (Jackson ImmunoResearch Laboratories, Inc.) or by alkaline phosphatase–conjugated antiimmunoglobulin antibodies (Promega). Quantification analysis (Vilbert Lourmat) was performed by scanning the membrane comparing the signal obtained with that obtained with anti-tubulin antibody (Amersham) for tissue and scraped mucosa or with anti–β-actin antibody (Sigma Chemical Co.) for brush border preparation. To perform histological analysis, organs from wild-type and mutant mice were dissected, fixed in formalin (10% vol/vol in PBS), ethanol dehydrated, and paraffin embedded. Sections of 5-μm width were prepared and stained with hematoxylin eosin–safranin according to standard histological procedures. Immunofluorescence studies were performed in cryostat sections of OCT-embedded tissues or in paraffin-embedded sections for fimbrin isoform immunodetection. 5-μm sections were prepared and fixed in 3% paraformaldehyde. After washing in PBS, permeabilization was obtained with 0.2% Triton X-100. Sections were incubated for 40 min with primary antibodies and 2% BSA, and after washing for 30 min with secondary fluorescent antibody. Control sections were obtained in the absence of primary antibody. The different antibodies that have been tested are as follows: villin , actin (phalloidine, dilution 1:3,000; Sigma Chemical Co.), ezrin , fimbrin (specific antibodies against the three isoforms of fimbrin, I- T- and L-fimbrin, have been produced in rabbit and then purified by affinity; our unpublished data), brush border myosin I (BBMI) (rabbit polyclonal, dilution 1:10; kindly provided by P.T. Matsudaira, Massachusetts Institute of Technology, Cambridge, MA), espin (rabbit polyclonal, affinity purified, dilution 1:200; kindly provided by J.R. Bartles, Northwestern University Medical School, Chicago, IL), E-cadherin (DECMA, rat monoclonal, dilution 1:100; Sigma Chemical Co.), sucrase isomaltase , dipeptidylpeptidase IV , neutral aminopeptidase (CD13, rat ascite, dilution 1:300; kindly provided by P. Auberger, Contrat Jeune Formation, Nice, France), alkaline phosphatase (rabbit polyclonal, affinity purified, dilution 1:500; our unpublished data), carcinoembryonic antigen (mouse monoclonal, not diluted; kindly provided by J.-L. Teillaud, Institute Curie). Small pieces of tissue (∼1–2 mm) were fixed for 2 h at room temperature in 2.5% glutaraldehyde and 2% paraformaldehyde in 80 mM cacodylate buffer, pH 7.2, 0.05% CaCl 2 . After washing with the same buffer, the tissue was postfixed for 30 min at 4°C with 1% osmium and 1.5% potassium ferrocyanure in 80 mM cacodylate buffer, pH 7.2, and then at room temperature for 1 h with 2% uranyl acetate in 40% ethanol. The samples were dehydrated in a series of graded ethanol solutions and then embedded in Epon. For brush border preparations, the procedure has been described previously . In brief, the brush border membranes were fixed for 30 min at room temperature in 0.1 M phosphate buffer, pH 7.0, with 3% glutaraldehyde and 0.2% tannic acid. Washing was performed for 10 min at room temperature with 0.1 M phosphate buffer, pH 7.0, containing 10% sucrose. Postfixation was done for 1 h at 4°C in 1% osmium in 0.1 M phosphate buffer, pH 6.0. The samples were then washed three times in water and processed as above. Silver sections were contrasted and then observed with CM120 Twin microscope (Philips). Semithin sections (0.2 μm) were stained with toluidine blue and were examined with a Leitz (DMRD) microscope. Images showing transverse sections through the microvilli and having a magnification of 22,000 were selected. Negatives were scanned on an Arcus II scanner (Agfa) to give a pixel size of 1.3 nm on the digitized image. All images were divided into boxes of 1,024 × 1,024 pixels to be processed using NIH image 1.60 (44 boxes for 8 wild-type animals, and 83 boxes for 12 villin-null mice). The Fourier transform and the power spectrum of each square area were calculated. Fourier analysis has allowed us to classify the specimens in three major categories (nonordered, slightly ordered, and well-ordered specimens) according to the characteristics of the power spectra of the transverse sections. About 1 cm of intestine was fixed as described above for transmission electron microscopy. The intestine was then opened and fixed, luminal side up, on pieces of cork. Dehydration was then performed in a series of graded ethanol solution. The samples were dried by the critical point method using liquid CO 2 and coated with gold palladium. Observation was performed with a scanning electron microscope (Jeol JSM 840A). Three series of experiments were conducted. After mouse anesthesia, a jejunum loop was in situ isolated, taking care not to injure the local vasculature. Drugs were applied either from the apex or from the basolateral side of the intestine. For the apical application of drugs, the intestinal loop was washed with PBS without Ca 2+ and Mg 2+ . Then, ∼700 μl of a solution of ionophore A23187 (10 μM [Sigma Chemical Co.] diluted in PBS) was injected in the lumen of the intestine (∼3 cm) between two clamps, for 10 min. After washing with PBS, this loop was separated in two parts with a surgical clamp, and only the lower part was injected with ∼300 μl of PBS containing 2 × 10 −4 M CaCl 2 for 30 min. This experiment was repeated in five wild-type and five villin-null mice. In separate animals, thapsigargin (1 mM; Sigma Chemical Co.) or ATP (1 mM; Sigma Chemical Co.) was injected in an isolated segment of the jejunal loop for 30 min. For each animal, a segment infused with only PBS served as control. For the basolateral infusion of drugs, the isolated loop was carefully placed in a Petri dish containing 1 or 10 μM carbachol (Sigma Chemical Co.) in PBS plus Ca 2+ for 20 min. This experiment was repeated in two wild-type and two villin-null mice. At the end of the experiment, the animal was killed with a lethal dose of anesthetic. The intestinal loop treated with the drug or PBS was cut and prepared as described above for transmission electron microscopy and immunofluorescence study. Experiments were performed in six awake animals (three wild-type and three mutants) after 24 h fasting with normal drinking. Then, a 60-min refeeding period was observed. The mice were then killed and the intestine was removed as described above for immunohistochemistry analysis. DSS (2.5%, wt/vol, mol wt 40,000; ICN Biomedical) was administered in the drinking water of 27 wild-type mice and 26 mutant mice for 13 consecutive days (total numbers for 5 independent experiments performed in parallel, with ∼ 5 wild-type and 5 mutant mice in each series). This procedure is known to induce colic epithelial injury . Survival of the animals was surveyed during a period of 13 d. The remaining living animals were killed at day 13, and the small and large intestines were sampled as described above for histological analysis. The survival curves were analyzed by Kaplan Meier transform of probability versus days of DSS treatment. A P value <0.05 is considered as significant. 1 of 150 G418-gancyclovir–resistant ES cell clones underwent homologous recombination at the villin locus as determined by Southern blot analysis . Hybridization with a neo probe failed to detect any additional sites of integration (data not shown). The targeted clone was injected into host C57Bl6 blastocysts, and the blastocysts were transferred to the uteri of pseudopregnant females. Germline transmission of the mutant allele was achieved with the targeted ES clone. The heterozygous mice did not display any obvious abnormalities in comparison with their wild-type littermates. To examine whether animals homozygous for the villin mutation were viable, heterozygous animals were intercrossed and genotypes of the progeny were determined by Southern blot or by PCR analysis. Mice homozygous for the villin mutation were detected among the intercross progeny . The genotypes of the progeny showed a good fit to Mendelian distribution (+/+: 84, 27%; +/−: 148, 48%; −/−: 74, 24%). Homozygous villin-deficient mice were indistinguishable from their heterozygous or wild-type littermates on the basis of size, activity, fertility, or aging. When interbred, villin-null females had litter sizes similar to their wild-type littermates. Animals were followed for as long as 2 yr and no obvious pathology developed. To verify the absence of villin protein in homozygous mice, we examined the small intestine, colon, and kidney. By immunofluorescence, no staining was observed in the null mutant mice contrasting with an apical brush border staining in the wild-type animals . Western blot analysis of the same tissues was performed using polyclonal antibodies which recognize either the head piece COOH-terminal domain or both this domain and the core of villin. No villin protein was detected in the mutant mice, whereas a significant amount was present in wild-type animals . By Northern blot analysis and reverse transcription PCR, no villin mRNA was detected in the homozygous animals. No aberrant transcripts could be detected (data not shown). Plasma values did not reveal any major differences between homozygous and wild-type animals (Na concentration: 147 mM ± 1.8, n = 17, and 145 mM ± 0.8, n = 10; glucose concentration: 14.9 mM ± 1.87, n = 7, and 14.3 mM ± 1.34, n = 5; creatinine concentration: 20.6 μM ± 3.79, n = 5, and 19.7 μM ± 3.14, in wild-type and villin-null mice, respectively, means ± SEM). This observation suggests, in a first approximation, that the transport functions of intestine and kidney were unaffected under basal conditions. This hypothesis could be indirectly correlated to the normal distribution in the intestinal epithelial cells of the major digestive enzymes. Indeed, the different enzymes—sucrase isomaltase, dipeptidylpeptidase IV, neutral aminopeptidase, and alkaline phosphatase—that have been studied by immunofluorescence have a normal localization in both wild-type and villin-null mice (data not shown). Careful examination of histological sections and of scanning electron microscopy pictures of the different parts of intestine, duodenum, jejunum, ileum, and proximal and distal colon did not detect any differences in histological appearance between wild-type and mutant mice (data not shown). At the cellular level, transmission electron microscopy revealed no difference on brush border microvilli structure in homozygous mice compared with wild-type . Large heterogeneity in the length of the microvilli was observed in both wild-type and mutant mice. Fourier analysis was used as a quantitative method to describe the long range organization of the microvillus array. Representatives of all the defined categories, nonordered, slightly ordered, and well-ordered specimens (see Materials and Methods), indicating intra- and interindividual variations in the organization of the microvilli, were found in both wild-type and homozygote animals with a similar median. The percentage of the well-ordered crystalline lattices was similar for homozygote (34/83, 41%) and wild-type (14/44, 32%) mice (χ 2 test, P > 0.5). These crystalline lattices were characterized by the distance between two neighboring microvilli (a and b) and the lattice angle between these directions (γ) . The mean values are a = 119 ± 1.4 nm (mean ± SEM), b = 109 ± 1.8 nm, and γ = 58 ± 1.6° in wild-type animals (14 sections, n = 8), and a = 114 ± 2.1 nm, b = 107 ± 1.8 nm, and γ = 59 ± 0.92° in mutant animals (34 sections, n = 12), indicating no obvious difference ( t test, P < 0.05) in microvilli organization between these animals. To examine whether other actin-binding proteins take over the bundling activity of villin, protein extracts from purified brush border preparation and immunofluorescence staining of intestine sections were performed. Semiquantitative Western blot analysis of the three fimbrin isoforms (I, T, and L), espin, and ezrin as well as immunofluorescence staining (fimbrin isoforms, espin, ezrin, and BBMI) did not reveal any major difference between wild-type and homozygous animals. However, a slight increase in the labeling intensity of espin was systematically noticed in the mutant mice. This observation could be related to a better accessibility to espin epitopes in the absence of villin. Indeed, semiquantitative analysis of Western blot performed on brush border preparation failed to demonstrate any increase in espin concentration in villin-null mice. These results indicate that these known actin-binding proteins of the brush border microvilli are neither overexpressed nor downregulated in the absence of villin. To examine Ca 2+ -dependent severing of actin in villin-null mice, both in vitro and in vivo approaches were undertaken. In vivo, pharmacological, physiological, and pathological conditions were explored. In vitro, when brush borders were isolated in the absence of Ca 2+ and purified on sucrose gradient, a marked difference was observed between wild-type and mutant mice. On average, a threefold increase in the yield of brush border recovery, calculated as the ratio of final protein content over initial mucosa weight, was obtained in the villin-null mice compared with wild-type. This observation suggests that the mutant mice brush borders were less fragile to mechanical cell disruption. Both wild-type and mutant mice presented a tightly packed array of microvilli and a well-conserved ultrastructure with a normal actin filament bundle . The addition of Ca 2+ in wild-type preparation (final concentration 10 −5 to 10 −3 M) resulted in a rapid alteration of brush borders where the microvillar core disassembled with the concomitant vesiculation of the surrounding membrane. In contrast, the morphology of mutant mouse brush border remained unchanged in the presence or absence of Ca 2+ as assessed by both optical Nomarski observation and transmission electron microscopy. These results demonstrated that, in the absence of villin, no fragmentation activity upon actin filaments occurred in response to increased Ca 2+ concentrations. In vivo, in pharmacological experiments, jejunum loops isolated in situ were used to test the effect of Ca 2+ . Different strategies known to increase intracellular Ca 2+ concentration were monitored using ATP, carbachol, an acetylcholine agonist, Ca 2+ ionophore A23187, or thapsigargin, a blocker of the endoplasmic reticulum Ca 2+ ATPase. In the brush border from wild-type small intestine, the major effect observed with each of these conditions was a disruption of the apical F-actin phalloidin labeling, as illustrated in Fig. 4C and Fig. E , using 10 μM carbachol basolateral treatment in comparison to the intense and broad F-actin labeling observed in the untreated wild-type animal . Only thin phalloidin labeling remained at the apical pole, probably related to the terminal web region; the basolateral F-actin labeling was unaltered. Slight differences were noticed using the various conditions to increase intracellular Ca 2+ concentration: carbachol basolateral treatment affected the apical labeling all along the villus, whereas the others drugs, administered in the lumen of the intestine, altered mostly the upper part of the villus, probably due to differences in accessibility of the drug. In these animals, the villin labeling was unaffected . In contrast, in the villin-null mice, whatever conditions were used to increase Ca 2+ , the phalloidin F-actin labeling was unaffected: a large continuous F-actin ribbon was observed along the villi similar to that observed in both control wild-type and villin-null mice . No significant alteration of the microvilli structure was observed by transmission electron microscopy on fixed tissues. This discrepancy between electron and light microscopy observations may be due to the different procedures and processing of the specimens (i.e., fixatives and freezing). Alternatively, rapidly reversible effects may have occurred, as it has been shown in primary culture of proximal tubule for the Ca 2+ -dependent effect of parathormone . To assess this hypothesis in our in vivo conditions, extensive kinetics studies have to be performed in a large series of animals. In vivo, in a physiological fasting/refeeding condition, most of the apical phalloidin labeling disappeared in wild-type animals, whereas in villin-null mice, it remained unaffected . This alteration affected the whole length of intestinal villi similarly in the three animals. In pathological experiments where colitis was induced by DSS administration, although some variation was observed in the series of five experiments, villin-null mice appeared to be significantly more sensitive to the injuring effect of the DSS treatment. This is illustrated by the fact that 64% of mutant animals are dead at day 13 in contrast to only 30% of wild-type animals . The median survival time was 10 d for the mutant mice; this parameter cannot be defined for the wild-type animals. Multiple and extended sites of epithelial injury were conspicuously found in the villin-null colons compared with wild-type animals . These lesions included obvious large ulcerations, glandular atrophy, and inflammatory changes including neutrophil infiltrate, edema, and fibrosis . In contrast, the wild-type colon lesions were less severe and limited to focal regions . No alteration was observed along the small intestine. Villin is one of the major structural actin-binding proteins associated with the actin cytoskeleton of the intestinal epithelial cell brush border microvilli. Among the actin-binding protein family, villin is the only member which has severing, capping, nucleating, and bundling in vitro activities. In vivo, the search for these properties needs to be performed at the level of the tissue, organ, or organism. In this study, we have addressed the biological role of villin by generating mutant mice lacking villin through homologous recombination in ES cells. In the villin-null mice, the ultrastructure of the intestinal brush border is not modified, suggesting normal bundling of the actin filaments. The other proteins expressed in the brush border (fimbrin, espin, BBM1, ezrin) are not overexpressed to compensate for the absence of villin. Interestingly, however, the severing properties of villin are not compensated in vivo, as demonstrated by the absence of actin fragmentation when the intracellular Ca 2+ concentration was increased in mutant animals. Villin contains at least three actin-binding sites, two of which are Ca 2+ dependent and located in the core domain. The third is situated in the head piece domain and is Ca 2+ independent. The in vitro activities of villin upon actin vary with the Ca 2+ concentration . At high Ca 2+ concentration (>10 −4 M), villin severs F-actin into short filaments. At lower concentration (10 −7 to 10 −6 M), villin caps the fast-growing end of actin microfilament and thereby prevents elongation. At the same Ca 2+ concentration, villin nucleates microfilament growth when added to actin monomers. At very low Ca 2+ concentration (<10 −7 M), villin has no effect on actin polymerization but bundles actin filaments. In fibroblast cell culture, synthesis of large amounts of villin in cells which do not normally produce this protein induces both the growth of microvilli on the cell surface and the redistribution of F-actin. Villin lacking one actin-binding domain located at its COOH-terminal end did not induce growth of microvilli or stress fiber disruption . Moreover, it has been demonstrated that a cluster of charged amino acid residues (KKEK) is crucial for the morphological activity of villin, indicating that this motif is part of an F-actin binding site that induces G-actin to polymerize . Similar bundling properties of villin were demonstrated with a different approach using an antisense mRNA strategy in a colonic adenocarcinoma cell line. Indeed, stable expression of a cDNA encoding antisense villin RNA and thus downregulation of endogenous villin messenger induced dramatic brush border disassembly . The key role of villin in the morphogenesis of microvilli has not been verified in vivo . Indeed, both the length and the structural organization of the microvilli, in the null mouse intestine and colon are indistinguishable from those in wild-type animals. Even a sophisticated mathematical analysis of the microvilli organization such as Fourier analysis fails to demonstrate any significant difference. However, the heterogeneity observed in both types of animals may have prevented us from detecting significant differences. Nevertheless, because of the large number of animals studied in this paper and in one published previously , we believe that had they existed, structural alterations should have been detected. The discrepancy between the in vivo studies and cell culture experiments is presumably due to the adaptation and plasticity of gene expression during organogenesis in vivo, properties that are lost or inefficient in in vitro systems. Other actin-binding proteins are expressed in the brush border of epithelial cells (I isoform of fimbrin, BBMI, ezrin, and espin). Fimbrin is required, with villin, to bundle actin filaments in vitro . Three fimbrin isoforms have been demonstrated with relatively specific cell expression: I in intestine and kidney epithelial cells, L in hemopoietic cells and many tumor cell lines, and T in various cells and tissues . In villin-null mice, I fimbrin exhibited a cellular distribution and a semiquantitative expression similar to those observed in wild-type animals. No expression or labeling of the T and L isoforms was observed in the brush border. In addition, no modification was noticed for espin, another actin-binding protein localized in the intestinal epithelial cells for which an actin bundling function has been demonstrated. The expression and distribution of the other actin-binding proteins present in the brush border (ezrin and BBMI) were also not modified. They do not bundle actin filaments but are involved in the link between the brush border cytoskeleton and the plasma membrane. Ezrin is known to play a structural and regulatory role in the assembly and stabilization of specialized plasma membrane domains . BBMI has been reported to connect laterally the microfilament core with the microvillus membrane . The different members of the gelsolin/villin family of actin regulatory protein, if they are able to bundle actin in vivo as suggested by the presence of homologous COOH-terminal head piece sequences, would be good candidates to compensate for the absence of villin and might explain the normal organization of actin microfilament bundles in villin-null mice. The Ca 2+ -regulated severing properties of villin have been established mainly in vitro. This study is the first report, to our knowledge, that supports a role for the villin-severing activity in vivo. In a normal situation, at the usual low intracellular Ca 2+ concentration, the severing properties of villin should not be effective. However, a local increase of Ca 2+ can occur in some physiological conditions, such as hormonal stimulation. In these conditions, in the cells expressing villin, severing of F-actin microfilaments would be induced, a phenomenon that can be part of the hormonal response. To test the role of villin in physiological or pathological situations, we have used different pharmacological or physiological (fasting/refeeding) strategies, to increase the intracellular Ca 2+ concentration. For each of them, a net decrease of the F-actin microfilament labeling with phalloidin was observed only in wild-type animals, a result that suggests a severing effect of villin. In fasting/refeeding experiments, Ca 2+ concentration should also increase. Indeed, in these conditions, catecholamine and digestive hormones increases are expected, and subsequently induce an increase in the intracellular Ca 2+ concentration. Other reports also support this hypothesis. In the ileum, it has been demonstrated that F-actin fragmentation is necessary for carbachol to inhibit NaCl absorption . The cholinergic agonist carbachol activates basolateral M3 muscarinic cholinergic receptors and then induces a biphasic increase in intracellular Ca 2+ concentration involving a phosphoinositol phospholipase/phosphokinase (PLC/PKC) cascade . The physiological effect of carbachol consists of an inhibition of the NaCl absorption and brush border Na + /H + exchange , presumably as a result of the internalization of the transport proteins (Donowitz, M., personal communication). Another situation in which modifications of intracellular Ca 2+ concentration occurs is during infection by microorganisms or during different conditions that induce cell injury (e.g., acid application, stress, wounding). During the intestinal infections by enteroaggregative or enteropathogenic Escherichia coli (EAEC, EPEC), vesiculation of the brush border has been observed . The increased Ca 2+ concentration induced by the bacterial infection might rearrange the cytoskeleton and activate Ca 2+ -dependent kinase(s) resulting in morphological changes such as microvilli effacement and pedestal formation in the host cells. Damage of the intestinal mucosa has also been investigated by the use of DSS, a chemical agent that induces acute and chronic experimental ulcerative colitis in mice . DSS also causes changes in the intestinal microflora. This alteration, which can be compared with pathological intestinal infection, together with inappropriate macrophage function or toxic effects on colonic epithelium, is a possible mechanism by which enteral DSS induces ulcerative colitis in experimental animals. The observation that the death probability was two times higher in villin-null mice compared with wild-type suggests that villin might be involved in the cell injury processes and/or in the cell repairing processes. The exact role of villin in these processes remains to be further investigated. A working hypothesis is that the binding of villin to F-actin microfilaments plays a key role in the dynamics of the actin cytoskeleton, and therefore it might be a major factor in cell organization and motility. Interestingly, the present result may also have clinical implications. Indeed, the etiology of the human hemorrhagenic colorectitis is still unknown. Considering the large extension of the colon lesions in villin-null mice after DSS, an alteration of villin, such as a villin gene mutation, could be an interesting etiologic hypothesis for this pathology. This study illustrates the complexity of the protein interactions at the cellular, tissue, organ, and animal levels. During mouse development, mechanisms might be used to compensate for the absence of villin and thus to organize well-structured microvilli in intestinal cells. In the adult mouse, this compensation persists and results in an apparently normal intestinal epithelium. We can postulate that fimbrin may play a role to compensate for the bundling activity of villin that leads to obtain normal morphogenesis, whereas no other actin-binding protein could efficiently provide the severing activity necessary for the dynamics of cortical actin microfilaments in enterocytes. This proposal can be evaluated in future experiments by creating villin- and fimbrin-null mice. Moreover, cellular apoptosis that occurred at the upper part of the villi was similar in villin-null mice and in the wild-type animals (data not shown). Thus, the equilibrium between living and apoptotic cells in the intestine is maintained under physiological conditions in the mutant mice. As illustrated in this work, after inducing cell injury by the administration of DSS, the lack of epithelial cell plasticity might lead to dramatic effects. Starvation, unbalanced diet, cell injury, local infections, and carcinogenesis are stresses that might affect the dynamics of brush border F-actin microfilaments. Indeed, it is well established that actin microfilaments are essential to remodel cell shape and to drive cell motility. Hence, external stimuli leading, for instance, to epithelial plasticity and cell repair could be required in a variety of conditions in which villin-null mice might be found deficient in the course of future investigations. | Study | biomedical | en | 0.999998 |
10459017 | Unless stated otherwise, cell culture media and supplements were obtained from GIBCO BRL and reagents were obtained from Sigma Chemical Co. Recombinant vimentin was expressed and purified by published protocols . Mutagenesis was carried out on cloned human vimentin in the pTZ18U vector (Muta-Gene Phagemid In Vitro Mutagenesis kit; Bio-Rad Laboratories). The 102C vimentin clone was generated by the deletion of the sequence encoding the NH 2 terminus of vimentin and by the introduction of a new initiation ATG codon immediately preceding the codon for asparagine-102. For the N410 vimentin clone, a stop codon was introduced after the codon for isoleucine 410. Both constructs were subcloned into the PET-7 vector for expression in Escherichia coli . For fimbrin expression and purification, BL21(DE3) cells were transformed and the cells were plated onto ampicillin plates. Colonies were inoculated into 2 liters of 2× YT media and shaken for 24 h at 37°C. The cells were harvested by centrifugation (4,000 rpm for 30 min) and suspended in 50 mM Tris, pH 8.0, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, and 25% sucrose, and frozen. The cell suspension was thawed, lysed by sonication, and centrifuged at 18,000 rpm for 30 min. The supernatant was dialyzed into 10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 mM sodium azide and loaded onto a DEAE-Sepharose fast flow column (Pharmacia Biotech) preequilibrated with the same buffer. After washing to remove unbound material, the column was eluted with a linear gradient of NaCl (0–300 mM). Peak fractions were pooled, concentrated, and loaded onto a Q-Sepharose column (Pharmacia Biotech) that was equilibrated in 10 mM Pipes, pH 7.0, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 0.1 mM PMSF, and 0.1 mM sodium azide. The protein was again eluted with a linear salt gradient (0–300 mM NaCl), pooled, and loaded onto a Sephacryl S200 HR gel filtration column (Pharmacia Biotech). The purified protein was concentrated on a microconcentrator (30-kD cut-off). Glutathione-S-transferase (GST) fusion constructs were generated by PCR cloning of human L-fimbrin. cDNA regions corresponding to L-fimbrin protein sequences 97-248, 243-385, 97-188, 97-142, 143-188, and 119-160 were amplified using synthetic oligonucleotide primers (Life Technologies, Inc.). A nucleotide tail coding for an in-frame BamHI restriction site was engineered into each 5′ PCR oligo; a nucleotide tail coding for an EcoRI restriction site and a downstream amber stop codon (TAG) was engineered into each 3′ PCR oligo. PCR-amplified DNAs were subcloned, via the engineered BamHI and EcoRI restriction sites, into a GST-vector, pGEX-4T2 (Pharmacia Biotech), directly downstream of the GST transcription region. GST-fimbrin fusion proteins were produced by inducing log phase DH5-α bacteria containing the GST construct with isopropyl-β- d -thiogalactopyranoside for 1.5 h, purifying the recombinant protein with glutathione agarose beads, and concentrating the eluate with a centriprep/centricon concentrator (Amicon). The cells used in these experiments were mouse macrophage cell lines P388D1 (ATCC TIB-63) and IC-21 (ATCC TIB-186). The cell lines were cultured in 85% RPMI-1640 medium, 15% FBS, and antibiotics (50 IU penicillin and 50 μg/ml streptomycin). Cell culture and maintenance techniques were performed as described by American Type Culture Collection. P388D1 cells were grown to confluence (adherent cells), washed once with PBS, scraped off the plate in PBS, and pelleted by centrifugation. P388D1 cells were also grown in suspension (nonadherent cells), washed twice in PBS, and pelleted by centrifugation. The cells were lysed on ice in 10 mM Pipes buffer, pH 6.8, containing 0.5% Triton X-100, 300 mM sucrose, 100 mM KCl, 3 mM MgCl 2 , 10 mM EGTA, 2 mM PMSF, and 50 μM sodium vanadate . After 3 min, the cells were centrifuged at 10,000 rpm for 10 min. Aliquots of the supernatant representing the Triton-extractable fraction were precleared with protein A conjugated to agarose beads. For immunoprecipitation of fimbrin, the precleared supernatants were incubated with different antifimbrin sera (736.5, 738.5, 739.5, and 163.3) or their respective prebleeds for 1 h. For immunoprecipitation of vimentin, the precleared supernatants were incubated with a combination of antivimentin sera (Chemicon International, Inc.; Sigma Chemical Co.). Protein A agarose was added and the extracts incubated for 1 h. The beads were washed four times with buffer A (0.1% Triton-X 100, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) and directly solubilized in the appropriate buffer for one- or two-dimensional analysis. Two-dimensional gel electrophoresis was carried out as described by O'Farrell et al. 1977 . Isoelectric focusing was carried out in the first dimension by using ampholytes pH 3-10 (Bio-Rad Laboratories), and setting a pH gradient from 4–8. Proteins were resolved in the second dimension on a 7.5% SDS–polyacrylamide gel. The antifimbrin complex immobilized on protein A agarose beads was solubilized in SDS-IEF buffer (0.5% SDS, 9.5 M urea, 2% ampholytes pH 3-10, and 5% 2-mercaptoethanol). After 10 min, an equal aliquot of Garrel's buffer (4% NP-40, 9.5 M urea, 2% ampholytes pH 3-10, and 5% 2-mercaptoethanol) was added and incubated at 37°C for 15 min. Samples were loaded at the basic end and allowed to electrophorese for 8,000 Vh. After isoelectric focusing was complete, the IEF gels were equilibrated in SDS sample buffer, and then electrophoresed in the second dimension. The gels were finally silver stained . The silver-stained spots were excised from the gel, washed, and processed as described by Shevchenko et al. 1996 . The protein was reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The gel piece was finally dried in a Speed-Vac and rehydrated at 4°C in digestion buffer (50 mM NH 4 HCO 3 ) containing trypsin (Boehringer Mannheim). Excess trypsin was removed and the gel incubated overnight at 37°C. Digested peptides were extracted with 5% formic acid and 50% acetonitrile. The pooled extracts were dried, dissolved in 5 μl of 5% formic acid and 50% acetonitrile, mixed in a 1:1 ratio with either α-cyano-4-hydroxy-trans-cinnamic acid or 3,5 dimethoxy-4-hydroxy cinnamic acid (Aldrich Chemical Co.), and then spotted onto the target plate. All mass spectra were obtained on the Voyager Elite mass spectrometer (DE-STR; PerSeptive Biosystems) operated in the reflector mode. A list of monoisotopic peptide masses was imported into PepFrag and MS-Fit that searched the Swiss-Prot or GenPept databases. The following parameters were used in searches: cysteines were modified by aminocarboxymethylation, proteolytic digestion with trypsin (3–4 incompletes), mass tolerance of 1 Da, and molecular weight range of 10 kD. In other experiments, proteolytic fragments of fimbrin and vimentin were identified by microsequencing of electroblotted fragments following previously published protocols . Peptides were sequenced using a microsequencer (model 477A; Applied Biosystems). The fimbrin and vimentin concentrations were obtained by measuring absorbance at 280 nm and by SDS-PAGE. Protein was mixed with Sulfo-NHS-Biotin (Pierce Chemical Co. ) at a molar ratio of 1:20 and coupled following the supplier's protocol. Unreacted biotin was removed and the derivatized protein concentrated on a 30-kD cut-off microconcentrator (Amicon). Typically, 100% of the molecules were derivatized as assayed by binding to streptavidin-agarose. The overlay binding assay was performed essentially as described by Merdes et al. 1991 . Electrophoresis of protein samples was performed in duplicate; one of the gels was stained and the other electroblotted onto polyvinylidene difluoride (PVDF) (Millipore Corp.). The blot was blocked with 1% nonfat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% Tween 20) for 1 h. For the fimbrin overlay assay, the blot was incubated overnight with labeled fimbrin in 1% nonfat dry milk in TBST. For the vimentin overlay assay, the blot was incubated overnight with labeled vimentin in 1% nonfat dry milk in a low ionic strength buffer (8.03 mM NaH 2 PO 4 , 1.47 mM KH 2 PO 4 , 0.2 mM CaCl 2 and 1% Triton X-100, pH 7.2). The blot was washed with TBST, incubated for 1 h with the ABC elite vector reagent (Vector Labs, Inc.), washed again with TBST, and finally developed using the ECL kit (Amersham). Purified vimentin was purchased (Cytoskeleton), whereas recombinant fimbrin and vimentin were expressed and purified as described above. Vimentin was kept in an unpolymerized state by disassembling filaments in 8 M urea, 5 mM sodium phosphate, pH 7.2, 0.2% 2-mercaptoethanol, and 1 mM PMSF. The sample was dialyzed overnight against 5 mM sodium phosphate, pH 7.4, containing 0.2% 2-mercaptoethanol and 0.2 mM PMSF. The protein concentration was adjusted to ∼1 mg/ml and incubated with precleared fimbrin (50 μg each) at 37°C for 1 h in 1 ml of low ionic strength buffer containing 8.03 mM NaH 2 PO 4 , 1.47 mM KH 2 PO 4 , 0.2 mM CaCl 2 and 1% Triton X-100, pH 7.2. Antifimbrin or preimmune serum was added and the proteins were incubated for an additional 1 h. Protein A–Sepharose was added for 1 h and the beads were finally washed three times with buffer A and directly solubilized in SDS–sample buffer. The samples were resolved on SDS-PAGE, electroblotted onto a PVDF membrane, and probed with an antivimentin HRP-linked antibody (Affinity Biologicals Inc.). Vimentin was diluted to a concentration of 0.5 mg/ml in a total volume of 100 μl using assembly buffer (6 mM phosphate buffer, pH 7.4, 3 mM KCl, 0.2% 2-mercaptoethanol, 0.2% PMSF). Polymerization was achieved by adding 3 μl of 5 M NaCl and incubating at 37°C for 1 h. For cosedimentation assays, preformed vimentin IFs were incubated for an additional hour with fimbrin maintained at concentrations from 0.25 to 1 mg/ml. In other experiments, vimentin and fimbrin were mixed together and IF polymerization, then initiated in the assembly buffer. The proteins were collected by ultracentrifugation at 100,000 g for 30 min and analyzed by SDS-PAGE. Purified vimentin (2.5 μg) was applied to nitrocellulose membranes. The blots were placed into wells of a 12-well tissue culture plate (Costar Corp.) and an overlay assay with labeled fimbrin (0.1–1 μM) was performed as described earlier. The amount of labeled protein that bound was estimated by densitometric scanning (GS-700 Densitometer; Bio-Rad Laboratories) and quantified using the molecular analyst software (Bio-Rad Laboratories). Nonspecific binding of biotinylated fimbrin was determined by measuring the amount of binding to nitrocellulose containing no vimentin. Specific binding was determined by subtracting nonspecific binding from the total binding. Scatchard analysis was carried out to estimate the K d of binding. Plot shown gives on the vertical axis bound/free fimbrin and on the horizontal axis bound fimbrin (pmols). The amount of fimbrin that bound in pmols was obtained from a standard curve of known amounts of biotinylated fimbrin directly spotted onto nitrocellulose. Small amounts of Lys-C (0.25 μg) were incubated with purified fimbrin (50 μg) under nondenaturing conditions in 50 μl of 25 mM Tris, pH 7.7, and 1 mM EDTA. Aliquots were removed at 1, 10, 15, and 30 min, quenched by the addition of PMSF, and then boiled in SDS–sample buffer. The digested products were resolved on SDS-PAGE gels that were either silver stained or electroblotted onto PVDF. For immunofluorescence studies, cells were allowed to attach onto glass coverslips (VWR Scientific) for 1–48 h, gently rinsed in PBS, and fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After three washes with PBS, the cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 2 min before immunostaining. To prepare detergent-extracted cells, adherent cells were first incubated with ice-cold 10 mM Pipes buffer, pH 6.8, containing 0.5% Triton X-100, 300 mM sucrose, 100 mM KCl, 3 mM MgCl 2 , 10 mM EGTA, 2 mM PMSF, and 50 μM sodium vanadate for 3 min, washed with the same buffer, and then fixed with 4% PFA. Nonspecific protein absorption was inhibited by incubating the cells for 1 h in PBS containing 3% BSA, 0.2% Tween 20, and 0.2% fish gelatin. Coverslips were incubated for 1 h at 37°C with affinity-purified rabbit–fimbrin antibody (737.4a) diluted 1:100 and goat–vimentin antibody (Sigma Chemical. Co.; Chemicon International, Inc.) diluted 1:50. The coverslips were washed with PBS and the cells were incubated for 1 h at 37°C with secondary antibodies, FITC-conjugated donkey anti–rabbit and Texas red–conjugated donkey anti–goat (Jackson ImmunoResearch Laboratories). After three washes with PBS, the coverslips were directly mounted in Vectashield (Vector Labs, Inc.) and examined. Cells examined for actin distribution were stained with rhodamine phalloidin (Molecular Probes Inc.). Cells were imaged by epifluorescence microscopy using a Nikon TE300 microscope with 60 and 100× oil immersion lenses. The images were recorded with a Hammatsu Orca CCD camera and analyzed with the Open Lab software program (Improvision Inc.). For deconvolution microscopy, the cells were viewed on a Nikon Eclipse 800 fluorescence microscope with a 100× oil immersion lens. Image stacks were recorded at 134-nm intervals (z series) with a Hammatsu Orca CCD camera and analyzed with the CellScan software (Scanalytics) or Metamorph imaging system (Universal Imaging Corp.) using the exhaustive photon reassignment algorithm. The images were reconstructed in three dimension using either the acquisition or Imaris software (BitPlane). Colocalized proteins were identified by using the colocalization algorithm in Imaris, where voxels containing the two different signals were extracted. The images were finally processed with Adobe Photoshop. In this paper, we investigated the mechanism that targeted fimbrin to podosomes and filopodia at the cell–substratum interface of adherent macrophage cells. Because cytoskeleton–matrix interactions could involve different sets of proteins than in the cortical cytoskeleton, our initial experiments examined whether fimbrin is in a precipitable complex with other proteins in adherent macrophage cells. In Triton-soluble extracts of adherent P388D1 cells, we found four polypeptides of molecular weights, 45, 55, 68, and 75 kD that coprecipitated in immune complexes with four different fimbrin-specific antisera . This interaction with fimbrin is specific, because exogenously added fimbrin inhibited the immunoprecipitation of the 45, 55, and 75 kD (not shown). Other polypeptides seen on the gel were also present in immunoprecipitates of the preimmune sera and, therefore, were not characterized. Having detected several potential fimbrin-binding proteins, we proceeded to identify them as actin (45 kD), vimentin (55 kD), and the heat shock protein, Hsp70 (75 kD). Identification was based on a search of sequence databases (PepFrag and MS-FIT) using the tryptic mass fingerprint of each polypeptide and confirmed by immunoblots using commercially available antisera that are specific for each protein (not shown). Finally, to confirm that fimbrin bound the proteins directly we also probed electroblots of two-dimensional gels with biotinylated fimbrin. On the blots, fimbrin bound actin, vimentin, and Hsp70 . Binding to these proteins was inhibited by an excess of unlabeled fimbrin (not shown). Although Hsp70 appeared to bind fimbrin with a higher affinity, we studied the fimbrin–vimentin interaction because of its potential role in modulating the assembly and structure of the actin and IF cytoskeletons. Because the fimbrin–vimentin complex is found in a Triton X-100 extract of adherent cells, we also investigated whether the complex was present in nonadherent cells. Although the total amount of vimentin in adherent and nonadherent cells is identical, we could immunoprecipitate a detergent soluble pool of vimentin only from adherent cells using vimentin-specific antibodies . Furthermore, a fimbrin-specific antibody detected fimbrin only in vimentin immunoprecipitates from adherent cells. Reciprocal immunoprecipitations with fimbrin-specific antibodies confirmed the presence of an extractable complex in adherent cells but not in nonadherent cells (not shown). This extractable fraction of vimentin in adherent cells was estimated to be ∼1% of the total protein. To understand the nature of the fimbrin–vimentin interaction, we first tested whether fimbrin bound preassembled vimentin filaments using a standard high speed pelleting assay. However, this assay was unable to detect fimbrin in pellets of vimentin filaments (not shown). Next, we tested the binding of fimbrin to unpolymerized vimentin subunits using an immunoprecipitation assay. From a purified mixture of fimbrin and vimentin, vimentin is detected in immunoprecipitates using a fimbrin antibody and not in the preimmune serum . To characterize this interaction more carefully, we measured the binding of soluble fimbrin to vimentin that was immobilized on nitrocellulose membranes. On a Scatchard plot , we estimated the K d of fimbrin binding to vimentin to be 0.25 μM. From the intercept at the x-axis, fimbrin binds 3.8 molecules of vimentin. A simple interpretation of the data is that fimbrin binds a tetramer of vimentin. Because the binding of fimbrin to unpolymerized vimentin is a novel finding, we proceeded to investigate whether the interaction can be localized to discrete domains in both proteins using proteolytic fragments as well as different deletion constructs. In an overlay assay , biotinylated fimbrin is shown to bind to full-length unpolymerized vimentin (WT) and vimentin deleted of its COOH terminus (N410), but does not bind to vimentin deleted of its NH 2 terminus (102C). Similar results were obtained with an overlay assay of the NH 2 - and COOH-terminal fragments of vimentin that were generated by 2-nitro-5-thiocyanobenzoate cleavage at the single cysteine residue present in the molecule (not shown). Binding between the two proteins was not affected by the ionic strength of the assay and comparable levels of binding were detected in either low (5 mM sodium phosphate) or high (150 mM NaCl) ionic strength solutions. These results suggest that fimbrin binds specifically to the NH 2 -terminal region of vimentin. To map the vimentin-binding region on fimbrin, we conducted overlay assays on fimbrin proteolyzed with lys-C . In these experiments, a low ionic strength buffer was used to keep vimentin from polymerizing. Biotinylated vimentin is seen to specifically bind to a 26-kD domain of fimbrin. The 26-kD fragment was sequenced and shown to be part of ABD1. To narrow the vimentin binding site on actin binding domain 1, we tested binding of biotinylated vimentin to various truncations of the ABD1 domain . Using the overlay assay, we found that vimentin binds any part of the CH1 domain that contained residues 143–188 ( Table ). Because the biochemical experiments strongly suggested that fimbrin is in a complex with vimentin subunits, we carried out a series of immunocytological studies to identify how the proteins were distributed and to determine where this complex is localized. If fimbrin and vimentin are in a complex, they should colocalize to the same structures and compartments in the cell. Using fluorescence microscopy, we first examined the distribution of filamentous actin (F-actin) as well as fimbrin and vimentin in two macrophage cell lines; highly differentiated, motile IC-21 cells and moderately differentiated, nonmotile P388D1 cells. In nonmotile P388D1 cells, phalloidin staining was localized in filopodia and cell surface microvilli and the cell body . However, P388D1 cells do not organize the actin cytoskeleton into podosomes, and the dotted pattern characteristic of podosomes was absent in these cells. On plated IC-21 cells , F-actin is detected in clusters of small dots, podosomes, or rosette adhesions at the leading edge of the cell, in the cell body, and in retraction fibers at the trailing end of the cell. This staining pattern is similar to that observed when primary mouse macrophages are stained for actin . Thus, the actin cytoskeleton is organized differently in the two cell lines. We examined the distribution of fimbrin and vimentin in the two macrophage cell lines by fluorescence deconvolution microscopy. Double staining of P388D1 cells revealed fimbrin in filopodia and in the perinuclear region of the cell , whereas vimentin localized to filopodia and the cell body . To obtain colocalized signals we imported image stacks for each fluorescent pattern into an image processing software, Imaris, and voxels (134 nm) that contained the two signals were extracted and displayed in yellow. Fimbrin and vimentin are seen to colocalize in the filopodia . In IC-21 cells, fimbrin is found in podosomes, retraction fibers, and in the perinuclear region of the cell , whereas vimentin stained podosomes, retraction fibers, and the cell body . The two image stacks were also processed in Imaris, and voxels containing the two fluorescent signals (displayed in yellow) show that the proteins colocalized in podosomes (arrow), retraction fibers, and in the perinuclear region . The colocalization of both proteins on the basal surface was confirmed from side views of a three-dimensional reconstruction of the cell (not shown). These results show that fimbrin and vimentin colocalize to actin-rich structures, filopodia, podosomes, and retraction fibers in two different macrophage cells. Our biochemical studies detected fimbrin and vimentin in the Triton X-100 extract of IC-21 and P388D1 cells. To locate those structures that contain this extractable complex, we also examined the distribution of fimbrin and vimentin after Triton X-100 treatment. Before extraction, the complex was examined on freshly plated cells. Macrophages that had just attached and were beginning to spread show vimentin and fimbrin staining primarily in the perinuclear region (not shown). Half an hour after plating, the two proteins were found in numerous common foci in regions that were actively spreading . However, the majority of the vimentin network is in the perinuclear region. 3 h after plating, the cells were well polarized and the complex was now restricted to foci in that region of the cell that was actively expanding and to retraction fibers at the trailing end of the cell . After Triton X-100 extraction, IC-21 cells show complete loss of staining of fimbrin and vimentin from both retraction fibers and podosomes . Similarly, in P388D1 cells, both proteins were less abundant or undetected in filopodia but were present in the cell body (not shown). Our studies describe an adhesion-dependent relationship between the actin and intermediate filament cytoskeletons. We present four independent observations that document a specific interaction between an actin cross-linking protein, fimbrin, and an IF protein, vimentin. First, the two proteins coprecipitate from Triton-X 100–extracted cells with either fimbrin- or vimentin-specific antibodies. Second, binding assays with recombinant proteins restrict the binding region between residues 143–188 of fimbrin and the head domain of vimentin. Third, the two proteins colocalize in podosomes, filopodia, and retraction fibers. Finally, both proteins are extracted from these structures by the same detergent treatment. These biochemical and morphological observations are strong evidence of a fimbrin–vimentin complex that is found in ventral structures where a macrophage adheres to the substratum. The association between fimbrin and vimentin, reported in this study, is a new finding. Based on previous studies, fimbrin was shown to bind and cross-link actin filaments. This function was consistent with its localization in actin bundles that support cell surface microvilli . However, early studies also detected fimbrin at the cell–matrix interface , which suggested that fimbrin had a function associated with cell adhesion. This suggestion was reinforced by our finding that during embryogenesis L-fimbrin is transiently expressed and localized to the basal surface of embryonic enterocytes, whereas T- and I-fimbrin and the majority of F-actin are in the developing brush border at the apical surface of the same cells . Because IFs are now recognized components of focal adhesions , interactions with fimbrin could explain how vimentin is differentially targeted to cell adhesion structures where it can assemble into IFs. In addition, association with vimentin that may be transported to adhesion sites in a microtubule-dependent mechanism can explain how fimbrin is targeted to adhesion sites where it can cross-link actin filaments into a bundle. The fimbrin–vimentin interaction is unusual because fimbrin binds a vimentin subunit and not a filament. Typically, cytoskeletal networks are cross-linked by proteins that bind polymerized filaments . For example, elegant immuno EM studies show plectin cross-bridges between assembled IFs and MTs . Plectin is also implicated in binding to actin filaments through an actin-binding domain located at the NH 2 terminus, whereas the IF-binding domain is found at the COOH terminus . However, this is not the case for fimbrin. Its interaction with a subunit of vimentin is suggested by the following: the 1:4 stoichiometry of binding , the absence of substantial fimbrin colocalization with vimentin filaments in the cell body, and the inability of fimbrin to bind polymerized vimentin in pelleting assays (Correia, I., and P. Matsudaira, unpublished data). The detergent-extracted fraction most likely includes the pool of unassembled vimentin subunits in the cell that are believed to be tetramers of vimentin. The identity of the vimentin subunit as a binding partner of fimbrin is also consistent with the low percentage (1%) of this species in the supernatant . Thus, the interaction with a vimentin subunit and not a filament suggests that the complex has a function other than to cross-link the actin and IF cytoskeletons. Binding to vimentin subunits places fimbrin in a unique position to influence the assembly and organization of both the actin and IF cytoskeletons. We speculate that the fimbrin–vimentin complex plays an early role in the assembly of the actin and vimentin cytoskeleton in filopodia and podosomes. This speculation is supported by our biochemical observation that the fimbrin–vimentin interaction in P388D1 cells is adhesion-dependent. The time course of the localization of fimbrin and vimentin in these structures, obtained by epifluorescence microscopy, also suggests that the complex is a transient structure that is involved early in cell adhesion. During the early stages of attachment of motile IC-21 cells, we detected costaining of fimbrin and vimentin in podosomes of expanding lamella. After 3 h, fimbrin does not become associated with the vimentin network of filaments . Because fimbrin does not bind assembled vimentin filaments in vitro other proteins may assist in anchoring the vimentin network at mature adhesion sites. These observations are relevant as studies obtained from time-lapse and interference reflection microscopy indicate a preformed complex of cytoskeletal proteins that nucleate the recruitment of adhesive molecules before anchoring the lamellipodia and filopodia to the substratum . Although the compositions of the nucleating proteins were not described, the fimbrin–vimentin complex is in the right place and at the right time to play this role. This model of fimbrin–vimentin function is compatible with earlier reports from Tao and colleagues , who described a similar interaction between desmin tetramers and calponin in smooth muscle cells. They proposed the idea that an IF subunit and an actin-binding protein may be involved in the assembly of IFs at dense bodies. We agree with their interpretation. In fact, it is alluring to consider that the interactions involve cell adhesions. As discussed in the following section, an interaction between an IF protein, desmin, or vimentin, and a protein with a CH domain, calponin, or fimbrin may represent a highly conserved function that may be related to the establishment of cell adhesion structures. The fimbrin–vimentin complex is biochemically distinguished from the rest of the actin and IF cytoskeletons by its extraction properties. The critical observation is that fimbrin and vimentin are extracted from a subset of structures (podosomes, filopodia, and retraction fibers) by detergent. Biochemical quantitation shows the detergent-extracted population represents ∼1% of the total vimentin in the cell. Most vimentin is in a detergent-insoluble fraction, and fluorescence microscopy localizes the insoluble vimentin to filaments in the cell body and around the nucleus. Thus, the vimentin–fimbrin complex found in early cell adhesion structures is biochemically and structurally distinct from the vimentin network in the cell body. This distinction may provide useful approaches for studying the function of the complex in live cells. The biochemical and structural properties of the fimbrin–vimentin complex may be explained by the location of fimbrin and vimentin binding sites on the proteins. Our overlay binding assays now tentatively map the region of fimbrin–vimentin interaction to residues 143–188 of the NH 2 -terminal actin-binding domain of fimbrin and the head domain of vimentin. These sites lie within interesting regions of both proteins. Fimbrin binds a pair of actin filaments using actin-binding domains that are located in the NH 2 - and the COOH-terminal regions of the molecule . Quantitative modeling of a fimbrin x-ray structure to an EM reconstruction of fimbrin bound to actin filaments predicts that actin binds fimbrin through interactions at the two CH domains in the NH 2 -terminal actin-binding domain, ABD1 . Vimentin also binds fimbrin on ABD1, opposite to the actin-binding site, but on the same side where the regulatory calcium-binding domain (residues 1–100) and the COOH-terminal actin-binding domain (ABD2) are located . This location poses interesting implications for actin bundling. Although we have not yet studied the ability of the fimbrin–vimentin complex to bind and cross-link actin filaments, we suspect that vimentin binding to fimbrin may interfere with the ability of fimbrin to cross-link actin. The fimbrin binding site lies on an important domain of vimentin. The NH 2 -terminal domain is absolutely essential for incorporation of vimentin subunits into protofilaments and higher order filaments, and deletion of this domain prevents assembly of vimentin filaments . Furthermore, phosphorylation of the IF network on the NH 2 - and COOH-terminal domains (head and tail domains) is a well established means for the disassembly of the IF network . Interestingly, preliminary studies carried out in vivo indicate that the fimbrin–vimentin complex is phosphorylated on vimentin and not on fimbrin (not shown). Although we have not localized the sites of phosphorylation, fimbrin bound to the NH 2 -terminal domain of vimentin may play a role in regulating vimentin assembly by either directly preventing incorporation of its subunits into filaments, or indirectly, by modulating the addition or removal of phosphate groups. | Other | biomedical | en | 0.999995 |
10459018 | Media, tissue culture reagents, and FCS were purchased from GIBCO BRL and Boehringer Mannheim. As a model system, we used EpH4 cells, a spontaneously immortalized mouse mammary epithelial cell line which displays a fully polarized epithelial cell phenotype . EpH4 cells were cultured on permeable filter supports at 37°C, 5% CO 2 , and 98% humidity in Eagle's medium, supplemented with 10 mM Hepes and KOH (pH 7.3), 50 IU/ml penicillin, 50 mg/ml streptomycin, and 5% FCS. For some of the immunolocalization experiments, Transwell™ filters , 24-mm-diam, 0.4-μm pore size, were used. Transepithelial electrical resistance (TER) of filter-grown EpH4 monolayers was measured using a Millicell Electrical Resistance System (Millipore Corporation) equipped with an Endohm-24-electrode (World Precision Instruments) according to standard procedures. Hybridoma clone IM7.8.1 (TIB-235; American Type Culture Collection) (producing rat anti–human/mouse pan CD44 antibodies) was propagated in medium with Ig-depleted FCS and secreted antibody were purified on protein G–Sepharose columns (Pharmacia). The anti–human annexin II antibody HH7 is a mouse monoclonal antibody recognizing murine annexin II . Anti-p11 antibody H21 reactive with a trans-dominant mutant of annexin II was previously described . Mouse monoclonal antibody cross-reacting with mouse annexin II and rabbit polyclonal antibody against caveolin were from Transduction Laboratories and mouse monoclonal antibody against actin as well as rat monoclonal against ZO-1 were purchased from Chemikon. Mouse monoclonal antibody H68.4 recognizing mouse transferrin receptor was a generous gift from Dr. J. Trowbridge. Secondary antibodies used for immunofluorescence were from Jackson ImmunoResearch Laboratories: Cy3-goat anti–rat (712-165-150), Cy3-goat anti–mouse (715-160-150), FITC-goat anti–mouse (115-095-100); and from Molecular Probes: Alexa488-goat anti–rabbit , Alexa488-goat anti–rat and Alexa488-goat anti–mouse . Protease inhibitors aprotinin, pepstatin, leupeptin, and Pefabloc SC were from Boehringer Mannheim. LipofectAMINE PLUS™ Reagent was from GIBCO BRL. Reagents for cholesterol depletion included digitonin and methyl-β-cyclodextrin both from Sigma-Aldrich. Rhodamine-phalloidin was purchased from Molecular Probes (R-415). Latrunculin A was a kind gift from Dr. J. Knoblich (IMP, Vienna). Confluent cell monolayers were washed twice with cold PBS, scraped in PBS and spun down at 2,000 rpm at 4°C. Cells were lysed in 200 μl of TN (25 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, cocktail of protease inhibitors, 10% sucrose, and 1% Triton X-100) on ice, and incubated for 30 min on ice. Samples were mixed with 400 μl of cold Optiprep™, transferred into SW60 centrifuge tubes and overlaid with 600 μl of each 35%, 30%, 25%, 20%, and 0% Optiprep™ in TN. The gradients were spun at 35,000 rpm in SW60 rotor for 12 h at 4°C. Fractions were collected from top to bottom of centrifuge tubes, proteins were precipitated according to Wessel and Flügge 1984 followed by Western blotting analysis. Only in case of the anti-panCD44 antibody (IM7.8.1) nonreducing SDS-PAGE was applied. The cholesterol sequestration in vivo was performed with M-β-CD in confluent cultures. Monolayers were washed once with PBS, then the prewarmed medium, containing 5 mM of M-β-CD, was added and cells were incubated for 15 min at 37°C. Cholesterol sequestration in isolated membrane fractions was achieved by treatment of pelleted membranes with varying concentrations of digitonin for 30 min on ice. The cholesterol content of pelleted membranes before and after treatment was determined by an enzymatic-fluorometric assay . Values were normalized to total membrane protein. Sparsely grown cells were incubated with anti-panCD44 antibody IM7.8.1 (10 μg/ml) diluted in normal Eagles's medium for 10 min at 37°C, followed by three washes in PBS, containing 1 mM CaCl 2 , 1 mM MgCl 2 , and 0.5% gelatin. Control cells were fixed in acetone and methanol before treatment with secondary Cy3-labeled goat polyclonal anti–rat antibodies to visualize CD44 staining. Clustering of CD44 was induced in the remaining cell samples by incubation with Cy3-labeled goat polyclonal anti–rat antibodies (20 μg/ml) in the normal Eagle's medium for 20 min at 37°C; thereafter, the cells were rinsed and fixed as above. All cell samples were then double stained with antibodies against annexin II or VIP21 and caveolin. The construction of the trans-dominant mutant of annexin II was described previously . Transient transfections of sparse EpH4 cells were performed using LipofectAMINE PLUS™ Reagent according to the manufacture's recommendations. Cells were permeabilized as described in Mackay et al. 1997 . In brief, confluent, filter-grown EpH4 cells were permeabilized at 37°C for 10–20 min using 0.003% digitonin in DK buffer (150 mM potassium glutamate, 10 mM Hepes and KOH, pH 7.3, 5 mM glucose, 2 mM MgCl 2 , and 0.4 mM EGTA), supplemented with an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, and 10 μg/ml creatine phosphokinase), DTT as an antioxidizing agent and a cocktail of protease inhibitors. 50 μM of GTPγS or 0.05–0.1 μM of rhodamine-phalloidin was added to the permeabilized cells where indicated. Cells were washed twice in PBS, fixed with acetone and methanol (1/1) at −20°C for 2 min, and air-dried. The cells were then washed with PBS and incubated in blocking buffer (0.5% gelatin in PBS containing 1 mM CaCl 2 , and 1 mM MgCl 2 ) at room temperature for 30 min in order to decrease nonspecific antibody binding. Subsequently, cells were incubated for 1 h at room temperature with primary antibodies diluted in blocking buffer, followed by three washes in blocking buffer. Secondary antibodies were applied in the same way. The samples were mounted in Moviol and processed for microscopy. All confocal microscopy images were obtained using a Leica TCS NT confocal microscope (Leitz). Images were processed using an Octane Workstation (Silicon Graphics, Inc.) using the Huygens (Scientific Volume Imaging), Imaris, and Colocalization (Zitplane AG) software packages. The sparse EpH4 cells grown on coverslips were washed briefly with fresh medium and incubated with FITC-labeled antistandard CD44 antibody IM7.8.1 (10 μg/ml) diluted in normal Eagles's medium for 20 min at room temperature. For the labeling of the transferrin receptor K + -depleted EpH4 cells were incubated with FITC-labeled transferrin (50 μg/ml) for 20 min on ice. The cells were rinsed with medium to remove nonbound antibody and the coverslips were assembled into the perfusion chamber (H. Saur Laborbedarf, Reutlingen, Germany) and connected to the peristaltic pump, allowing the exchange of the medium and maintaining cells at 37°C. Initial imaging, photobleaching of the selected regions of the plasma membrane, and recording of the fluorescent recovery were performed in a Leica TCS NT confocal microscope system (Leitz). The bleached areas had a surface area of ∼40 μm 2 . The fluorescence recovery was recorded immediately using a time-lapse option of the system. The data obtained were analyzed using TCS NT quantification software. The intensities of the bleached and recovered regions were normalized in regard to the fluorescent intensity of the independent region in the same samples. The percentage of recovery was calculated as Ir /Ii, multiplied by 100%, where Ir is the intensity of the photobleached region after recovery and Ii is the intensity of the selected region before photobleaching. In fibroblasts, ≤50% of CD44 is resistant to Triton X-100 extraction . However, in Madin-Darby canine (MDCK) and bovine (MDBK) kidney epithelial cells CD44 was completely soluble in Triton X-100 and no association of CD44 with the cytoskeleton could be demonstrated . We used the Triton X-100 solubility assay to explore a possible cytoskeletal association of CD44 in the EpH4 mammary epithelial cell line. The majority of CD44 remained in the insoluble fraction after Triton X-100 extraction at concentrations up to 1% (data not shown). We tested whether Triton X-100 insolubility of CD44 could be a consequence of its localization in Triton X-100–insoluble membrane domains . Therefore, we performed floatation experiments in Optiprep™ gradients in the presence of Triton X-100. In this gradient, detergent-insoluble, glycosphingolipid-rich membrane fractions representing the lipid rafts will float to the interphase between the 0% and 20% Optiprep™ layers. Solubilized proteins or cytoskeleton-associated, detergent-insoluble proteins remain at the bottom of the gradient. In EpH4 cells, a fraction of CD44 isoforms floated to low density fractions, together with the detergent-resistant glycosphingolipid-rich membranes . There were several bands detected by anti-panCD44 antibody (IM7.8.1) by Western blot, due to the fact that there are several CD44 isoforms expressed in EpH4 cells, as revealed by RT-PCR (data not shown). However, all isoforms partitioned into the lipid raft fractions with the same efficiency. The total amount of CD44 that could be recovered from lipid raft fractions under experimental conditions described above was determined as a result of three independent experiments, indicating that in a steady state ∼35% of CD44 partitions into lipid rafts as assessed by Optiprep™ gradient floatation, followed by Western blot . Caveolin/VIP21, a known cholesterol-binding protein, was found in the same lipid raft fractions. A second lipid-binding protein, annexin II, also efficiently partitioned to this fraction . However, addition of the Ca 2+ -chelating agent EDTA to the homogenization buffer reduced this efficiency to <10%, while CD44 distribution over the gradient did not change (data not shown). This suggested a Ca 2 +-dependent association of annexin II with cholesterol-rich membranes. In contrast, the transferrin receptor, a transmembrane protein localized to clathrin-coated pits, was always found to be associated with heavier fractions and never occurred in lipid rafts, confirming the correct partitioning of different membrane proteins in the gradient . Our biochemical analysis suggested that CD44 occurred in detergent-insoluble lipid rafts, together with caveolin/VIP21 and annexin II. Both proteins are located basolaterally in EpH4 cells . However, they may be incorporated into different subtypes of basolateral lipid rafts. Caveolin/VIP21 is a structural component of caveolae , but a minor fraction is localized within the entire plasma membrane . In EpH4 cells, CD44 isoforms mostly failed to colocalize with caveolin/VIP21, indicating that CD44-containing lipid rafts represent structures distinct from caveolae . However, CD44 was found to colocalize with annexin II along the basolateral plasma membrane domain . Colocalization of annexin II with CD44 in the lateral membrane of EpH4 cells was dependent on an intact lipid environment. To reduce the plasma membrane cholesterol concentration, we treated cells with the cholesterol-sequestering drug, M-β-CD , before fixation. M-β-CD forms stable inclusion complexes with cholesterol by incorporating it into its hydrophobic cavity. Typically, treatment of confluent EpH4 monolayers reduced cholesterol from 25 to 9.5 μg cholesterol/mg membrane protein as revealed by an enzymatic fluorometric assay . Upon depletion of membrane cholesterol by M-β-CD, both CD44 and annexin II redistributed to the entire plasma membrane, including the apical domain and were no longer colocalized (data not shown). One of the possibilities to selectively analyze microdomains enriched in CD44 is to use in vivo antibody labeling of CD44 followed by cross-linking of CD44–antibody complexes with secondary antibodies in living cells. In sparsely grown EpH4 cells labeled with primary anti-CD44 antibodies followed by immediate fixation, both CD44 and annexin II were diffusely distributed all over the plasma membrane . After cross-linking of CD44 in intact cells with anti-CD44 plus secondary antibodies, we observed that most of CD44 accumulated in big patches on the plasma membrane. Interestingly, these patches also contained annexin II, apparently recruited into these complexes at the cytosolic face of the plasma membrane . In contrast, no significant colocalization of CD44 and caveolin/VIP21 was observed using anti-caveolin/VIP21 antibodies, either before or after antibody cross-linking of CD44 . These results were consistent with our hypothesis that CD44-containing lipid rafts were distinct from the caveolae submembranous compartment. We previously found that the colocalization of CD44 and annexin II in lipid rafts was impaired after depletion of the cells from cholesterol by M-β-CD (data not shown). Therefore, it was of interest to analyze if M-β-CD would also impair the formation of patches after CD44 antibody cross-linking and prevent the inclusion of annexin II into these patches. Interestingly, the ability of CD44 to form clusters upon antibody cross-linking was significantly reduced by pretreatment of cells with 5 mM M-β-CD. Smaller clusters formed upon cross-linking failed to recruit annexin II . In most cell lines studied so far, annexin II exists as a heterotetramer bound to its ligand p11. The chimeric p11–annexin II protein inducing the aggregation of endogenous annexin II and p11 was previously described . In brief, it is a chimeric protein comprising the NH 2 -terminal domain of annexin II fused to the NH 2 terminus of p11. As a result, this mutant has two binding sites for p11 and one for annexin II. p11 dimers bound to this chimera would accommodate additional sites for binding of chimera and/or annexin II–p11 complexes, leading to the formation of the large aggregates containing annexin II, p11, and the chimeric protein. In nonpolarized EpH4 cells transiently expressing the mutant, the appearance of large annexin II–p11-positive clusters underneath the plasma membrane was observed . The extent of aggregate formation was dependent on the levels of expression. Most interestingly, CD44 was also redistributed in the plasma membrane of transfected cells, largely colocalizing with trans-dominant annexin II . Actin cytoskeleton was rearranged in transfected cells, and the ends of the actin bundles were often found colocalizing with the clusters of trans-dominant mutant . To further characterize CD44/annexin II–containing lipid rafts, we tested whether the stability of these complexes with respect to cholesterol depletion was dependent on direct or indirect interaction with the actin cytoskeleton. The first approach was to test if active GTP-binding proteins were required for CD44-containing lipid raft stability, using a nonhydrolyzable GTP analogue (GTPγS). To allow the use of GTPγS, we adapted a method to generate semipermeabilized cells described by Mackay et al. 1997 to our polarized, filter-grown EpH4 cells. For this, EpH4 cells were permeabilized by very low concentrations of digitonin (0.003%), a known cholesterol-sequestering detergent . This treatment depleted cholesterol from EpH4 cells twice as efficient than M-β-CD (from 25 to 3.9 μg cholesterol/mg membrane protein, see above). The permeabilized cells were supplemented with an ATP-regenerating system, a cocktail of protease inhibitors, and with DTT as antioxidant. Under these conditions we could observe the redistribution of CD44 and annexin II over the entire plasma membrane . However, when nonhydrolyzable GTPγS was added to the cells during digitonin treatment no redistribution to the apical membrane occurred, and CD44 and annexin II continued to be colocalized in the basolateral plasma membrane domain . One obvious consequence of arresting small Rho-GTPases in their active state (as caused by GTPγS) is the stabilization of filamentous actin cables , as suggested by our observation that this treatment resulted in appearance of thicker bundles of stress fibers and a more prominent subcortical actin ring (not shown). Thus, a possibility to explain the above results was that GTPγS treatment stabilized CD44-containing lipid rafts in the basolateral plasma membrane through their enhanced interaction with stabilized, underlying actin cables under these experimental conditions. To test for this possibility, we chose an alternative way to stabilize F-actin filament bundles, using the fungal toxin phalloidin. The phalloidin-stabilized actin filaments still retain some of their functional properties; for example, they are able to move on solid-phase myosin substrates . This experiment was performed in the same digitonin-semipermeabilized EpH4 cell system as described above. While digitonin permeabilization caused redistribution of CD44 in the control cells , treatment of the cells with phalloidin during digitonin-induced cholesterol depletion fully mimicked the activity of GTPγS. CD44 remained undetectable on the apical surface of phalloidin-treated cells after the depletion of plasma membrane cholesterol . At the same time, the subcortical actin ring was stabilized . These results suggest that the stability of lipid rafts containing CD44 also requires intact actin filament bundles and/or an interaction of the proteins present in the lipid rafts with the underlying cytoskeleton. Looking for the biochemical evidence that CD44-containing lipid rafts interact with the actin cytoskeleton, we decided to treat EpH4 cells with the actin cytoskeleton-disrupting drug latrunculin A before the lipid rafts floatation experiment. To our surprise, the proportion of CD44 in the detergent-insoluble, glycosphingolipid-rich membrane fractions increased dramatically , as compared with the mock-treated control . These results indicate that a significant bulk of CD44-containing lipid rafts is not capable of floating up to the light lipid rafts fractions due to its interaction with the heavy cytoskeleton elements. This experiment further implied that the vast majority of CD44 molecules in EpH4 cells is localized to the lipid rafts. To obtain complimentary evidence that CD44-containing lipid rafts do indeed interact with the underlying actin cytoskeleton, we employed a technique known as fluorescence recovery after photobleaching (FRAP). In brief, CD44 molecules on the surface of living cells were labeled with FITC-labeled anti-panCD44 antibody IM7.8.1. The selected regions of the plasma membrane were bleached and the subsequent recovery of the bleached regions was recorded. The absolute values obtained were normalized by comparison with the fluorescent intensities in an independent region of identical size. These lateral diffusion measurements indicated that CD44 molecules were virtually immobilized in the plasma membrane of control cells, as compared with the transferrin receptor, a transmembrane protein of clathrin-coated pits or free lipid dye BODIPY-FL-sphingomyelin (data not shown). The FRAP measurements of the lateral mobility of the transferrin receptor were performed in K + -depleted cells in order to prevent receptor internalization . However, the lateral mobility of CD44 was notably improved in the cells pretreated with either M-β-CD or actin disrupting drug latrunculin A . For instance, the percentage of recovery of the photobleached region in control cells after 105 s was ∼19%, in comparison with 28% and 40% in M-β-CD–treated and latrunculin A–treated cells, respectively. This finding indicates that lateral mobility of CD44 molecules in the plasma membrane is dependent on the presence of the plasma membrane cholesterol and an intact actin cytoskeleton, again suggesting the direct interaction of lipid raft–associated CD44 with underlying cytoskeleton. In unpolarized, sparsely seeded epithelial cells, CD44 distributes over the entire plasma membrane, including microvilli, and areas of cell-to-cell contacts. As epithelial cells polarize CD44 redistributes to the basolateral surface of the plasma membrane and all isoforms looked at so far localize exclusively within this plasma membrane domain . In MDCK cells the localization and restriction of the standard form of CD44 (CD44s) to the lateral cell surface depends on a critical dileucine motif in the cytoplasmic tail of the protein . This motif serves as a dominant localization signal since it is also able to redirect the normally apical protein placental alkaline phosphatase (PLAP) to the basolateral plasma membrane . In polarized EpH4 mammary epithelial cells, the standard and several variant CD44 isoforms are expressed endogenously and all localize to the basolateral plasma membrane domain. Previous studies in polarized MDCK cells showed that CD44 was completely Triton X-100 soluble . However, we found in EpH4 cells that a majority of CD44 could not be extracted by Triton X-100 at 4°C. Other authors reported previously that CD44 insolubility can be attributed to its partition into Triton X-100–insoluble lipid microdomains in certain cell types . To test this hypothesis, we floated Triton X-100–insoluble material on Optiprep™ density gradients and were able to show that in EpH4 cells a significant portion of all CD44 isoforms was present in lipid rafts. How could CD44 partition into basolateral lipid rafts in epithelial cells? CD44 might first partition into basolateral TGN-derived vesicles, due to its active basolateral sorting signal. Upon arrival at the basolateral membrane domain, CD44 might then partition into lipid rafts due to the intrinsic properties of its transmembrane domain. Alternatively, CD44 might partition into cholesterol-rich membranes already in the TGN, causing these vesicles to be sorted basolaterally instead of apically, due to the dihydrophobic sorting motif Leu331/Val332. This scenario would result not only in proper basolateral sorting of CD44, but would also recruit cholesterol-rich membranes to the basolateral surface, allowing CD44 to serve as an assembly anchor for basolateral lipid rafts. One of the proteins expected to localize to cholesterol-rich membrane domains was caveolin/VIP21. However, the bulk of CD44 did not colocalize with caveolin/VIP21 in EpH4 cells and the observed minor colocalization did not occur in deep plasma membrane invaginations indicative of caveolae. A considerable degree of overlap has been previously observed between CD44 and annexin II in Triton X-100–extracted fibroblasts, as revealed by immunofluorescence analysis . We observed a colocalization between CD44 and annexin II in EpH4 cells. In addition, the depletion of membrane cholesterol by M-β-CD abolished this colocalization, causing redistribution of both CD44 and annexin II over the entire cell surface, including the apical plasma membrane. This drug preferentially extracts cholesterol from the outer leaflet of the plasma membrane and partially liberates transmembrane and GPI-anchored proteins from lipid rafts, leaving caveolae virtually intact . Together, these observations suggest that CD44 partitions into lipid rafts but not into caveolae. Annexin II is a Ca 2+ -binding protein present in a wide variety of cells and tissues which can associate with actin filaments and membranes. Within cells, annexin II occurs either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100–related protein, p11 . Annexin II has been suggested to be involved in membrane transport, both the regulated exocytic pathway and the endocytic pathway . In addition, annexin II is a major cellular substrate for the src-family of protein kinases which partition into lipid rafts in various cell types . Also it has been implicated that annexin II might interact with the submembranous actin network with this activity depending on its ability to form the heterotetrameric annexin II–p11 complex and on the presence of intact Ca 2+ -binding sites . It has been shown previously that interaction of the annexin II–p11 heterotetramer with the plasma membrane, but not with the submembranous cytoskeleton, induces the tyrosine phosphorylation of annexin II, presumably exposing the tyrosine-phosphorylation site as a consequence of induced conformational changes . Moreover, recent work indicated that approximately half of the cellular annexin II Ca 2+ independently binds to BHK membranes where it may be involved in organizing lipid microdomains . Interestingly, this pool of annexin II is released upon cholesterol sequestration. In EpH4 cells only a minor fraction of annexin II was associated with detergent-insoluble membranes in a Ca 2+ -independent manner (data not shown), while the bulk of cellular annexin II was bound to rafts in the presence of Ca 2+ . Our findings also clearly agree with the earlier observation that the annexin II– p11 tetramer is significantly enriched in the Triton X-100–insoluble fraction of MDCK cells in the presence of Ca 2+ . It is tempting to speculate, that the interaction of annexin II with lipid rafts in living cells may be regulated by elevated intracellular Ca 2+ concentrations and/or phosphorylation. Interestingly, another member of the annexin family, annexin XIIIb was also found in the Triton X-100–insoluble fraction of MDCK cells. Annexin XIIIb is associated with the apical plasma membrane, enriched in apical transport vesicles and is involved in apical delivery of TGN-derived transport vesicles . Thus, annexin II and annexin XIIIb associate with Triton X-100–insoluble fractions from different cellular locations. These data suggest that, at least in MDCK and EpH4 cells, different annexins might organize rafts and/or modulate raft function in distinct locations; e.g., by stabilizing inner leaflets of sphingolipid-cholesterol rafts . Artificial clustering of the smallest 85-kD isoform of CD44 (CD44s) has previously been shown to promote binding of the protein to soluble hyaluronic acid (HA) . In rat pancreatic carcinoma cells, CD44 splice variants, but not the standard CD44, form molecular aggregates within the plasma membrane. It was postulated that the regulation of clustering, mediated by either the presence of variant exons and/or glycosylation, allows cells in turn to regulate the binding properties of CD44 to HA . It was also shown that Cys286 within the transmembrane domain of CD44 was required for TPA-induced covalent dimerization of CD44 and enhanced HA binding . These observations prompted us to cluster CD44 on the cell surface by antibodies. This clustering of CD44 induced efficient coclustering with annexin II. Significant amounts of annexin II at the cytoplasmic face of the plasma membrane were recruited into these clusters. Most importantly, both clustering of CD44 by antibodies and recruitment of annexin II to these clusters were dependent on the presence of membrane cholesterol. The fact that the antibody-induced patches were practically devoid of caveolin/VIP21 again confirmed that CD44–annexin II lipid rafts were distinct from caveolae. Neither did CD44 colocalize with the transferrin receptor (not shown), an integral membrane protein of clathrin-coated pits which is virtually excluded from lipid rafts . The possibility of artificial clustering of annexin II was previously described . Expression of an annexin II–p11 chimera, acting as a trans-dominant mutant, causes aggregation of annexin II–p11 tetramers. This therefore interferes with the normal distribution of annexin II, and may also alter annexin II–dependent proteins and/or structures. We observed the formation of annexin II–positive clusters underneath the plasma membrane of transiently transfected cells. These complexes also contained CD44 and, interestingly, the actin cytoskeleton was rearranged. Actin bundles were either surrounding or coclustering with annexin II aggregates. In previous work using the same mutant it was reported that in MDCK cells the formation of annexin II–p11 aggregates did not affect the actin-based cell cortex, but translocated early endosomes . The difference between aggregates formed in MDCK and EpH4 cells and their morphological consequences can be attributed to the different cell types used and distinct experimental techniques. Our observation that annexin II aggregation caused both coclustering of CD44 and rearrangements of the actin cytoskeleton strongly suggested that annexin II might participate in a functionally important clustering of CD44-containing lipid rafts in the plasma membrane and their coupling to the actin cytoskeleton. In fibroblasts, CD44 was previously found to interact with proteins of the ERM (ezrin-radixin-moesin) family, which are peripheral actin cross-linkers . In turn, ERM proteins directly interact with Rho GDP dissociation inhibitor (RhoGDI). This reduces RhoGDI activity and thus activates Rho subfamily members . These results suggest that members of the ERM family as well as RhoGDI and RhoGDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which in turn regulate reorganization of actin filaments underneath the plasma membrane. For instance, RhoA appears to regulate actin stress fibers at the basal membrane representing a ground state from which epithelial structures are built, whereas Rac1 activity appears to be required to assemble circumferential actin structures at the lateral membrane which affect the distribution of actin-associated membrane proteins . In line with this, members of the ERM protein family could reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac in digitonin-permeabilized fibroblasts . CD44 interacting with ERM can provide the necessary link to the plasma membrane . We performed immunoprecipitation analysis with anti-CD44 antibodies in EpH4 cells, but we were unable to detect the presence of ERM proteins or ankyrin/fodrin in immunoprecipitates with anti-panCD44 antibodies (data not shown). However, the rearrangement of the actin cytoskeleton after expression of trans-dominant annexin II mutant suggested the possible involvement of annexin II in linking of CD44-containing lipid rafts to the cytoskeleton. This prompted us to ask if changes in actin cytoskeleton structure or stability might alter the integrity and localization of CD44–annexin II lipid rafts. Therefore, EpH4 cells were treated with digitonin to deplete them from cholesterol and at the same time render them permeable to GTPγS, a nonhydrolyzable analogue of GTP, which locks GTP-binding proteins in their active state. GTPγS is a pleiotropic reagent affecting many different classes of GTP-binding proteins. However, one of the major consequences of treating the cells with GTPγS is an increased actin polymerization. Adapting the method described by Mackay et al. 1997 to our fully polarized EpH4 cells, disruption of the association between CD44 and annexin II as well as their redistribution to the apical cell surface could be completely prevented by GTPγS, maintaining colocalized CD44 and annexin II in the basolateral plasma membrane domain despite of the cholesterol depletion. GTPγS also enhanced the formation of stress fibers and the formation of a pronounced subcortical actin ring (data not shown). Stabilization of filamentous actin itself by phalloidin also protected CD44–annexin II membrane complexes from dissociation and redistribution by cholesterol depletion, suggesting a direct involvement of stabilized actin filaments in this process. One of the direct possibilities to address the question whether CD44-containing lipid rafts interact with the actin cytoskeleton are the measurements of FRAP. Surprisingly, CD44 exhibited distinct recovery rates in different sites of the sparsely grown, nonpolarized EpH4 cells. The mobility rate was lower in the ventral and trailing regions of the cells than at the leading edge. This difference may reflect weaker coupling of CD44 to the underlying cytoskeleton in the dynamic leading edge region. Interestingly, our results are in line with the observations by Jacobson et al. 1984 . The diffusion coefficient of an 80-kD glycoprotein that they studied and which was identified several years later as CD44, was threefold higher near the leading edge of motile cells compared with the trailing region. However, when the fluorescence recovery rates were measured in arbitrary regions of the cells, typically ventral or trailing, it was clear that the extent of the lateral mobility of CD44 was dependent on the state of the actin cytoskeleton and lipid composition of the plasma membrane. Thus, the lateral mobility of CD44 was significantly enhanced when either the actin cytoskeleton was disrupted by latrunculin A or the plasma membrane cholesterol was depleted by M-b-CD. This observation would support our hypothesis that CD44 interacts with the actin cytoskeleton in a cholesterol-dependent manner. In addition, there is significantly more CD44 floating to the lipid rafts fraction after the disruption of the actin cytoskeleton by latrunculin A, which again argues for the interaction of CD44 with actin taking place in the lipid rafts. Annexin II could fulfill a cross-linker function in several ways. First, it might directly link CD44 with actin, which could be achieved by its interaction with CD44 on a protein–protein level. However, our experiments do not point in this direction. Alternatively, annexin II can either modify rafts, therefore, ensuring the possibility of their interaction with the cytoskeleton via different means, or, due to its high affinity to lipids, cross-link entire raft structures to the underlying actin cables. Clearly, more work will be required to resolve these exciting questions. | Study | biomedical | en | 0.999997 |
10459019 | MDCK cells are a canine kidney-derived nontransformed epithelial cell line that are maintained in DME (GIBCO BRL), supplemented with 5% FBS. A1N4 cells are a human mammary nontransformed epithelial cell line that are grown in IMEM, supplemented with 0.5% FBS, 0.5% hydrocortisone, 5 μg/ml insulin, and 10 ng/ml EGF . These cells synchronize in G 0 in the absence of EGF. The wild-type (WT) and S37A mutant (S37A) β-catenin plasmids were described previously . The bacterial chloramphenicol acetyltransferase gene driven by the CMV promoter of the pcDNA 3 plasmid (Invitrogen Corp.) served as the negative control (CON). For stable transfections, 800,000 MDCK cells were plated per 100-mm tissue culture plate. The next day, 15 μg of the various plasmids were transfected using the lipofectamine PLUS method (GIBCO BRL): 32 μl lipofectamine and 45 μl PLUS reagent. All of the plasmids included the neomycin-resistance cassette for selection. 48 h later, the cells were split 1:20 and cultured for 2 wk in the presence of 500 μg/ml of Geneticin (GIBCO BRL). An approximately equal number of colonies grew up for each transfected plasmid. For each transfection, all of the colonies were trypsinized and combined to give stable cell pools. Whole cell and cytoplasmic lysates were made and immunoblotting performed as described previously . Cells were grown to confluence in 4-well BIOCOAT chamber slides (Falcon Plastics). Cells were washed twice in PBS and fixed in 4% paraformaldehyde in PBS for 10 min. Cells were then permeabilized in 0.2% Triton X-100, 4% paraformaldehyde in PBS for 10 min. After washing in PBS, cells were blocked in 3% ovalbumin for 1 h. The chambers were incubated with primary antibodies overnight at 4°C. After washing in PBS five times for 5 min each, fluorescein- or Texas red-conjugated secondary antibodies were added for 1 h. Primary and secondary antibodies were diluted in 6% normal goat serum. After removal of the secondary antibody, the chambers were washed five times for 5 min in PBS, and the chambers removed. The cells were mounted with Vectashield (Vector Labs, Inc.). The anti–β-catenin and anti-p27 mAbs were from Transduction Laboratories. The antihemagglutinin mAb (HA-11) was purchased from Berkeley Antibody Co., Inc. A second high affinity anti-HA mAb was purchased from Boehringer Mannheim Corp. . The anti–E-cadherin (SHE78-7) mAb was purchased from Zymed Labs, Inc. Peroxidase- and fluorescein-labeled secondary antibodies were purchased from Kirkegaard and Perry Laboratories, Inc. The Texas red-labeled secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. In 12-well dishes, cells were transfected with 0.5 μg of the TOPFLASH LEF/TCF reporter plasmid and 0.005 μg of the constitutively expressed Renilla luciferase, as a normalization control. As a negative control, cells were transfected with the FOPFLASH reporter plasmid in which the LEF/TCF binding sites have been mutated. The cells were lysed and assayed for Firefly and Renilla luciferase activities using the STOP & GLO assay (Promega Corp.). All results are normalized to the Renilla luciferase activity. For each cell pool, 150,000 cells were suspended in 3 ml DME + 5% FBS, and warmed to 37°C. 300 μl of a prewarmed (52°C) 3% agarose/PBS solution was mixed with the cell suspension and then layered into 3 wells of a 6-well plate (1 ml/well), which were previously coated with 1 ml of 0.6% agarose in DME. The agar was allowed to solidify at room temperature for 20 min before 3 ml of growth medium was added to each well. The medium was changed every three days. After 14 d, the colonies were counted by an Omnicon 3600 Colony Counter and photographed. To have an equal number of cells plated at the first time point, 10,000 CON, and 5,000 WT and S37A cells were plated per well of 12-well plates. At each time point, the cells were washed once in PBS and trypsinized in 1 ml trypsin/versene (GIBCO BRL). The single cell suspension was counted on a Coulter Counter set at 10 μm min with 20-μm maximum diameter. Each data point was performed in triplicate. For each cell pool, 100 cells were plated onto each of three 100-mm tissue culture dishes in DME + 5% FBS. 4 d after plating, the colonies were photographed at 400×. After 8 d, the cells were washed with PBS, stained with crystal violet, and washed with water. The colonies were counted and then photographed. The plating efficiency is the mean number of colonies per dish/100 cells plated per dish. Cells were cultured in 6-well plates 3 d after confluence. The cells were washed twice in PBS and 2 ml of fresh medium was added to each well. 24 h later, the shed cells were removed with medium and counted on a Coulter Counter, as described. Two flow cytometric assays were used. Cells were washed in PBS and trypsinized. Cells were washed in PBS and pelleted. After removing the wash buffer, the pellet was vortexed and resuspended in 0.1 ml of citrate/DMSO buffer (250 mM sucrose, 40 mM Na 3 C 6 H 5 O 7 2H 2 O, 5% DMSO, pH 7.60). The pellets were then frozen at −80°C. The cells were then processed as in Vindelov et al. 1983 . Cells were washed once in PBS and trypsinized. Trypsinized cells were pelleted at 1000 g and washed in 5 ml cold PBS. After a second centrifugation, the cells were resuspended in 0.5 ml cold PBS and fixed by dripping in 1.5 ml cold 100% ethanol, while slowly vortexing the cell suspension. After at least 1 h at 4°C, the cells were stained with propidium iodide and DNA content was measured by flow cytometry. The ethanol fixation method was also used for the flow cytometric analysis of apoptosis. A1N4 cells were plated in 100-mm tissue culture dishes and grown overnight to ∼40% confluency. The cells were washed three times in PBS and then maintained in the absence of EGF for 46–50 h. This synchronized >95% of the cells in the G 0 /G 1 phase of the cell cycle. To stimulate reentry into the cell cycle, EGF-containing medium was added back to the cells. Parallel dishes were analyzed at each time point for β-catenin protein (whole cell or the cytoplasmic pool) and for the cell cycle distribution. 50,000 A1N4 cells were plated per well of 12-well dishes and transfected with 1 μg of the TOPFLASH reporter plasmid and 0.01 μg of the Renilla control plasmid by the calcium phosphate method. The cells were then synchronized by EGF starvation (G 0 /G 1 ) or 1 μM nocodazole (G 2 /M), or treated with the proteosomal inhibitor ALLN, which stabilizes β-catenin. The cells were collected and the luciferase measurements were made as described. Confluent cells were trypsinized into a single cell suspension. 700,000 cells were plated in 150-mm tissue culture dishes coated with 0.8% agarose, to which they could not attach. At the various time points, the cells were collected, washed in PBS, and any cell aggregates were dispersed by trypsinization. Cells were then analyzed for apoptosis using three separate assays. Samples were analyzed by flow cytometry (see Cell Cycle Analyses, Ethanol Fixation). In this analysis, the hypodiploid peak constituted the apoptotic population. Samples were stained with fluorescein-labeled AnnexinV and propidium iodide (Trevigen) according to the manufacturer's protocol, and analyzed by flow cytometry. The two AnnexinV positive quadrants of the analysis were taken as the apoptotic fraction. Cells were fixed in 10% formalin for 10 min and stained with Hoechst #33258 (25 μg/ml in PBS) for 10 min at room temperature in the dark. Cells were placed on a glass slide and analyzed by fluorescence microscopy. 750,000 CON, and 500,000 WT and S37A cells were plated in T75 tissue culture dishes. 26 h later, the flasks were exposed to 5 Gy of γ-irradiation. Another group of flasks received a mock irradiation (0 Gy). At 8 and 24 h after irradiation, the cells were trypsinized and their cell cycle profile was determined. To investigate the effects of β-catenin on normal cellular function, MDCK cells were stably transfected with constitutively expressed β-catenin transgenes that have been engineered to contain a COOH-terminal HA tag. In addition to WT β-catenin, a construct harboring a previously described serine to alanine point mutation at residue 37 (S37A) was used, which encodes for a β-catenin protein largely resistant to ubiquitination . The cells used are pooled stable transfectants; that is, after selection with G418, all of the drug resistant colonies resulting from each transfection were combined. These will be referred to as cell pools. As a negative control, a cell pool expressing the bacterial chloramphenicol acetyl transferase gene was generated (CON). Stable cell pools were generated to avoid the phenotypic artifacts that can result from the selection and propagation of individual clones derived from single transfected cells. We found that MDCK cells are especially prone to clonal morphological variation. When examined by immunoblotting, expression of the HA tag was detectable only in the cell pool expressing the more stable S37A mutant . We believe that epitope inaccessibility and antibody insensitivity result in the poor detection of the HA-tagged β-catenin and, consequently, the HA tag was undetectable by immunoblotting in untreated WT cells. To demonstrate that the WT cells were capable of expressing HA-tagged β-catenin, all three cell pools were treated with the histone deacetylatase inhibitor sodium butyrate to nonspecifically increase gene expression. This treatment resulted in clearly detectable expression in the WT cells and very high expression in the S37A cells, whereas the CON cells lacked expression under both conditions. Sodium butyrate treatment was not used in any other experiments in this study. In untreated cells, a similar pattern was seen by immunofluorescence microscopy. Using an antibody specific for the HA tag and a fluorescein-labeled secondary antibody, staining was detectable in the S37A cell pool , but was difficult to detect in the WT cells (data not shown). To demonstrate the HA tag in the WT cells, a high affinity anti-HA antibody (Boehringer Mannheim) and a Texas red-conjugated secondary antibody was used to increase the sensitivity of the assay. Under these conditions, expression of the HA-tagged protein was clearly demonstrable in most of the WT cells , even in the absence of butyrate, whereas expression was not evident in the CON cells . A β-catenin specific antibody revealed a normal staining pattern in all three cell pools . Whole cell lysates do not exhibit any significant increase in total β-catenin levels (data not shown) because MDCK cells express a large amount of endogenous β-catenin, most of which is complexed with E-cadherin at the cell membrane. However, it is the cytoplasmic pool that is involved in β-catenin signaling and an increase in this pool was evident in both WT and S37A expressing cells, as compared with the CON cell pool . To confirm that β-catenin was being functionally overexpressed in both the WT and S37A cell pools, LEF/TCF-dependent nuclear signaling was measured using the TOPFLASH reporter construct . This reporter consists of four consensus LEF/TCF binding sites placed upstream of the cFos minimal promoter. As a negative control, a similar reporter construct (FOPFLASH), in which the LEF/TCF binding sites have been mutated, was used. Even though the HA tag was not easily detected in the untreated WT cell pool, LEF/TCF signaling is elevated well above the control in both the WT and S37A cell pools. Overexpression of β-catenin in MDCK cells previously was shown to alter cell morphology. The stable cell pools used in this report have essentially the same morphology as the MDCKs expressing an inducible form of NH 2 terminally truncated β-catenin . The WT and S37A cell pools are less efficient at forming tight colonies of cells, as compared with CON cells . In addition, the cells along the edges of the WT and S37A colonies tend to extend projections more readily, giving them a more mesenchymal morphology. The morphology of these cell pools also varied at high density. In contrast to their appearance at lower density, the WT and S37A cells appeared to be more tightly adherent to each other (data not shown). This is supported by the fact that these cells are significantly slower to round up when trypsinized during normal cell passaging. To confirm that expression of the β-catenin transgenes did not prevent strong intercellular adhesion, the ability of the WT and S37A cells transepithelial resistance was measured in the presence and absence of Ca 2+ . Both the WT and S37A cells formed a strong barrier in the presence of Ca 2+ (>1,000 ohms/chamber) that was completely diminished in the absence of Ca 2+ . These results are consistent with what is seen in normal epithelial cell lines and confirms strong cadherin-mediated adhesion. To characterize the distribution of these cells in the cell cycle, DNA/flow cytometry analysis was performed on these cells during exponential growth phase. Both of the β-catenin overexpressing cell pools had a reduced proportion of G 0 /G 1 cells and an increased proportion of S and G 2 cells, as compared with the control cells . This suggests that either a greater proportion of the WT and S37A cells are cycling or the G 1 phase of the cycle is shorter in duration than it is in the CON cells. Growth curves demonstrated a significant difference between the β-catenin overexpressing cells (WT and S37A) and the CON cells . The curves depicting the growth of the WT and S37A cell lines diverged from that of the CON cells, demonstrating that the alterations in cell cycle distribution resulted in increased growth. Also, overexpression of β-catenin increased saturation density of these cells . Together with the demonstration that the WT and S37A cells proliferate more rapidly at confluence , it is clear that β-catenin overexpression significantly diminishes the property of contact inhibition of growth. Interestingly, in every replication of this experiment, the number of cells in the WT and S37A wells was elevated (up to 50%) above the CON cells at the first time point of the growth curve. To determine if a difference in plating efficiency might explain the discrepancy in the cell number on the first day of the growth curves, 100 cells were plated per 100-mm tissue culture dish in three dishes for each cell pool. The colony count provides a rough estimate of the plating efficiency of the cells. This experiment revealed a small (but not statistically significant) difference in plating efficiency that may contribute to the consistent differences in cell number, but does not explain them entirely . We believe that the combination of increased plating efficiency and elevated proliferation rate account for the differences seen at the first time point. An obvious increase in the rate of colony growth in the β-catenin overexpressing cells was more dramatic. The colonies from the WT and S37A cells were many fold larger than those from the CON cells. The morphology of these clones provides one explanation for the difference in colony size . Whereas the CON cells formed tightly adhesive, epithelioid colonies , the WT and S37A cells formed a large number of colonies containing a more scattered, mesenchymal phenotype . The morphological changes suggest that enhanced motility may contribute to this dramatic increase in colony size, but this is speculative. Also, the reduced adhesiveness in the WT and S37A cells may promote large colony formation by avoiding the contact inhibitory effect of tight cell–cell adhesion. In addition, other data suggest that the WT and S37A cells have an increased proliferative rate, even in the presence of strong intercellular adhesion . The reduction in proliferative rate that nontransformed cells experience at high cell density has been termed contact inhibition of growth. Although this is a widely recognized phenomenon, the signaling mechanisms involved remain unknown. To address this, the MDCK cell pools were grown to confluence and cell cycle parameters were monitored. Pilot experiments revealed that the WT and S37A cells shed more cells into the medium than CON cells. To quantify this effect, cells that were two to three days after confluence were washed twice with PBS, and fresh medium was added. The medium was collected from the wells on the next day and the suspended cells were counted. The number of shed cells was markedly elevated in the WT and S37A cells, as compared with the CON cells . In these experiments, shedding of the S37A cell pool was consistently higher than in the WT cell pool. The hypothesis that a higher proliferative rate was responsible for the difference in cell shedding was tested by performing cell cycle analysis of these cells grown three days after confluence. This analysis demonstrated that the WT and S37A cells had a higher proportion of S phase and G 2 phase, and a lower percentage of G 0 /G 1 phase, as compared with the CON cells . This cell cycle profile is precisely what would be expected if the WT and S37A cells were proliferating more rapidly than the CON cells, and is consistent with other experiments in which the G 1 /S checkpoint control regulates contact inhibition . Presumably, in the absence of additional space to attach to the culture dish, the newly formed cells are shed into the medium. One important aspect of cell cycle regulation is cell cycle blockade after DNA damage. These blocks, which occur at the G 1 /S and G 2 /M transitions, presumably allow the cell to repair its DNA before the damage-induced errors become permanent . We postulated that β-catenin overexpression might alter the DNA damage-induced late G 1 block of the cell cycle in the MDCK cells. The three cell pools were γ-irradiated with 0 or 5 Gy. Eight hours after irradiation, all of the cell pools show some G 1 /S and G 2 /M cell cycle blockade . However, while CON had very few S-phase cells (5.96%), the WT and S37A cells retained a significant number of cells in S phase (15.26 and 14.99%). 24 h after irradiation, 25.2 and 21.4% of the WT and S37A cells, respectively, were in S phase, compared with 0.77% of CON cells. These data demonstrate that the radiation-induced G 1 /S block is strongly attenuated by the overexpression of β-catenin and indicates that elevated β-catenin might lead to the accumulation of DNA damage and increased incidence of other mutations. The previously described block of G 1 /S progression by APC in normal cells points to a role of endogenous β-catenin in the regulation of cell cycle progression in nontransformed cells . Together, with our demonstration that even the modest elevations of β-catenin described in this study can regulate cell cycle progression, this led us to investigate its level of expression throughout the cell cycle. Preliminary experiments were performed with parental MDCK cells that were partially synchronized in early G 1 by serum starvation. Parallel wells of cells were collected at various time points after release from G 0 by the addition of serum to make whole cell or cytoplasmic lysates for analysis of β-catenin protein levels. Although total β-catenin protein did not vary appreciably during the cell cycle, cytoplasmic β-catenin levels increased significantly from G 1 to S phase (data not shown). The increase began in late G 1 and continued through S phase. These pilot experiments led us to examine this phenomenon in the A1N4 cell line, which is easily synchronized in early G 1 by the removal of EGF from the growth medium. Like MDCK cells, cytoplasmic levels of β-catenin protein increased in late G 1 and continued to rise in S phase , whereas total cell β-catenin did not vary (data not shown). Densimetric scanning revealed a 23-fold increase in cytoplasmic levels from early G 1 /G 0 to S phase . As a control, the blot was reprobed for cyclin dependent kinase inhibitor, p27 . As expected, variations in p27 were inversely related to β-catenin. To determine if this oscillation in cytoplasmic β-catenin led to fluctuations in β-catenin–LEF/TCF signaling, A1N4 cells were assayed for TOPFLASH activity after being synchronized in G 1 phase or G 2 /M phase of the cell cycle. The level of β-catenin–LEF/TCF signaling corresponded with the levels of cytoplasmic β-catenin measured by Western blotting . The elevation in signaling at G 2 /M was greater than that induced by treatment with the proteosomal inhibitor, ALLN. These data indicate that oscillations in β-catenin signaling may be involved in the normal regulation of cell cycle progression. The ability of cells to proliferate in the absence of attachment to a solid substrate correlates well with the transformed, tumorigenic phenotype. To assess the oncogenic capacity of β-catenin in vitro, cells were suspended in 0.3% agar and allowed to grow for two weeks. The ability of the WT and S37A cells to form colonies in soft agar was clearly enhanced relative to the CON cells . Although the CON cells do exhibit a background level of colony formation, expression of the β-catenin transgenes resulted in a 10–20-fold increase in the number of colonies and an obvious increase in colony size . Multiple experiments did not demonstrate a significant difference between the WT and S37A cell pools. This is the first demonstration that full-length β-catenin, WT and S37A mutant, has transforming capacity. When nontransformed epithelial cells are deprived of attachment to an extracellular matrix for an extended period of time they undergo apoptosis . This suspension-induced apoptosis has been termed anoikis. In the soft agar growth experiments, it appeared that most CON cells die when suspended in soft agar. However, the remaining cells did contribute to a background rate of colony formation. To investigate the possibility that β-catenin increases the colony-forming capacity of MDCK cells by preventing anoikis, cells were cultured on a cushion of 0.8% agar in normal growth medium, collected at eight hour intervals over a 24-h period, and assayed for apoptosis. Microscopic examination of the cells after 16- and 24-h incubations revealed that the majority of the WT and S37A cells were larger and more refractile to light than the CON cells (data not shown), suggesting that the CON cells were preferentially undergoing apoptosis. These preliminary results were confirmed by DNA/flow cytometry and AnnexinV staining of cells that had been kept in suspension for 0, 8, or 16 h . Both methods showed that anoikis was significantly inhibited by β-catenin overexpression. The results of further analysis of the flow cytometry and AnnexinV data for the percentage of hypodiploid and AnnexinV-positive cells, respectively, are compiled in Table . The DNA/flow cytometry data revealed that the percentage of hypodiploid cells was markedly and consistently lower in the WT and S37A cells relative to the CON cells. However, these data significantly underestimate the percentage of apoptotic cells in the CON samples at the 16 h time point, as the disintegrating apoptotic cells were lost from the analysis. The AnnexinV assays appeared to retain these cells and probably give a more accurate estimate at 16 h. As a third independent method of measuring apoptosis, nuclear morphology of cells before and after suspension was analyzed by Hoechst staining. In contrast to the nonsuspended cells, which all had normal nuclear morphology , most of the suspended CON cells displayed characteristically shrunken apoptotic nuclei . In contrast, the nuclei of the majority of WT and S37A cells displayed a normal morphology . A fraction of the cells (∼1/4) were apoptotic, which is consistent with the AnnexinV and flow cytometry results. Interestingly, a minority of CON cells were found to be associated with clumps of five or more cells. Most of these cells displayed normal nuclear morphology. This was a clear demonstration that cell–cell adhesion can prevent apoptosis induced by suspension, and this probably caused us to underestimate the percentage of apoptosis among the suspended CON cells by the AnnexinV and flow cytometric methodologies. These data demonstrate that β-catenin overexpression may promote soft agar colony formation of MDCK cells by the promotion of cell cycle progression and the inhibition of anoikis. It is suspected that the cadherin-associated protein β-catenin promotes the process of carcinogenesis . The data that support this hypothesis include the following observations: it associates with and is downregulated by the tumor suppressor APC; it transduces (at least partly) the oncogenic Wnt growth factor signal to the nucleus; it is mutated in a significant number of human cancers; and, overexpression of an NH 2 terminally truncated form of β-catenin in the epidermis of transgenic mice produced well-differentiated hair tumors . However, no studies provide direct evidence for the transforming potential of full-length β-catenin. In addition, no investigations have addressed the question of which cellular processes β-catenin may regulate to effect cellular transformation. This report characterizes phenotypic alterations that result from β-catenin overexpression in a nontransformed epithelial cell line. Effects are seen in the regulation of three important cellular activities/properties: proliferation, apoptosis, and morphology. It demonstrates that modest β-catenin overexpression significantly enhances the ability of these cells to proliferate, especially in situations that would normally inhibit the cell cycle at the G 1 /S transition. Most striking is the demonstration that it promotes growth in soft agar, a phenotype closely correlated with tumorigenicity. Most nontransformed cells require adhesion through integrin receptors to extracellular matrix components to transit through the G 1 phase of the cell cycle . In addition, suspension of normal, attachment-dependent cells blocks them late in G 1 phase. β-Catenin overexpression also resulted in increased proliferation of cells at high cell density. The mechanism by which high cell density inhibits proliferation is unknown, but also involves a block in late G 1 . The presence of cell–cell adhesion, the reduction of cell-substrate adhesion, and the depletion of growth factors have all been implicated . β-Catenin's dual activities as a regulator of cadherin-mediated cell–cell adhesion and as the transducer of a mitogenic signal implicate it in this regulatory process. Both cadherin and α-catenin can inhibit β-catenin signaling in other experimental systems . Together, with the results of the present study, these data support the hypothesis that cell–cell adhesion promotes the formation of cadherin/β-catenin/α-catenin complexes and that these complexes negatively regulate β-catenin signaling, which discourages cell cycle progression. However, the fact that proliferation is reduced at high cell density, as compared with sparsely plated cells, even in the WT and S37A cells, suggests that other mechanisms are also involved . The cell cycle analyses and growth curves in this study demonstrate that β-catenin overexpression can significantly alter the proliferative rate of these cells. The distribution of the WT and S37A cells is weighted heavily toward S phase and away from G 1 . When considered along with the other cell cycle data, it appears that β-catenin overexpression expedites the G 1 /S transition in MDCK cells. The easing of the barrier to G 1 /S transition manifests as a difference in cell growth on plastic, as growth curves of the β-catenin overexpressing cells diverged significantly from the control cells. β-Catenin overexpression also has a notable effect on cell morphology. The MDCK cell line is a nontransformed epithelial line that has very strong intercellular adhesion and extends cell membrane extensions only to a limited degree. β-Catenin overexpression converts MDCKs into a more mesenchymal cell type . At low density, cell–cell adhesion is reduced and the cells take on a more spindly, stretched shape. This change in morphology is reminiscent of an epithelial to mesenchymal transition . EMTs are developmentally important cellular conversions, especially during gastrulation, the point in development at which β-catenin knockout mouse embryos are aborted. Also, an EMT has been suggested to underlie the progression from benign tumor to metastatic carcinoma . Indeed, it previously has been suggested that β-catenin signaling may regulate this process . The absence of anoikis is another characteristic of transformed cells. The present study and others have shown that MDCK cells are very dependent on attachment to the extracellular matrix for survival . After 16 h in suspension, the majority of CON cells were apoptotic, as measured by three independent methods. The expression of the WT and S37A β-catenin transgenes markedly retards this process, allowing ∼75% of the single cells to survive. This is a vigorous inhibition of anoikis. Taken together, the proliferation, anoikis, and morphology data demonstrate that these cells are clearly transformed by β-catenin. These in vitro results suggest that overexpression of full-length β-catenin should promote tumorigenesis in vivo. Two separate studies have demonstrated the effect of tissue-specific overexpression of an NH 2 terminally truncated form of β-catenin. Expression of the truncated form of β-catenin in the epidermis of transgenic mice by Gat et al. 1998 resulted in the formation of two types of hair follicle-related tumors. Taken together with the present study, these results strongly suggest that full-length forms of β-catenin are important mediators of oncogenesis in vivo. Interestingly, a study by Wong et al. 1998 , in which an NH 2 terminally truncated form of β-catenin was overexpressed in the intestinal epithelium of transgenic mice, produced conflicting results. Proliferation of the intestinal epithelial cells in these animals was stimulated 1.5–3-fold, in accordance with the results of the present study. However, the elevated proliferation rate was balanced by an increase in apoptosis, the net result being no change in intestinal villus height. To explain the discrepancy between these results and our own, we suggest that β-catenin overexpression can protect cells only from certain apoptotic signals. It is possible that the compensatory mechanism by which the authors suggested that the transgenic mice might have maintained their cell census in the face of increased proliferation is mediated through the stimulation of β-catenin–insensitive apoptosis. It is also possible that full-length β-catenin has signaling capacities that are lost when its NH 2 terminus is removed. The results presented in the present study also differ from those published previously by Young et al. 1998 . They reported that overexpression of the Wnt-1 growth factor transformed Rat-1 fibroblasts while expression of the S37A mutant form of β-catenin we described previously had no effect. Two differences between the two studies may explain the conflicting results. First, the morphological effects we describe may only be detectable in an epithelial cell type. Second, the studies of Young et al. 1998 were carried out without serum, whereas the present ones were done with serum. It is possible that Wnt-1 activates parallel signaling pathways (in addition to β-catenin signaling) that may circumvent the need for serum to stimulate proliferation. β-Catenin's position lower in the pathway may preclude the activation of such parallel pathways and, therefore, it is unable to stimulate proliferation of Rat-1 fibroblasts in the absence of serum. The cell cycle blocks that characterize the response of cells to DNA damage are important for the maintenance of genomic integrity. To prevent the permanent incorporation of mutations induced by various DNA damaging stimuli, the cell cycle can pause at the G 1 /S and the G 2 /M transitions . During these delays, the cell assesses the damage to its DNA and either repairs the damage or destroys itself. Premature reentry into the cell cycle may result in the accumulation of mutations to oncogenes and tumor suppressor genes, which would increase the likelihood of cellular transformation and cancer. The data from this study suggest that β-catenin overexpression may result in the premature reentry of cells into the cell cycle after γ-irradiation–induced DNA damage, and thereby promote the accumulation of oncogene mutations and carcinogenesis. An association between apoptosis and the APC/β-catenin axis has been suggested previously. Reexpression of the APC gene in a tumor cell line that lacks WT APC resulted in the induction of apoptosis within 24 h . Since one of the functions of APC is to downregulate β-catenin, it is possible that β-catenin itself is a regulator of apoptosis. Our demonstration that β-catenin alone significantly protects cells from anoikis strongly implies that it can be a potent inhibitor of apoptosis. Also, during the process of apoptosis, caspase-3 can cleave β-catenin protein . One purpose of this cleavage may be to destroy the antiapoptotic β-catenin signal within the cell and thereby hasten the completion of the apoptotic process. The caspase-mediated cleavage of focal adhesion kinase (FAK) is thought to function in this manner . It has been postulated that the induction of apoptosis by the loss of appropriate extracellular matrix attachment (i.e., anoikis) is a means of protecting the organism from improper cell growth . Anoikis is prevented by integrin-mediated signaling. Several enzymes have been implicated as being downstream of integrins in this signal transduction pathway. These include FAK, phosphoinositide-3-kinase, protein kinase B/Akt, and integrin-linked kinase . The present report suggests that β-catenin may also lie downstream of integrins. Several integrin-stimulated signaling pathways might lead to the induction of β-catenin signaling. One possible connection between integrins and β-catenin is the integrin-activated, antiapoptotic kinase PKB/Akt. PKB is known to inhibit the activity of glycogen synthase kinase 3-β, a serine kinase that functions directly to reduce β-catenin protein and signaling . It is possible that the result of these two inhibitory interactions is that activation of PKB by integrin signaling functions to positively activate β-catenin signaling. The data presented in this report describing the effects of β-catenin overexpression are similar to previous reports describing the effects of ILK . ILK is a 59-kD serine kinase that was first described as a β 1 -integrin-associated kinase. ILK overexpression causes cells to undergo an EMT and promotes their growth in soft agar. This is associated with an increase in LEF-1 protein levels. As a result of increased LEF-1, β-catenin becomes completely localized to the nucleus and β-cat-LEF/TCF signaling increases significantly. In addition, loss of cell attachment to the underlying ECM was shown to result in a dramatic reduction in LEF protein. In a separate study, ILK directly phosphorylated and inhibited the activity of GSK-3β. This may constitute another mechanism by which integrin signaling may result in increased β-catenin–LEF/TCF signaling. Anoikis results from the interruption of integrin-mediated signaling . In addition to ILK, the integrin-associated nonreceptor tyrosine kinase FAK may also be involved in the transduction of these signals because FAK signaling suppresses p53-dependent apoptosis . Ilic et al. 1998 also demonstrated that an atypical protein kinase C isoform (PKCλ/ι) is required for this p53-dependent apoptotic pathway, since inhibition with both chemical PKC inhibitors and a dominant–negative construct protect FAK-defective cells from apoptosis. Previously, we reported that an atypical PKC isoform was involved in regulating β-catenin degradation . Inhibiting atypical PKC activity using the same chemical PKC inhibitors used by Ilic et al. 1998 resulted in the inhibition of the ubiquitination and degradation of β-catenin. In addition, treatment of cells with these PKC inhibitors increases β-catenin–LEF/TCF signaling (unpublished results). Taken together with the present study, it is possible that the inhibition of PKCλ/ι or another atypical PKC may increase β-catenin stability and signaling, leading to the suppression of p53-mediated apoptosis . The c-myc promoter is also regulated by the APC/β-catenin signaling pathway . The upregulation of c-myc by β-catenin may constitute one mechanistic link between β-catenin and tumor formation. c-myc is potent oncogene that regulates cell cycle progression. However, c-myc overexpression cannot induce cellular transformation on its own. In fact, when overexpressed alone, c-myc markedly increases the susceptibility of cells to apoptosis . To transform cells, c-myc requires an accompanying survival signal to prevent cells from undergoing apoptosis. Advancement through the G 1 phase of the cell cycle can result in either progression into S phase or apoptosis, depending on the presence or absence of certain survival signals, for example, IGF-1 . In addition to stimulating c-myc, β-catenin may transduce the requisite antiapoptotic signal that would permit cell cycle progression. The increase of cytoplasmic β-catenin protein before S phase during the cell cycle may serve this purpose in normal cells . Additionally, β-catenin would protect against anoikis if overexpressed in epithelial cells. Our data do not demonstrate any reproducible phenotypic difference between the WT and S37A expressing cells, except in the measurement of protein expression and in cell shedding at confluence. It is important to note that in both the WT and S37A cell pools, the level of cytoplasmic β-catenin protein and β-catenin–LEF/TCF signaling is elevated relative to the CON cells. This implies that a modest increase of cytoplasmic β-catenin can result in significant changes in signaling and cellular transformation and that overexpression of the wild-type gene alone is sufficient. This may also explain how the relatively small increase in endogenous cytoplasmic β-catenin that occurs before the onset of S phase may regulate the G 1 /S transition in the normal cell cycle . However, it is interesting to note that the increase in signaling above CON levels and the difference between the WT and S37A cells are relatively small when compared with other published results . It is possible that the fact that this study was performed with cells that stably express a constitutively active transgene is responsible for both phenomena. We believe that the very high levels of β-catenin expression and signaling that can be achieved in nontransformed cells by transient transfection is not conducive to their survival and propagation. If true, selection pressures against very high expression would: result in the production of stable cells expressing only moderately elevated β-catenin protein and signaling; and, limit the extent to which the S37A mutation could stimulate signaling above WT β-catenin. In addition, some studies have used different β-catenin mutants, which may be more active. It is plausible that some of the phenotypic alterations induced by β-catenin overexpression could be the result of altered cadherin function and independent of β-catenin signaling. However, the fact that these cells display strong intercellular adhesion at high density and retain the ability to generate tight junctions (as measured by electrical resistance across the monolayer in culture) demonstrates that E-cadherin function remains intact. The APC/β-catenin signaling pathway has been implicated in a large number of epithelial cancers . In most cases, mutations in either APC or β-catenin result in stabilization of β-catenin protein and elevated β-catenin–LEF/TCF signaling. However, it is not clear what role this pathway has in normal cells. In this study, we demonstrate that β-catenin is a potent oncogene. All of the major phenomena that characterize cellular transformation, that is, soft agar growth, altered morphology, inhibition of apoptosis, and stimulation of cell cycle progression, can be induced by the modest overexpression of β-catenin in a nontransformed epithelial cell line. This clearly indicates that β-catenin can play a direct role in the process of carcinogenesis and that a major component of APC function is its downregulation. These data suggest that, as an early event in the progression of colorectal cancer, activation of β-catenin signaling promotes adenoma formation by promoting proliferation and survival of epithelial cells in the abnormal tissue architecture of a tumor mass. In addition, it may also promote the accumulation of mutations and cancer progression by attenuating the DNA damage-induced G 1 cell cycle block. | Study | biomedical | en | 0.999997 |
10459020 | All chemicals were obtained from Sigma Chemical Co. unless otherwise stated. The antibodies U1, U13, and U14 were obtained from Ivan Gout (Ludwig Institute, London, United Kingdom). The antibody to the COOH terminus of p85 was obtained from Transduction Laboratories, and the antibody to p110 was obtained from Autogen Bioclear. The Sto x22 antibody to clathrin was a kind gift of Julian Downward (Imperial Cancer Research Fund, London, United Kingdom). p85 DNA was obtained from a human cDNA clone by PCR and then fused to the COOH terminus of MUT2 GFP cDNA . GFP-p85 contained all the 724 amino acids of the human p85α, GFP-2SH2 contained amino acids 321–724, GFP-CSH2 contained amino acids 615–724, and GFP-NSH2 contained amino acids 321–474. The chimeric cDNAs were cloned into the CMV promoter-driven plasmid pcDNA3.1/Zeo (Invitrogen), and microinjected at 0.1 ng/nl into the nuclei of C18 cells which had been serum starved (DMEM supplemented with 0.5% FCS) for 18 h. C18 (NIH-3T3 based), Cos-7, A431, and MCF-7 cells were cultured in DMEM supplemented with 10% FCS. For transfection, 5 × 10 6 Cos-7 cells were electroporated (0.3 kV, 250 μF, 0.4 mm cuvette) with 10 μg of either the GFP-p85, GFP-2SH2, or pcDNA3.1/Zeo as a control. After 48 h, the cells were lysed at 4°C in Triton buffer (50 mM Tris, pH 7.4, 5 mM EGTA, 1% Triton X-100, 150 mM sodium chloride, 25 mM benzamidine, 10 μg/ml leupeptin, 0.2 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM PMSF) for immunoprecipitation. The fusion proteins were in vitro transcribed and translated using a TNT-coupled reticulocyte lysate system (Promega) with [ 35 S]methionine labeling. The protein sizes were deduced using SDS-gel electrophoresis (10% gel) and autoradiography. A 50-μl TNT reaction was sufficient for six immunoprecipitations. The reaction was diluted in 2.5 ml of PBS, and 400 μl of the protein mix was immunoprecipitated for 2 h with antibodies bound to protein A–Sepharose. The immunocomplexes were washed four times with RIPA buffer (PBS with 1% Triton X-100, 0.1% SDS) and once with PBS/0.2% Triton X-100 before being eluted with SDS-PAGE buffer at high temperature. GFP-p85 and GFP-2SH2 proteins from transiently transfected Cos-7 cells were immunoprecipitated using an antibody to GFP (gift of Dave Shima, Imperial Cancer Research Fund, London) to distinguish them from the endogenous p85. Proteins were separated on a 10% SDS-polyacrylamide gel and subjected to Western blotting with an antibody to p85 (1:250; Transduction Laboratories). The filter was then stripped for 1 h at 60°C with 2% SDS, 100 mM mercaptoethanol in 62.5 mM Tris, pH 6.8. This was then blocked and reprobed with an antibody to the p110 catalytic subunit (1 μg/ml; Autogen Bioclear). Detection was by incubation with horseradish peroxidase–conjugated secondary antibody and visualization was by enhanced chemiluminescence (Amersham). C18 cells in 9-cm dishes (80% confluent) were stimulated with 5 × 10 −7 M EGF for 2.5 min to bring about phosphorylation of the erbB-3 chimera. The cells were lysed in Triton X-100 buffer at 4°C. The chimera was precipitated for 2 h with 5 μg of EGF receptor 1 (EGFR1) which reacts with the extracellular domain of EGFR and 2.5 mg of protein A–Sepharose. Immunocomplexes were washed and analyzed as above. The phosphorylation status of the chimera was assessed using an antibody specific for phosphotyrosine (PY20, 1:1,000; Transduction Laboratories). Microscopy was performed using a Zeiss Axiovert 135 microscope using a 63× objective lens with fluorescein and Cy3 filters. The images were captured with a CCD camera and processed using IPLab and Photoshop 4.0 software. Fluorescence intensity was normalized to the first time point in the films. The temperature was maintained throughout at 37°C using an environmental chamber. For immunofluorescence, cells were grown on glass coverslips. They were fixed at 4°C for 15 min with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and incubated with 1:50 antivinculin antibody (Sigma Chemical Co.). Antibody detection was by reaction with Cy3-conjugated goat anti–mouse IgG for 20 min at room temperature. Localization of clathrin was performed essentially as for vinculin using the anticlathrin mouse mAb Sto x22 at 1:100. Immunostaining for the chimera was performed using the EGFR1 mAb as described by Gullick et al. 1985 . A number of constructs were made containing different portions of p85 fused to GFP: the full length p85 protein (GFP-p85), the two SH2 domains (GFP-2SH2), the NH 2 -terminal SH2 domain (GFP-NSH2), and COOH-terminal SH2 domain (GFP-CSH2). The constructs were in vitro transcribed and translated in the presence of [ 35 S]methionine, and the resulting proteins were immunoprecipitated with antibodies to GFP and p85 to check the integrity of the encoded proteins . GFP-p85 was immunoprecipitated with an antibody to GFP, the inter–NSH2-SH3 domain (U1), the SH3 domain (U13), and the COOH-terminal SH2 domain (Transduction Laboratories) . GFP-2SH2 was immunoprecipitated with antibodies to GFP and the NH 2 - (U14) and COOH-terminal SH2 domains (Transduction Laboratories) . GFP-CSH2 and GFP-NSH2 were immunoprecipitated with anti-GFP and COOH- and NH 2 -terminal SH2 domain antibodies, respectively . GFP-p85 was able to bind weakly to protein A–Sepharose in the absence of antibody , possibly due to exposure of hydrophobic sequences in the unfolded protein. The COOH-terminal SH2 antibody immunoprecipitated GFP-p85 and GFP-2SH2 well but GFP-CSH2 very weakly, which suggests that the antibody recognizes the larger protein with a higher affinity. p85 binds the p110 catalytic subunit between its two SH2 domains to form the holoenzyme. To determine whether the GFP-tagged proteins engineered to encode this region retained their ability to bind p110, the full length and the 2SH2 constructs were transiently transfected into Cos-7 cells. The expressed proteins were immunoprecipitated with an antibody to GFP to distinguish them from the endogenous p85 and were then analyzed by SDS-PAGE and immunoblotting. The proteins were blotted with an antibody against p85 and then reprobed with an antibody to p110 . In both the full length and 2SH2 transfected cells, but not the mock transfected cells , the p110 subunit was coimmunoprecipitated, indicating that the addition of GFP to their NH 2 termini did not interfere with their ability to form heterodimers. The behavior of the GFP-p85 proteins in live cells was assessed by digital fluorescence microscopy. A stable NIH-3T3 cell line (C18) was used which expressed low levels of endogenous murine EGFRs (<2,000/cell) and a high level of a chimera of the extracellular domain of human EGFR fused to the cytoplasmic domain of human c-erbB-3 (130,000/cell) . Activation with EGF has been shown previously to recruit p85 to the chimera , probably as a result of transphosphorylation by endogenous EGFRs because the erbB-3 receptor is thought to be kinase defective . Intranuclear microinjection of the C18 cells with the GFP-p85 construct resulted in visible expression as early as 2 h after injection. The protein was distributed throughout the cytoplasm and was excluded from the nucleus . The GFP-tagged 2SH2, CSH2, and the NSH2 domains were distributed throughout the cytoplasm but were also present to a lesser extent in the nucleus . The GFP-p85 and GFP-2SH2 proteins, but not the smaller constructs, were also localized at discrete points at the periphery of the cell , which were identified as focal complexes by colocalization experiments with an antibody to vinculin , demonstrating that both of the SH2 domains have to be present for this localization to occur. This peripheral localization of p85 was also apparent in the epithelial cell lines, A431 and MCF-7, after microinjection with GFP-p85 . Addition of 500 nM EGF to C18 cells expressing the GFP-p85 protein resulted in recruitment of p85 to the cell surface where it formed patches . The EGFR/c-erbB-3 chimera was also visualized after 500 nM EGF addition using an anti-EGFR antibody. Fig. 5A and Fig. B , shows that the receptors cluster to form the same patching pattern. To confirm that p85 is localizing to the receptor and not to another cellular site, rhodamine EGF was used to mark the external part of the receptor. Incubation of injected cells with 50 nM rhodamine EGF for 5 min resulted in patching indistinguishable from unlabeled ligand. Paired rhodamine/GFP images of the cells were then taken. A similar pattern was obtained for the rhodamine and GFP images, demonstrating that the majority of p85 relocalizes to the receptor . Expression of the GFP-p85 protein in mock-transfected NIH-3T3 cells also resulted in focal complex localization in unstimulated and stimulated cells, but no patching was seen after EGF addition, indicating that p85 was only recruited to the erbB-3 chimera and not to endogenous EGFR homodimers (data not shown). Recruitment to the receptor was first visible after 30 s and was observed up to 1 h after EGF addition. During this time no visible internalization occurred. Cells were costained with anticlathrin antibodies to demonstrate the lack of coated vesicles or early endosomes associated with the patches . There was also no change over 1 h after ligand stimulation in the localization of the tagged PI 3-kinase at the focal complex sites. EGF addition to A431 cells expressing the GFP-p85 protein also resulted in translocation to the membrane and patching . In A431 cells, the EGFR is normally present at the base of microvilli. Immediately after 500 nM EGF addition, the surface of the cells and the microvilli undergo ruffling, hence GFP-p85 recruitment to the receptor at the base of the microvilli is visible as a wave pattern. After 10 min, when the ruffling has subsided, the familiar patching pattern was observed. The patches are roughly the same size as those formed in C18 cells, which is to be expected as they have similar levels of erbB-3 present . Although internalization was not generally apparent in this cell line, certain individual cells showed signs of internalization after 1 h of EGF incubation . Visualization of the clustering of the p85–receptor complex also enabled us to identify changes in cluster formation in response to ligand concentration. At 500 and 50 nM EGF, clustering occurs in patches over the complete surface of the C18 cells within 2–3 min. At 5 nM EGF, however, the clustering occurred reproducibly at the cells' periphery . At the lower EGF concentration of 0.5 nM and 50 pM no patches were observed . The mechanism by which p85 translocates to the membrane after activation is not known. Relocation of p85 could be by diffusion or could be regulated by its lipid products or by interaction with the actin cytoskeleton . PI 3-kinase activity has been suggested to be involved in actin cytoskeleton–dependent translocation of src to the cell membrane because of its affinity for v-src and also because of its role in protein trafficking . To address whether p85 redistribution is controlled in a similar manner, cells expressing GFP-p85 were incubated with cytochalasin D to disrupt the actin cytoskeleton . Although cytochalasin D is not thought to depolymerize all cellular actin, phalloidin staining of the treated cells showed that most was disrupted and the cell shape was visibly distorted. Despite this treatment, p85 was still able to translocate to the membrane after EGF addition . Treatment of GFP-p85–expressing cells with Ly294002, an inhibitor of PI 3-kinase activity, also did not affect p85 movement . These data suggest that p85 translocation to the membrane is not dependent on the catalytic activity of the enzyme or the integrity of the actin cytoskeleton. However, relocalization was inhibited by the EGFR tyrosine kinase inhibitor AG1478 at a concentration (1 μM) sufficient to completely inhibit receptor tyrosine phosphorylation , demonstrating that this activity is required. Thus, although SH2 binding is required it does not appear to be sufficient for relocalization since EGF addition to cells expressing either the two SH2 domains or the NH 2 - or COOH-terminal SH2 domain alone did not result in patching (data not shown). Direct visualization of p85 within live cells results in positional information about the signaling molecule that cannot be easily obtained by traditional biochemical methods. We have observed a focal complex localization for PI 3-kinase in resting cells, which is dependent on the binding of the two SH2 domains of p85. Focal adhesions not only act as structural links between the extracellular matrix and the actin cytoskeleton but also transduce biochemical signals from the extracellular matrix . Clustering of cell surface integrins is associated with the tyrosine phosphorylation of a number of focal adhesion proteins. In fibroblasts these include focal adhesion kinase, tensin, p130 cas , and paxillin . These proteins can then interact with signaling molecules which contain SH2 domains, such as src kinases , csk, and adapter proteins, such as crk and Grb2 . p85 association with focal adhesion kinases via its SH2 domains in vitro has been described previously . p120 cbl , an adapter protein, has also been reported to bind PI 3-kinase in macrophages and in PC12 cells. In PC12 cells, p120 cbl was thought to link PI 3-kinase to the EGFR independently of the presence of erbB-3 . However, we did not see any evidence of EGFR/PI 3-kinase linkage in NIH-3T3 cells without chimera after EGF stimulation (data not shown), although this may be due to very low levels of EGFR expression. In macrophages, p120 cbl has been reported to link PI 3-kinase to the integrin signaling pathway . Both focal adhesion kinase and p120 cbl are potential p85 binding sites because they both have at least two pYXXM motifs . Association of PI 3-kinase with other focal adhesion proteins such as src kinase has been described, but these interactions are mediated via SH3 domains and cannot be responsible for the binding seen during these experiments . It is interesting that PI 3-kinase is only associated with focal complexes at the periphery of the cells, and not to focal adhesions underneath the cell. Focal complexes share many constituents with focal adhesions but they are morphologically distinct . PI 3-kinase at these complexes may be involved in the signal pathways that are responsible for membrane ruffling and cell migration, especially since it is known to be involved in hepatocyte growth factor–induced cell scattering of MDCK cells . Such focal complexes are under the control of Rac, which has been identified as a downstream target of PI 3-kinase . The focal complex localization of p85 indicates that PI 3-kinase is directly involved in signaling through these complexes and not just as an upstream regulator. After 500 nM EGF stimulation, GFP-p85 is rapidly translocated to the EGFR/erbB-3 receptor dimers which then form patches. No other cellular relocation was observed. In C18 cells, the patches appear to remain at the cell membrane with no obvious receptor internalization occurring. The patches were also not associated with clathrin, which suggests that the receptors were not being internalized into endosomal vesicles. Heregulin is slowly internalized and degraded when it is added to cells that express high levels of erbB-3 and erbB-2 receptors . Experiments using a similar EGFR/erbB-3 chimera in NIH-3T3 cells have also shown that internalization is very limited and the half-life of the receptor remains unchanged after the addition of EGF . EGFR is the only member of the type 1 growth factor receptor family that can internalize normally , and in A431 cells certain individual cells did show signs of receptor/p85 internalization. The difference in receptor trafficking between the cell lines could be due to their receptor status. C18 cells have 60 times more (chimeric) erbB-3 receptor than EGFR. Upon EGF stimulation the predominant receptor present in the receptor/GFP-p85 clusters will be erbB-3, which slows down the dimer internalization. In A431 cells, there is 10 times more EGFR present than erbB-3. After EGF stimulation of these cells, the predominant receptor in the clusters will be EGFR, which might explain why internalization appears to occur after 1 h of ligand stimulation. Note that this is slower than expected for EGFR expressed alone, but as PI 3-kinase binds only to erbB-3, we are presumably only following internalization of erbB-3/EGFR heterodimers. p85 remains visible in the clusters at the cell membrane for up to 1 h. After 45 min, the erbB-3 chimera is fully dephosphorylated and mitogenic signaling should be attenuated . PI 3-kinase has been implicated in the trafficking of receptors in mammalian cells; indeed, the presence of p85 binding sites on the platelet-derived growth factor receptor is required for its trafficking and degradation , and p85 has been reported to be internalized with the receptor . Wortmannin, a PI 3-kinase inhibitor , can also affect other processes, such as transferrin receptor recycling and transcytosis . It can also induce changes in the morphology of endosomes . PI 3-kinase lies upstream of early endosomal antigen 1, which binds to endosomes , and upstream of Rab5, which regulates early endosome fusion . These data suggest that a continuous p85 presence may be involved in the trafficking of the erbB-3 receptor, which is probably recycled rather than degraded . The translocation of p85 to the receptor is not dependent on PI 3-kinase activity or the actin cytoskeleton; thus, its binding to erbB-3 is more likely due to random collision rather than active relocation. However, it is dependent on the tyrosine phosphorylation of the cytoplasmic domain of erbB-3, which allows SH2 interactions. It is generally believed that the two SH2 domains are all that are required for receptor binding . However, the results presented here suggest that the NH 2 -terminal portion of the protein (containing the SH3, Bcr, and proline-rich domains) is also important in localizing p85 to the erbB-3 receptor, possibly due to self association allowing the recruitment of additional copies to the complex, or by stabilization of p85 at the receptor by binding to other signaling molecules. PI 3-kinase itself has many functions in different intracellular systems. Our results demonstrate that in NIH-3T3 cells it exists at focal complexes and probably as an inactive cytoplasmic pool which is drawn to the cell surface by growth factor receptors in response to extracellular signals. Direct visualization of the subcellular location of second messengers using this technique will allow similar experiments in other cell types and in response to other stimuli to determine if these are common effects. In these environments, low affinity interactions, possibly not measurable by biochemical techniques, may be observed with activators, substrates, and downstream molecules. Finally, since ligand binding does not accelerate internalization of c-erbB-3 (or c-erbB-2 and c-erbB-4), it is not clear why receptor clustering occurs in this system. It is also not known if ligand-unoccupied as well as occupied receptors are present in these patches. It is possible that such behavior may control signal sensitivity by mechanisms such as those described recently by Bray et al. 1998 . It is also possible that aggregation of second messenger molecules or the juxtaposition of different second messengers may permit combinatorial signaling. Light-based systems should be helpful in exploring such models. Moreover, they might be utilized to act as screens for promising pharmacological activators or inhibitors. | Other | biomedical | en | 0.999998 |
10459021 | Transgenic mice expressing the TIMP-1 (Ts + ) or TAg (TAg + ) transgenes in liver were generated and bred as described previously . Single transgenics were crossed to generate four categories of littermates designated as wild-type controls (TAg − /Ts − ), TIMP-1 controls (Ts + ), TAg controls (TAg + ), and double transgenic TIMP-1–overexpressing (TAg + /Ts + ) mice. Female littermates were killed at specified ages, and the liver tissue was processed and embedded or flash frozen for analyses. Liver tissue was homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 1.0% NP-40, 150 mM NaCl, 0.5 M PMSF, 1 mM EDTA, 10 μg/ml pepstatin, 10 μg/ml leupeptin) at 4°C. Samples were centrifuged for 10 min at 16,000 g , the supernatants collected, and the protein content determined by the Bradford assay. Aliquots containing 2.5 mg of protein were adjusted to a volume of 500 μl. TAg was immunoprecipitated by adding 0.5 μg/ml of anti-SV40 large T small t antibody (clone PAb 108; PharMingen) and 50 μl of Gamma Bind Plus Sepharose (Amersham Pharmacia Biotech), and rocking for 2 h at 4°C. Immunoprecipitates were collected by centrifugation in a refrigerated Eppendorf centrifuge and washed three times with NET/gel buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin, 0.02% sodium azide). The samples were subjected to 10% SDS-PAGE and electroblotted to Hybond N nylon membrane (Amersham Pharmacia Biotech). The membranes were sequentially probed with primary antibodies PAb 108, anti-p53 (clone PAb 421; obtained from Dr. S. Benchimol, Ontario Cancer Institute), and anti-RB (clone G3-245; PharMingen), followed by hybridization with peroxidase-conjugated anti–mouse IgG antibodies and ECL chemiluminescence (Amersham Pharmacia Biotech). Total RNA isolated from individual liver tissue samples (20 μg) was electrophoresed in formaldehyde agarose gels, subjected to Northern blotting and sequential hybridizations with [α- 32 P]dCTP-labeled and random primed cDNA probes for murine IGF-II (cDNA obtained from Dr. G. Bell, University of Chicago, Chicago, IL), rat IGFBP-3 (cDNA, obtained from Dr. A. Herington, Queensland University of Technology, Brisbane, Australia), 18S ribosomal RNA, and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were performed. The latter two probes were used to control for equal loading and transfer of samples. Paraffin sections of formalin-fixed liver tissue were generated as described previously . Clone PAb 108, biotinylated goat anti–mouse IgG, and streptavidin-peroxidase conjugate (Zymed) were used for TAg immunohistochemistry, and peroxidase-conjugated anti–proliferating cell nuclear antigen (PCNA) antibodies (Dako) were used to detect proliferating cells. The detection of bound antibody was carried out using diaminobenzidine (Kirkegaard & Perry), which forms a reddish-brown pigment at sites of peroxidase activity. Digoxygenin-labeled (Boehringer Mannheim) IGF-II riboprobes were synthesized using the rat IGF-II cDNA, which was cloned in Bluescript KS. In situ hybridization was performed as described previously . Specific signal appears as purple pigment. Liver samples were homogenized as for Western blotting. Equivalent amounts of protein from each sample (40 μg) were subjected to 10% SDS-PAGE followed by electroblotting to Hybond N. Membranes were blocked by incubating for 1 h in blocking buffer (10% BSA in TBS, 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl) followed by overnight hybridization with 50,000 cpm/ml 125 I–IGF-II (Amersham Pharmacia Biotech) in blocking buffer. Membranes were washed three times for 15 min with TBS, air dried, then subjected to autoradiography. Liver samples were prepared as for Western blotting. Protein from each sample (40 μg) was subjected to SDS-PAGE in a 10% polyacrylamide gel containing 1.0 μg/ml recombinant human IGFBP-3 (kindly provided by Dr. C. Maack, Celtrix Pharmaceuticals, Santa Clara, CA). Following electrophoresis, gels were washed for 30 min with 2.5% Triton X-100. Gels were equilibrated with transfer buffer (50 mM Tris-HCl, pH 8.0, 5 mM CaCl 2 ), then the proteins were subjected to capillary transfer onto PVDF membranes as described previously . The transfer was performed either in the absence or presence of 10.0 μg/ml recombinant human TIMP-1 (kindly provided by Dr. H. Nagase, University of Kansas, Kansas City, KS). Transfers took place overnight at 37°C. Under these conditions, only proteolytic fragments of IGFBP-3 transfer from the substrate-gel to the membrane. Intact IGFBP-3 (30 kD) impregnated in the polyacrylamide matrix remains in the gel . Following transfer, the PVDF membrane was probed with polyclonal antibody against IGFBP-3 . Liver tissue was homogenized as for Western blotting, except that NP-40 and proteinase inhibitors were not added. 50 μg of protein from each sample was brought up to a volume of 28 μl with homogenization buffer containing 50 μM CaCl 2 . 125 I–IGFBP-3 (50,000 cpm/ml; Diagnostic Systems Laboratories) was added, and the samples were incubated for 5 h at 37°C, followed by SDS-PAGE, electroblotting, and autoradiography . Liver protein homogenates were prepared as above. Total levels of IGF-II were quantified according to a published protocol . In brief, 10 μl from each sample was dot blotted onto nitrocellulose in triplicate. The membrane was incubated in blocking buffer and then with mAb against IGF-II. The IGF-II concentrations were determined from a standard curve generated with known quantities of purified recombinant IGF-II (kindly provided by Eli Lilly and Co.). To assess the relative levels of dissociable IGF-II, equivalent quantities of total IGF-II from each sample (1.2 μg) were incubated with IGFBP-4–conjugated Affigel overnight at 4°C with gentle rocking. Samples were centrifuged for 10 min at 1,000 g and the pellets washed three times with TBS. The pellets were then incubated with 0.5 M acetic acid for 5 min to elute IGF-II bound to the immobilized IGFBP-4. Supernatants were collected and dried under vacuum. Samples were resuspended in TBS and the relative levels of dissociable IGF-II determined using the IGF-II dot blot assay. To validate that increasing the molar ratio of IGFBP-3 to IGF-II affects IGF-II binding to the IGFBP-4 Affigel affinity media, increasing amounts of recombinant IGFBP-3 were mixed with a constant amount of recombinant IGF-II and the mixtures were subjected to the IGFBP-4 chromatographic separation, as described above. Frozen liver tissue samples were homogenized in TBS containing 1% Triton X-100, 1 mM PMSF, 100 μM Na 3 VO 4 , 1 mM EDTA, 10 μg/ml pepstatin, 10 μg/ml leupeptin. Aliquots from each supernatant containing 5.0 μg of protein were immunoprecipitated as above, using antibodies against the IGF-IR (clone aIR3; Calbiochem). Immunoprecipitates were then incubated with kinase reaction buffer (10 mM Hepes, pH 7.4, 5 mM MnCl 2 , 5 mM MgCl 2 , 100 μM Na 3 VO 4 , 50 mM NaF) containing 2.0 μCi/μl [γ- 32 P]ATP either with or without 50 μg Poly (Glu, Tyr) 4:1 (Sigma Chemical Co.) for 30 min at 37°C. Samples were electrophoresed on 10% SDS-PAGE gels, dried onto Whatman 3 MM paper, and autoradiographed. Liver proteins were isolated as for the receptor kinase assays. For insulin receptor substrate (IRS)-1, 2.5 mg of protein from each sample was immunoprecipitated with polyclonal antibodies against rat IRS-1 (Upstate Biotechnology) as described above. The immunoprecipitates were then Western blotted and the membranes probed sequentially with antiphosphotyrosine (clone 4G10; Upstate Biotechnology) and anti–IRS-1 antibodies. For mitogen-activated protein kinase (MAPK), replicate 10% SDS-PAGE gels were run with 40 μg of protein from each sample loaded on each gel. Following electroblotting, membranes were probed with antibodies specific for phospho-MAPK or MAPK (New England Biolabs). Autoradiographs were scanned using a Molecular Dynamics Densitometer. Absorbance was quantified using ImageQuant ® software. Statistical significance was determined using t test. For all gel electrophoresis, each lane corresponds to a tissue sample taken from an individual mouse. All samples were obtained from 185-d-old female mice unless indicated otherwise, as we determined previously that TIMP-1 modulation significantly affected TAg-induced preneoplastic proliferation at this age . We have shown previously that transgenic TIMP-1 expression does not affect TAg oncoprotein levels in double transgenic mice that coexpress TAg and TIMP-1 transgenes (TAg + /Ts + ) . Equivalent TAg protein levels in both TAg + and TAg + /Ts + animals are confirmed here in Fig. 1 a (top panel). Since TAg binds to and inactivates the tumor suppressor gene products p53 and Rb to induce hyperplasia , we investigated whether TAg interactions with these tumor suppressor proteins are altered by TIMP-1 overexpression. The amount of p53 and Rb that coimmunoprecipitated with TAg protein was examined by Western blotting and was found to be similar between TAg + and TIMP-1–overexpressing (TAg + /Ts + ) livers, as shown in Fig. 1 a (middle and bottom panels). This indicates that TAg interactions with p53 and Rb proteins are intact, and unaffected by the elevation of TIMP-1. IGF-II is a fetal mitogen in rodents, and its transcription is normally repressed in adult tissues by p53 . However, focal reactivation of IGF-II and its localization to proliferating cells during TAg-induced tumorigenesis have been reported in two independent transgenic tumor models . Cellular proliferation and tumor development have been shown to be profoundly inhibited in one such model when crossed onto an IGF-II–null background , revealing the fundamental importance of IGF-II reactivation in TAg-induced tumorigenesis. IGF-II reactivation has been frequently observed during TAg-induced hepatocarcinogenesis . To investigate whether IGF-II was reactivated in our hepatocellular carcinoma model, we examined its spatiotemporal expression in the livers of experimental and control littermates. IGF-II mRNA was not detected by Northern blot analysis of liver tissue from wild-type mice of all ages, or in the livers of TAg + mice before 165 d of age. However, from 170 d of age, multiple IGF-II transcripts commonly observed in mouse and human tissues were expressed . Fig. 1 c shows that IGF-II mRNA was expressed in liver tissue from TAg + and TAg + /Ts + littermates at 185 d of age. Densitometric analysis of the major IGF-II transcript (4.2 kb) confirmed that expression was comparable in TAg + and TAg + /Ts + littermates (3.3 ± 1.2 vs. 3.8 ± 0.6, n = 5 per group). The timing of IGF-II reactivation coincided with TAg-induced liver enlargement, and remained unaffected by TIMP-1 overexpression. In contrast to IGF-II mRNA reactivation, IGF-I mRNA expression remained very low and unchanged during all stages of TAg tumorigenesis (data not shown). The liver-specific C reactive protein promoter, which directs TAg expression , resulted in uniform TAg expression in almost all hepatocytes by 185 d of age, as shown by immunohistochemistry . In adjacent sections, in situ hybridization revealed IGF-II mRNA production by most hepatocytes , and extensive hepatocytic hyperplasia was detected by PCNA immunostaining . The liver of a 185-d-old double transgenic TIMP-1–overexpressing (TAg + /Ts + ) littermate demonstrated the same ubiquitous expression patterns of both the TAg oncoprotein and IGF-II mRNA . In contrast, probing the adjacent sections with anti-PCNA antibody revealed far less proliferation in the livers of TAg + /Ts + mice compared with TAg + mice. In our previous study using morphometric analysis, we found this proliferation to be significantly suppressed in TIMP-1 transgenic liver tissue . Together, the results demonstrate that the effects of TIMP-1 are exerted downstream of IGF-II reactivation, but before cell proliferation. For further analyses of the effects of TIMP-1 on IGF-II bioactivity, we chose to use liver specimens from 185-d-old mice. This choice was based on our previous pilot study encompassing 20–250 d, in which we found that hepatocyte proliferation was maximal (>65%) in TAg + mice at 185 d of age, and was inhibited 3.3-fold in TAg + /Ts + littermates at this age . The difference in proliferation was most accentuated at this age, and therefore we anticipated that in vivo analysis of molecular factors in the IGF-II signaling pathway would be most clearly resolved at this point in time. The above observations that IGF-II is reactivated in both TAg + and TAg + /Ts + livers , yet hepatocyte proliferation is only prevalent in TAg + tissue at 185 d , further supported the use of this age group for IGF-II bioactivity studies. IGFPBs regulate IGF activity by sequestering free IGFs, thus preventing ligand–receptor interactions. Previous studies from one of our laboratories have demonstrated that MMPs, the primary proteinases inhibited by TIMP-1, can degrade IGFBPs both in vitro and in vivo . Since the amount of high-affinity, intact IGFBP may be regulated in part by MMP-mediated proteolysis, we compared the levels of intact hepatic IGFBPs using 125 I–IGF-II Western ligand blotting. Of the hepatic IGFBPs that bound 125 I–IGF-II on ligand blots, only IGFBP-3 (42–46-kD doublet) levels were strongly affected by TIMP-1 modulation . The levels of this binding protein, which is also the major serum carrier protein for IGFs , were increased by more than twofold in the livers of TIMP-1–overexpressing mice (TAg + /Ts + ) compared with levels in TAg + littermates . TIMP-1 overexpression did not affect the levels of IGFBP-4 (24–26 kD), although there were notable minor elevations of a doublet of proteins at 28–32 kD that, based on molecular mass, likely represent IGFBP-1, -2, and/or -5 . In contrast to IGFBP-3 protein levels, Northern blot analysis showed that TIMP-1 overexpression did not affect the levels of IGFBP-3 mRNA , attributing the differences in IGFBP-3 protein levels to posttranscriptional events. MMP-1, -2, -3, and -9 have been shown to proteolytically cleave IGFBP-2, -3, and -5, a process inhibited by TIMP-1 in vitro . Here, IGFBP-3 substrate zymography was used to identify IGFBP-3–degrading proteases in liver homogenates. We found two IGFBP-3–degrading activities with molecular masses of ∼62 and ∼84 kD . Both activities were substantially reduced in the presence of recombinant TIMP-1 , demonstrating that the IGFBP-3–degrading proteinases were MMPs. Next, we determined whether a decreased proteolysis of IGFBP-3 is evident in liver tissue obtained from TIMP-1–overexpressing mice. Liver homogenates were analyzed for their ability to degrade 125 I–IGFBP-3 into smaller molecular weight species, as described in Materials and Methods. There was significantly less degradation of 125 I–IGFBP-3 by liver homogenates from TIMP-1–overexpressing (TAg + /Ts + ) mice compared with TAg + littermates . These data suggest that TAg can induce MMPs that are capable of degrading IGFBP-3, and that coexpression of TIMP-1 can reduce MMP activity, thereby inhibiting IGFBP-3 degradation. Together, these actions allow for a net increase in tissue IGFBP-3 protein levels. We determined whether the increased IGFBP-3 levels in the TIMP-1–overexpressing liver tissue affected the amount of dissociable IGF-II. Fig. 4 a shows that using a dot blot procedure developed to quantify total IGF-II , recombinant IGF-II can be measured linearly over a range of concentrations (0.4–3.2 μg). Next, dissociable IGF-II was measured by IGFBP-4–conjugated Affigel affinity chromatography. Using recombinant IGF-II and recombinant IGFBP-3 in different molar ratios, we were able to confirm that increasing molar ratios of IGFBP-3 reduced the amount of IGF-II that bound to the Affigel . To measure difference in levels of dissociable IGF-II in liver samples, we first quantified total IGF-II using the dot blot assay. Aliquots containing equivalent amounts of total IGF-II were subjected to the affinity chromatography procedure. TIMP-1 overexpression resulted in a sixfold decrease in dissociable IGF-II levels in the livers of TAg + /Ts + mice compared with their control TAg + littermates . This demonstrates that despite an equivalent extent of IGF-II reactivation, the level of dissociable or bioavailable IGF-II is reduced in TIMP-1–overexpressing animals. A reduction in the levels of dissociable or bioavailable IGF-II should result in decreased signaling through the IGF-IR pathway in the livers of TAg + /Ts + mice. Unlike postnatal IGF-II inactivation that occurs in normal rodent liver, the IGF-IR is expressed at constitutive levels in adult mouse liver, and IGF-II exerts its mitogenic effect through this receptor . Therefore, IGF-IR kinase activity, as well as the phosphorylation status of the downstream signaling effectors, IRS-1 and MAPK, were assessed. IGF-IR immunoprecipitated from TAg + /Ts + liver tissue exhibited lower autophosphorylation (data not shown) and showed reduced kinase activity on an exogenous substrate , compared with receptor from TAg + controls. Moreover, tyrosine phosphorylation of IRS-1, which binds to IGF-IR following activation, was also reduced in TIMP-1–overexpressing livers . Sequential probing of the same blot with an antibody against nonphosphorylated IRS-1 showed that IRS-1 protein levels were not altered in TIMP-1–overexpressing animals . Phosphorylation of the downstream signaling molecules, the MAPKs extracellular signal regulatory kinase (Erk)-1 and Erk-2, was also reduced in TIMP-1–overexpressing liver tissue , whereas the absolute levels of these proteins were unaffected . These data provide direct evidence that the protein levels of the IGF-IR downstream signaling mediators were not altered, but that signaling from the IGF-IR was attenuated in TIMP-1–overexpressing transgenic tissue. IGFs are critical growth factors involved in growth, transformation, and tumorigenesis and act through the IGF-IR . Studies have shown that cells null for the IGF-IR do not display the normal increase in proliferation in response to growth factors or serum as seen in normal cells, and that all phases of the growth cycle are prolonged . Indeed, in several cell lines, abrogation of the IGF-IR has resulted in enhanced apoptosis. Furthermore, IGF-IR–null cells cannot be transformed by TAg , activated Ha- ras , a combination of both, or by the overexpression of other growth factor receptors. And finally, a number of studies have shown that ablation of this receptor in tumor cell lines significantly decreases their tumorigenic potential in vivo. Together, these data demonstrate that interruption of the ligand–receptor interaction between IGFs and the IGF-IR effectively disrupts several aspects of the tumorigenic process. In nature, under homeostatic circumstances, little or no IGFs are present in the free or bioavailable form due to their sequestration by one or more of the six known high-affinity IGFBPs. Because IGFBPs demonstrate equal or higher affinities for IGFs than does the IGF-IR, little or no IGFs are normally available to interact with receptors. Recent studies have begun to elucidate mechanisms by which IGFs can be released from IGFBPs so they may interact with cell-surface receptors and exert their mitogenic and metabolic effects. These studies have demonstrated that a primary phenomenon invoked to release IGFs from IGFBPs is through decreasing the affinities of IGFBPs for IGFs. The best-characterized mechanism involved in decreasing the affinities of IGFBPs is proteolytic degradation . Proteolytic cleavage has been demonstrated for at least five IGFBPs, IGFBP-2 to IGFBP-6, and occurs in several physiologic as well as pathologic circumstances , yet little is known of the identity of these IGFBP-degrading proteinases in vivo, or the mechanisms that regulate the proteolytic cleavage. In vitro, we and others have provided evidence that production of proteinases, a common feature of transformed cells , can result in degradation of IGFBP–IGF complexes, releasing IGFs to interact with cell surface receptors, thereby triggering proliferation of target cells. We have also shown recently that in vitro, MMPs function as IGFBP-3– and IGFBP-5–degrading proteinases . In addition to degrading IGFBPs, extensive data also support the role of MMPs in tumorigenesis, angiogenesis, and metastasis . In the current studies, we now demonstrate the importance of MMP-mediated IGFBP degradation in neoplastic proliferation in vivo, and a means of controlling this degradation. We have used a double transgenic, TAg-based tumor model to determine directly whether TIMP-1 inhibits IGF bioactivity in vivo to suppress hepatocyte proliferation that leads to tumorigenesis. We selected this model as it provided important features: (a) IGFs are crucial mitogens for TAg-induced transformation, proliferation, and tumorigenesis ; and (b) transgenic TIMP-1 overexpression in this model substantially inhibits TAg-induced proliferation and hepatocellular carcinoma , making it possible to measure in vivo differences in molecular factors and IGF-IR signal transduction in transgenic tissue. A systematic analysis has now been undertaken to explore connections that might exist between TAg expression and IGF-II bioactivity on cell proliferation to define the molecular mechanisms behind TIMP-1–mediated tumor suppression in vivo. The events explored herein and the results are summarized in Fig. 6 , and are discussed below. First, an examination of p53 and Rb in these mice showed that TIMP did not interfere with the molecular interactions between TAg and these tumor suppressor proteins. Next, we found that IGF-II expression was indeed reactivated at the onset of cell proliferation, similar to the model of TAg-induced pancreatic tumorigenesis , indicating a key mitogenic role for IGF-II in our tumor model. IGF-II reactivation is frequent in TAg-induced liver tumorigenesis , as well as in liver tumor formation induced by oncogene or growth factors . Furthermore, our data showing that TIMP-1 significantly inhibited hepatocellular proliferation despite IGF-II reactivation suggested that TIMP-1 might directly act at a posttranscriptional level to modulate IGF-II activity. Our investigations reveal the novel finding that hepatic TIMP-1 overexpression specifically inhibits IGFBP-3 proteolysis, leading to a significant elevation of hepatic IGFBP-3 levels. We have determined that MMPs induced during TAg tumorigenesis appear to function in degrading IGFBP-3. Furthermore, we demonstrate that dissociable IGF-II levels are decreased in TIMP-1–overexpressing hepatic tissue. The physiologic consequence of reducing dissociable IGF-II levels (e.g., a reduction in IGF-II bioactivity) was confirmed by demonstrating a significantly reduced signal transduction from the IGF-IR, as measured by reduced IGF-IR kinase activity and tyrosine phosphorylation levels of IRS-1 and MAPK. Thus, despite TAg-induced reactivation of IGF-II in the liver, the transgenic TIMP-1–mediated increase of IGFBP-3 levels blocks TAg-induced hepatocyte proliferation by effectively reducing bioavailable IGF-II levels. Together, our data provide direct evidence that the inhibition of extracellular proteolysis by TIMP-1 attenuates the bioactivity of the tumor-inducing growth factor IGF-II. Based on previous studies in transgenic systems, a relationship has begun to emerge between TIMP/MMP expression within a tissue and the tissue's susceptibility to tumor development. We have demonstrated that overexpression of TIMP-1 in the liver inhibits hepatocellular carcinoma , and its elevation in the skin compromises the ability of transplanted lymphoma cells to grow as a primary tumor , whereas a reduction of TIMP-1 in these tissues augments tumor development . Consistent with these observations, the overexpression of MMP-3 (stromelysin-1) in mammary tissue leads to spontaneous mammary tumor development and the transgenic expression of MMP-I (type I collagenase) in skin augments carcinogenesis . In addition, the ablation of MMP-7 (matrilysin) impairs colorectal tumor development in the min mouse tumor model . In many of these studies, ectopic TIMP or MMP expression results in altered cellular proliferation. For example, hepatic TIMP-1 overexpression inhibits hepatocyte hyperplasia , MMP-3 overexpression leads to mammary epithelial hyperplasia , and MMP-1 overexpression to epidermal hyperproliferation . Despite the many reports indicating that shifts in the extracellular proteolytic balance have a strong influence on early tumor development and on cell proliferation within the afflicted organ, the molecular mechanisms for these effects have remained elusive. Having previously excluded the effects of TIMP-1 on hepatocyte apoptosis , our present investigation provides in vivo evidence of a link between the inhibition of extracellular proteolysis, reduced growth factor bioavailability, and reduced cellular proliferation. We have shown previously that this reduction in cellular proliferation precedes suppressed tumor development in this model . TIMPs have traditionally been considered regulators of cell invasion and motility by virtue of their ability to inhibit MMP-mediated ECM degradation. Now it is recognized that in addition to the effects on ECM structural proteins, extracellular proteolysis has the potential to control the release of growth factors tethered to the ECM , the processing of soluble growth factor binding proteins, i.e., IGFBPs , and the processing of cell surface molecules such as membrane-bound TNF-α, the Notch receptor in Drosophila , and the FGF receptor 1 . Although these and other studies support the overall concept that extracellular proteolysis can broadly influence cell proliferation, behavior, and fate, the observations were made in in vitro systems. To our knowledge, our data are the first in vivo evidence to substantiate the importance of proteolytic degradation of a tethering or binding/carrier protein for a growth factor in the pathological development of cancer, and of its regulation by the natural inhibitor, TIMP-1. Our data demonstrate that the dynamics of IGF-II bioactivity can be altered in vivo by proteolytic modulation, and emphasize that factors other than ECM proteins constitute important physiological targets of MMP-mediated cleavage within the extracellular microenvironment. The importance of IGFs in cellular transformation has been demonstrated by findings that cells which do not express IGF-IR cannot be transformed by any of a number of dominant oncogenes . Indeed, TAg was unable to transform IGF-IR–null fibroblasts, despite the presence of various growth factors present in serum . Thus, IGF-IR is necessary for TAg-mediated transformation to take place. Consistent with these findings, TAg expression has been shown to reactivate IGF-II gene transcription in vivo. IGF-II reactivation occurs in pancreatic beta cells transgenically expressing TAg , and here we show that IGF-II reactivation occurs in hepatocytes expressing TAg transgene coincident with the onset of hepatocyte hyperplasia. Similar to our finding, other investigators have reported that TAg expression in the liver tissue coincides with reactivation of IGF-II gene transcription and this reactivation has been associated with neoplastic transformation . Such reactivation of IGF-II has also been reported during c- myc– and TGF-α–induced hepatocellular carcinomas in transgenic mice . The consequence of IGF-II reactivation as it relates to neoplastic transformation has only been addressed recently in TAg models of pancreatic cancer and hepatocellular carcinoma. In the former study, the investigators bred TAg transgenics into an IGF-II–null background , whereas in the latter TAg transgenics were bred into an Igf2 (+/−) background . In both instances, tumor size and incidence were decreased, strongly suggesting a role for IGF-II in the pathogenesis of these two tumor types. Furthermore, several transgenic lines overexpressing IGF-II demonstrate that IGF-II is associated with tumor formation, including mammary tumors, lymphomas, and hepatocellular carcinomas . Although these studies provide compelling support for IGF-II as a causal mitogen in the tumorigenesis evidenced in the current animal model, final resolution of this hypothesis must await studies examining our findings in models that are either null or upregulated for IGF-II. Such studies are currently being pursued in our laboratories. Both IGF-I and IGF-II are widely implicated in promoting several human cancers, including liver, prostate, and breast cancer, and their expression correlates with poorer prognosis . Furthermore, in transgenic models, IGF-I and IGF-II are causally implicated in tumorigenesis . These studies point to IGF bioactivity as a target for cancer therapeutics. Our results indicate that TIMP-1–like biomolecules or synthetic MMP inhibitors may be promising candidates for the therapeutic modulation of IGF dosage in novel clinical strategies. Alternatively, strategies to alter specific IGFBP levels or the production of proteinase-resistant IGFBPs may prove to be effective therapeutic interventions. A distinct feature of all of these approaches will be to target the bioavailability rather than the production of a growth factor. The results presented here provide compelling evidence for a novel mechanism by which endogenous TIMPs contribute to the cellular microenvironment. We demonstrate that the inhibition of extracellular proteolysis in vivo impairs the activity of a specific growth factor responsible for hyperplasia during TAg-induced tumorigenesis. Because TIMPs are also capable of inhibiting invasion, metastasis, and angiogenesis , all of which are promoted by IGF action , the combined outcome of TIMP-1 elevation may be to suppress multiple stages of tumor development, maintenance, and progression. | Study | biomedical | en | 0.999996 |
10459022 | The human rhabdomyosarcoma line RD was obtained from American Type Cell Collection. RD cells were transfected with integrin α3 cDNA to yield RD-A3 as described previously 71 . RD-C9-1 and RD-C9-2 are stable, unsorted lines independently derived by transfecting RD cells with full-length CD9 cDNA (generated by reverse transcriptase PCR) that was cloned into the expression plasmid pZeoSV (Invitrogen). Likewise, RD-A3C9-1 and RD-A3C9-2 are independently prepared stable transfectants derived from RD-A3 cells. All RD cells and transfectants were cultured in DME supplemented with 10% FCS. To induce differentiation and syncytia formation, subconfluent RD cells were cultured in 2% FCS. The mouse myogenic cell line C2C12 8 was obtained from American Type Cell Collection and maintained in DME supplemented with 20% FCS (growth medium). To induce differentiation of confluent C2C12 cells, growth medium was replaced with DME containing 2% horse serum (differentiation medium) 57 . To avoid loss of fusion competence, C2C12 cells were passaged only a few times before use. Rat anti–mouse mAbs KMC8, anti-CD9; R1-2, antiintegrin α4; MFR5, antiintegrin α5; KMI6, antiintegrin β1; KM114, anti-CD44; R35-95, rat IgG2a negative control antibody; and hamster anti–mouse mAbs, HMβ1-1 and Ha2/5, antiintegrin β1, were all obtained from PharMingen. Hamster anti–mouse CD81 mAb 2F7 was purchased from Southern Biotechnology. Rat anti–mouse mAb CY8.2, antiintegrin α7, was a gift from Dr. Randall Kramer, University of California, San Francisco, San Francisco, CA. Rabbit polyclonal antibody against the integrin α3A cytoplasmic tail was provided by Dr. J.A. McDonald (Mayo Clinic, Scottsdale, AZ). Mouse anti–human N-CAM mAb, NCAM-OB11; anti–N-cadherin mAb, GC-4; and anti-pan cadherin cytoplasmic tail mAb, CH-19, all of which cross-react with mouse, were purchased from Sigma Chemical Co. Mouse mAbs MY-32, anti–myosin heavy chain (MHC); and DE-U-10, antidesmin, were also obtained from Sigma Chemical Co., and mouse antiactin mAb, C4, was purchased from ICN Biomedicals, Inc. Mouse anti–human mAbs were against integrin α5, A5-PUJ2 55 ; CD9, BU16 (Biodesign International), DU-ALL-1 (Sigma Chemical Co.), C9-BB 6 ; and CD81, M38 20 . C2C12 cells were plated onto a 96-well tissue culture plate at 20,000 cells/well and cultured in growth medium. At confluence, growth medium was replaced by differentiation medium, in the presence of various mAbs. Numbers of myotubes in four independent 2.6-mm 2 fields were scored by microscopy as described 68 . Elongated, glossy myotubes were easily distinguished from unfused myoblasts . In some experiments, numbers of myotubes per well that were longer than 250 μm were determined using the program Scion Image 1.60 (Scion Corp.), to acquire and analyze computer images from an Axiovert 135 microscope (Carl Zeiss) (as described in detail elsewhere; Stipp, C.S., and M.E. Hemler, manuscript submitted for publication). C2C12 cells were plated onto an 8-well chamber Permanox slide (Nunc) and precoated with mouse laminin (GIBCO BRL). Confluent cells were cultured in differentiation medium (with or without anti-TM4SF antibodies) for 6 d and then fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100, 0.1% sodium citrate. Nuclei of apoptotic cells were stained by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) 21 , using In Situ Cell Death Detection Kit (Boehringer Mannheim) according to the manufacturer's instructions. Immunofluorescence was analyzed using an Axioscop (Carl Zeiss). For DNA ladder analyses, C2C12 cells were cultured for 6 d in differentiation medium, and then myotubes were detached from mixed myoblast/myotube cultures by treatment with 0.01% trypsin, 0.004% EDTA for 15 min as described previously 5 . DNAs were extracted using the Apoptotic DNA Ladder Kit (Boehringer Mannheim) according to the manufacturer's instructions, electrophoresed in 2% agarose gel, and stained with 0.5 μg/ml ethidium bromide. C2C12 cells were lysed in lysis buffer (1% Brij 96 or Brij 99, 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin) for 1 h at 4°C. Insoluble materials were pelleted at 12,000 rpm for 10 min, and the cell lysates were precleared twice by incubation with protein G (Amersham Pharmacia Biotech) for 30 min at 4°C. Immune complexes were formed by addition of mAbs and collected onto protein G beads, followed by four washes with immunoprecipitation buffer. After elution from beads with Laemmli sample buffer, proteins were separated on SDS-PAGE, transferred to nitrocellulose membranes, and blotted with biotinylated antibody followed by peroxidase-conjugated ExtraAvidin (Sigma Chemical Co.) and visualized with Renaissance Chemiluminescent Reagents (DuPont). Whole cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and then preblotted with PBS containing 0.05% Tween 20 and 3–5% nonfat dry milk. Proteins were then sequentially immunoblotted with primary antibody followed by peroxidase-conjugated secondary antibody, and visualized with Renaissance Chemiluminescent Reagents. For flow cytometry, cells were incubated with negative control mAb or specific mAb, washed three times, and then incubated with FITC-conjugated goat anti–mouse Ig. Stained cells were analyzed using a FACScan™ (Becton Dickinson). Fluorescence with negative control mAb was subtracted to give specific mean fluorescence intensity (MFI) units. To evaluate the role of a representative TM4SF protein on myogenic cells, we monitored CD9 expression on mouse C2C12 cells undergoing myogenic cell differentiation. At confluence, C2C12 cells begin fusing into myotubes and express muscle-specific proteins under the control of muscle-specific transcription factors, including MyoD, myogenin, Myf-5, and MRF4 38 . This differentiation is accelerated by medium containing low levels of serum. The level of CD9 protein was upregulated as C2C12 cells approached confluence, and reached a peak at ∼1 d after incubation in differentiation medium . In contrast, muscle-specific proteins desmin and MHC were upregulated at a later stage. Control antiactin blots showed comparable amounts of protein loaded on each lane . After 1 d of C2C12 myoblast differentiation, TM4SF proteins CD9 and CD81 could be coimmunoprecipitated with several different β1 integrins, including α3β1, α5β1, and α7β1 (not shown). Although there have been many reports of constitutive TM4SF–integrin association (for review see references 24 and 26 ), there has been little evidence that such complexes can be regulated. Here we show that formation of integrin–CD9 complexes is highly regulated, particularly in the early phase of C2C12 differentiation . During subconfluent growth, relatively little CD9 was detected in a β1 immunoprecipitation , and little β1 was present in a CD9 immunoprecipitation , even though β1 and CD9 were clearly present. However, as cells reached confluence and began to differentiate, complex formation was clearly upregulated. β1 integrin–CD9 complexes reached a maximum at ∼1 d after culture in differentiation medium . Complex formation was sustained, or decreased slightly, during the later stages of differentiation . Densitometric quantitation of immunoblotted β1 and CD9 levels confirmed a marked increase in the ratio of β1-associated CD9 relative to total immunoprecipitated β1, with the peak ratio ( r = 2.0) occurring after 1 d of differentiation. Likewise, there was an increase in CD9-associated β1 relative to total immunoprecipitated CD9, again with the peak ratio ( r = 0.5) occurring after 1 d of differentiation. Notably, these peak ratios were 7–15-fold greater than ratios obtained when cells were 40% confluent. In another experiment, immunoprecipitation of CD81 yielded associated β1 and CD9 proteins, with peak association again occurring at ∼1 d after differentiation (data not shown). In a control experiment, after 1 d of differentiation mature integrin β1 protein was present in immunoprecipitates of CD9 or CD81 , but not cadherin or N-CAM. Both mature and immature precursor forms of β1 were present in C2C12 cell lysate and in a β1 immunoprecipitate . We used mAbs KMC8 and 2F7 to determine (by flow cytometry) that TM4SF proteins CD9 and CD81 were both present on 100% of C2C12 cells, at levels ∼100–1,000-fold above background (data not shown). The addition of mAbs to either CD9 or CD81 caused a marked delay in the formation of myotubes, and both mAbs together showed an additive inhibitory effect, as seen in three different experiments . A photo illustrating the delay caused by anti-CD9 plus anti-CD81 mAb at day 3 is shown in Fig. 4 A. In contrast to the anti-TM4SF antibodies, antiintegrin anti-α4, anti-α5, and anti-β1 antibodies did not delay myotube formation. Likewise, no delay was caused by anti-CD44 or anti-CD44 plus anti-α5 together . However, anti-α5 and anti-β1 mAbs did cause myotubes at day 3 to be significantly shorter than myotubes treated with control mAb, anti-CD44 mAb, or anti-α4 mAb . mAbs to other integrin subunits were not tested for the following reasons: an anti-murine α3 mAb is not yet available, the anti-α7 mAb was not available in sufficient quantity, and the α6 subunit is only weakly expressed on C2C12 cells. Anti-CD9 and anti-CD81 antibodies, either alone or in combination, had no effect on myoblast proliferation as C2C12 cells grew to confluence in growth medium over a 5-d period (data not shown). Thus, anti-TM4SF antibody effects on myotube formation are not an indirect consequence of inhibition of myoblast proliferation. Anti-CD9 and anti-CD81 mAbs not only caused a delay in myotube formation, but also accelerated myotube degeneration . Specific examples of myotube degradation at day 10 are shown in Fig. 4 B. Again, the effects of anti-CD9 and anti-CD81 mAb were additive . Again, myotube maintenance was not affected by control mAb, anti-α4, anti-α5, anti-CD44, or anti-β1 mAbs . In Fig. 3 B, mAbs were not added to cultures until day 5, when peak numbers of myotubes were already formed. Nonetheless, in subsequent days the numbers of myotubes again were diminished upon addition of anti-TM4SF mAb, with the effects of anti-CD9 and anti-CD81 mAbs being additive. This result excludes the possibility that delayed myotube formation is responsible for the subsequent degeneration of myotubes. Effects of an anti-CD9 mAb (KMC8) were even more dramatically obvious when elongated myotubes (>250 μm) were analyzed rather than total myotubes. As shown in Fig. 5 , ∼50% fewer elongated myotubes were formed in the presence of 20 μg/ml KMC8 at day 3. By day 5, the inhibitory effect was less pronounced, with ∼25% fewer elongated myotubes being formed. The most dramatic effect of mAb KMC8 was seen after maximal elongated myotube formation had already occurred (days 6–8). For example, at day 8, ∼90% fewer myotubes were maintained when 10–20 μg/ml KMC8 was present . These anti-CD9 mAb effects were clearly dose dependent. To assess antibody effects on myotube attachment, C2C12 myoblasts were allowed to differentiate into myotubes in the presence of antibodies for 5 d, and then detached myotubes were removed by washing. Counting of myotubes present before and after washing (in quadruplicate) revealed that myotube detachment was relatively unaffected by antibody incubation (no antibody, 29% detachment; control IgG, 27% detachment; anti-α4, 32% detachment; anti-α5, 34% detachment; anti-CD44, 33% detachment; anti-CD9, 30% detachment; anti-CD81, 31% detachment; anti-CD9 plus anti-CD81, 18% detachment). Thus, failure to maintain myotubes did not appear to result from direct promotion of detachment by anti-CD9 or anti-CD81 mAbs. In another experiment, neither anti-CD9 nor anti-CD81 mAb had any effect on confluent C2C12 myoblast cell adhesion to tissue culture plastic, or to surfaces coated with fibronectin or mouse laminin 1. Likewise, these antibodies had no consistent effect on either random C2C12 cell motility or transwell haptotaxis towards fibronectin or laminin (data not shown). In a separate experiment, a culture of confluent myoblasts was disrupted by scratching with a plastic tip. Again, anti-CD9 and anti-CD81 antibodies had no effect on the rate of myoblast migration into the vacated area, or on the alignment of myotubes in that area. A previous report demonstrated that antiintegrin β1 mAb, CSAT, inhibited both morphological differentiation (myotube formation), and biochemical differentiation (meromyosin expression) in chicken embryo myoblasts 47 . Here we demonstrate that a combination of anti-TM4SF CD9 and CD81 mAb did not alter biochemical differentiation of C2C12 cells , marked by the appearance of either MHC or desmin. In contrast, an anti-β1 mAb did substantially lower the appearance of MHC, consistent with previous anti-β1 mAb effects on biochemical differentiation 47 . Control antiactin immunoblots showed that equal amounts of protein were loaded in each lane. Anti-CD9 and anti-CD81 mAbs induced early degeneration of C2C12 myotubes . Some of these myotubes showed cellular blebs (data not shown), which is suggestive of apoptosis. Thus, we used the TUNEL method to confirm whether degenerating myotubes were apoptotic. Fluorescein-labeled UTP was incorporated into nuclei of degenerating myotubes after 6 d in the presence of anti-CD9 or anti-CD81 mAb . In contrast, myotube nuclei were not stained when either control mAb or no mAb was present. Also, no unfused myoblasts were stained in any of the cultures. Because only a fraction of the total myotubes appeared to undergo apoptosis, we did not expect to see a strong DNA laddering effect. Nonetheless, we separated myotubes from unfused myoblasts, extracted DNA, and studied apoptosis-associated internucleosomal fragmentation by electrophoresis . Albeit at a low level, DNA laddering was evident in myotubes cultured in the presence of anti-CD9 or anti-CD81 mAb . In contrast, the control lane did not show DNA laddering although the same amount of DNA was loaded . In additional studies, apoptosis in a mixed myotube/myoblast preparation was determined by measuring histone-associated DNA fragments (Cell Death Detection ELISA PLUS Kit; Boehringer Mannheim). After only 4 d of differentiation, anti-CD9 and anti-CD81 mAb each yielded ∼1.1–1.2-fold enhancement of apoptosis, and both antibodies together yielded only ∼1.5-fold enhancement of apoptosis. However, after 6 d of differentiation, anti-CD9 and anti-CD81 mAb each yielded ∼1.5-fold enhancement of apoptosis (compared with control mAb), and both antibodies together yielded ∼3-fold enhancement of apoptosis. Together these results suggest that anti-TM4SF mAbs induce early apoptosis of C2C12 myotubes, but not unfused myoblasts. For further evaluation of TM4SF protein function in muscle-derived cells, we transfected CD9 into the human myoblast-derived RD sarcoma cell line. Although confluent RD cells continue growing, they constitutively express myogenic transcription factors, and undergo a limited and abortive myogenic differentiation 11 28 65 . Here we analyzed RD cell transfectants as they became confluent after ∼6 d in 2% FCS. An RD cell line overexpressing CD9 (RD-C9-2) became substantially more multinucleate than untransfected RD . Likewise, RD cells transfected with CD9 plus integrin α3 subunit (RD-A3C9) became substantially more multinucleate than RD cells transfected with α3 alone . Some of these multinucleate cells were myotubes, but most of them were apolar, giant cells that resemble virus-induced syncytia . Using time-lapse video microscopy, we confirmed fusion between a multinucleate giant cell and a mononucleate cell (data not shown). Despite differences in syncytia formation, all of the RD transfectants proliferated at essentially the same rate as RD cells. Quantitation revealed that two distinct RD lines overexpressing CD9 (RD-C9-1, RD-C9-2) formed approximately fourfold more syncytia than untransfected, mock-transfected (RD-Z), or control transfected (RD-A3) cells . Also, two distinct RD lines expressing both α3 integrin and CD9 (RD-A3C9-1, RD-A3C9-2) showed approximately eightfold more syncytia than control RD cells, including RD-A3 cells expressing α3 alone . CD81 overexpression studies were not carried out because CD81 is already highly expressed on RD cells. Effects of TM4SF overexpression on longer-term syncytia maintenance could not be determined, as syncytia became obscured and displaced by unfused tumor cells that were continually growing and preferentially adherent. In a separate experiment, CD9-transfected fibrosarcoma cells 6 were cultured for 5–8 d as they became confluent in 2% FCS. No syncytia were observed for this cell line during this time. Anti-CD9 mAbs, BU16 and DU-ALL-1 , substantially delayed the appearance of RD-C9-1 cell syncytia. However, no delaying effect was seen with another anti-CD9 mAb, C9/BB, which recognizes a different CD9 epitope. An anti-CD81 mAb, M38, also delayed syncytia formation, whereas a control antiintegrin α5 mAb, A5-PUJ2, did not affect syncytia formation. Despite the strong expression of CD9, CD81, and other TM4SF proteins on skeletal muscle 50 58 , the role of TM4SF proteins during myogenesis had not been studied previously. Here we found that CD9 and CD81 are also abundant on murine myoblast C2C12 cells, and we provide strong evidence for the involvement of at least two TM4SF proteins (CD9 and CD81) during myoblast differentiation. First, CD9 expression was upregulated during the early phase of myogenic differentiation of murine C2C12 cells. Second, anti-CD9 and anti-CD81 antibodies substantially delayed fusion of C2C12 myoblast cells and RD rhabdomyosarcoma cells. Third, CD9 overexpression promoted cell fusion in four independently transfected myoblast-derived RD cell lines. In contrast to muscle-specific proteins MHC and desmin, CD9 expression was upregulated at a much earlier stage of myoblast differentiation. In addition, anti-TM4SF antibodies caused a delay in cell fusion without altering biochemical differentiation of C2C12 cells (as evidenced by a lack of effect on MHC or desmin expression). Thus, CD9 expression appears neither to participate in myogenic transcription factor regulation of other proteins, nor to be regulated by myogenic transcription factors. Also, the delay in fusion seen here was not due to altered myoblast proliferation, although antibodies to CD9 and CD81 have been reported to affect lymphocyte proliferation 62 67 . Likewise, delayed fusion did not appear to arise from promotion of cell detachment, inhibition of attachment, or altered C2C12 cell migration or alignment. Instead, we hypothesize that TM4SF proteins may delay or inhibit myogenesis at an early stage, by a mechanism that may directly influence cell fusion. Fusion was delayed in two different cell types, upon treatment with three different anti-CD9 mAbs and two different anti-CD81 mAbs. In addition, RD cell fusion was promoted upon transfection of CD9. These results are consistent with TM4SF proteins contributing to fusion, perhaps by engaging in protein–protein interactions with each other, or with other proteins. In this regard, TM4SF proteins are reported to associate laterally with many other cell surface proteins 24 40 61 73 . At present, there is no evidence for TM4SF proteins having counterreceptors that would participate in cell–cell interactions. Although CD9 (and α3 integrin) promoted fusion in RD cells, no syncytia formation was seen in CD9-transfected HT1080 fibrosarcoma cells, which are known to contain CD9–α3β1 integrin complexes 6 . Thus, CD9 and α3 integrin by themselves are not sufficient to promote fusion, as the rhabdomyosarcoma RD cells must contain specific proteins or other factors required for cell fusion. The TM4SF effects on myoblast fusion shown here are reminiscent of anti-TM4SF antibody effects and CD9 overexpression effects on virus-induced syncytia formation 17 20 39 72 . For example, the delay in myoblast fusion seen here with anti-CD81 and anti-CD9 antibodies could be mechanistically related to the delay in feline immunodeficiency virus (FIV) production caused by anti-CD9 mAb 17 . Also, a more distantly related TM4SF protein called peripherin/rds was reported to promote fusion of lipid vesicles in vitro 9 . Besides directly contributing to fusion through potential protein–protein interactions, TM4SF proteins might also contribute to myoblast fusion by regulating cellular signaling. In this regard, CD9 and CD81 associate with phosphatidylinositol 4-kinase 7 and with PKCα and PKCβII (Zhang, X., and M. Hemler, manuscript in preparation). Consistent with this, PKC plays a key role in myoblast fusion 16 , and PKCα is present in both myoblasts and myotubes 29 . We found that CD9 on C2C12 cells associates with various β1 integrins, including α3β1, α5β1, and α7β1 and that CD9 associated with α3β1 on α3-transfected RD cells. These results are consistent with integrin–TM4SF protein associations seen on many other cell lines 6 22 24 26 33 40 51 52 . Importantly, we have provided firm evidence for the upregulation of CD9–β1 integrin complexes in coordination with the onset of myoblast differentiation. As far as we know, there have been few if any prior demonstrations of physiologically relevant integrin–TM4SF complex regulation. The regulated formation of TM4SF–integrin complexes suggests that they could play a role during myogenesis. Indeed, even though α3 integrin alone did not effect RD cell fusion, it potentiated the effects of CD9, such that twice as many syncytia were formed. Possibly, integrin expression could contribute to myoblast fusion by altering the distribution and/or signaling functions of CD9 in a way that does not affect integrin-dependent cell adhesion or motility. The α3β1 integrin is absent from adult striated muscle, but is variably present in fetal skeletal muscle 4 43 44 , whereas the α7β1 and α5β1 integrins are prominent on myoblasts and developed skeletal muscle 14 37 59 60 . Although integrins may modulate TM4SF protein contributions during myogenesis, there are several critical differences between the roles of TM4SF proteins and integrins. First, anti-CD9 and anti-CD81 antibodies caused a delay in myoblast fusion, whereas antiintegrin α4, α5, and β1 antibodies did not cause a delay in the appearance of the peak number of myotubes. Second, anti-α5 and anti-β1 antibodies caused myotubes at day 3 to be significantly shortened, but anti-CD9 and anti-CD81 did not have this effect. Third, anti-CD9 and anti-CD81 mAbs had no effect on biochemical differentiation of C2C12 cells (defined by MHC or desmin appearance), whereas an anti-β1 mAb did markedly inhibit MHC appearance, consistent with previous anti-β1 mAb effects on biochemical differentiation in chicken embryo myoblasts 47 . Fourth, antiintegrin antibodies may affect myogenesis largely by blocking myoblast cell adhesion and/or motility 15 32 47 77 . In contrast, we have not found anti-TM4SF antibodies, or CD9 overexpression to have an effect on cell adhesion here or elsewhere (e.g., see references 6 , 41 , 78 ). Likewise, we saw no effect of anti-CD9 or anti-CD81 mAbs on C2C12 cell motility, despite reports showing TM4SF proteins contributing to the motility of other cell types 2 31 49 75 78 . In conclusion, integrins may associate with TM4SF proteins and modulate their functions during myogenesis, but for the most part their specific functional contributions appear to be very distinct. Aside from CD9 costimulation of T cells leading to apoptosis 63 , there have been few reports linking TM4SF proteins to apoptosis. Here we used three different methods to demonstrate that anti-CD9 and anti-CD81 antibodies promoted apoptotic degradation of C2C12 myotubes after they were formed. The antibodies did not trigger myoblast apoptosis. Also, if anti-CD9 and CD81 mAbs were not added until myoblast fusion had already occurred, they still resulted in apoptotic degradation. Thus, delayed cell fusion was not a prerequisite for apoptotic degradation. Conversely, apoptotic degradation did not contribute to a delay in cell fusion. The delay in fusion was most obvious during days 1–3 of differentiation, whereas apoptotic degradation was not obvious at day 3 and was not readily detectable until at least day 5. In previous reports, myotube degradation and apoptosis in vitro and in vivo were associated with disrupted expression and localization of integrin α7β1, and loss of adhesion to laminin-2/4 42 68 69 . In vivo muscle cell degradation, possibly due to diminished cell adhesion, also occurred in skeletal muscle containing high numbers of cells lacking α5 66 . However, because neither CD9 nor CD81 appears to regulate myotube adhesion, or to promote myotube detachment, they must regulate apoptosis by a different mechanism, perhaps by modulating cell signaling (as discussed above). Although myoblast precursors may be more generally susceptible to apoptosis than terminally differentiated myotubes 70 , we did not observe unfused myoblast apoptosis resulting from anti-CD9 and anti-CD81 mAb treatment. Thus, TM4SF protein modulation of apoptosis appears to apply selectively to myotubes, and not myoblasts. Other cell surface proteins such as N-cadherin, M-cadherin, N-CAM, meltrin, VCAM, and CDO have also been implicated in the process of myoblast fusion (see Introduction). However, in comparison to CD9 and CD81, perturbation of these other proteins causes not just a delay, but a more substantial inhibition of myoblast fusion. In addition, these other proteins have not been linked to apoptotic degradation of myotubes. Although antibodies to N-cadherin, α4 integrin, and β1 integrin inhibited myoblast fusion, deletion of these genes did not adversely affect muscle formation 13 27 76 . Thus, contributions of each of these proteins could be compensated by the functions of other related proteins. It is well established that TM4SF proteins including CD9, CD63, CD81, CD82, and CD151 can form complexes with each other within cellular membranes 1 6 31a . Thus, besides CD9 and CD81, some of these other TM4SF proteins might possibly also be involved in myoblast fusion. However, definitive analysis of the role of other TM4SF proteins on C2C12 cells will not be readily achieved until the appropriate antimurine mAbs become available. It remains to be determined whether deletion of genes for any TM4SF proteins will have an adverse effect on myoblast fusion in vivo. In this regard, there were no abnormal phenotypes at birth in muscular systems of CD81-deficient mice (Tsitsikov, E.N., J.C. Gutierrez-Ramos, and R.S. Geha, unpublished observations). Given that several different TM4SF proteins may form complexes with each other, it seems highly possible that loss of one particular TM4SF protein may be compensated by others. In conclusion, we have shown that TM4SF proteins CD9 and CD81 may play key roles during myoblast fusion, as seen in both murine C2C12 cells and human RD cells. Furthermore, CD9 and CD81 may also contribute to the protection of myotubes from apoptosis. | Study | biomedical | en | 0.999998 |
10461418 | Choledochal cyst is a congenital dilatation of the bile duct. This congenital disease is relatively rare in western countries and more than two thirds of the cases were reported in Japan 1 ) . Anomalous union of pancreaticobiliary duct (AUPBD) has been regarded to be the etiological factor of the choledochal cyst 2 ) . However, choledochal cyst is not always associated with AUPBD and the presence of AUPBD without choledochal cyst has been increasingly recognized recently, probably because of the advances in hepatobiliary imaging techniques. Therefore, some authors suggest that these two anomalies should be considered separately 3 ) . There have been many reports about choledochal cyst or AUPBD, but their cases were mainly infants or neonates. Moreover, cholangiography was obtained by the percutaneous transhepatic route or intraoperatively 4 ) . Thus, the pancreaticobiliary junction might have been fully evaluated in previous series. While most of the choledochal cysts were observed in infants, it can also be found in adults 5 ) . All of our cases had choledochal cyst diagnosed in adulthood and the presence or absence of AUPBD was confirmed by endoscopic retrograde cholangiopancreatography (ERCP). The number of the cases was 44, the largest series having been collected from a single institution, except the ones from Japan. Moreover, there were rare reports in English literature which compared clinical features of choledochal cyst according to the presence of AUPBD. The purpose of our study was to compare the clinical characteristics of 44 cases with adulthood choledochal cyst according to the presence or absence of AUPBD. Furthermore, we tried to clarify the significance of AUPBD in patients with choledochal cyst. From August 1990 to December 1996, 52 cases (1.03%) were diagnosed as having choledochal cyst out of 5,037 ERCP referrals. The diagnosis of choledochal cyst was made as a localized non-proportional dilatation of bile duct after exclusion of tumor, stone or inflammation as a cause of the dilatation 6 ) . All of the patients in our series were more than 16 years of age. Of the 52 choledochal cyst cases, we selected 44 cases, of which the pancreaticobiliary junction was clearly visualized. Choledochal cyst was classified as I, II, III, IVa, IVb, V according to Todani’s classification. Type I is cystic or diffuse dilatation of extrahepatic bile duct, type II is a diverticulum in the extrahepatic bile duct, type III is choledochocele, type IVa is multiple cystic dilatation of intra-and extrahepatic bile duct, type IVb is multiple cystic dilatation of extrahepatic bile duct and type V is multiple cystic dilatation of intrahepatic bile ducts (Caroli’s disease) 7 ) . AUPBD was defined as the anomalous union of pancreaticobiliary duct system at a distance > 15 mm from the papilla of Vater 8 ) This anomaly was divided into type II and II according to Kimura’s classification. Type I AUPBD looks as though the pancreatic duct joins the bile duct, which is the major duct, whereas in type II, it looks as though the bile duct joins the pancreatic duct, which is the major duct 8 ) . The cases with choledochal cyst were divided into those associated with AUPBD (n=28, AUPBD-present group) and those without (n=16, AUPBD-absent group) and clinical characteristics were compared between the two groups. Furthermore, in the AUPBD-present group, characteristics were also compared according to Kimura’s classification of AUPBD. The angle of the pancreaticobiliary junction was measured as viewed frontally. Statistical analysis was made by Fisher’s exact test and Mann Whitney U test. Of the 44 choledochal cyst cases, 17 cases had type I cysts, 1 had type II, 6 had type III, 18 had type IVa, 1 had type IVb, and 1 had type V. The AUPBD-present group was 28 (64%), while the AUPBD-absent group was 16 (36%). AUPBD was observed only in type I and IV patients, whereas it was not shown in type II, III, V patients ( Table 1 ). Age distribution of choledochal cyst was 7 in 16–19 years, 7 in 20–29 years, 11 in 30–39 years, 7 in 40–49 years, 5 in 50–59 years and 7 in 60–69 years ( Table 2 ). There were 15 males and 29 females (M:F ratio, 1:1.9). Comparing the characteristics according to the AUPBD-association, female cases were more observed in both groups, whereas the mean age of the AUPBD-present group was 49.2 years, younger than that of the AUPBD-absent group, although it was not statistically significant ( Table 3 ). Gallstone diseases were associated in 18 (41%) patients with choledochal cyst (n=44). The location of the gallstones was 11 in the cyst, 5 in the gallbladder and 2 in the intrahepatic duct. Pancreatic stones were shown in 2 (5%) patients. Acute inflammation was observed in 31 (70%) cases. They were cholecystitis (n=12), cholangitis (n=8) and pancreatitis (n=11). Malignant neoplasm occurred in 9 (20%) cases: gallbladder in 3, common bile duct in 4 and pancreas in 2 cases. All the cancers in the common bile duct arose from the cyst wall and were adenocarcinoma, pathologically. The difference in the incidence of associated diseases according to the presence of AUPBD was as follows. The incidence of gallstone disease in the AUPBD-present group did not differ from that in the AUPBD-absent group, while acute inflammation occurred more frequently in the AUPBD-present (26/28, 93%) than in the AUPBD-absent group (5/16, 31%) (p<0.01). Malignant neoplasm developed only in the AUPBD-present group (9/28, 32%), more often than in the AUPBD-absent group (0/16, p<0.05) ( Table 3 ). Pancreatic disorders (pancreatic stone, pancreatitis or pancreatic cancer) occurred in 12 of 28 cases (43%) in the AUPBD-present group, whereas only in 1 of 16 (6%) cases of the AUPBD-absent group (p<0.05, Table 3 ). According to Kimura’s classification, AUPBD (n=28) was divided into type I (n=12) and II (n=16), but we could not find any difference in associated diseases between the two groups ( Table 4 ). The maximal diameter of the common bile duct was higher in type II AUPBD, but it was not statistically significant. However, the angle between the biliary and pancreatic duct was higher in type II (85.1°) than in type I (45.2°)(p<0.05, Table 4 ). At the Children’s Hospital of Los Angeles, choledochal cyst was diagnosed in 0.5 patients per year 10 ) . However, in our institution (3 rd referral center), we experienced 53 cases during 6 years (incidence of about 9 per year). Our data suggest that choledochal cyst is more prevalent in Korea than in western countries. It also implies that the incidence of choledochal cyst may be higher not only in Japan but also in other oriental countries than in western countries 11 ) . Since the earlier report by Babbit et al. 12 ) about the frequent association of AUPBD with choledochal cyst, AUPBD has been regarded as an etiological factor of choledochal cyst 2 ) . However, the rate of AUPBD association with choledochal cyst was from 33% to 100% according to the reports 4 , 13 – 15 ) . It might be due to the difference in the characteristics of the selected cases. According to our results, AUPBD was associated only with type I and IV choledochal cyst ( Table 1 ). Therefore, the rate of AUPBD can be affected by the number of cases with type I or IV. Furthermore, Todani et al. 6 ) sub-classified type I choledochal cyst into type 1a, Ib, Ic, and suggested that AUPBD may not be associated in type I. This also implies that the association rate of AUPBD can be influenced even by the sub-classification in type I choledochal cyst. Moreover, Matsumoto et al. 16 ) divided choledochal cyst into childhood-type and adulthood-type. Association of AUPBD was observed in almost 100% of childhood-type, whereas less frequently found in adulthood-type. The difference of association rate in many reports, including ours, may be explained by the difference in the characters of the cases included. Although AUPBD was considered as an etiological factor of choledochal cyst 2 , 12 ) , the pathogenetic mechanism of choledochal cyst may not be explained solely by AUPBD because AUPBD was not found in all of the choledochal cyst cases. Our data also showed that AUPBD was not always associated with choledochal cyst cases (28/44, 64%). The increasing reports of AUPBD without choledochal cyst support the notion 17 ) . In AUPBD, a union of the pancreatic and biliary ducts is located outside the sphincter of Oddi. Therefore, two-way regurgitation occurs. Pancreatic juice refluxes into the common bile duct, or bile regurgitates into the pancreatic duct because the action of the sphincter muscle does not functionally affect the union 18 ) . Because the intraductal pressure is generally higher in the pancreatic duct than in the bile duct, pancreatic juice regurgitates into the biliary tract resulting in the pancreatic enzyme activation and subsequent recurrent inflammation. These may give rise to metaplastic and, finally, malignant change of the biliary epithelium 19 ) . Furthermore, after recurrent inflammation of the bile duct, the pressure in the bile duct rises and bile may reflux into the pancreatic duct causing various pancreatic disorders, including acute or chronic pancreatitis, pancreatic stone or pancreatic cancer 18 ) . The incidence of malignant diseases in choledochal cyst is said to be 2.5%–15%, 15 times greater than the control population without choledochal cyst 20 ) . In our series, cancer developed in 9 of 44 (20%). The incidence in our series might be higher than in other reports because all of our cases were in adulthood. It is well known that the incidence of cancer in choledochal cyst increases with age 21 ) . Moreover, cancer developed only in the AUPBD-present group, which implies that AUPBD may be a more important contributing factor than the choledochal cyst itself. Flanigan 22 ) pointed out that only 57% of cancers occurred in choledochal cyst were located in the cyst wall and the rest of cancers developed in bile duct other than the cyst wall. Moreover, Nagorney et al. 23 ) also suggested that malignant neoplasm developed in choledochal cyst is not always located in the cyst wall. In one of their cases, cancer developed in the remaining bile duct after complete cyst excision. These data imply that risk factors causing cancer in choledochal cyst are more than the choledochal cyst itself. The cancers developed in our series were located in gallbladder (n=3), common bile duct (n=4) and pancreas (n=2). Only 4 cases occurred in the cyst wall. Recent reports of gallbladder cancer in AUPBD cases without choledochal cyst suggest that AUPBD is more important for the carcinogenic process than the choledochal cyst itself 24 ) . In 35 cases of choledochal cyst described by Yoshida et al., 8 cases developed cholangiocarcinoma 25 ) . AUPBD was associated with all of these 8 cases, which supports our notion that AUPBD may be the major contributing factor for cancer development. Suda et al. examined AUPBD in 34 bile duct cancer patients, 24 gallbladder cancers and 171 controls without biliary disease 26 ) . They observed AUPBD in 8 of 34 cholangiocarcinoma, 4 of 24 gallbladder cancer, but none in controls. They suggested that AUPBD is one of the pathogenetic factors in biliary malignancy. In our results, acute inflammatory condition, such as cholecystitis, cholangitis or pancreatitis, was more prevalent in the AUPBD-present than in the AUPBD-absent group. The younger age in the AUPBD-present group suggests that patients in this group might visit hospitals earlier because of more severe symptoms. One of the factors worth mentioning is pancreatic diseases associated with choledochal cyst. There had been several reports about the association of pancreatitis, pancreatic stone or pancreatic cancer in choledochal cyst 20 , 27 – 29 ) . In the previous reports, however, they did not analyze the data considering AUPBD. In one Japanese report, acute pancreatitis occurred in 30 (17%) of the 176 cases with AUPBD 30 ) . Activated pancreatic enzymes, after entering the biliary tract, may cause cholangitis, gallstone and cholangiocarcinoma 31 ) . Likewise, these enzymes may reflux back into the pancreatic duct and cause various pancreatic disorders, such as acute or chronic pancreatitis and pancreatic cancer 32 ) . In our results, pancreatic disorders developed more frequently in the AUPBD-associated group ( Table 3 ). These high incidences of malignancy and inflammatory diseases associated with AUPBD also have therapeutic implications for choledochal cyst. In cases of choledochal cyst with AUPBD, cholecystectomy also should be performed in addition to cyst excision because the incidence of gallbladder cancer is very high. Moreover, surgical procedure for correction of AUPBD should be added. Biliary diversion from the pancreatic juice (pancreaticobiliary disconnection) may be needed for prevention of bi-directional reflux of pancreatic and bile juice 23 , 24 ) . In this regard, cholecystectomy along with the resection of dilated bile duct and the biliary diversion from pancreatic juice should be performed in cases with choledochal cyst and AUPBD. Komi et al. 35 ) subdivided AUPBD associated with choledochal cyst into several categories and suggested that pancreatitis could not be prevented by cholecystectomy, cyst excision and hepaticojejunostomy in certain subgroups. Furthermore, they suggested that, in cases with AUPBD showing dilated common channel or accessory pancreatic duct, sphincteroplasty or pylorus preserving pancreaticoduodenectomy should be needed in addition to the previously mentioned procedures. Schreiber et al. 36 ) described that AUPBD may be observed as two clinical manifestations. One is the biliary tract disease, such as acute cholecystitis, cholangitis and cholangiocarcinoma. The other one is caused by stasis of pancreatic fluid due to anomalous drainage in the common channels leading to periductal and interlobular fibrosis as a histological sign of chronic pancreatitis. Thus, Schreiber et al. suggested that resection of the anomalous junction and hepaticojejunostomy with a Roux-en-Y anastomosis may resolve both pancreatic reflux into the biliary system and stasis of the pancreatic secretion. The claim by Schreiber et al. has something to do with that of Komi et al. suggesting that hylorus-preserving pancreaticoduodenectomy may be recommended in certain cases of AUPBD. AUPBD frequently associated with choledochal cyst may have an implication not only as an etiological factor but as an associated disorder leading to a grave clinical course. In this regard, we should make an effort to confirm the presence of AUPBD in patients with choledochal cyst. Moreover, adequate surgery may be required to prevent the occurrence of cancer. Cancer associated with choledochal cyst may often be in an advanced stage when detected. Curative resection may be difficult 20 ) . Prevention, therefore, may be the best way, if possible. AUPBD associated with choledochal cyst may be a very important factor that affects the clinical course, surgical planning and prognosis. | Study | biomedical | en | 0.999996 |
10461419 | The pathogenic role of H. pylori in chronic gastritis and its association with duodenal ulcer (DU) are well established 1 – 3 ) . Therefore, the 1994 NIH Consensus Development Conference recommended eradication of H. pylori in all patients with documented peptic ulcer disease 4 ) . The dramatic effect of H. pylori -eradication on the natural course of DU disease has been sufficiently well reported by now 5 – 9 ) . However, published studies on BGU relapse are scarce, and its follow-up periods do not exceed 12 months 10 – 14 ) . There are a few differences between DU and BGU: positivity rates of H. pylori in BGU patients have been reported at 70–90% 15 ) , lower than that of DU, 95%–99% 1 – 3 ) . The reason for this difference may originate from the fact that nonsteroidal anti-inflammatory drugs (NSAIDs) are another major cause of BGU. These facts may play a role in the pattern of recurrence in BGU and DU. However, there is still a considerable lack of knowledge on the post-therapeutic course of BGU disease. We conducted this study to investigate how the BGU recurrence rate is reduced by eradication of H. pylori in a 2 year follow-up. This study was performed for H. pylori -positive 65 patients with active BGU, who were enrolled between October 1995 and September 1996, and followed up for 2 years. In forty patients, H. pylori was eradicated by triple therapy: omeprazole 20mg once a day, clarithromycin 500mg twice a day and amoxicillin 1.0g twice a day. The non-eradicated group consisted of 19 patients in whom the triple therapy was not conducted and 6 patients in whom H. pylori was not eradicated with the triple therapy. Patients with pregnancy or lactation, treatment with colloidal bismuth subcitrate or antibiotics within 3 weeks of gastroscopy, severe concomitant diseases and a history of previous gastric surgery were excluded. In addition, patients were excluded if they were under maintenance acid-suppressive therapy. Six biopsy specimens were taken within 3cm of the pyloric ring before beginning, and 4 weeks after completion of triple therapy. The biopsy specimens were analyzed with CLOtest, microscopy of Gram stained mucosal smear, culture and histology after H&E staining as described in detail elsewhere 16 , 17 ) . A patient was regarded as H. pylori -positive if one or more of the four aforementioned test methods demonstrated H. pylori colonization of the gastric mucosa. Follow-up gastroscopy was performed 6, 12, 18 and 24 months after treatment, or whenever the ulcer symptom recurred, for evaluation of ulcer recurrence. BGU recurrence was defined as endoscopically confirmed recurrent ulcer after endoscopically proven healing of the initial ulcer. By definition, superficial erosions were not considered to be ulcers. Four H. pylori tests were conducted in the eradicated group whenever follow-up gastroscopy was taken. Clinical factors, such as age, gender, smoking, alcohol, past history of BGU and ingestion history of NSAIDs were evaluated. For statistical analysis, continuous variables were analyzed by Student’s t test, and categorical variables by Chi-square test and Fisher’s exact test. A p value of <0.05 was considered to be significant. The mean age of the non-eradicated group was 51.6 ± 13.0 years and that of the eradicated group 50.6 n-eradicated group was 51.6 ± 13.0 years and that of the s taken. Clinical factors, such as age, gender, smoking, alcohol, past history of BGU and ingestion history of NSAcated group. Smoking history was found in 16 patients (64%) of the non-eradicated group, and in 20 patients (50%) of the eradicated group. Alcohol history was found in 11 patients (44%) of the non-eradicated group and in 26 patients (65%) of the eradicated group. Past BGU history was found in 17 patients (68%) of the non-eradicated group and in 18 patients (45%) of the eradicated group. Ingestion history of NSAIDs was found in 6 patients (24%) of the non-eradicated group and in 6 patients (15%) of the eradicated group. There was no statistical difference between these two groups in age, gender, smoking, alcohol, past history of BGU and ingestion history of NSAIDs. BGU recurrence was found in 15 patients (60%) of the non-eradicated group: 12 of 19 patients (63.2%) in whom triple therapy was not conducted and 3 of 6 patients (50%) in whom H. pylori was not eradicated with triple therapy. In comparison, BGU recurrence was found in 4 patients (10%) of the eradicated group, which was significantly lower than that of the non-eradicated group ( p <0.001, Table 1 ). In the 25 non-eradicated patients, the BGU recurrence rate was 16% (4 patients) within 6 months, 40% (10 patients) within 1 year, 56% (14 patients) within 18 months and 60% (15 patients) within 2 years . The respective recurrence rates in the 40 eradicated patients were 0%, 7.5% (3 patients). 10% (4 patients) and 10%, respectively . The recurrent ulcer sites in the 15 BGU patients of the non-eradicated group were the same as the ulcer sites of the initial diagnosis. In two patients, the number of ulcers increased to 3 and 5, including the initial one. The recurrent ulcer sites in the 4 recurred patients of the eradicated group were the same as those of the initial ulcer. The mean age of the recurred group was 65.3 ± 15.2 years, which is significantly older than that of the non-recurred group, 49.1 ± 10.9 years ( p <0.05, Table 2 ). The two female patients who had BGU recurrence and NSAIDs history were very old (67 and 83 years old), and this brought about a significant increase of the mean age for the recurrence group. Two patients were male (sex ratio, 1:1) in the recurred group, and 31 patients (sex ratio, 6.2:1) in the non-recurred group. Smoking history was found in 3 patients (75%) of the recurred group and 17 patients (47.2%) of the non-recurred group. Alcohol history was found in 1 patient (25%) of the recurred group and 25 patients (69.4%) of the non-recurred group. Past BGU history was found in 3 patients (75%) of the recurred group and 15 patients (41.7%) of the non-recurred group. Ingestion history of NSAIDs was found in 3 patients (75%) of the recurred group and 3 patients (8.3%) of the non-recurred group. There was no statistical difference between these two groups in gender, smoking, alcohol, past history of BGU and ingestion history of NSAIDs. When BGU recurrence was found in the 4 patients of the eradicated group, one patient (25%) was found to be H. pylori positive and two patients (50%) irregularly took NSAIDs due to arthritis before BGU recurring. The BGU recurrence in these 3 patients occurred within 1 year. In the remaining one patient, H. pylori was still negative and he denied ingestion history of NSAIDs when BGU recurred within 18 months. In the 36 non-recurred patients, only one patient (2.8%) was found to be H. pylori positive again in 1 year. H. pylori infection and NSAIDs are very important risk factors for peptic ulcer 18 ) . The positivity rates of H. pylori in patients with DU and BGU were somewhat different: that is, they have been reported to be 95–99% in DU 1 – 3 ) , but 70–90% in BGU 15 ) , which is lower than in DU and shows a wider variation than in DU. These results suggest that NSAIDs as the cause of BGU might be more significant than DU 19 ) . We have previously shown that the H. pylori infection rate and the rate of NSAIDs history were 82.8% and 26.1% of BGU patients, respectively, and 91.1% of the BGU patients had either H. pylori infection or NSAIDs history 20 ) . In contrast, H. pylori infection rate of patients with DU was 94.2% 9 ) , higher than that of BGU patients. In addition, even though the major cause of DU and BGU is H. pylori , their pathogenensis is thought to be different. That is, if the H. pylori infection occurs when the gastric acid secretion from parietal cell is high, H. pylori resides and multiplies in the antrum, avoiding the body, causing chronic antral gastritis and DU 21 ) . However, if H. pylori infection occurs in the low acid secretory state, such as malnutrition, immaturity, and intercurrent infection, pangastritis and multifocal gastric atrophy occur, and BGU or stomach cancer can develop 22 , 23 ) . Actually, H. pylori density was higher in the antrum than in the body for DU patients, but it was somewhat reversed for BGU patients 24 ) . In addition, the body is more adequate for H. pylori detection in BGU and stomach cancer patients, but it was reversed in DU patients 25 ) . These differences between DU and BGU may cause a difference in their pattern of recurrence. Combined data from 30 pilot and controlled studies show an overall DU relapse rate of 61% (range, 20–100%) in patients who remain H. pylori -positive, compared with 3% (range, 0–22%) in patients free of H. pylori 10 , 26 , 32 ) . This wide variance in the study results may be caused by several factors: absence of documented initial ulcer disease and ulcer healing; unknown H. pylori status at the time of ulcer relapse or at the conclusion of the study 10 , 27 – 29 ) and the assessment for cure of the infection by H. pylori clearance instead of H. pylori eradication, leading to high rates of H. pylori recrudescence and ulcer relapse rates 10 , 29 – 32 ) . Only a few studies have reported gastric ulcer relapse rate in relation to H. pylori status with a follow-up period of 1 year 10 – 14 ) . Gastric ulcer relapsed in 47–55.6% of patients who remained H. pylori -positive compared with 3–7% of the cured patients 10 – 14 ) . Because H. pylori infection is associated with ulcer disease, ulcer relapses will also be related to recurrent infection 1 , 33 ) . It is therefore crucial that H. pylori eradication is documented accurately. Nevertheless, studies performed with appropriate diagnostic accuracy still report H. pylori -negative ulcer relapses 10 , 12 , 14 , 29 , 32 ) . In these cases, the (occult) use of aspirin or NSAIDs may account for recurrent ulcers in the absence of H. pylori infection 12 , 34 ) . In one study where patients taking aspirin or NSAIDs were excluded, recurrence of DU or BGU was completely prevented by successful H. pylori eradication for up to 9.8 years (mean follow-up: 2.5 years) 26 ) . In the present study, the BGU recurrence rates in the 25 non-eradicated group were 16% within 6 months, 40% within 1 year, 56% within 18 months and 60% within 2 years . The respective recurrence rates in the 40 eradicated patients were 0%, 7.5% (3 patients), 10% (4 patients) and 10%, respectively , which were significantly lower than those of the non-eradicated group (p<0.001). We also investigated the similar study in DU patients with a 4 year follow-up 35 ) . When BGU recurrence pattern is compared with that of DU, there were two kinds of difference between BGU and DU. One is that the recurrence rate of BGU patients in the non-eradicated group looked lower than that of DU patients. That is, in a control group, comprising 31 patients with DU who were not treated with H.pyiori eradication regimen, the DU recurrence rate was 61% within 1 year, 81% within 2 years, 84% within 3 years and 90% within 4 years 35 ) . The recurrence rate of BGU in the non-eradicated group in the present study shows 40% within 1 year and 60% within 2 years, and these rates are lower than those of the DU group. The other is that the main cause of recurrence looks different in DU and BGU. In the 45 patients with DU in whom bacteria had been eradicated, DU recurrences were 0% within 1 year, 4% within 2 years, 13% within 3 years and 18% within 4 years, and all of them were found to be H. pylori positive again 35 ) . Moreover, no DU recurrence was found in the patients who remained H. pylori negative. In BGU patients, the recurrence rate of the eradicated group was 7.5% (3 patients) within 1 year and 10% (4 patients) within 2 years, which looks like slightly higher than that of the DU group. Among these 4 recurred BGU patients, only one patient was found to be H. pylori positive again within 1 year, and two patients had NSAIDs ingestion history. In the remaining one patient in whom BGU was found to recur within 18 months after eradication, H. pylori was still negative and there was no history of NSAIDs ingestion. These results suggest that BGU recurrence is caused by NSAIDs or H. pylori reinfection, although DU recurrence after eradication of H. pylori nearly depends on H. pylori reinfection. In the present study, most of the recurrent ulcer sites in the 19 recurred BGU patients were the same as the ulcer sites of the initial diagnosis, regardless of eradicated (4 patients) or not (15 patients). In two cases of BGU-recurred patients of the non-eradicated group, the number of ulcers increased to 3 and 5, including the initial one. These results suggest that the original weak mucosal point, which had already appeared as an ulcer, is the persistent weak site where BGU can easily recur even after healing, regardless of whether the reattacking cause is H. pylori or NSAIDs. In conclusion, the eradication of H. pylori in patients with BGU reduces the recurrence of BGU similar to DU. However, the major causes of BGU recurrence appear to be NSAIDs ingestion and reinfection of H. pylori , which is different from DU in which H. pylori reinfection is the main cause. | Review | biomedical | en | 0.999997 |
10461420 | The increasing use of more intensive chemotherapeutic regimens to achieve maximal antitumour activity has produced severe and prolonged neutropenia in many patients 1 , 2 ) . Life-threatening infection is a significant complication in patients undergoing intensive myelosuppressive therapy for treatment of malignancy. Neutropenia was known almost 3 decades ago as a major predisposing factor for the development of infection in patients with cancer. Absolute neutrophil counts less than 1000 cells/mm 3 are associated with over 70% of septic episodes in neutropenic host. Fatality rates range from 30% to 70%, depending on the degree and the duration of neutropenia 2 , 3 ) . Early empiric therapy with broad-spectrum bactericidal antibiotics is now standard practice in treating the cancer patients with febrile granulocytopenia 1 , 4 ) . Imipenem is an antibacterial agent of the carbepenem class of beta-lactams with a very broad spectrum of activity that includes most gram-negative and gram-positive pathogens, including aerobes and anaerobes, and with marked activity against species producing beta-lactamases 1 , 5 , 6 ) . Sulbactam is a beta-lactamase inhibitor which has been combined with ampicilin and cefoperazone 7 ) . Sulbactam itself has limited antibacterial activity against some aerobic gram-negative bacilli (AGNB) which include non-aeruginosa Pseudomonas spp. and Acinetobacter spp 8 , 9 ) . In combination with cefoperazone, it extends the spectrum of the latter antibiotic to some anaerobes, including Bacteriodes fragilis, and many beta-lactamase-producing AGNB (10). Aminoglycosides have played an important role, especially in the treatment of gram-negative rod bacteremia in these granulocytopenic patients. Aminoglycosides are rapidly bactericidal and show concentration dependent killing, a feature that favors regimens that achieved high peak serum concentrations 11 , 12 ) . Several studies regarding imipenem-cilastatin and sulbactam-cefoperazone plus amikacin as initial therapy for febrile neutropenic cancer patients had been reported 6 , 13 – 16 ) . The purpose of our study was to compare the efficacy of empiric therapy with imipenem-cilastatin monotherapy and the combination of sulbactam-cefoperazone plus amikacin in the treatment of presumed bacterial infection in neutropenic cancer patients. We performed this study at Ondokuz Mayis University Hospital, Samsun. Patients were eligible if they had liquids or solid tumours, neutropenia and fever (defined by at least two oral temperature readings above 38.0 °C at least 4 h apart within a 24-h period, single oral temperature above 38.5 °C) in the absence of an obvious noninfectious cause of fever, such as administration of blood products or cytotoxic drugs. The patients were informed about antibiotic regimens. Initial assessment included history and physical examination, urinalysis, complete blood counts, absolute neutrophil counts, blood biochemistry and chest X-ray. Specimens for bacterial and fungal cultures were collected from nose, throat, urine, stool, sputum (if available), blood and any other appropriate sites before commencement of antibiotic treatment. The antimicrobial prophylaxis was not used before initial treatment. Fifteen patients were randomly allocated to receive either imipenem-cilastatin (500 mg i.v. every 6 h) or a combination of sulbactam-cefoperazone (2 g i.v. every 12 h) plus amikacin (15 mg/kg/day, on average 500 mg i.v., every 12 h). The response to empiric therapy was evaluated at 72 h and therapy was modified only if the patient did not respond, or deteriorated, if there were adverse reactions, or if a resistant pathogen was isolated. If fevers persisted after five days of antibotic therapy, empirical antifungal therapy (fluconazole, 200 mg PO or IV every 24 hours) was started. Therapy was generally continued until the patient was free of symptoms of infection for 5 days or granulaocyte count increased to >1000/mm 3 . Antibiotic-related nephrotoxicity was diagnosed when the serum creatinine level increased to 0.5 mg/dl from baseline in the absence of other causes of renal dysfunction or other nephrotoxic drugs. Antibiotic-related hepatotoxicity was indicated when the serum aspartate and alanine aminotransferase increased 2-fold from baseline in the absence of other causes of hepatic dysfunction or hepatotoxic drugs. During a 12-month period, 30 patients with episodes of fever and granulocytopenia were enrolled in this study. Clinical characteristics of the evaluable patients are shown in Table 1 . Of the 30 evaluable episodes, 15 were treated with an imipenem-cilastatin monotherapy and a combination of sulbactam-cefoperazone plus amikacin. Age and underlying disease were comparable. Numbers of culture-positive episodes were 10 and 11, respectively, in the two regimens and the overall rate of positive cultures was 73%. The sites of infection and the causative organisms are listed in Table 2 . An early clinical evaluation after 72 h of empiric therapy showed that 60% of patients responded favorably to the initial empiric therapy. Table 3 shows the status at early evaluation. All of the patients in whom culture-negative episodes were observed were successfully treated with the initial regimen. One patient who had herpetic infection used acyclovir. There were no statistically significant differences between the treatment groups in patient outcome for the microbiologically documented, clinically documented, or possible infection groups (p>0.05). In 9 (60%) of imipenem-cilastatin groups and 9 (60%) of sulbactam-cefoperazone plus amikacin groups, an outcome of success without modification of therapy was achieved. The addition of vancomycin was the most frequently used modification in both groups. There were no major adverse effects requiring a change of antibiotics. However, two patients developed drug induced diarrhea and emesis in the group receiving the imipenem-cilastatin. None of these was considered serious or severe. Granulocytopenia has been closely associated with cancer and its treatment 4 , 17 ) . Despite the poor inflammatory reaction in these patients, the onset of fever usually represents an infection in the body 1 , 13 , 18 ) . Confirmation of infection usually involves a delay of some days before all the laboratory results are available. The primary objective of empiric antibiotic therapy is to protect many patients with cancer from the immediate cause of death 2 , 18 ) . In order to reduce infection-related morbidity and mortality, a number of treatment concepts have been utilized during the past 3 decades. Therefore, the antibiotic regimens must be selected against the major known pathogens 1 , 3 , 18 , 19 ) . Since the 1970’s, the most frequently used empiric antibacterial therapy has consisted of two or three-drug regimens, usually combining a cephalosporin, an antipsedomonal penicilin or both with an aminoglycoside 1 , 18 ) . The rationale for this approach is based on the wide range of potential gram-positive and gram-negative organisms necessitating coverage and the evidence that combination therapy results in additive or synergistic bactericidal activity. The availability of newer, broader spectrum cephalosporins, carbapenems and monobactams has led to considerable controversy regarding the use of these agents as monotherapy for empiric antibacterial treatment of the febrile compromised host 20 , 21 ) . Most investigators have shown that antibiotic combinations of aminoglycoside with a beta-lactam for synergy and avoidance of the emergence of resistant bacteria are more effective than monotherapy in the treatment of febrile neutropenic patients 22 , 23 ) . Monotherapy with imipenem-cilastatin has been previously demonstrated to be effective in noncomparative trials. Comparative studies with imipenem-cilastatin versus piperacillin plus amikacin and ceftazidime plus amikacin have shown that imipenem-cilastatin is equal to other combinations 24 , 25 ) . These results have demonstrated that imipenem/cilastatin monotherapy could be a practical alternative regimen 6 , 13 , 14 , 24 ) . The incidence of infection due to gram-positive organisms has increased markedly in patients with cancer 26 , 27 ) . In our study, 73% of the episodes had a positive culture; gram-positive pathogens accounted for 62% of the isolates. The response rate (60%) was the same for the two regimens. Each regimen covers the organisms most frequently isolated in neutropenic patients. Toxicity related to antibiotics were minimal in both groups. In summary, this study demonstrated that monotherapy of imipenem-cilastatin and the combinations of sulbactam-cefoperazone with amikacin were equally effective in the treatment of febrile episodes in neutropenic cancer patients. | Review | biomedical | en | 0.999997 |
10461421 | The atherosclerotic process that results in coronary artery disease (CAD) is a generalized process that may involve the entire vasculature 1 , 2 ) . The relation between the presence of aortic plaque in the thoracic aorta and the development of cardiovascular disease has been called to attention recently. Previous roentgenographic studies have suggested a relation between aortic plaque and CAD but have lacked clinical utility 3 , 4 ) . With improved noninvasive imaging techniques now available it is necessary to determine whether atherosclerotic aortic plaque can be reliably detected and correlated with coronary artery disease in a clinically useful manner. Transesophageal echocardiography (TEE) offers high resolution imaging for evaluation of thoracic aortic disease. The degree of atherosclerotic alteration in the thoracic aortic intima can be reliably determined using TEE imaging 5 , 7 ) . Previous analyses have shown that the presence of atherosclerotic plaque in the thoracic aorta correlated closely with systemic embolism and vascular disease 8 – 12 ) and retrospectively a marker of CAD 13 – 15 ) . Many cardiologists still frequently recommend pre-operative coronary angiography for patients requiring valve surgery because of the difficulties in non-invasive visualization of CAD 16 ) . The value in the prediction of CAD in detecting atherosclerotic aortic plaque by TEE may be attractive and have great influence on routine pre-operative cardiac catheterization 17 – 19 ) . In this study, we prospectively assessed the accuracy of TEE detection of thoracic aortic plaque for predicting the absence or presence and severity of CAD in a series of consecutive patients undergoing open heart surgery. One hundred thirty-one patients underwent TEE with aortic imaging in the operating room at St. Paul’s Hospital between January 1996 and May 1998. All patients included in this study underwent cardiac catheterization with coronary angiography for one of the following indications: angina (n=53), postmyocardial infarction (n=18), valvular heart disease (n=46) and congenital heart disease (n=14). Introperative two-dimensional TEE was performed using ultrasound equipment (Acuson 128XP) with 5-MHz biplane transducer. All studies were recorded on super VHS videotape for subsequent review and analysis. After aortic imaging, the transducer remained in place to assist in cardiac monitoring during surgical procedure. Aortic intimal changes were graded on a scale of I to IV proposed by Fazio et al 13 ) . The thoracic aorta was considered normal with respect to atherosclerotic disease and classified as grade I when the intimal surface was smooth and continuous without lumen irregularity or increased echodensity. Grade II changes consisted of increased echodensity of the aortic intimal surface, which was smooth and continuous without lumen irregularity or thickening. Grade III changes consisted of focal or linear increased density of intima associated with lumen irregularity and thickening or ulceration. Grade IV changes consisted of intimal thickening and lumen irregularity associated with protruding thrombus or highly echodense material that induced shadow artifact (consistent with calcification). Atherosclerotic aortic plaque was defined as a lesion with grade III or grade IV changes . All 131 studies on the recorded tapes were graded by two independent cardiologists with experience in TEE. Any discrepancy was resolved by consensus. Cardiac catheterization with coronary angiography was perfomed by the Judkins technique. Angiographic films were interpreted by the angiographers, who had no knowledge of the echocardiographic results. Coronary artey disease was defined as ≥ 50% reduction of the luminal diameter (75% reduction in luminal area) in the left anterior descending, left circumflex or right coronary arteries. The number of vessels with significant stenosis was recorded. Left main coronary artery disease with ≥ 50% reduction of the luminal diameter was considered to be two-vessel disease involving left anterior descending and left circumflex arteries. Angiographic and corresponding echocardiographic data were complied on a 2×2 contingency table to allow calculation of sensitivity and specificity as well as positive and negative predictive values. Discrete variables were analysed by the chi-square test, and a two-tailed t test was used to compare continuous variables. For incremental data, the Speaman correlation analysis was applied. Multiple logistic regression analysis was used to determine whether aortic plaque was a statistically significant predictor of significant CAD independent of age, gender and coronary risk factors. A P value <0.05 was considered statistically significant. The study group consisted of 73 men and 58 women with an average age of 54±10 years (range 17 to 75 years). 89 (68%) had one or more risk factors. Hypertension was present in 29%, 33% of patients had hypercholesterolemia, 41% smoked cigarettes, 16% had diabetes mellitus and 31% had obesity. Seventy-six (58%) of 131 patients were found to have obstructive CAD. Of this group, 11 patients had one-vessel disease, 25 had two-vessel disease and 40 had three-vessel disease. Ten patients had left main coronary artery stenosis and seven of them were classified as having three-vessel disease. TEE detected atherosclerotic plaque in the thoracic aorta in 71 of the 76 patients with obstructive CAD but in only 10 of the 55 patients without obstructive disease. Fifty of our 131 patients did not have aortic plaque on TEE. Forty-five of these 50 patients did not have obstructive CAD; the other 5 had obstructive disease ( Table 1 ). The discovery of atherosclerotic plaque in the thoracic aorta on TEE had 93% sensitivity for obstructive coronary artery disease. The specificity or “negativity in health” was 82%. The positive predictive value of plaque for obstructive CAD was 88% and negative predictive value was 90%. The accuracy of TEE as a test to predict obstructive CAD was 89% in our study. Of the 113 patients without previous myocardial infarction, atherosclerotic plaque was detected in the thoracic aorta in 54 of the 59 patients with CAD, and in 10 of the 54 patients without obstructive disease . Thus, it was not present in 49 patients. Forty-four of these 49 patients(90%) did not have significant obstructive coronary disease. Therefore, in patients without previous myocardial infarction, the presence of thoracic atherosclerotic plaque on TEE study identified CAD with a sensitivity of 92%, specificity of 88%, and positive and negative predictive value of 84% and 90%, respectively. Of the 11 patients with single-vessel CAD, 9 (82%) had aortic plaque as did 24 (96%) of 25 patients with two-vessel disease and 38 (95%) of 40 with three-vessel disease. All 10 with left main obstructive CAD had aortic plaque on TEE ( Table 2 ). There was a significant relation between the different grades of thoracic aortic atherosclerosis and the severity (number of obstructed vessels) of coronary disease . Aortic plaque was a predictor of obstructive CAD at a statistically significant level . We also compared age, gender and the coronary risk factors for obstructive CAD. By univariate analysis, age, sex, hypertension, smoking and diabetes mellitus were significant predictors of obstructive CAD ( Table 3 ). Multivariate logistic regression analysis including age, sex, hypertension, smoking, diabetes and aortic plaque revealed two independent predictors of significant CAD: aortic plaque and sex. Aortic plaque was the most significant independent predictor ( Table 4 ). Obstructive CAD was a predictor of thoracic aortic plaque at a statistically significant level . Age, sex, hypertension and smoking were also significantly associated with aortic plaque ( Table 5 ). By multivariate logistic regression analysis, age , sex , smoking and CAD remained statistically significant predictors of thoracic aortic plaque. Advances in non-invasive diagnostic techniques over the last decade have enabled accurate assessment of patients with valvular heart disease. Quantitation of hemodynamics, ventricular function and detailed description of valve leaflet thickening, calcification and mobility by Doppler echocardiography have allowed an appropriate selection of patients for valve repair or valve replacement without the need for pre-operative cardiac catheterization and without compromising theirs clinical outcome 17 ) . However, detection of important coexistent, but asymptomatic CAD has remained the Achilles’ heel of non-invasive methods used in the pre-operative evaluation of patients with valvular heart disease. Pre-operative detection of CAD in patients undergoing valve surgery has been regarded as a prerequisite to avoid peri-operative coronary events that might compromise patient outcome 16 ) . This has been particularly important in elderly patients in whom the prevalence of risk factors for CAD increases the likelihood of adverse cardiovascular events in the peri-operative period and has resulted in the recommendation of routine pre-operative coronary angiography. The atherosclerotic process that results in CAD is not restricted to the coronary vasculature. In previous roentgenographic studies, the presence of calcified atherosclerotic aortic plaque on chest X-ray film was associated with an increased risk of cardiovascular events and death 3 , 4 ) . However, the low resolution of X-ray limits the possibility of detecting aortic plaque 13 ) . TEE provides high-resolution imaging of thoracic aorta and is a useful method of evaluating thoracic aortic atherosclerosis, aneurysms and dissections. Many studies have shown that the presence of atherosclerotic plaque in thoracic aorta, detected by TEE, correlated closely with systemic embolism 8 – 12 ) . The purpose of our study was to determine whether TEE imaging of noncoronary vascular structures could accurately predict the presence and degree of CAD detected angiographically. Our results show that the thoracic aortic plaque detected on TEE study appeared to be a useful marker for predicting the presence and severity of CAD. These findings are consistent with the observation of Fazio et al 13 ) , and further substantiate the concept that atherosclerosis is a generalized process involving predominantly medium-sized muscular arteries 10 , 12 ) . In this study, although age and some risk factors correlated significantly with the presence of CAD, the predictive values of these variables were lower than those of thoracic aortic plaque detected by TEE examination. Furthermore, multivariate regression analysis revealed thoracic aortic plaque was the most significant independent predictor of CAD. This study proved that the absence of atherosclerotic plaque in the thoracic aorta, as detected by TEE, is highly specific for angiographically normal coronary arteries and has a high negative predictive value. If coronary risk factors are also taken into account, the positive predictive value of thoracic aorta atherosclerosis improves without an adverse effect on the negative predictive value. The high negative predictive accuracy of obstructive CAD in this study may have important clinical significance. Our results suggest that the decision to perform cardiac catheterization and coronary angiography should individually take into account risk factors and, particularly, TEE detection of thoracic aortic plaque. TEE detection of complex aortic plaque may also help to avoid embolic complication when a patient is referred for cardiac catheterization because systemic embolism arising from atherosclerotic debris of the thoracic aorta has been described as following invasive procedures involving the aorta 20 – 22 ) . Thus, in a recent study, the risk of systemic embolism caused by a guiding wire during transfemoral catheterization was 0.1% overall but 27% in patients with complex atherosclerotic plaques and debris 23 ) . The identification and location of plaque on TEE may help decrease the morbidity of catheterization. In conclusion, our results indicate that TEE detection of atherosclerotic plaque in the thoracic aorta is useful in the noninvasive prediction of the presence and severity of coronary artery disease. Clinical implications: In patients with valvular heart disease, the absence of thoracic aortic plaque on TEE studies may predict normal or minimal atherosclerotic coronary arteries. In selected patients, TEE examination may avoid the need to perform cardiac catheterization and coronary angiography. This can be extremely important for patients with unstable hemodynamic conditions in whom invasive assessment requires risky procedures. Selective use of cardiac catheterization and coronary angiography will also lower the cost of management and preoperative evaluation of patients with valvular heart disease. A large prospective study using a multiplane transducer is recommended before coming to definitive decision-making conclusions. | Study | biomedical | en | 0.999996 |
10461422 | Acetaminophen, a widely-used analgesic, is known to cause lethal hepatic necrosis at high doses. This acetaminophen-induced hepatotoxicity is not a result of the parent compound but is mediated by its reactive metabolite N -acetyl- p -benzoquinone imine (NAPQI) 1 ) . After low acetaminophen doses, NAPQI is inactivated by conjugation to glutathione 2 , 3 ) . However, in acetaminophen overdose, or other circumstances which lead to depletion of glutathione 2 ) , the reactive metabolite binds covalently to hepatic proteins, which may initiate the development of hepatic necrosis 4 – 7 ) . In humans, three cytochrome P450 enzymes, cytochrome P4502E1 (CYP2E1), cytochrome P4501A2 (CYP1A2) and cytochrome P4503A4 (CYP3A4), have been known to be involved in acetaminophen activation 8 – 10 ) , and most (30–78%) of this biotransformation is mediated by CYP2E1 8 ) . Recently, it was reported that cyp2e1 knockout mice are resistant to acetaminophen-induced hepatic necrosis 11 ) . Also, many drugs or chemicals, which inhibit CYP2E1 activity, are known to prevent acetaminophen-induced liver injury in mice or rats 12 – 15 ) . However, most of these chemicals have limited data on safety in human beings. Chlormethiazole is a sedative and anticonvulsive drug widely used in the treatment of alcohol withdrawal in Europe. Recently, it was reported that chlormethiazole inhibits not only the CYP2E1 transcription but also the catalytic activity of the enzyme in humans 16 ) . Therefore, we investigated the effect of chlormethiazole on acetaminophen-induced liver injury in mice. Female C57BL/6 mice (15–20 g body weight) were fed a standard diet up to 4 h before the experiment. Only drinking water was then allowed until 1 h after administration of acetaminophen, when food was reinstated. Acetaminophen (Sigma Chemical Co., St. Louis, Mo.) was dissolved in 40°C physiologic saline (25 mg/mL) and injected to the mice at doses ranging from 200 to 600 mg/kg intraperitoneally. Chlormethiazole was dissolved in 5% dextrose water (8 mg/mL) and administered at a dose of 60 mg/kg intraperitoneally. Each group in this study consisted of eight mice. Six groups of mice were studied: Group 1, mice were given equivalent volume of 5% dextrose water and normal saline to obtain control samples; Group 2, 5% dextrose water 30 min before 400 mg/kg of acetaminophen; Group 3, chlormethiazole 30 min before 400 mg/kg of acetaminophen; Group 4, 400 mg/kg of acetaminophen and then chlormethiazole 2 h after acetaminophen injection; Group 5, 5% dextrose water 30 min before 500 mg/kg of acetaminophen; Group 6, chlormethiazole 30 min before 500 mg/kg of acetaminophen. 24 h after acetaminophen injection, blood was collected for determination of serum aminotransferase activities and the mice were killed. The livers were immediately removed and sections of the livers were fixed in 10% formalin and embedded in paraffin for histologic examination. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by the method of Reitman and Frankel with kits obtained from Sigma Chemical Company 17 ) . Paraffin sections were stained with hematoxylin-eosin, according to standard procedures. Coded histologic sections were examined under light microscopy by a blinded observer. The extent of necrosis was graded from 0 to 4+ as follows: Histologically normal sections were graded 0. Minimal centrilobular necrosis was graded 1+; more extensive necrosis confined to centrilobular regions was graded 2+; necrosis extending from central zones to portal triads was graded 3+; and massive necrosis of most of the liver was graded 4+ 18 ) . In a separate experiment, 5% dextrose water and acetaminophen at doses ranging from 200 to 600 mg/kg were injected into mice of 5 groups. In another 4 groups of mice, chlormethiazole and acetaminophen at doses ranging from 200 to 500 mg/kg were injected. Animals were observed for 48 hours and the number of surviving animals at 48 h was counted. Two separate experiments with the same protocol were performed. Three groups of mice were studied: Group 1, mice were given 5% dextrose water and normal saline; Group 2, 5% dextrose water 30 min before 400 mg/kg of acetaminophen; Group 3, chlormethiazole 30 min before 400 mg/kg of acetaminophen. Mice were sacrificed 6 h after acetaminophen administration. The livers were immediately removed and homogenized in ice-cold 1.15% (w/v) KCl solution. This homogenate was used to measure the extent of hepatic lipid peroxidation by the thiobarbituric acid method as described by Ohkawa et al 19 ) . Results are expressed as median with range. Nonparametric statistical procedures were used, and the significance of differences between groups was evaluated using the Mann-Whitney U test. In mice receiving acetaminophen only, there was a striking elevation of serum transaminases at a dose of 400 mg/kg. Pretreatment with chlormethiazole 30 min before 400 mg/kg of acetaminophen completely inhibited acetaminophen-induced liver injury (median 118.5 U/L, range 75 to 142 for AST; median 49 U/L, range 41 to 64 for ALT). Furthermore, in animals receiving chlormethiazole 2 h after acetaminophen, the mean AST and ALT levels were less elevated , reaching only 20% of the value of the acetaminophen-only group (p < 0.001). Significant but less marked protection was observed when acetaminophen was injected at a dose of 500 mg/kg . Typical extensive perivenular necrosis was observed in mice receiving acetaminophen only. Nearly all mice pretreated with chlormethiazole 30 min before 400 mg/kg of acetaminophen had histologically normal livers (p < 0.001). Mice treated with chlormethiazole 2 h after acetaminophen also had less severe necrosis than those receiving acetaminophen only . Fig. 3 shows the survival rate at doses ranging from 200 to 600 mg/kg of acetaminophen. Whereas more than 50% of mice died at 500 mg/kg of acetaminophen, all the mice pretreated with chlormethiazole survived at the same dose. There was no mouse with elevated hepatic malondialdehyde level in the group pretreated with chlormethiazole (median 39.7 pmole/mg protein, range 32.4 to 47.4), compared with those of controls (median 39.4 pmole/mg protein, range 35 to 51.9). Elevated levels were observed in about half of the mice treated with acetaminophen only (median 44.5 pmole/mg protein, range 35.1 to 109.6). However, the differences were not significant, statistically . Acetaminophen, a widely used analgesic, is known to cause potentially lethal hepatic necrosis. Hepatotoxic effect is expected in nonalcoholic patients with doses higher than 7.5 g. An overdose of 13 to 25 g is often lethal 20 ) . However, acetaminophen hepatotoxicity can occur in some individuals who ingest quantities within the therapeutic range. Alcoholics, with their induced cytochrome P-450 activity (especially, CYP2E1), attendant malnutrition and low glutathione levels, are predisposed to the toxic effects of acetaminophen. Prescribed doses as low as 4 g/d for 2 days have been associated with hepatotoxicity, and long-term use of as little as 2.6 to 3.9 g/d can result in significant hepatotoxic injury in alcoholics 21 ) . Concomitant use of isoniazid or barbiturate is also associated with increased hepatotoxic effect 22 ) . Approximately 75% of an administered therapeutic dose of acetaminophen undergoes sulfation or glucuronidation; 5% to 10% is oxidized by the cytochrome P-450 enzymes to a toxic metabolite, N -acetyl- p -benzoquinone imine. Normally, N -acetyl- p -benzoquinone imine is conjugated with glutathione and safely excreted. With an overdose, increased oxidation occurs, glutathione becomes depleted and toxic N -acetyl- p -benzoquinone imine causes hepatocellular injury 23 ) . In humans, three cytochrome P450 enzymes, cytochrome P4502E1 (CYP2E1), cytochrome P4501A2 (CYP1A2) and cytochrome P4503A4 (CYP3A4), have been known to be principal catalysts of acetaminophen activation, and most (30–78%) of this biotransformation is mediated by CYP2E 18 – 10 ) . As discussed above, cyp2e 1 knockout mice are known to be resistant to acetaminophen-induced hepatic necrosis 11 ) . Many drugs or chemicals that inhibit CYP2E1 activity are known to prevent acetaminophen-induced liver injury in mice or rats 12 – 15 ) . Cimetidine has been shown to prevent acetaminophen-induced hepatocellular injury in rats. However, the dose given in that experiment was 120 mg/kg, 18 times higher than the usual therapeutic dose in humans 18 ) . Additionally, studies performed with human microsome suggested a need for 5–10 times higher cimetidine concentrations than putative therapeutic concentrations 24 ) . Subsequent clinical trials have failed to prove the efficacy of cimetidine against acetaminophen-induced hepatic injury in humans 20 , 25 ) . Other chemicals, such as cobalt chloride, methoxsalen or propylene glycol have been shown to prevent acetaminophen-induced hepatic injury in animal experiments, but most of these chemicals have limited data on safety in human beings 12 – 15 ) . In the present study, chlormethiazole attenuated acetaminophen-induced hepatic injury in mice. However, it could not completely prevent hepatic injury at higher doses of acetaminophen. Other P-450s, such as CYP1A2 or CYP3A4 having a higher Km for acetaminophen, may be responsible for the toxicity at high doses of the drug. Chlormethiazole was reported to inhibit CYP2E1 transcription as well as CYP2E1 catalytic activity in humans 16 ) . However, one study performed in vivo in rats showed no significant effect of chlormethiazole on CYP2E1 catalytic activity, but showed selective transcriptional suppression of CYP2E1 26 ) . Therefore, the rat model seems to be not suitable to evaluate the therapeutic efficacy of chlormethiazole in acute acetaminophen-induced hepatic injury. It is not known whether chlormethiazole inhibits CYP2E1 catalytic activity at the constitutive level in mice. However, it can be speculated that chlormethiazole probably inhibits the CYP2E1 catalytic activity in mice because our data showed its protective effect against acute acetaminophen-induced hepatic injury. If chlormethiazole merely inhibits the CYP2E1 transcription, it is unlikely that chlormethiazole can nearly abolish hepatic injury in acute acetaminophen poisoning. Further studies are needed to confirm this assumption. The dose of chlormethiazole given in this study produced mild sedation without respiratory depression in mice. At this dose, chlormethiazole nearly abolished acetaminophen-induced liver injury in mice. Since it was reported that a single administration of 192 mg of chlormethiazole in healthy human controls dramatically inhibits CYP2E1 catalytic activity 16 ) , the dose needed to prevent acetaminophen-induced liver injury may be within the usual therapeutic dose (1.5 g/d) in humans. Lipid peroxidation has been observed after acetaminophen administration in vivo and in vitro in mice and rats 27 , 28 ) . Although elevated levels were observed in about half of mice treated with acetaminophen, hepatic malondialdehyde levels among experimental groups in this study were not different statistically. The discrepancy between data from our study and previous studies may result from different doses of acetaminophen and different duration of starvation before acetaminophen administration. However, the possibility that lipid peroxidation may not be prerequisite for acetaminophen-induced hepatic injury cannot be excluded because mice with normal hepatic malondialdehyde level in the acetaminophen group also showed gross and histological evidence for hepatic damage. Other studies also have showed the late appearance of products of lipid peroxidation 29 ) . Theoretically, chlormethiazole may be useful in preventing the CYP2E1-mediated generation of toxic intermediates. A very recent study has shown the protective role of chlormethiazole against ethanol mediated liver damage 30 ) , and our study also showed the protective role against acetaminophen-induced liver injury. Because chlormethiazole inhibits the generation of toxic metabolites, it is unlikely that chlormethiazole can substitute for N -acetylcysteine, which replaces the depleted glutathione stores necessary to prevent accumulation of toxic metabolite 23 ) , in acetaminophen overdose. In clinical practices, treatment with N -acetylcysteine within 10 hours of acetaminophen usually prevents severe liver damage. Severe hepatic necrosis usually develops when treatment is delayed 31 ) . So it is unlikely that patients who cannot benefit by N -acetylcysteine can benefit from chlormethiazole. Chlormethiazole may be useful in patients who cannot tolerate N -acetylcysteine side-effects such as vomiting or anaphylactoid reaction, or as a combination therapy with N -acetylcysteine. It may also be useful in situations when inhibition of CYP2E1 activity is beneficial. Administration of chlormethiazole before acetaminophen in alcoholic patients may be an example. In conclusion, we have shown that chlormethiazole effectively reduces acetaminophen-induced liver injury in mice. Further studies are needed to assess its role in humans. | Study | biomedical | en | 0.999997 |
10461423 | Both metabolic 1 , 2 ) and vascular defects 3 – 5 ) have been implicated in the pathogenesis of diabetic neuropathy, but the precise mechanisms causing peripheral neuropathy have not been elucidated. Nerve conduction is impaired in overt diabetic neuropathy by a combination of structural and metabolic defects in the peripheral nerve 6 ) . Many studies attribute the slowing of nerve conduction in diabetic rats to alterations in nerve Na + and Na + -related metabolism mediated by a reversible Na + , K + -ATPase defect 1 , 7 , 8 ) The decreases in Na + , K + -ATPase activity and motor nerve conduction velocity (MNCV) in diabetic rats have been reported to be ameliorated by treatment with aldose reductase inhibitors 8 – 11 ) , dietary myo-inositol supplementation 12 ) , gangliosides 13 , 14 ) , prostaglandin E 1 15 ) , anti-oxidants 16 or essential fatty acids 17 , 18 ) . More recent experiments showed that there was a positive correlation between cAMP content and Na + ,K + -ATPase activity, and MNCV and Na + ,K + -ATPase activity in the rat sciatic nerve 19 ) . Haemodynamic abnormalities of the peripheral nerve have also been suggested as a major cause of the functional deterioration in the neuropathies observed in streptozotocin (STZ)-induced diabetic rats 3 , 20 . In addition to marked biochemical and functional abnormalities, morphological changes have been demonstrated in the nerves of diabetic rats 21 ) . Cilostazol(6-[4-cyclohexyl-1H-tetrazol-5-yl)butyl}3,4-dihydro-2(1H)-quinolinone]), a potent phosphodiesterase inhibitor, increases the cAMP content of the sciatic nerves of rats 19 ) . In this study, we examined the effects of cilostazol on biochemical, functional and morphological aspects of experimental diabetic neuropathies induced by STZ in the rat. Eight-week-old male Sprague Dawely rats (Charles River, Japan) were acclimatized to their new environment for one week before the experiment. After an overnight fast, the rats were rendered diabetic by i.p. injection of STZ (35 mg/kg) (Sigma Chemical Co., St. Louis, MO, USA) in 10mM citrate buffer, pH 4.5. A diabetic rat was identified by a nonfasting tail-vein plasma glucose level exceeding 16.7mM/L in the first week after injection with STZ. Diabetic rats at 8 weeks after STZ treatment were divided into two groups: the first group was given a pelleted diet containing 0.03% cilostazol(15mg/kg/day) (Otsuka Pharmaceutical Co. Tokyo, Japan) for another four weeks and the second group was the untreated age-matched diabetic rats. Age-matched normal rats were used as non-diabetic controls. Motor nerve conduction velocity (MNCV) was measured before and after treatment with the drug. Rats in each group were killed by cardiac puncture under light ether anesthesia in the 12th week after STZ-treatment for the measurement of cAMP and light and electron microscope examination of sciatic nerve samples. MNCVs were measured in the sciatic nerves of rats using a Nuropack II (Nihon Kohden Co., Tokyo, Japan), as previously reported. 26 ) . Briefly, animals were lightly anesthetized with an i.p. injection of 40 mg/kg pentobarbital sodium. Body temperature was monitored using a rectal probe and maintained at 37 °C with a warming pad. Sciatic-tibial NCV was determined non-invasively by stimulating proximally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimulation. The proximal and distal latencies of the compound muscle action potentials, recorded via bipolar electrodes from the first interosseous muscle of the hind paw, were measured from the stimulus artifact to the onset of the negative M-wave deflection, subtracted, and divided into the distance between the stimulating and recording electrodes, giving a value for MNCV in m/s. Nonfasting plasma glucose was measured in tail-vein blood samples, using Beckman glucose analyzer II (Beckman Instruments, Inc., Fullerton, CA). Sciatic nerves were removed from the rats under ether anesthesia and weighed. The whole or prepared nerves were immediately boiled for 5 min with sodium acetate buffer (pH 4.0), homogenized using a glass homogenizer, and then centrifuged at 2000 × g 5 min. The supernatant was used for cAMP determination by radioimmunoassay (RIA) (Incstar Co., Stillwater, Minnesota, U.S.A.) On the day after the final MNCV determination, non-fasted animals were anesthetized with ether. Midthigh segments of the sciatic nerve were surgically removed and fixed in a 2.5% cacodylate-buffered glutaraldehyde fixative, dehydrated and embedded in Epon. Embedded sciatic nerves were sectioned and examined microscopically. Ultrathin cross sections were stained with toluidine blue. Results are expressed as means±SD. Multiple between-group comparisons were performed using one-way analysis of variance (ANOVA). Statistical comparisons in three groups were made using Student’s t test. The correlation between cAMP content and MNCV was analyzed by linear regression analysis. As shown in Table 1 , blood glucose levels were significantly elevated and body weights significantly decreased compared to normal control rats 12 weeks after STZ administration. cAMP levels were decreased in the sciatic nerves of the diabetic rats. Administration of cilostazol (weeks 9–12) had no effect on blood glucose levels and body weights but significantly increased cAMP levels of sciatic nerves compared to age-matched, untreated diabetic rats. However, cilostazol treatment did not restore cAMP levels of diabetic rats to those of age-matched normal control rats. As shown in Table 2 , significant reduction in MNCVs were observed in diabetic rats compared to those in age-matched normal rats at the 8th and 12th weeks after STZ treatment (p<0.001). Cilostazol treatment significantly increased MNCV in the sciatic nerves of diabetic rats but they were not restored to the levels noted in age-matched normal control rats. As shown in Fig. 1 , there was a positive correlation between cAMP content and MNCV in the sciatic nerve at 12 weeks after STZ treatment (r=0.86; P<0.001). cAMP and MNCV in untreated diabetic rats were scatterd in the lower left part of the curve and cilostazol treatment shifted the data points to the upper right part of the curve. Morphological changes were examined in the sciatic nerve by light and electron microscopy. In the peripheral nerves of the diabetic rat, there was reduction in the number of myelinated fibers and there appeared to be more small fibers in the nerve when compared to non-diabetic control rats. Myelinated fiber density and size distribution were restored after treatment of cilostazol . In electron micrographs, a thickening of capillary walls and duplication of basement membranes in the nerves of diabetic rats were noted. These vascular abnormalities were restored towards normal after cilostazol treatment, . The streptozotocin (STZ)-induced diabetic rat is the most commonly used animal model of human diabetic neuropathies. Early changes in nerve function are characterized by reduced nerve conduction velocity in this animal model 3 , 22 , 23 ) . Diabetic rats of eight weeks’ duration show a significant reduction in sciatic MNCV which can be normalized by insulin treatment and near normoglycemia 24 ) . Acute hyperglycemia induced by STZ elicits activation of the polyol pathway with sorbitol accumulation, myo-inositol depletion and reduction of Na + ,K + -ATPase activity 25 , 26 ) The decrease in myo-inositol causes depletion of phosphoinositides, followed by poor calcium mobilization and impaired protein kinase C activity, eventually resulting in impaired Na + , K + -ATPase activity 27 ) . Thus, nerve Na + /K + pump activity is reduced; fibers become Na+ loaded, Na + channels become inactivated, and MNCV decreases. Decreasd Na + ,K + -ATPase activity and motor nerve conduction velocity(MNCV) have been reported to be restored by aldose reductase inhibitors 8 – 11 ) , dietary myo-inositol supplementation 8 , 12 ) , gangliosides, 13 , 14 ) prostaglandin E 1 15 ) , anti-oxidants 16 ) or essential fatty acid 17 , 18 ) in diabetic rats. Recent studies have shown that cAMP levels are decreased in the peripheral nerves of STZ-diabetic rats 19 ) . cAMP is one of the intrinsic intracellular modulators which regulates Na + ,K + -ATPse activity or the Na + /K + pump in various tissues 28 , 29 ) . Therefore, the possibility was suggested that increased cAMP may improve MNCV by restoration of Na + ,K + -ATPse activity. It has been reported that cilostazol increased cAMP content in the nerves of diabetic rats and improved MNCV without changing myo-inositol levels, and the positive correlation between cAMP content and Na+,K + -ATPse activity was noted also 19 ) . In addition to marked biochemical and functional abnormalities, morphological changes have been demonstrated in the peripheral nerves of STZ-induced diabetic rats 21 ) . As reported by other investigators, STZ in the present study also increased blood glucose levels and reduced body weights of the rats. In addition, the cAMP level and MNCV of sciatic nerves were decreased by STZ as reported earlier. 19 Blood glucose and body weights were unaffected by cilostazol treatment but cAMP levels and MNCV were significantly increased compared to untreated diabetic rats but were not restored to the levels observed in age-matched, normal control animals as previously reported 19 ) . This discrepancy may be explained by the different strains of rats used to produce the diabetic animals or by differences in the duration and/or the severity of the experimental diabetes. Since rats with STZ-induced diabetes were not treated with insulin, we intentionally used rats with a milder diabetic state to ensure survival of the animals to the end of this prolonged study. A positive correlation between cAMP content and MNCV in the sciatic nerve was also observed. Whether the association of a reduced cAMP content with decreased MNCV implies the involvement of cAMP in motor neuron function is unknown at the present time. Reduced cAMP levels could modulate the function of motor neurons and precipitate the neuropathic condition. Clear mechanisms by which cAMP enhances MNCV are unknown, but it is possible that an increased cAMP content could affect cAMP-dependent protein kinase activity, followed by an enhancement of Na + ,K + -ATPase activity in the sciatic nerve. It has been reported that protein kinase C agonists normalized Na + ,K + -ATPase activity in the diabetic rabbit nerve 27 , 30 ) and cAMP stimulates protein kinase C activity in cultured renal cells 31 ) . In addition to various metabolic factors, reduced nerve blood flow, caused by rheological changes coupled with vasa nervorsum microangiopathy, leads to endoneural hypoxia of sufficient magnitude to impair nerve function 3 , 20 ) . Some experimental studies have reported that hyperglycemia-induced blood flow reduction and the resultant endoneural hypoxia were important factors underlying nerve conduction deficits early in the development of diabetic neuropathy 3 ) . The alteration of nerve blood flow, accompanied by nerve ischemia in diabetic rats, may be related to functional abnormalities 3 ) and the reduction of Na + ,K + -ATPase activity observed 32 ) . Uehara et al 33 ) reported that the anti-platelet effects of cilostazol improved nerve blood flow, increased mean myelinated fiber size and enlarged the lumen of endoneural microvessels in diabetic rats. In our study, STZ reduced myelinated fiber size and there appeared to be more small fibers, thickening of capillary walls and duplication of basement membranes in the endoneural microvessels of sciatic nerves when compared to those of the non-diabetic, control rats. The myelinated fiber density, size distribution and vascular abnormalities were restored after treatment of cilostazol. It is possible that correction of nerve ischemia by cilostazol may result in the prevention of structural changes also. Cilostazol is a potent phosphodiesterase inhibitor, which increases the cAMP content in the sciatic nerve of rats 19 ) . Our study revealed that cilostazol can elevate cAMP levels and prevent impairment of MNCV in the sciatic nerves of STZ-induced diabetic rats, functionally and morphologically. This suggests that there is a relationship between the decreased rate of nerve regeneration and the decreased cAMP contents, which may be a possible pathophysiological link in the peripheral motor nerves of diabetic rats. Cilostazol may be useful for the prevention of diabetic neuropathies from functional and structural aspects of its action. | Study | biomedical | en | 0.999999 |
10461424 | Hemophagocytic syndrome (HS) is a systemic disease characterized by fever, pancytopenia, hepatosplenomegaly, lymphadenopathy, coagulopathy and histologically proliferation of histiocytes in the lymphoreticular system 1 , 2 ) . It is usually associated with viral infection such as EBV, cytomegalovirus and adenovirus in the immunocompromised patients 3 , 4 ) . Recently, it has been reported that EBV-associated HS is frequently observed in lymphoma of T-cell lineage and shows fatal outcome 5 , 6 ) . It is difficult to distinguish HS from malignant histiocytosis (MH) and providing the suggestion of previously diagnosed MH may include HS in T-cell lymphoma. MH is a systemic malignancy derived from cells of the mononuclear phagocytic system and the essential diagnostic features include disseminated and progressive proliferation of morphologically atypical histiocytes and presence of phagocytosis by these neoplastic cells. HS has the similar clinical manifestations, but the cytologic atypia in hemophagocytic cells is not present 7 ) . Nasal angiocentric lymphoma(AL) has been classified as a peripheral T-cell lymphoma, but recently classified as nasal or nasal type T/NK cell lymphoma and highly infected with EBV 8 , 9 ) . MH-like HS does not infrequently occur in nasal AL and usually has a rapidly fatal course 8 ) . We recently experienced a patient with nasal AL in whom, as a terminal event, a syndrome mimicking MH developed. In this study, we retrospectively analyzed AL with HS which had initially been suspected of having MH to assess the clinical significances and the pathogenetic association with EBV, and that would help to change our concept of MH and distinguish HS from MH. From 1987 to 1996, a total of 42 patients admitted to Catholic Cancer Center were established to have nasal AL. Ten of 42 patients showed HS during the clinical course of AL. The diagnosis of HS was based on a combination of the following clinicopathologic features: ① fever and splenomegaly, ② cytopenia of at least two hematopoietic series, and ③ over 2% of hemophagocytic histiocytes in the bone marrow 1 , 10 ) . All patients underwent computed tomography of PNS and abdomen, plain chest X-ray and bone marrow examinations; they were staged according to the Ann-Arbor system 11 ) . The clinical and laboratory records were reviewed retrospectively. The histopathologic diagnosis of AL was done by examination of paraffin-embedded tumor section with hemotoxylin-eosin stain. For immunophenotypical study, sections of the paraffin-embedded blocks were cut at 6 μm thickness and stained with specific monoclonal antibodies by the avidin-biotin complex (ABC) peroxidase methods described previously 12 ) . Mouse anti-human T-cell(CD45RO)(DAKO® Carpinteria, CA) and Mouse anti-human B-cell(IgG2a)(DAKO® Carpinteria, CA) were used for immunophenotyping of T and B lineage, respectively. Serologic tests of IgG and IgM Abs against EBV-viral capsid antigen (VCA), early antigen (EA) and Abs against EBV nuclear antigens (EBNA) were performed in three available patients using the indirect immunofluorescence methods as previously described 13 ) . The EBV RNA in situ hybridization studies were performed in 10 patients using biotinylated 26bp oligonucleotides (Research Genetics) complementary to the most abundant early RNA sequence in EBV infections. 6 μm sections cut from paraffin block, placed on organosilane pretreated glass slides, were deparaffinized, dehydrated, predigested with pepsin solution and hybridized for 30 min at a concentration of 0.5 μg/ml of probe. After washing and blocking of endogenous peroxidase, detection was accomplished using streptavidin alkaline-phosphatase conjugate followed by development of signal with stable fast red TR/stable naphthol phosphate(Research Genetics) and counterstaining with hemotoxylin. A red color within the nucleus over background levels was considered as a positive reaction. A known EBV-positive neoplasm was a positive control and an EBV-negative lymphoid tissue was a negative control in each run. The duration of overall survival was from the date of diagnosis of AL to the date of death or last follow-up. Survival duration from the diagnosis of HS was from the date of diagnosis to the date of death. The basic clinical features and scheme of all patients were summarized in Table 1 . There were 6 male and 4 female patients with median age of 43 years (range 23–63 years). The initial clinical stage of lymphoma were stage I (2 cases), II (3 cases), III (3 cases) and IV (2 cases). Five patients had HS as initial manifestation, three at the time of relapse of lymphoma, and two during the clinical remission of lymphoma. Four patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy and six had only supportive care. All cases showed rapidly fatal outcome and there was no significant difference depending on the methods of management. The median survival time from the diagnosis of HS of all patients, supportively cared patients and patients with CHOP, was 18 days (range 2–44 days), 18 days (range 2–27 days), and 19.5 days (range 14–44 days), respectively. The estimated median survival from the date of original diagnosis of AL was 4.1 months (range 2 days–36.5 months). Polymorphic cellular composition was observed in six cases and the other 4 cases had relatively monomorphic composition. Angiocentricity was observed in 4 cases, and necrosis was in all cases. Immunophenotypical studies showed that all cases expressed T-cell phenotype. Representative histologic pictures of lymphoma and bone marrow of HS are shown in Fig. 1 , 2 , and 4 , 5 , respectively. The serological tests in three available patients were as follows; positive anti-EBNA Ab, positive anti-VCA IgG Ab(titer over 1:640), and positive anti-EA IgG Ab(1:320), which indicated the active infection of EBV. In all cases, the EBV transcripts were seen in the nudei of the atypical cells by in situ EBER hybridization . We recently experienced a patient with MH-like HS in nasal AL as a terminal event. HS has the similar clinical manifestations of MH and is difficult to distinguish from MH. Recently reassessment of patients previously diagnosed as having MH revealed that most cases were lymphoma of T-cell lineage or other, such as a virus-associated HS 14 ) . Retrospective analysis in our center revealed that 11 of a total 16 patients , with MH previously diagnosed, were MH-like HS. Nine of 11 patients, initially suspected as MH, were caused by nasal AL(data not shown). Nasal AL which has the histologic feature of angiocentricity has been classified as a peripheral T-cell lymphoma but, recently, it is proving that it may include true natural killer (NK) cell lineage 8 , 9 ) . Although angiocentricity is common to these tumors, it is not universally seen and the term AL has proved confusing. Therefore, it is proposed that the term ‘nasal or nasal type T/NK cell lymphoma’ should replace AL as a choice 8 ) . In this study, we only used pan T-cell marker(CD45RO) to distinguish from B-cell and all showed T-cell phenotype and it did not perform NK-cell phenotying due to lack of fresh specimens. More phenotypical studies including immunophenotyping and T-cell receptor gene rearrangement would be needed to distinguish the true T-cell lymphoma from NK-cell lymphoma. In this histologic study, necrosis and cellular polymorphism were a more common feature than angiocenricity and monomorphism, respectively. HS was originally described in immunocompromised patients with viral infection 3 ) . Among various infectious agents, herpes virus, especially EBV, has been frequently implicated in the pathogenesis of VAHS 3 , 15 ) . EBV is a ubiquitous Herpes virus with tropism for B-lymphocytes and oropharymgeal epithelium and has a strong association with endemic Burkitt’s lymphoma, B-cell lymphoproliferative lesions in immunocompromised patients and nasopharyngeal carcinoma 13 , 16 ) . But recently, it appears that EBV has also been linked to about 40% of peripheral T-cell lymphoma and Hodgkin’s disease 17 – 19 ) . EBV is also more regularly detected in more than 80 % of nasal AL and, in these tumors, virtually all tumor cells harbour the virus. In this study, the presence of EBV was detected in all cases, particularly in most of the atypical lymphoid cells by the in situ hybridization, and the serologic test in three case indicated active infection. Although a causal relationship between EBV and AL is still undefined, the characteristic clinicopathologic features strongly suggest that EBV may contribute to the lymphomagenesis and the biologic features of AL and in situ hybridization and serologic study would be helpful for diagnosis and prediction of AL with HS 8 , 13 ) The mechanism of HS in AL is not fully understood yet, but it is thought to be caused by the cytokines, especially interferon-γ, tumor necrosis factor and interleukin-1 released from EBV infected lymphocytes 20 , 21 ) . Further evaluation of the EBV-viral oncogenes and the microenvironment in HS would be needed to define the pathogenesis and the role of EBV in these tumors. HS contributes to the high mortality of AL and usually has rapidly fatal outcome 6 , 22 ) . It is difficult to predict HS in the course of nasal AL, but symptomatic recurrence and the histologic progression of the primary nasal lesion may be candidates. So, the repeated biopsy of the nasal lesion in symptomaticaly recurrent patients would be essential. In this study, most HS was developed in the active disease-status, but also even in the clinical remission of two cases. We could not confirm the postmortem pathologic staging of all cases due to lack of the autopsy-specimen. However, Jaffe et al. 7 ) had reported that malignant lymphoma with erythrophagocytosis simulating MH had prevalence of lymphoma-involvements in lymph nodes, spleen, liver, lung, skin and kidney and hepatosplenomegaly was a common feature in all cases of HS, even in the patients with clinical remission. Therefore, pathologic staging, including laparoscopic biopsy, would be needed for the exact staging of lymphoma with MH-like HS. There is no effective treatment for HS and, in literatural review, the combination of high-dose intravenous immunoglobulin and etoposide or high dose steroid were effective in some cases of HS 2 , 6 , 23 ) . Smith et al. 24 ) described a young patient successfully treated with CHOP chemotherapy in the early phase of the clinical course. But, in this study, all patients showed as rapidly progressing into the fulminant course despite CHOP chemotherapy or palliative steroid pulse therapy. It seems that more aggressive treatment is needed in this condition and further investigation of the pathogenesis and biology of AL with HS should be pursued in order to improve the prognosis. | Study | biomedical | en | 0.999995 |
10461425 | Hantavirus and related viruses are the causative agents of Hemorrhagic Fever with Renal Syndrome and Hantavirus Pulmonary Syndrome (HFRS/HPS) 1 – 3 ) . Despite extensive studies on etiologic virus and epidemiology, there is no consensus on the pathogenesis of HFRS and HPS. In HFRS/HPS, the major manifestations depend on the vascular change. The change might be the result of the infection of endothelial cells with Hantaan virus, but it is not known to what extent changes in endothelial function are from virus infection or from immune response to virus infection 4 , 5 ) . Clinically, there is a varying degree of disseminated intravascular coagulation (DIC) which was evident in the early phase of the illness. It is believed, also, that DIC would be the consequence, at least in part, of functional changes of endothelium resulting in clinical syndrome 6 ) . We had reported the possible role of the endotheial cells in the development of HFRS 7 – 9 ) . In inflammatory responses, the expression of various adhesion molecules on the endothelial cells is important. After the initial tethering on the endothelial surface, ICAM-1 expression is believed to be critical in the recruitment and trafficking of mononuclear cells into inflamed lesion through it 10 , 11 ) . The pathology of HFRS showed a variable degree of inflammatory cell infiltration in various tissues 12 , 13 ) We believed that the vascular endothelial cells would express various kinds of adhesion molecules when they got infected with Hantaan virus and these molecules would play a key role in the establishment of inflammatory lesions in HFRS. This study investigated the role of adhesion molecule in pathogenesis of Hantaan virus related disease. The expression of ICAM-1 antigen on the cell membrane of human umbilical vein endothelial cells (HUVECs) was assessed by immunohistochemistry, and ICAM-1 mRNA in the endothelial cells was assessed by in situ hybridization after Hantaan virus infection. The materials used in this study were come from the following companies. Gelatin, DEPC, PBS, 20 × SSC, formamide, paraformaldehyde and HBSS from Sigma (St. Louis, MO, (I.S.A.); FBS and EGM (Endothelial cell growth medium) with 2 % FBS and supplements from Clonetics (San Diego, Ca, U.S.A.); TNF- α from Biosource (Camarillo, CA, U.S.A.); ICAM-1 monoclonal antibody, In Situ Hybridization Work station and BCIP substrate kit from R&D (Minneapolis, MN, U.S.A.); Anti-mouse alkaline phosphatase immunohistochemistry kit and Fast Red substrate kit from Dako (CA, U.S.A.); Crystal Mount from Biomeda (Foster City, CA, U.S.A.). Plasticwares including Thermanox® and glasswares including Superfrost® were purchased from Fisher Scientific (Atlanta, GA, U.S.A.). HUVECs were used in this study. HUVECs had been harvested from a single donor, confirmed by their characteristic expression of vWF Ag and stored after initial propagation in liquid nitrogen until use. The cells were propagated again on the 0.2 % gelatin coated surface with EGM with 15 % FBS. Growth media was fed every 3 days until they reached confluence. For the immunohistochemistry, the cells were transferred to the 0.2 % gelatin coated Thermanox® in the 24-well plates. For the in situ hybridization, cells were propagated on the 0.2 % gelatin coated 6-well plate. Prototype of Hantaan virus from (Department of Microbiology, College of Medicine, Chung-Ang University, Seoul, Korea) was infected on the HUVECs. Virus was propagated on the Vero E-6 cells for 13 days. After clearing of culture supernatant by centrifugation, virus solution was stored in liquid nitrogen until use. The infectivity of the virus solution was assessed by classic plaque assay. Virus was adsorbed with the dose of 0.2 – 0.5 PFU/HUVECs for 60 min. after washing twice with HBSS. After removal of remained virus solution, cells were fed with maintenance media (growth media with 5 % FBS) and maintained for 3 hours, 6 hours, 12 hours, 1 day, 2 day and 3 days, respectively. The virus solution irradiated for 5 × 10 5 rad was treated in same way to prepare the negative control, and TNF- α was added into media in the concentration of 5 ng/mL for the positive control. The cells were fixed at once with cold 4 % paraformaldehyde in PBS for 20 min. Before the fixation, the cells on the Thermanox® were washed twice with HBSS and fixed with an ample amount of fixative. The Thermanox® were dehydrated in graded alcohol and stored in −70°C until use. For the immunohistochemistry, cells were rehydrated in graded alcohol and kept in PBS for 10 min. For immunohistochemistry, anti-ICAM-1 monoclonal antibody was reacted in 1:3,000 dilution with 5 % skim milk in PBS for 16 hours at 4′C. After a wash with PBS − 0.2 % Tween 20 twice, 300 uL of link antibody was applied for 30 min. at room temperature. After a wash with PBS-Tween 20 twice again, 300 uL of alkaline phosphatase conjugate was applied again and reacted for 30 min. at 37°C. For the coloring reaction, Fast Red substrate was applied for 5 to 15 min. along the intensity of color reaction. Each Thermanox® was fixed on the glass slide and mounted with Crystal Mount after the counter-staining with Meyer’s hematoxylin. For in situ hybridization, the cells were trypsinized from the 6-well plates and washed with RNAse free PBS twice. Cells were fixed for 20 min. with cold 4 % paraformaldehyde and cells were washed with RNAse free 3 × PBS once and 1 × PBS twice. Cells were suspended in appropriate volume of RNAse free PBS and smeared on the Superfrost® slides. After drying for an hour, slides were treated with 2 × SSC and DEPC water briefly. The cells were reacted with 300 uL of prehybridization/hybridization solution for 60 min. at 37°C. Remaining solution was removed and cells were reacted with 300 uL of digoxigenin labeled ICAM-1 probe cocktail in the concentration of 200 ng/mL for 16 hours at 42′C. After serial wash with 4 × SSC/30 % formamide, 4 × SSC/30 % formamide and 0.2 × SSC/30 % formamide twice, respectively, slides were washed with modified TBS (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM MgCl2, 0.1 % BSA and 0.1 % Triton). Slides were reacted with sheep anti-digoxigenin/alkaline phosphatase conjugate for 30 min. at room temperature in the dilution of 1:600. After a wash with modified TBS twice, 300 uL of revealing agent (substrate 5-bromo-4-chloro-3-indolylphosphate (BCIP) was overlayed and reacted for 8 hours at 37°C. Slides were mounted with Crystal Mount after a brief rinse with distilled water and countersigned with 0.1 % Malachite Green. To improve the preparation, the above procedure was repeated on the cells grown on the Thermanox® 12 hours after Hantaan virus inoculation, 6 hours after TNF-stimulation in the concentration of 200 ng/mL and 12 hours after radiation sterilized Hantaan virus inoculation, respectively. By classic plaque assay, the infectivity of our virus solution was calculated as the following equation and it was 2.6 × 104 PFU/mL. We could count plaques clearly when we had overlaid Neutral Red-Noble Agar on day 13 and waited for 2 days. We could not find any positive cells when we had inoculated radiation sterilized virus solution. With Hantaan virus inoculation, we could find a small number of scattered positive cells throughout the cell layer after 12 hours. After 24 hours, the positive islands against Hantaan antigen increased in number and started to distribute all over the monolayer . Three days after inoculation, the positive cells increased markedly in their number and distribution . On day 7, almost all cells were demonstrated to have Hantaan antigen by immunohistochemistry. Hantaan virus inoculation expressed ICAM-1 antigen after 6 hours and ICAM-1 expression increased with time. ICAM-1 antigen expressed high between 12 hours and 24 hours of postinoculation. The percentage of positive cells is between 5 % and 10 %. After day 1, the expression decreased abruptly and was barely seen in the day 4 specimen. As a positive and negative control, we stimulated HUVECs with TNF- with the concentration of 200 ng/mL for 6 hours. Irradiated sterilized Hantaan virus solution was on HUVECs and referred to as a negative control. When Hantaan virus was inoculated, we could observe positive reaction after 4 hours that increased in intensity and extent along the course. The positive reaction was evident between day 1 and day 2 and tended to decrease . We used TNF- α stimulated HUVEC for 8 hours in the concentration of 200 ng/mL as positive control . As negative control, we employed radiation sterilized Hantaan virus and did hybridization 12 hours later. In the cells grown on the Thermanox®, the expression of mRNA of ICAM-1 was evident in comparison to that of radiation sterilized virus infected cells and the number of positive cells was comparable to that of TNF- α stimulated cells. Hantavirus and related viruses are the causative agents of HFRS and HPS. Despite extensive clinical-epidemiological and virologic studies, there is no consensus on the pathogenesis of HFRS and HPS. The agent designated Hantaan virus is a single stranded RNA virus belonging to the family Bunyaviridae, genus Hantavirus. The genus consists of at least five species; Hantaan virus (Korean Hemorrhagic Fever), Seoul virus, Puumala virus (Nephropathia Epidemica), Porogia virus (Balkan HFRS) and Prospect Hill virus 14 , 15 ) In the People’s Republic of China, over 100,000 cases of HFRS are reported annually and the incidence is increasing. Hundreds of cases of nephropathia epidemica have occurred annually in Finland and other Scandinavian countries since the 1930’s. Antibody studies indicate worldwide distribution. Antibodies in human sera have been found in South and North America (including Hawaii and Alaska), Southeast Asia and Africa. Furthermore, laboratory acquired cases have been reported in Russia, Korea, Japan and Europe, with rats implicated in Korea, Japan and the United Kingdom 15 , 16 ) . The mortality rates are between 3 to 32 percent in the former Soviet Union and between 7 and 15 percent in China. During the outbreak in the New Mexico-Arizona-Colorado region, two-thirds of the cases were fatal. Between April 1951 and December 1976, the overall case fatality ratio in Korea was 6.6 percent 15 ) . Although it is not clear how the clinical syndrome, including fever, shock, bleeding tendency, renal failure and hypopituitarism happens in HFRS, it is believed that the basic functional disturbances depend on the vascular changes. The vascular changes in HFRS may be a result of the infection of endothelial cells with Hantaan virus, but it is not known to what extent changes in endothelial function are from virus infection or from immune response to virus infection. There is no doubt that there is variable degree of DIC in the early phase of HFRS, Clinically, DIC was evident in all patients who visited the hospital 5 days from the onset of the illness 17 , 18 ) . DIC is believed to be a consequence, at least in part, of functional changes of endothelium resulting in kinin activation and clinical syndrome 6 , 9 ) . The most plausible initiating mechanism of DIC is the exposure of pro-coagulant activity from the endothelial cells by the infection of the virus or from the endothelium damaged with immune complex or platelet-anti-platelet antibody complex. We had reported the possible role of the endothelial cells in the development of HFRS. We were able to localize viral antigens in the endothelial cells and demonstrated the changes of anti-coagulant characteristics of endothelial cell membrane after Hantaan virus infection 7 – 9 ) Pathology also showed edema and destruction and other evidence of injury on the endothelial cells of small vessels in HFRS 12 ) . Hantaan antigen also had been found in the endothelial cells of brain, lung, heart, liver and kidney of autopsy cases 9 , 13 ) . The expression of various adhesion molecules on the endothelial cells are important in the initiation of inflammatory reaction and its propagation into adjacent tissues. For the establishment of inflammation in specific tissue, there should be tropism between circulating inflammatory cells and vessels in those tissues. It should help inflammatory cells to be localized in a specific area. ICAM-1 expression is critical in the recruitment and trafficking of mononuclear cells into inflamed lesion through it 11 ) . The pathology of HFRS showed a variable degree of inflammatory cell infiltration in various tissues. The kidney is infiltrated heavily with mononuclear cells along with the destruction of tubular epithelial cells and medullary interstitial hemorrhage and varying degrees of vascular changes 12 , 13 ) . We believed that the vascular endothelial cells would express various kinds of adhesion molecules when they got infected with Hantaan virus, and these molecules would play a key role in the establishment of inflammatory lesions in HFRS. Three members of the immunoglobulin gene family are expressed on endothelial cells and are involved in leukocyte emigration and activation, ICAM-1, ICAM-2, and VCAM-1, with a fourth member, ICAM-3, expressed on leukocytes. ICAM-1, ICAM-2 and ICAM-3 account for all known binding of LFA-1 to cells. The differences in tissue distribution of the three LFA-1 ligands suggest that it is unlikely their functions completely overlap. Binding affinity differs among the different ICAM-species, with ICAM-2 and ICAM-2 binding to LFA-1 expressing cells more weakly than ICAM-1. Expression of ICAM-1 was found to be markedly elevated in response to interleukin 1 (IL-1), tumor necrosis factor (TNF), interferon- γ (IFN-r) and bacterial lipopolysaccharide (LPS) in a wide variety of cells, including endothelial cells. ICAM-1 and/or ICAM-2 is thought to be responsible for the basal binding of lymphocytes to unstimulated HUVECs, indicated by the ability of MoAb against CD18 to inhibit this binding almost completely 19 ) . In this study, the changes of ICAM-1 were investigated on the Hantaan virus infected HUVECs by immunohistochemistry and in situ hybridization. ICAM-1 expression was evident in the early hours of infection on HUVECs. The number of ICAM-1 positive cells increased with time during the 12 or 24 hours after infection. Although there was some variation in the proportion of positive cells, 5 to 10 % of HUVECs were positive after 12-24 hours. The number of positive cells exceeded by far in comparison to gamma irradiated virus. They had been employed as negative control. They distributed in patch and the number of ICAM-1 positive cells increased and reached the peak after 12–24 hours of post-inoculation. As would be expected by the broad tissue distribution of ICAM-1, these protein play a more general role in the immune response than just emigration of leukocytes out of the vasculature. ICAM-1 plays an accessory, antigen independent role in these adhesive interactions. One such accessory role in which ICAM-1 functions is to enhance antigen dependent T cell activation. The presence of ICAM-1 on the surface of the antigen presenting cell results in a shift in antigen dose response curves, such that the T cell becomes activated by 10 to 100-fold lower concentrations of antigen 20 ) . ICAM-1 may also provide coactivation signals for bacterial superantigen activation of T lymphocytes 21 ) . Other activities which may be involved include T cell dependent B cell activation, cytotoxic T lymphocyte mediated killing of target cells, natural killer cell mediated killing of target cells, neutrophil induced oxidative damage of target cell and T lymphocyte development. VCAM-1 was known to elicit a co-stimulatory signal to resting T cells. When Hantaan virus infected endothelial cells up-regulated the expression of ICAM-1 and, possibly, other adhesion molecules, they could recruit the circulating mononuclear cells and platelets. Endothelium and attached mononuclear cells and platelets could provide a vast area of phospholipid surface and it could lead into the activation of a coagulation system and DIC. Recruiting mononuclear cells from the circulation would express tissue factor activity along with the infection of the endothelium, and it would contribute to the establishment of DIC. It deserves further study. This study has a few pitfalls. We did not pursue any dynamic change. In vivo, stimulated endothelium can express adhesion molecules that increase the adhesion of monocytes and results in the production of IL-1, TNF and TF. TNF- α increases the expression of adhesion molecules and recruits mononuclear cells upon the endothelial cells. The recruited cells are able to express tissue factor and they can amplify the coagulation process, too. They may secure the concentration of cytokine high in the lesion and can influence mononuclear cell adhesion in various ways and induce pro-coagulant activity. We did not study the kinetics of various cytokines and their effects on the HUVECs infected with Hantaan virus. It may establish the local effect and response of HUVECs. In conclusion, the human umbilical vein cells infected with Hantaan virus express ICAM-1 for circulating inflammatory mononuclear cells, and it may contribute organ specific inflammatory processes, including kidney, heart and CNS in HFRS. | Study | biomedical | en | 0.999998 |
10461426 | Helicobacter pylori is now recognized as the major pathogenic factor for the development of chronic gastritis type B, peptic ulcer, and is strongly associated with gastric adenocarcinoma and lymphoma. H. pylori infection is a worldwide problem and more than 50% of the world’s population were infected with H. pylori . The prevalence of H. pylori infection in Korea was more than 70% of the population and virulence factors such as cagA or vacA of the H. pylori isolated from Korean adults revealed high levels compared with those from western countries 1 ) . To understand the pathogenesis of H. pylori infection and to develop novel therapies and vaccines, an adequate animal model to reproduce the various aspects of H. pylori disease is required. Early attempts to colonize rodents with H. pylori were unsuccessful 2 , 3 ) . The first models of H. pylori infection were large animals such as gnotobiotic piglets 4 ) , monkeys 5 ) and mice which do not express normal immune systems like euthymic germ-free mice 6 ) and athymic nude mice 7 ) . These animal models can not be used easily to study immune response or to develop vaccines against H. pylori infection because they are more expense and difficulty for handling than small-sized animals, such as mice. H. felis or H. mustalae , which are different from H. pylori , have been used to infect mice 8 ) or ferrets 9 ) , respectively. However, these animal models do not mimic H. pylori infection in man and subsequent pathologic features because those Helicobacter species do not have VacA and other virulence factors required for the induction of inflammation and ulcers 10 ) . Recently, there has been some success in the development of a mouse model using human strains of H. pylori 11 , 12 ) . Unfortunately, there has not been any report about the mouse model infected with H. pylori in Korea. Considering the geographic differences in the prevalence of virulence factors such as cagA or vacA of H. pylori isolated from Korean adults, compared with those from western countries 1 , 13 ) , the establishment of a mouse model infected with H. pylori isolated from Korean adults is needed to investigate the pathogenesis of H. pylori infection and to develop the vaccines. The present study describes the first attempt to establish a mouse model by direct inoculation of fresh H. pylori isolates to specific pathogen-free BALB/c mice without long-term adaptation. H. pylori was isolated from the antral biopsy specimens of patients with duodenal ulcer at Seoul National University Hospital. Biopsy specimens were placed in Brucella broth (Difco Laboratories, Detroit, MI, USA) immediately and homogenized with a tissue grinder. The homogenate was then inoculated on selective agar [GC medium base (Difco Laboratories). 0.024% yeast extract, 1% hemoglobin, 1% IsoVitaleX, 5 mg/L vancomycin, 1 mg/L mycostatin, 5% sheep blood]. The plates were incubated for 5–7 days at 37°C under microaerophilic conditions (5% O 2 , 10% CO 2 and 85% N 2 ) in a CO 2 incubator . Pure culture isolates were examined by Gram stain and biochemical assay such as urease test 14 ) . Isolates were finally confirmed as members of the genus Helicobacter by a polymerase chain reaction (PCR) as described below. For a long-term storage, isolates were stored in Brucella broth containing 15% (v/v) glycerol and kept at −70°C. DNA was extracted from the H. pylori isolates with proteinase K, sodium dodecyl sulfate and hexadecyltrimethyl ammonium bromide (Sigma, St. Louis, MO, USA). The cell lysate was extracted in sequential steps with equal volumes of phenol, phenol/chloroform/isoamylalcohol (25:24:1) and chloroform. DNA was then precipitated with isopropanol. The DNA pellet was washed with 70% ethanol, dried and resuspended in sterile Tris-EDTA buffer (pH 8.0). Total RNA of H. pylori was extracted by the acid guanidinium thiocyanate-phenol-chloroform method 15 ) (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0; 0.4% sarcosyl, 0.1 M 2-mercaptoethanol). The RNA was dissolved in diethyl pyrocarbonate-treated distilled water, and quantitated at absorbance of 260 nm. One μ g of each RNA of H. pylori isolated from human and mouse was used for single-strand cDNA synthesis with oligo(dT) 15 primer (Promega, Madison, WI, USA) and Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD, USA) as described previously 16 ) . A number of PCRs were performed as described previously 17 ) to characterize the H. pylori isolates obtained from humans or mice. These were Helicobacter -specific PCR, ureA , cagA and vacA PCRs and random amplified polymorphic DNA (RAPD) PCR 18 ) . Each PCR primer was designed on the basis of published sequences of H. pylori 1 , 16 , 19 , 20 ) as shown in Table 1 . The primer sequence used for RAPD PCR was based on the publication by Lee et al. 12 ) (5′-AACGCGCAAC-3′). Amplification of H. pylori genomic DNA sequences was carried out in a volume of 50 μ l containing PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.3)], 1.5 mM MgCl 2 , 200 M dNTP, 0.5 M primers, 2 U of Taq polymerase (Perkin-Elmer, Norwalk, CT, USA) and 100 ng of bacterial DNA or cDNA. Each reaction mixture was amplified for 33–39 cycles (shown in Table 2 ). In RAPD PCR, the MgCl 2 concentration was increased to 3 mmol/L and 20 pmol of a single primer was used. A sample omitting DNA or cDNA was included in every reaction as a negative control. Each PCR was performed with hot-start procedure 21 , 22 ) and the final extension was performed at 72°C for 10 min after the completion of the amplification cycles using a thermal cycler (Perkin Elmer). PCR products were separated in 2% NuSieve agarose gel (FMC Bioproducts, Rockland, ME, USA) and identified using ethidium bromide stains. Twenty-two, specific pathogen-free, 6-week-old female BALB/c mice were divided into two groups (4 of control and 18 of experiment). Three of frozen cagA +/ vacA + H. pylori strains (strain # 99, # 232 and # 234) and one of fresh clinical cagA +/ vacA + H. pylori isolates (strain # 7) were used in this study. Each H. pylori strain was cultured under microaerophilic conditions and was harvested in the sterile phosphate buffered saline (PBS, pH 7.4) and mixed by equal density. Mice were infected with the H. pylori mixture as described previously 23 ) . Briefly, all animals were fed on a commercial diet and given water ad libitum . Mice were fasted overnight except for water. Mice were inoculated intragastrically with 10 9 colony forming units (CFUs) of H. pylori mixture or PBS after treatment with 0.2 ml of 0.2 M NaHCO 3 intragastrically to neutralize gastric acidity. These procedures were repeated two or more times with a 2-day interval. Mice were sacrificed 1, 2, 4 or 6 weeks after the last inoculation. The stomach of each mouse was bisected longitudinally. A specimen for rapid urease test (CLO test, Delta West Pty Ltd, Western Australia) was obtained from one half of the stomach and the remains of it were used for gastric scrapings for H. pylori cultures. They were fixed in buffered 10% formalin, using standard procedures, embedded in paraffin, sectioned at 4 ml, and stained with hemotoxylin & eosin for histology and Warthin-Starry silver to assess the level of bacterial colonization 24 ) . The glandular mucosae of the body, antrum and pylorus were examined histologically for a variety of inflammatory responses, epithelial changes and the presence of H. pylori . ELISA was performed for the assessment of immune response in H. pylori -infected mice as described previously 25 , 26 ) with minor modification. Briefly, to obtain the antigen to coat the microtiter plate, colonies of H. pylori were sonicated for three 30 sec bursts with 30 sec resting periods in an MSE Soniprep 150, and ultracentrifuged at 100,000 × g for 60 min (Beckman TL-100, Palo Alto, CA, USA). The antigen was diluted in 0.1 M carbonate buffer (pH 9.6) to a final concentration of 10 μ g/ml. Microtitre plates were coated with 100 μ l/well of antigen solution and incubated overnight at 4°C, washed three times with PBS containing 0.05% Tween-20 and then blocked with 200 μ l/well of 1% bovine serum albumin in PBS/Tween-20 at room temperature for 2 hours. After washing the plates, 100 μ l of each serum sample diluted 1:75 in PBS were added to wells in duplicate and the plates were incubated at room temperature for 2 hours. The plates were then washed three times and incubated with 100 μ l/well of a goat anti-mouse IgG peroxidase conjugate (Pierce, Rockford, IL, USA) and diluted 1:700 in 1% BSA-PBS at room temperature for 2 hours. After three washings, 100 μ l/well of a substrate solution containing p-nitrophenyl phosphate in diethanolamine-MgCl 2 buffer was added to each well. The reaction was stopped with 50 μ l/well of 2 N H 2 SO 4 , and read the absorbance read using Wellscan microplate reader at OD450 nm (Dynatech MR 700, Alexandria, VA, USA). Specific pathogen-free BALB/c female mice were inoculated with H. pylori . Mice were sacrificed 1, 2, 4 and 6 weeks after the last inoculation. Gastric specimens from infected mice were processed for urease test and histologic evaluation. The positivity of CLO tests in gastric specimens obtained from mice sacrificed one and two weeks after the last inoculation was only 40% but 75% in those from 4 and 6 weeks. ( Table 3 ). As presented the above, the positivity of CLO test was gradually increased from 4 weeks after treatment with H. pylori , which indicates that the colonized H. pylori proliferated slowly and was located in the stomach. The CLO test in the control group was negative during the experimental period. In the first week after the last inoculation, no definite presence of bacteria and infiltration of inflammatory cells were shown in H. pylori -infected group . However, the inflammatory infiltrates of lymphocytes, plasma cells and polymorphonuclear leukocytes in the antrum and corpus mucosa were gradually increased from 2 weeks after the last inoculation of H. pylori . At six weeks after the last inoculation, the gastric lesions were characterized by definite inflammatory infiltrates accompanied by the disruption of gastric architecture and distinct H. pylori colonization . Control mice showed no evidence of significant infiltration of inflammatory cells during the entire experimental period . Isolates from infected mice showed typical morphology and biochemical characteristics of H. pylori as the same as the original H. pylori strain (data not shown). Furthermore, the Helicobacter -specific PCR showed that the mouse and human isolates were identical to H. pylori . The original human clinical isolates and the mouse isolates were also positive for cagA and vacA by PCR . A comparison of the genomic DNA by RAPD PCR was performed for the mouse isolates and four original human clinical isolates which were used to infect the mouse. The mouse isolates gave an identical band pattern to only one (strain # 7) of the four human isolates . These results suggest that strain # 7 may be the one that is able to colonize the mouse gastric mucosa among all the human isolates tested. The experimentally infected mice showed serum antibody response to the colonizing strain that could be detected by an ELISA of mice sera from the first week of infection. Anti- H. pylori IgG antibody levels increased markedly from the second week after the last inoculation with H. pylori and reached the plateau after the fourth week. In contrast, the negligible serum antibody response was shown in the control group during the entire experiment period . H. pylori has been unambiguously implicated in the etiology of chronic gastritis and the recurrence of peptic ulceration in humans. Despite the high prevalence of H. pylori infection, serious gastric diseases characterized by gastric or duodenal ulcers are noted in a relatively small fraction of the H. pylori -infected population: To understand how H. pylori infects and occasionally causes serious diseases, and to investigate the immunologic mechanisms of H. pylori infection more precisely, it is necessary to develop the animal model infected with H. pylori . In the present study, we successfully established an animal model by oral challenges with a fresh H. pylori isolate to BALB/c mice without a long-term adaptation. Based on the result of urease tests, more than a half of the challenged mice were presumed to be infected by H. pylori . A striking feature of infection was the increasing serum IgG response against H. pylori sonicate antigen; infected mice showed a systemic antibody response to the infected strain from the first week of infection. Serum IgG response was apparent in all mice by 6 weeks post-infection although the CLO positive was only 55% in all infected mice. We speculate that it may be due to a peculiar colonization pattern of H. pylori in gastric mucosa. H. pylori has acquired particular properties of colonization of the unique ecological niche on the surface of gastric epithelial cells and the distribution of H. pylori with associated inflammation are often patchy 27 , 28 ) . Such a patchy distribution of H. pylori colonization can lead to sampling error and subsequently, the false negative results of microscopic examination, culture and rapid urease test. Furthermore, the process of colonization with H. pylori in the gastric mucosa may take week 29 ) . The organism must enter the stomach, survive brief exposure to acid, traverse the mucous layer 30 ) , attach to epithelial cell receptors 31 ) , adapt its physiology to the hostile host environment and thereby establish its niche. In this study, only the bacteria of a negligible number were found in the gastric antrum. However, the colonization with numerous bacteria was observed in the body and fore-stomach transition zones, accompanied with the disruption of gastric gland architecture. With light microscopy, the bacteria were observed in large numbers in the mucus overlying the epithelial cells and at the top of the gastric pits. It is closely similar to the colonizing pattern of H. pylori in humans. The infiltration of inflammatory cells, such as lymphocytes and plasma cells, in the antrum and corpus mucosa was gradually increased from 2 weeks after the last inoculation of H. pylori . In contrast, the infiltration of polymorphonuclear leukocytes was not prominent in the lamina propria until 4 weeks post-infection. However, it became evident at 6 weeks, although with only a few numbers. In the present study, both H. pylori isolates from human (before inoculation to mouse) and mouse showed identical patterns of virulence factors such as vacA1 , vacA2 , vacA3 and cagA . By RAPD PCR, the mouse isolate exhibited an identical band pattern to only one (fresh isolate # 7) of the four human isolates. This result suggests that the fresh isolate plays an important role in the colonizing process compared to long-term stored strains. Consecutive inoculation of ICR mice with H. pylori recovered from the stomach of BALB/c mice also showed systemic antibody response to the colonizing strain (data not shown). The present study shows that a mouse model of H. pylori infection is successfully established. This model can be utilized for animal experiments of H. pylori , such as vaccine studies, screening for novel therapies, and investigation of the mechanisms of pathogenesis. Although some problems remain to be solved, such as to develop the strain with high colonizing ability and to examine a variety of mice strain, this mouse model will provide opportunities for studies on the interrelationship between bacteria and the host with respect to colonization and the ecology of bacteria in the stomach. It will also facilitate investigations of the mechanisms of H. pylori -associated diseases, including peptic ulcer, gastric cancer and gastric lymphoma. | Study | biomedical | en | 0.999995 |
10461427 | More than 60% of patients with liver cirrhosis suffer from malnutrition 1 ) , and this may adversely affect morbidity and mortality in liver cirrhosis 2 – 4 ) . Proper nutrition may reduce complications and increase survival rates 5 – 7 ) , though accurate, quantitative nutritional assessment is difficult in patients with liver cirrhosis; conventional markers, such as weight, serum albumin levels and peripheral lymphocyte counts, are influenced by non-nutritional factors 8 ) . Changes in one or more body components not only provide early detection of malnutrition but also show the pattern of specific tissue loss, and this might provide insight into the disease process and information regarding outcome and prognosis 9 , 10 ) . The methods used include anthropometric assessment, bioelectric impedance analysis, isotope dilution, the measurement of total body potassium, dual energy x-ray absorptiometry (DEXA) and neutron activation 11 ) . Neutron activation analysis, DEXA or deuterium oxide dilution accurately reflect changes in body composition associated with chronic liver disease 9 ) . Because of availability problems, cost and the amount of radiation involved, the clinical use of neutron activation analysis is limited 12 ) . DEXA measures three body components: fat mass, lean soft tissue mass (comprising muscle, inner organs and body water) and bone mineral content, and is a quick, simple method for body composition analysis. Its advantages are the relatively low radiation dose, excellent reproducibility and high precision 11 , 12 ) . In clinical practice, the most reliable method of nutritional assessment is anthropometric measurement. It is simple, and may be used as a bedside tool 13 ) , but its drawback is the potential of underestimation of fat loss in cirrhotic patients with subcutaneous fluid retention 9 ) . Measurements need to be validated by comparison with the results obtained by reference methods. In this study, anthropometric measurements and DEXA were chosen because the former are quick, and easily performed at the bedside, and the latter accurately reflects changes in body composition associated with chronic liver disease 11 ) . We analysed the changes of body composition of patients with liver cirrhosis and compared them with those for healthy control subjects. Body composition was determined using anthropometry for fat and fat-free mass and DEXA for body fat, lean soft tissue mass and bone mineral content. The anthropometric findings for lean and fat tissue mass in cirrhotic patients were compared with those for total fat mass and lean soft tissue mass measured by DEXA. Sixty-six patients (58 males, 8 females) with liver cirrhosis and 94 healthy controls (49 males, 45 females) were enrolled in this study. Their mean age was 51.8 (range 30–67) years and 52.9 (range 40–68) years, respectively. Control group consisted of volunteers who were considered healthy on the bases of history taking, physical examination and blood chemistries. The liver cirrhosis was diagnosed with liver function tests, abdominal CT or ultrasonography and clinical evidence of portal hypertension such as varices, splenomegaly or ascites. Patients with recent gastrointestinal hemorrhage, infection or malignancy were excluded. The severity of ascites was graded as mild or non-existent (detectable only by ultrasonography), moderate (obvious but less than tense) or severe (tense). In the cirrhotic group, ascites was mild or non-existent in 20 patients, moderate in 35 and severe in 11. In those with ascites or edema, all evaluation was made after disappearance of ascites and edema with medical treatment. Liver cirrhosis was virus-related in 44 cases and alcohol-related in 22. Child-Pugh scores showed that 25 were Child-class A, 24 were class B and 17 were class C. Anthropometry was performed in all cases, but DEXA was measured only in 37 cirrhotic patients and 39 controls who agreed with that test. The retrograde analyses of clinical parameters, such as age, body weight, body mass index and Child-classes detected no difference between the DEXA group and the non-DEXA group in both patient and control groups ( Table 1 ). Anthropometric measurements of mid-arm circumference (MAC) and triceps skinfold thickness (TSF) were obtained using a skinfold caliper (Fabrication Enterprises Incorporated, New York, USA) and a flexible steel tape. Measurements were taken midway between the tip of the acromion and olecranon process of the left arm, while the patient stood in a relaxed position 17 ) . Upper mid-arm muscle area (MAMA) and upper mid-arm fat area (MAFA) were calculated as 14 ) , MAMA ( cm 2 ) = ( MAC − TSF · π ) 2 / 4 MAFA ( cm 2 ) = upper arm area − MAMA = ( MAC ) 2 / 4 π − MAMA Using a total-body DEXA scanner (XR-26, MARK II), fat mass, lean soft tissue mass and bone mineral content were measured, and DEXA data and those of MAMA and MAFA measured by anthropometry, were compared between cirrhotic patients and healthy controls. Correlations between fat mass vs. MAFA and lean soft tissue mass vs. MAMA in patients and controls, which had been performed by anthropometry and DEXA, were evaluated using Pearson’s correlation coefficient. To evaluate the clinical parameters related to depleted body components in cirrhotic patients, the numbers of patients, whose MAFA values were lower than the fifth percentile of controls, were counted and correlations with Child-classification and the severity of ascites were analysed. A p value of less than 0.05 was considered significant. In cirrhotic patients of both sexes, fat mass and MAFA were lower than in sex-matched controls . The number of cirrhotic patients with measured values below the fifth percentile of normal controls was 21 (31.8%) on the basis of MAFA and 15 (40.5%) on the basis of fat mass Fat depletion was more severe in Child-class C patients and those with severe ascites . In cirrhotic patients and normal controls, lean soft tissue mass and MAMA were comparable . The number of patients with cirrhosis with measured values below the fifth percentile of normal controls was six (9.1%) according to MAMA and 0 (0%) according to lean body mass. Bone mineral content in cirrhotic patients and normal controls was comparable . In 37 cirrhotic patients and 39 controls, on which anthropometry and DEXA had been performed, MAFA and fat mass showed close correlation , whereas for MAMA and lean soft tissue mass, correlation was poor . This study, which investigated body compositional changes in patients with cirrhosis, demonstrated that there was a reduction in one or more body compositions in more than 40% of patients. It was very striking that the majority of patients showed predominant fat depletion despite maintaining their lean soft tissue mass and bone mineral content. Changes in body composition measured by anthropometry revealed as MAMA being impaired more severely than MAFA in males, whereas the opposite was true in females 13 , 15 ) . Crawford et al. reported that the majority of patients, who maintained body cell mass but had reduced body fat, were Child’s class A 10 ) . It is well known that body adipose deposits is the most important fuel source for patients with insufficient calorie intake 16 , 17 ) . A moderate nutritional disturbance affects only the fat component; in patients with liver cirrhosis, intestinal fat absorption may be abnormal 18 ) . There are some methodologic problems in the evaluation of body cell mass in patients with liver cirrhosis. For example, the usefulness of DEXA for determining body cell mass in liver cirrhosis is controversial 19 , 20 ) . Because of extracellular water retention, lean soft tissue mass may not represent body cell mass, even if there was no evidence of overt ascites or fluid retention 21 ) . One study 9 ) compared different methods for assessment of body composition in patients with liver cirrhosis, and found that in Child-class C patients, DEXA gave higher values for lean soft tissue mass than did neutron activation analysis. Anthropometry, furthermore, did not accurately reflect changes in fat-free mass. As shown by Bhatla et al. 22 ) in patients on continuous ambulatory peritoneal dialysis, DEXA included excess body water in lean soft tissue mass and, where there is overhydration, may mask malnutrition. Some authors, however, have recommended DEXA for body composition analysis in stable dialysis patients 23 , 24 ) or those with liver cirrhosis 25 ) after ascites has been controlled. Prijatmoko et al 9 ) . showed that DEXA can detect advanced protein depletion in patients with liver cirrhosis. The methods used in this study, DEXA and anthropometry, tend to overestimate fat-free mass. Changes in the hydration status of soft tissues can alter attenuation of the dual energy source, resulting in incorrect calculation of the amount of lean tissue. With regard to bone mineral content in cirrhosis, the findings differ. Prijatmoko et al 9 ) . showed that this was maintained, but other investigaors found that it was reduced 26 , 27 ) . MAMA and MAFA measured by anthropometry represent muscle mass and fat and, to validate this system for fat mass measurement, we compared MAFA with fat mass. As observed previously 9 , 28 , 29 ) , MAFA values correlated closely with those of fat mass, but those of MAMA correlated poorly with lean soft tissue mass. The nutritional status of cirrhotic patients with severe ascites and those who were Child-class C was more impaired, as reported by others 5 , 13 , 14 , 30 ) . Closer nutritional evaluation of patients with severe ascites and/or severe hepatic dysfuction is therefore required. In summary, we have shown that, in more than 40% of patients with liver cirrhosis, there were reductions in the amount of stored fat, and that these changes were more severe in Child-class C patients and those with severe ascites. Fat mass estimated according to MAFA correlated closely with fat mass measured by DEXA. In view of its easy bedside use, MAFA is recommeded for the measurement of fat mass in cirrhotic patients. | Review | biomedical | en | 0.999997 |
10461428 | In patients with sepsis and septic shock, the coagulation and fibrinolytic system are independently activated. Tissue factor released from endothelial cells which are stimulated by endotoxin and inflammatory mediators and the consumption of natural coagulation inhibitor antithrombin III, protein C and protein S activate the coagulation system 1 – 3 ) . Fibrinolysis is regulated by both activators and inhibitors released from endothelial cells 4 ) . Although the levels of plasminogen activator antigen are increased, fibrinolysis is dominated by increased levels of type 1 plasminogen activator inhibitor (PAI-1) 5 , 6 ) This imbalance between coagulation and fibrinolysis predisposes to the development of disseminated intravascular coagulation, fibrin deposition and microthrombi. Fibrin deposition and complement activation can cause extensive vessel wall damage and may be associated with multiple organ failure 2 , 3 ) . There have been several clinical studies concerning biochemical evidence of activation of coagulation and inhibition of fibrinolysis in sepsis 4 , 6 ) . Thrombin-antithrombin III(TAT) and plasmin- α 2 -antiplasmin(PAP) complexes are known as highly sensitive and specific markers of coagulation and the fibrinolytic system 7 , 8 ) . Recently, it has been reported that the imbalance between coagulation and fibrinolysis described as TAT/PAP ratio leads to mortality and organ dysfunction in patients with sepsis 9 , 10 ) . In this study, we investigated what kind of changes within the hemostatic system are related to the severity of illness and the prognosis in patients with sepsis. We prospectively studied 32 patients with sepsis (20 male, 12 female) admitted from August 1996 to July 1997. Age (mean ± SD) was 59.4 ± 17.8 years. The diagnosis of sepsis was according to the criteria described by the American College of Chest Physician/Society of Critical Care Medicine(ACCP/SCCM) Consensus Conference held in 1991 11 ) . The diagnosis of sepsis was made if two of the four following criteria were fulfilled: temperature > 38°C or < 36°C, respiratory rate > 20/min or PaCO 2 < 32mmHg, white blood cell count > 12,000/mm 3 , or < 4,000/mm 3 , or immature neutrophil > 10% with evidence of infection. The septic shock was defined as the sepsis-induced hypotension or vasopressor dependency and the presence of signs of hypoperfusion, such as lactic acidosis, altered mental status or oliguria. The origins of infection were pulmonary (22), urogenital (4), wound (4), and abdominal (2) foci. Bacteremia was noted in 9 cases (4 Gram-positive, 5 Gram-negative organisms), septic shock in 8 cases and acute respiratory distress syndrome in 5 cases. 12 of 32 patients expired mainly due to multiple organ failure. The patients with underlying cardiovascular disease, pregnancy and liver disease, as well as those on drugs affecting the hemostatic system, such as wafarin, were excluded. Control values were obtained from 20 healthy volunteers (M:5 F:15, Age: 32.4 ± 5.7 years) among the laboratory and technical personnel. The age of the control group was not matched with the patient group; this limitation may not significantly influence the result of this study because the fibrinolytic enzyme activity does not show much variation according to age, but is only slightly decreased in elderly 12 ) . The clinical status of the patients was assessed using the APACHE III scoring system 13 ) . APACHE III score was obtained within 24hrs after clinical determination of the sepsis. If more than 3 parameters were missing, the case was excluded. APACHE III score of the sepsis patients was 60.3±39.0(range: 9 – 176). Blood samples were collected within 24hrs after the clinical diagnosis of sepsis. Venous blood was collected in 5-ml tubes (Vacutainer, Becton-Dickinson, Plymouth, UK) containing 0.5ml sodium citrate(0.13 mol/L). Plasma was separated by centrifugation at 1,500 × g for 15 min at 4°C within 30 min, and stored at −70°C until assay. Plasma TAT and PAP were measured by commercially available test kits according to the instructions of the manufacturers. Plasma TAT was measured by a sandwich ELISA (Enzygnost-TAT, Behringwerke AG, Marburg, FRG) which uses polyclonal rabbit antibodies against human thrombin and peroxidase-conjugated antibodies to human antithrombin III. Plasma PAP was measured by a one-step sandwich ELISA (Enzygnost-PAP, Behringwerke AG, Marburg, FRG) using monoclonal antibodies (PAP-6) against human PAP and peroxidase-conjugated antibodies to plasminogen. Student’s t test and chi-square test were used for comparison of the mean values in the various groups. In addition, Pearson’s correlation coefficients were calculated for the different variables. All results with p values of less than 0.05 were considered as statistically significant. Plasma TAT and PAP in 32 patients with sepsis were 15.1±16.8ng/mL and 771.3±380.3ng/mL and the values in 20 controls were 2.8±1.1ng/mL and 240.4±69.7ng/mL . Although plasma TAT and PAP in patients with sepsis were significantly higher than those in controls, the TAT/PAP ratio (21.3±19.0, expressed as TAT/PAP × 1,000) in patients with sepsis was not significantly different from that (12.3±4.4) in 20 controls. Plasma TAT and PAP in 20 survivors were 11.1±11.2ng/mL and 857.1±364.1ng/mL and those in 12 nonsurvivors were 21.7±22.3ng/mL and 628.4±378.1 ng/mL. The TAT/PAP ratio (34.4±21.4) in nonsurvivors was significantly higher than that (14.4±13.8) in survivors . Plasma TAT and PAP in 8 patients with shock were 11.3±9.0ng/mL and 602.1±495.0ng/mL and those in 24 patients without shock were 16.3±18.7ng/mL and 827.7±327.3ng/mL. The TAT/PAP ratio (27.2±22.5) in patients with shock was not significantly different from that (20.1±18.5) in patients without shock. No significant differences in TAT, PAP and TAT/PAP ratio were shown according to the presence of causative organisms, bacteremia, acute respiratory distress syndrome or other organ failure (data not shown). APACHE III score (93.5±41.8) in nonsurvivors was significantly higher than those (38.8±17.6) in survivors. In contrast to plasma TAT and PAP, Plasma TAT/PAP ratio in patients with sepsis was closely correlated with APACHE III score(r=0.47, p=0.008) . Plasma TAT was significantly correlated with plasma PAP (r=0.44, p=0.001) and negatively correlated with fibrinogen (r=−0.43, p=0.017) . Dividing the patients with sepsis into two groups according to the d-dimer levels, Plasma TAT (20.2±21.7ng/mL) and PAP in patients with d-dimer level equal to or greater than 500ng/mL were significantly higher than plasma TAT (12.4±13.5ng/mL) and PAP(626.8±357.0ng/mL) in patients with d-dimer level less than 500ng/mL. Despite the advances in antibiotic therapy and cardiopulmonary support, sepsis with septic shock remains as a highly lethal condition In sepsis, endotoxin of Gram-negative bacteria and capsular mucopolysaccharides of Gram-positive bacteria trigger the inflammatory reaction and hemostatic cascades. Endothelial cells which are stimulated by endotoxin and inflammatory mediators, express tissue factor and factor XII, resulting in activation of the coagulation pathway and consumption of the natural coagulation inhibitors, antithrombin III, protein C and protein S, also activate the coagulation system 1 , 2 , 14 ) . Platelets, aggregating to the endothelial cells damaged by protease or complement, are actively involved in the coagulation pathway 4 , 15 ) . Fibrinolysis is regulated by both activators and inhibitors which are released from endothelial cells. Although the levels of plasminogen activators are increased following activation of coagulation, fibrinolysis is relatively impaired compared with the activity of coagulation, and increased levels of type I plasminogen activator inhibitor (PAI-1) is suggested to play this role. This imbalance between coagulation and fibrinolysis predisposes to the development of disseminated intravascular coagulation (DIC), fibrin deposition and microthrombi. Fibrin deposition and complement activation can cause extensive vessel wall damage and may be associated with multiple organ failure 6 , 16 , 17 , 18 ) . In previous reports, many hemostatic parameters, including prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, d-dimer, fibrin degradation product (FDP), plasminogen and plasminogen activator inhibitor (PAI), were studied in patients with sepsis and showed various activation patterns 3 , 4 ) . Thrombin and plasmin are rapidly neutralized and stabilized by antithrombin III and α 2 -antiplasmin to form TAT and PAP, respectively. So, TAT and PAP are known as stable, accurate and sensitive markers of coagluation and fibrinolysis system 7 , 8 ) . Compared with FDP, d-dimer and antithrombin III, they have been known to be more useful parameters in the evaluation of disseminated intravascular coagulation 7 , 8 , 16 ) . The balance of coagulation and fibrinolysis can be expressed as TAT/PAP ratio. Some variations of TAT/PAP ratio from the imbalance between coagulation and fibrinolysis have been shown according to the underlying diseases associated with disseminated intravascular coagulation. The TAT/PAP ratio was high in sepsis, intermediate in solid tumor and low in hematologic malignancy such as acute promyelocytic leukemia. High TAT/PAP ratio in patients with sepsis means excessive activation of coagulation and more vulnerability to microthrombi, microembolization and eventual multiple organ failure 16 , 17 ) . In previous studies, plasma TAT, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and PAI-1 in patients with sepsis were reported to be elevated 17 , 18 ) . Lorente et al. 9 ) reported that plasma TAT had a close relationship with prognosis, although plasma TAT shows no significant correlation with APACHE score or multiple organ failure score, and that higher PAI-1 contributed to poor outcome. Recently, Kidokoro et al. 10 ) reported that plasma TAT and PAP in patients with sepsis were elevated and high TAT/PAP ratio from the imbalance between coagulation and fibrinolysis led to the onset of organ dysfunction. Philippe et al. 6 ) reported that plasma PAI-1 levels in patients with sepsis, causing relative impairment of fibrinolysis, were closely related with the severity and outcome. In our study, both TAT and PAP in patients with sepsis were obviously elevated. It was shown that the high TAT/PAP ratio in patients with sepsis was closely related with APACHE III score and outcome although the relationship of TAT/PAP ratio with organ dysfunction was not clearly shown, which might be due to the small sample size. The suppression of the fibrinolytic system was more prominent in nonsurvivors than survivors. In conclusion, both coagulation and the fibrinolysis system were activated in patients with sepsis and the imbalance between coagulation and fibrinolysis is closely related with the severity and outcome of patients with sepsis. | Study | biomedical | en | 0.999998 |
10461429 | Sweet’s syndrome (SS) is an acute febrile neutrophilic dermatosis characterized by fever, leukocytosis, tender cutaneous plaques or nodules and dense infiltration by neutrophils. Some cases have been reported as an association with malignant neoplasms 1 ) and autoimmune diseases, e.g., Behçet’s disease 2 ) , Sjogren’s syndrome 3 ) and rheumatoid arthritis 4 ) . Dermatomyositis (DM) is an inflammatory muscular diseases of unknown cause. Many lines of evidence suggest that both cellular and humoral mechanisms play a role in the pathogenesis of dermatomyositis 5 , 6 ) . But dermatomyositis, one of the rare autoimmune diseases, was not reported as an associated disease of SS. We describe an interesting case of SS associated with DM. Diagnosis was made by skin biopsy, and subsequent clinical resolution occurred after institution of prednisolone. It is necessary to be concerned that in patients with several autoimmune diseases, including DM, SS is present as an associated finding. A 57-year-old man with known DM was admitted to our hospital due to an 8-day history of fever, chills and painful cutaneous lesions on neck, left arm and trunk. The fever did not respond to antibiotics and non-steroidal antiinflammatory drugs. He denied having any other recent illness or symptoms preceding this acute episode of symptoms. On admission, he appeared acutely ill and his body temperature was 39.7°C, blood pressure 120/70 mm Hg, pulse rate 100/min, respiratory rate 25/min. On physical examination, he was febrile, alert, and well-oriented. He exhibited numerous erythematous, indurated, slightly tender plaques ranging in size from 0.7 to 6 cm, located on the posterior neck, left arm and trunk. Vesicles and/or pustules were present on the surface of some plaques . There were no petechiae, purpura or ulcerated or necrotic area, and the lesions did not blanch with pressure. Oral cavity and ophthalmologic examination was done, and revealed no abnormalities. About 12 months ago, he was admitted to our hospital due to fever and proximal muscle weakness of both extremities, erythematous/violaceous rash over the eyelids and erythematous, scaling rash over the knuckles of both hands. Laboratory test revealed elevated muscle enzymes: AST 95 IU/I (normal value: NV 5-40 IU/I), ALT 112 IU/I (NV 5-35 IU/I), CK 259 IU/I (NV 32-187 IU/I), LDH 649 IU/I (NV 218-472 IU/I), aldolase 37.9 U/ml (NV 2.0-8.0 U/ml). Antinuclear antibody was present in a homogenous pattern in a titer of 1:80. The Westergren ESR and cold reactive protein (CRP) was elevated. The patient’s symptoms and laboratory results were suggestive of inflammatory myopathy and electromyographic evaluation (EMG) and muscle biopsy were done. EMG showed typical findings of idiopathic myopathy, irritability of myofibrils (fibrillation potentials) on needle insertion and, at rest and short duration, low amplitude, polyphasic potentials on contraction. Histologic findings of muscle biopsy showed an area of degeneration and necrosis of myofibers in association with interstitial lymphocytic and histiocytic cellular infiltration . Chest roentgenogram showed fine reticulonodular infiltration on both lower lungs and a biopsy of the lung revealed usual interstitial pneumonia. The upper gastrointestinal endoscopy and a barium enema examination for underlying neoplasm of DM were all normal. Because of the relation of the findings to the criteria of DM, he was diagnosed as DM. High dose of steroid (prednisolone 1 mg/kg/day) was started. Proximal muscle weakness was slowly improved and abnormal laboratory findings were normalized, so the dose of steroid tapered progressively. At a dose of prednisolone 30 mg/day, he was discharged and followed up. From 3 months ago, the daily prednisolone dose was tapered to 5 mg/day by himself. Recently, he stopped medication, and the above-mentioned skin lesion and fever developed. Laboratory investigations revealed an elevated white blood cell count of 28,200/mm 3 with 83% polymorphonuclear leukocytes, 3% bands, 8% lymphocytes, 3% monocytes and 3% eosinophils, a platelet count 441,000/mm 3 . The Westergren ESR was 100 mm/hr and cold reactive protein (CRP) 127 mg/l (NV: < 5 mg/l). The serum urea nitrogen, creatinine, amylase, bilirubin, uric acid and alkaline phosphatase and uric acid were all normal. The levels of muscle enzymes showed as follows: AST 56 IU/I, ALT 69 IU/I, CK 99 IU/I, LDH 349 IU/I, aldolase 17.9 U/ml. Antinuclear antibody was present in a titer of 1:40. Cryoglobulin, C3 and C4 complement levels, and hepatitis B surface antigen and antibody were all normal or negative. The urinalysis did not show any abnormal findings. Blood and urine cultures were negative. Histologic examination of a biopsy specimen of one of the cutaneous plaques revealed dermal edema and perivascular infiltration, consisting predominantly of neutrophils and some small number of lymphocytes . But blood vessels had not findings of vasculitis. So, he was diagnosed as Sweet’s syndrome and flare-up of dermatomyositis. Prednisolone 50mg/day was instituted and a dramatic improvement of the skin lesions without scarring appeared within 2 weeks. Muscular weakness slowly improved with the above dose of steroid, and prednisolone was tapered. He remains without new skin lesions, fever and other symptoms four months later, while receiving tapering doses of steroids. Sweet’s syndrome was first described in 1964 by Sweet 7 ) , and several reports appeared. It is characterized by fever, neutrophilic leukocytosis and painful, erythematous cutaneous plaque with dense dermal infiltration with neutrophils. The cause of SS is still unknown, but hypersensitivity reactions to bacterial, tumor, autoantigen were suggested as may being involved in the pathogenesis of SS 8 ) . SS has been noted as usually occuring in middle-aged women and often presents within two weeks of an antecedent respiratory traction infection 7 , 9 ) . Conditions underlying SS have included malignancies and autoimmune diseases. Malignant neoplasms have been reported in approximately 10–20 % of the reported cases of SS 8 ) . Myeloproliferative disorders, including acute myelogenous leukemia, chronic myelogenous leukemia and multiple myeloma are the most commonly associated malignancing 8 ) . In addition to association with malignant neoplasms, SS has been reported in autoimmune diseases, such as Behçet’s disease 2 ) , Sjogren’s syndrome 3 ) , rheumatoid arthritis 4 ) , Reiter’s syndrome 10 ) , subacute cutaneous lupus erythematosus 11 ) , drug-induced lupus erythematosus 12 ) , ANA-negative lupus-like syndrome 13 ) and undifferentiated connective tissue disease 14 ) . In a search of the literature, we did not discover any other cases of these two diseases occurring together. Our patient is the first case of SS associated with DM. The skin lesions in DM may be a malar-like rash of the face which involves the nasolabial area (an area often spared in systemic lupus erythematosus) and erythematous/violaceous rash over the eyelids and erythematous and scaling rash over the knuckles and dorsum of the hand. When typical, the skin lesions of DM are often virtually pathognomonic of this disease. Skin biopsy of early, active lesions of DM showed epidermal atrophy, liquefaction and degeneration of the basal cell layer, perivascular infiltration of lymphocytes and histiocytes in the upper dermis and a striking vasculopathy of small vessels 15 ) . The patient exhibited herein did not show evidence of the above findings and vasculitis on skin biopsy. Histologic findings and distribution of skin lesions of SS are clearly distinct from those of DM. Also, the clinical course of skin lesions in SS is different to that of DM. In SS, the skin lesions are usually recovered within several days after the institution of steroid therapy. But cutaneous rash in dermatomyositis is a more indolent course with steroid therapy. The relationship between SS and DM may be proposed as several kinds: first, SS is an acute prodrome phase of flare-up of DM; second, the two diseases are different and occur together by chance; third, SS can be associated with several underlying diseases, of which DM and malignancy are one disease. We did not hold any one of the above three opinions. The association of malignancies and DM is well known, so the possibility of association between SS and underlying malignancy of DM may be suspected. But initial evaluations for malignant underlying DM are negative and the patient has not any evidence of malignancy during 12 months duration of DM. The onset of SS coincided with the flare-up of DM. Aggravation of muscle weakness and elevation of muscle enzymes may be evidence of flare-up of DM. This may be indirect evidence that SS and DM may be associated in this patient. In the light of immunoregulatory abnormalities and immune complex phenomena and disease onset, consistent with precipitation by viral and bacterial infections in DM, the association of SS and DM reported further supports the hypothesis that hypersensitivity or immunological abnormality is involved in the pathogenesis of SS. Further concern about SS as an associating disease of several autoimmune diseases, including DM, is needed. | Other | biomedical | en | 0.999997 |
10461430 | Appendiceal abscesses represent 2.3% of all the cases of acute appendicitis 1 ) and usually present a mass in the right lower quadrant 2 ) . Occasionally, abscesses develop in the right subhepatic space, right subdiaphragmatic space and right pararenal space or liver 3 ) . However, an appendiceal abscess located in the epigastric space has never been reported. The management of appendiceal abscess is controversial 4 ) . Recently, the ultrasonic or CT guided percutaneous puncture technique has offered an alternative to conventional surgical treatment 3 , 5 ) . We experienced a patient with an epigastric appendiceal abscess, demonstrated by abdominal CT scan and barium enema, which resolved spontaneously. A 49-year-old man was referred to our institution for further evaluation and management of an intraabdominal abscess with the air-fluid level located outside of the greater curvature of the stomach, demonstrated on the abdominal CT scan checked 5 days before the referral . He experienced left upper quadrant abdominal pain with fever and chills for 15 days without any preceding trauma and was managed at a private clinic without improvement of symptoms. Upon abdominal examination, an adult fist-sized, ill-defined, round, nonmovable and tender mass was palpated at the left epigastrium. No abnormalities were observed in CBC, urinalysis, stool examination, serum amylase and liver chemistry. We treated the patient with intravenous antibiotics and bed rest. On the second day of hospitalization, an gastroscopy was performed which showed a bulging external mass effect with adherent purulent material and mucosal friability around a fistula-like lesion at the posterior wall of the mid-gastric body . We took biopsy specimens which showed necrotic tissues along with an intact gastric mucosa. On the third day of hospitalization, a barium enema was performed and showed a hyperrotated cecum in the left upper quadrant and nonvisualization of the appendix with mucosal irregularity and focal luminal narrowing at the surrounding distal transverse colon on prone position film consistent with a hyperrotated cecum and appendiceal abscess. On the fifth day of hospitalization, after conservative management, the tender mass subsided. A follow-up gastroscopy, showed a focal regenerating edematous mucosa without the bulging effect noted in the initial examination . A follow-up CT scan disclosed the abscess pocket had subsided with surrounding inflammatory change only. The patient was discharged on the tenth day of hospitalization and has remained well for over 6 months. The palpable mass associated with acute appendicitis may consist of phlegmon or abscesses of various sizes 4 ) . 2–3% of the patients with appendicitis who are admitted to the hospital have abdominal masses 4 , 6 – 8 ) and appendiceal abscesses represent 50–89% of the patients with an appendiceal mass 4 , 9 – 12 ) . Complicating abscesses have been reported in 2.3% of all cases of acute appendicitis 1 ) . The main manifestations of acute appendicitis are fever, a palpable mass and leukocytosis 4 ) . Patients often have a palpable mass in the right lower quadrant (the “appendix mass”) 2 ) . Rarely does the abscess form in the right subhepatic space, right subdiaphragmatic space, liver or posterior pararenal space distant to the right lower quadrant. Therefore, the value of aggressive radiologic work-up and follow-up can not be overemphasized in suspected appendiceal abscesses, especially those present in locations remote from the appendix 5 ) . Jordan has reported that the distinction between an appendiceal mass and an appendiceal abscess could not be made by considering the patient’s duration of symptoms, temperature and white blood cell count when the patient was admitted 4 ) . Careful follow-up, routine barium enema study, ultrasonography and abdominal CT scanning would prevent misdiagnosis and delayed treatment 13 ) . Ultrasonography has made it possible to distinguish an abscess from the phlegmon without operation 14 ) and the abdominal CT scan has proven to be of considerable clinical value in characterizing periappendiceal inflammatory masses and in determining the relative size of the liquefied versus nonliquefied component 15 – 17 ) . Despite extensive clinical experiences, the surgical management of appendiceal abscesses remains controversial 4 , 7 , 10 ) . However, most authors agree that the initial treatment must be conservative management, including bed rest, nasogastric suction, systemic antibiotics and drainage, rather than early surgery 7 , 11 , 13 ) . Initial conservative management with intravenous antibiotics, with or without percutaneous drainage of the abscess, is prudent, safe and effective 17 ) and shows a high success rate (80–90%) and low morbidity rate (15%) 7 , 12 , 18 ) compared to the high complication rate of early surgery (15–50%) 2 , 4 , 7 , 9 ) . Guided percutaneous drainage is an effective alternative to surgical drainage 3 , 5 ) Nonoperative treatment and, if possible, ultrasonic percutaneous drainage of a verified abscess are safe procedures with few complications and late sequelae 12 ) . Recently, the ultrasonic percutaneous puncture technique has offered an almost atraumatic alternative to conventional surgical treatment 14 , 19 ) . Also, CT guided percutaneous abscess drainage is an effective alternative to surgery 7 , 17 ) . The recurrence of appendicitis after conservative treatment is between 4–80% 2 ) or 0–20% 13 ) . Sixty-six percent of the recurrent cases occurred within 2 years of the initial attack 13 ) . The abscess often spontaneously resolves or drains into the intestine 14 ) with a low recurrence rate 11 ) . Our case exhibited fever, chills and an epigastric abscess. At first, we suspected a pancreatic abscess because of its location, and managed initially with antibiotics and planned percutaneous drainage. However, the radiological studies, especially the CT scan and the barium enema, revealed an appendiceal abscess in the left upper quadrant of the abdomen due to a hyperrotated ceum. The abscess resolved spontaneously, and we think that the abscess drained into the stomach through a small fistula between the stomach and abscess cavity. The patient has remained well for over 6 months. | Clinical case | biomedical | en | 0.999996 |
10461431 | It has been claimed that tertian malaria had been completely eradicated in the latter part of 1970 in Korea 1 ) . But since 1993, this tertian malaria has reemerged to infect soldiers and civilians living near the armistice line 2 ) , resulting in 1,724 cases in 1997 3 ) . Also, imported malaria from an overseas trip has become a medical problem to the extent of calling attention. Reports have appeared calling attention to what has been termed primaquine resistance in Plasmodium vivax in Korea 4 ), 5 ) . We present a case of primaquine resistant tertian malaria which was thought to be relapsed because of loss of radical cure at initial treatment. A 50-year-old man presented on November, 1997 with high fever, chills and splenomegaly. Three weeks before admission, he had traveled to Ethiopia where he had remained for 10 days without prophylactic chemotherapy against malaria. The temperature was 39°C, the pulse was 100/min and the respiration rates were 22/min. The blood pressure was 110/80 mmHg. A physical examination was normal, except for a nontender, firm splenomegaly exceeding 5cm below the costal margin. An ultrasonogram of the abdomen revealed enlargement of the spleen and slight hepatomegaly. Laboratory examinations showed hemoglobin 16.1 gm/dl, white blood cell count 7,510/mm 3 , platelet count 49,100/mm 3 , alkaline phosphatase 248 IU/L, alanine aminotransferase 165 IU/L, aspartate aminotransferase 89 IU/L. At that time, microscopic examination of a blood smear was said to show malaria (P.vivax) and a diagnosis of malaria was made. After treatment with chloroquine (750 mg as a loading dose and then 350 mg each 6, 24, 48 hours later), he felt well but subsequently did not receive primaquine because of the physician’s mistake. After about three months, he became ill with fever and chills. Laboratory examinations showed hemoglobin 16.5 gm/dl, white blood cell count 4,150/mm 3 , platelet count 26,300/mm 3 . Microscopic examination of a stained blood smear for malarial organism (38,180/ μl ) was positive. The patient was given a conventional three-day course of chloroquine with relapsed malaria. On the following day, he became afebrile and reevaluation of blood smears showed decrease in the number of parasite counts(3,480/ μl ). He subsequently was given primaquine (15 mg base daily for 14 days) with improvement. The test of G-6PDH (glucose-6-phosphate dehydrogenase) deficiency was negatived(11.7 U/g). About three months after the first relapse, however, he revisited the hospital with fever, headache and chills. Physical examination showed no change, except that the spleen was felt 4cm below the costal margin. Peripheral blood examination showed variable forms of P.vivax with reduction in platelet count (97,000/mm 3 ). He was given a diagnosis of primaquine resistant malaria. The patient was treated with chloroquine in the same dose as the in first attack. On the next day, he became afebrile and smear for malarial organisms had become negative. He subsequently received primaquine (22.5 mg daily for 21 days) without side effects from the drug. One month later, he was well and the spleen was no longer palpable. Symptoms have not relapsed during 5 months of observation. Malaria is the world’s most important parasitic infection. Although it has been eradicated from temperate zones, increasing numbers of travelers from temperate areas each year visit tropical countries where the disease remains a major cause of morbidity and death. Relapse occurs with P.vivax infections because delayed developmental forms of the parasite, called hypnozoites, are present in the liver. Hypnozoites can mature months to years later to cause clinical disease. After maturing and multiplying in the liver, the parasites enter a red blood cell for further development. Clinical disease is produced when parasites are released from red blood cells. A radical cure is achieved for P.vivax infections by the use of both blood-stage antimalarial drugs and primaquine, which is the only available drug that can destroy hypnozoites. To provide radical cure of P.vivax malaria, treatment must be prescribed to eliminate the liver stage of the parasites, otherwise intermittent relapses with clinical symptoms will continue. Usually, after acute phase therapy with chloroquine, patients with P.vivax are treated with the standard course of primaquine, i.e. 15 mg base/day for 14 days, as curative therapy. Primaquine-resistant strains have been reported from Papua New Guinea, South-East Asia and Central and South America. Cases of primaquine treatment failure have also been reported in patients who visited Kenya, Sudan and Ethiopia 6 ) . The interval between primary infection and relapse varies with differing strains of P.vivax-Chesson strain from Papua New Guinea relapsing rapidly, at intervals of 28 days; strains from Southern China may relapse on an approximately annual basis. Such strains appear to show intrinsic differences in sensitivity to 8-aminoquinolines, the agents most commonly used to eradicate the liver stage. Studies carried out in the Western Pacific Region soon after World WarII, and predominantly involving the Chesson strains, indicated that a regimen of primaquine base 15 mg/day for 14 days led to failure in over 30 % of cases 7 ) . It would appear that this investigation regimen was selected solely on the basis of limiting potential toxicity. Common practice over subsequent decades within this region has been to use a regimen of primaquine base 22.5–30 mg/day for 14 days to ensure a high chance of parasite eradication 8 ) . In contrast, however, studies during the Vietnam War determined that strains from South-East Asia were more primaquine-sensitive and were almost entirely eradicated with primaquine base 15 mg/day for 14 days 9 ) . Strains from South-East Asia and Central and South America have also been reported to relapse after standard regimens of primaquine 10 )– 12 ) . Most authorities agree that it is reasonable to treat patients who acquire their infections from these areas with a higher dose regimen 13 ) . To reduce side effects, a regimen of 15 mg daily of primaquine base for 28 days has been used instead of 30 mg base daily for 14 days, under the assumption that it is the total, cumulative dose of primaquine that is important in eliminating hypnozoites. Unfortunately, there are no data to support the longer duration regimen. Human and animal studies have only used total doses scheduled over a shorter time interval (60 mg base daily for 7 days versus 30 mg base daily for 14 days) in showing equivalent cure rates 14 – 15 ) . It is curious that primaquine resistance emanates from areas where higher doses of primaquine have been used for longer periods. Whether this increased anti-malarial load is a risk factor for primaquine tolerance is uncertain. Animal and human studies of sub-therapeutic doses of primaquine in P.vivax infections resulted only in resistance in the erythrocytic stages of the life cycle 16 ) . While it has been suggested that the apparent increasing resistance to primaquine in P.vivax actually reflects unrecognized chloroquine-resistance in this parasite, at present, recurrence of vivax parasitemia after primaquine therapy and > 28 days from initial infection more often represents primaquine rather than chloroquine failure. Cause of the first relapse in our patient is thought to be inadequate regimen rather than chloroquine resistance: primaquine was not prescribed at initial infection. Second attack was due to primaquine resistance, since the peripheral blood smear of our patient was normal up to three days after the treatment (chloroquine) and relapse took place after three months. Moreover, he had traveled to Ethiopia where primaquine treatment failure has been reported 6 ) . Primaquine may cause nausea and abdominal pain, particularly if taken on an empty stomach and, more important, oxidant hemolysis with methemglobinemia, anemia and, sometimes, hemoglobinuria. Patients with a deficiency of glucose-6-phosphate dehydrogenase are particularly vulnerable to oxidant hemolysis, and primaquine is contraindicated in patients with severe form of the deficiency. In places where mild variants of glucose-6-phosphate dehydrogenase deficiency are common, primaquine (0.8 mg of base per kilogram; adult dose, 45 mg) should be given once a week for six weeks for a radical cure 17 ) . It is imperative to establish the most appropriate regimen with primaquine for the curative treatment of relapsed vivax malaria after the completion of chloroquine-primaquine therapy. | Other | biomedical | en | 0.999996 |
10461432 | Xanthogranulomatous cholecystitis (XGC) is an uncommon, focal or diffuse destructive inflammatory disease of the gallbladder that is assumed to be a variant of conventional chronic cholecystitis. This entity was first described in 1970 by Christen and Ishak as fibroxanthogranulomatous inflammation 1 ) . More recently, these terms, together with other labels, such as ceroid granuloma, ceroid-like histiocytic granuloma of the gallbladder 2 ) and biliary granulomatous cholecystitis 3 ) , have been abandoned in favor of XGC, a descriptive term first used by McCoy et al 4 ) . XGC is characterized grossly by irregular wall thickening of the gallbladder associated with the formation of yellowish nodules. Histologically, the nodules are predominantly composed of abundant lipid-laden macrophages, inflammatory cells and fibroblasts. The pathogenesis of XGC is uncertain, but histologic evidence of chronic inflammation is generally seen and gallstones are present in a large majority of cases. It has been suggested that XGC is initiated by a chronic inflammation and obstruction. Occasionally, XGC may closely mimic a gallbladder cancer or lead to complications such as perforation, abscess and fistula. We report a case with XGC mimicking gallbladder cancer in a hemophilia patient. A 36-year-old male was admitted to our hospital with a 10-day history of right upper quadrant pain with fever. 15 years ago, he was first diagnosed as having hemophilia A, and was followed up in the department of Hematology. On admission, the patient was febrile and had tenderness on deep palpation to the right upper quadrant. Rebound tenderness or hepatosplenomegaly was not found. Laboratory studies showed a white blood cell count 15800/mm 3 , hemoglobin 9.3 g/dL, ESR 141 mm/hr, and CRP 11.3 mg/dL, AST 27 U/L, ALT 16 U/L, ALP 430 U/L. Coagulation profiles were PT 12.8sec (control 11.8sec), aPTT 58.4sec (control from 28 to 40sec) and factor VIII 7.5%(50–150%). Ultrasonographic examination showed an inhomogenous, hypoechoic wall thickening of the gallbladder with internal small stones and no dilatation of intrahepatic bile ducts . Computed tomogram(CT) revealed a well-marginated, uniform, marked wall thickening of the gallbladder with multiseptate enhancement . There was no regional lymph node enlargement or focal mass in the liver. Magnetic resonance imaging (MRI) demonstrated diffuse wall thickening of the gallbladder by viewing high signal foci with signal void lesions . After factor VIII replacement, exploration was done. On operation, the gallbladder wall was thickened and the serosa were surrounded by dense fibrous adhesions which were often extensive and attached to the adjacent hepatic parenchyma. There was a small-sized abscess in the gallbladder wall near the cystic duct. Dissection between the gallbladder serosa and hepatic parenchyma was difficult. On intraoperative cholangiogram through the cystic duct after cholecystectomy, there was no evidence of remaining stone or bile duct dilatation. Cross sections through the wall revealed multiple yellow-colored, nodule-like lesions ranging from 0.5–2 cm. There were also multiple black pigmented gallstones ranging from 0.5–1 cm . The pathologic findings showed the collection of foamy histiocytes containing abundant lipid in the cytoplasm and admixed lymphoid cells . Histologically, it was confirmed as XGC. The patient was discharged on postoperative day 10 without complications. Xanthograulomatous cholecystitis (XGC) is a rare condition of chronic inflammatory disease of the gallbladder. In extensive reports, there is a slightly female predominance, which probably reflects the increased incidence of cholecystitis in women 5 , 6 ) . The lesions occur in a wide age range, but the incidence is higher in the sixth and seventh decades of life 6 , 7 ) . In 78–98% of the patients, gallstones have been demonstrated by ultrasonography 8 ) and obesity and/or diabetes mellitus are common 7 ) . The pathogenesis of XGC is unclear, although the role of lipid and bile is thought to be important. Previous reports have suggested that the important process is the extravasation of bile into the gallbladder wall, either from ruptured Rokitansky-Aschoff sinuses or focal mucosal ulceration 2 , 5 ) . Gallstones are present in most patients. Therefore, obstruction by stones and intraluminal stasis of bile have also been implicated as other important factors 9 ) . The true incidence of XGC is difficult to establish because this disease is apparently a rare condition, although retrospective estimates of the incidence in cholecystectomy specimens range from 0.7 7 ) to 9.0 percent 10 ) . Clinical manifestations of XGC are usually those of acute or chronic cholecystitis, but some patients present anorexia, nausea, vomiting, right upper quadrant pain and mass, suggesting gallbladder cancer. XGC may be difficult to distinguish clinically from acute or chronic cholecystitis; radiologically, it is difficult to distinguish from gallbladder cancer. Therefore, before the operation, differential diagnosis of XGC and gallbladder cancer by percutaneous needle biopsy might be helpful in planning the appropriate operative procedure. However, as XGC has the potential for fistula formation, Robert et al 6 ) . reported that percutaneous needle biopsy is probably contraindicated, and it may be necessary to confirm the benign nature of the lesion by intraoperative frozen section diagnosis. The most useful indication of percutaneous needle biopsy would, perhaps, be in advanced gallbladder cancer, as unnecessary laparotomy could be avoided. But XGC imitates gallbladder cancer in various ways. Also, there appears to be a positive association between the two lesions. One group reported gallbladder cancer in nearly 10 percent of resected specimens of XGC, higher than the expected incidence of gallbladder cancer in the general population 11 ) , and another reported a 10 percent incidence of XGC in reviewed cases of gallbladder cancer 12 ) . The reason for this association is not clear. It means that both XGC and gallbladder cancer are complications of gallstone and inflammation of the gallbladder, or it may suggest that tissue disruption by cancer facilitates extravasation of bile into the gallbladder wall 13 ) . Despite the possible association between XGC and gallbladder cancer, XGC is not believed to be a premalignant lesion 14 ) . Howard et al 15 ) , reported that intraoperative cultures of the bile and gallbladder have been positive usually for E. coli , Klebsiella , and/or Enterococcus and, less frequently, for Pseudomonas, Serratia and Staphylococcus aureus . Roberts et al. 6 ) reported that there was a high rate of postoperative infective complication, with one subphrenic abscess and three wound infections (one fatal), two of the patients with fistula. They are likely to be complicated by the presence of dense fibrous adhesions, abscess and adherence of the gallbladder to adjacent structures. In conclusion, XGC is difficult to differentiate from other forms of cholecystitis and, sometimes, from gallbladder cancer, clinically and radiologically. XGC may be a high risk of postoperative wound infection and other septic complications because of frequent adhesion and abscess formation. | Other | biomedical | en | 0.999998 |
10461433 | Insufficiency fractures occur in weakened bones unable to withstand even the stresses of normal daily activities. Spine, pelvis and long bones of lower extremities are common sites of insufficiency fracture 1 ) . Cases of sternal insufficiency fracture have been rarely reported in an elderly patient with osteoporosis or chronic obstructive pulmonary disease (COPD) taking corticosteroid 2 ) Osteoporosis often leads to thoracic kyphosis secondary to thoracic compression fracture. Thoracic kyphosis may cause a bending stress on the sternum, which may result in sternal insufficiency fracture 3 ) . To make the diagnosis of sternal insufficiency fracture, spontaneous fracture of sternum secondary to secondary neoplasm, lymphomatous infiltration and myelomatosis must be excluded 4 – 6 ) . It has been emphasized that sternal insufficiency fracture should be considered in the differential diagnosis of acute chest pain in the elderly 7 – 8 ) . Bone scintigraph contributes to the early diagnosis and chest computed tomography can confirm the diagnosis and rule out the possibilities of metastatic spread 9 ) . Therapy for underlying causes, as well as pain control, must be included for the treatment of sternal insufficiency fracture. We describe here a case of sternal insufficiency fracture in a patient with long-standing COPD who had osteoporosis and thoracic kyphosis. A 72-year-old woman, a smoker with a 15 years history of COPD, was admitted to our hospital because of anterior chest pain for 20 days. She denied any trauma history. She has been managed with corticosteroids after she was diagnosed as having COPD. Recently, her dyspnea was aggravated with the onset of acute chest pain. On examination, she appeared acutely ill. Body temperature was 37°C, respiration rate 30/min and blood pressure 120/70 mmHg. Auscultation of the chest showed coarse breathing sound without rale and heart sound was normal. There was a tenderness on the sternum. Her initial laboratory findings were as follows: hematocrit 42.1%, leukocyte count 11,600 × 10 9 /mm 3 (neutrophil 71.8%, lymphocyte 20%, monocyte 7.4%, eosinophil 0.3%), platelet count 235,000 × 10 9 /mm 3 . The values for glucose, liver function, renal function, muscle enzyme and electrolytes were normal. Urinalysis was normal. Arterial blood gas on room air revealed pH 7.443, PO 2 of 64.7 mmHg, PCO 2 of 38.9 mmHg, bicarbonate 26 mmol/L and O 2 saturation of 93.7%. The alveolar-arterial oxygen difference was 29.2 mmHg. Electrocardiogram revealed no definite abnormality. Lateral radiograph of the sternum showed buckling fracture of the upper body of the sternum , but her previous lateral radiograph of the sternum obtained 3 months ago revealed normal appearance. Lateral chest radiograph demonstrated compression fracture of the 5th and 8th thoracic spine and osteoporosis through the spine . Bone scintigraph showed increased uptake at the upper body of the sternum, costovertebral junction of both 2nd and 4th ribs, and compression fracture with hot uptake at 2nd, 3rd lumbar vertebrae . Chest CT revealed cortical breakage at the posterior aspect of the sternum with soft tissue swelling . Lumbar spine bone density measured by dual energy x-ray absorptiometry was reduced to 0.735 g/cm 2 (T score;−3.21) at the level of L2. Her chest pain and dyspnea were declined by the administration of calcitonin and analgesics. Stress fractures are classified as fatigue and insufficiency fracture depending on the amount of stress applied to bone and on the elastic properties of the bone. Insufficiency fracture occurs when the elastic resistance of bone is inadequate to withstand the stresses of normal activity. The spine, pelvis and lower extremities are commonly affected by these fractures 1 ) . Spontaneous sternal insufficiency fractures have been rarely reported in elderly patients 2 , 3 , 7 – 10 ) . The sternum is an integral part of the thoracic cage, and slight movement at the manubirosternal joints aids expansion of the chest during inspiration. The stresses of sudden forward angulation of the thoracic spine in violent flexion are transmitted to the sternum by the ribs and clavicles. Bone loss with age and loss of elasticity associated with ossification of the costal cartilage are major predisposing factors for sternal insufficiency fracture. Bones weakened by osteopenia have insufficient elastic resistance to withstand even the minimal stress of normal daily activity. Sternal insufficiency fracture, which has been attributed to chronically applied flexion compression stress to the sternum caused by accentuated thoracic kyphosis, results from acute flexion-compression stress applied to the weakened sternum, particularly during forward bending 9 ) . The degree of sternal displacement may vary with the extent of the kyphosis. But Chen et al. reported that sternal insufficiency fracture may occur with or without thoracic kyphosis 10 ) . They classified sternal insufficiency fracture into two types- nonbuckling or buckling- and reported that all buckling fractures were associated with thoracic kyphosis, whereas nonbuckling fracture may be presented with or without thoracic kyphosis. Our patient had buckling type fractures with thoracic kyphosis. Hameed et al. reported spontaneous sternal fractures in four patients with chronic obstructive pulmonary disease taking corticosteroid and suggested that corticosteroids treatment and limited mobility secondary to their lung disease caused accelerated trabecular bone loss, which, in turn, lead to progressive thoracic kyphosis and deforming stress to the sternum 2 ) . They also suggested that coughing may also cause sternal fracture. Our patient has suffered from COPD and has been taking corticosteroid for 15 years. In most cases, sternal insufficiency fractures occur at the junction between the superior third and the two inferior thirds of the sternal body 7 ) . If sternal fractures were accompanied by osteolysis and adjacent soft-tissue swelling, pathologic fracture must be considered 4 ) . Spontaneous sternal fractures have been also reported in patients with myeloma, metastatic cancer, lymphomatous infiltration, during labour or athletic activities. In our patient, pathologic fractures could be excluded on the basis of clinical findings and absence of osteolysis. Patients with sternal insufficiency fracture usually have symptoms of chest pain 4 , 6 , 11 , 12 ) . However, patients with buckling sternal insufficiency fracture may be asymptomatic. The chest pain may be similar to that of myocardial infarction, acute pericarditis, pulmonary embolism and reflux esophagitis. Cardiopulmonary disorders as underlying causes of chest pain could be ruled out by the electrocardiograph, laboratory findings and clinical course in our patient. Dyspnea in COPD patients with sternal insufficiency fracture can be aggravated by paradoxical movement of the upper fractured fragment. If sternal insufficiency fracture is clinically unsuspected, radiological investigation and accurate diagnosis might be delayed 2 ) . Sternal insufficiency fracture can be confirmed by lateral chest radiographs which provide information about the location of the fracture, the stage in the process of callus formation, additional thoracic vertebral fractures and thoracic kyphosis 10 ) . Bone scintigraphy is a very useful diagnostic tool in the diagnosis of insufficiency fracture in various sites, such as the sternum 13 ) . Bone scintigraphy, which shows an intense horizontal or oblique uptake, contributes to an early diagnosis. In most cases, increased uptake on bone scan is observed within 72 hours at the fracture sites, but the bone scan may detect insufficiency fracture as early as 1 day after occurrence. It is also useful in monitoring the healing process through gradual decrease in uptake intensity. The minimum time for return to normal is 5 months and, in 90% of patients, the abnormal uptake disappears within 2 years 14 ) . Computed tomography provides information on the location, extent and stage of repair and helps exclude the possibility of infection and malignancy 15 ) . Bone biopsy must be considered only in cases which are suspicious of malignancy and infection. Abundant granulation tissue with reactive bone and hyaline cartilage are the main histological features in bone biopsy specimens from the insufficiency fracture 14 ) . Analgesics or NSAID treatment can relieve the pain. Calcitonin therapy seems to be effective through both its analgesic and anti-osteoclastic effects 16 ) . In addition, the underlying causes for osteoporosis should be recognized and specifically treated. In summary, sternal insufficiency fracture should be included in the differential diagnosis of acute chest pain or aggravating dyspnea in COPD patients, and radiologic studies, such as lateral chest radiography, bone scintigraph or CT, must be considered. | Review | biomedical | en | 0.999999 |
10465644 | Chimeric Tac-furin protein constructs TTF and TFF in the plasmid pCDM8.1 incorporating the cytomegalovirus promoter were obtained from Michael Marks (University of Pennsylvania, Philadelphia, PA) , and Juan Bonifacino (National Institutes of Health, Bethesda, MD) . TRVb-1 cells are a CHO cell line which lacks endogenous transferrin receptors but stably expresses the human transferrin receptor . TRVb-1 cells were cotransfected with the Tac-furin plasmids and the pMEP plasmid (encoding resistance to hygromycin) using the LipofectAMINE reagent system (GIBCO BRL). Cells expressing Tac-furin constructs were selected by culturing in 200 U/ml hygromycin. Clonal populations of the TTF and TFF expressing cells were isolated for quantitative analyses. TRVb-1/Tac-TGN38 cells were described previously . TRVb-1 cells expressing Tac-furin chimeras or Tac-TGN38 were propagated as described previously , except that TRVb-1/Tac-furin cells were grown in the presence of 200 U/ml hygromycin. Monoclonal antibodies (IgG1) against Tac were purified from ascites fluid prepared from the hybridoma cell line 2A3A1H (ATCC) using protein G affinity chromatography. Antibodies were conjugated to Cy3 (Cy3–anti-Tac) (Amersham North America), Alexa 488 (A488–anti-Tac) (Molecular Probes), or fluorescein isothiocyanate (FITC–anti-Tac) (Molecular Probes) according to the manufacturers' instructions. For some experiments, antibodies were labeled with Na 125 I as described previously . Low density lipoproteins (LDLs) labeled with DiI were prepared as described . Human apotransferrin (Sigma Chemical Co.) was iron-loaded as described previously and conjugated to Cy3, Cy5 (Amersham), or Alexa 488 according to the manufacturers' instructions. NBD-C 6 -ceramide [N-(∈-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)- D -erythro-sphingosine] and fixable 70-kD dextrans conjugated to rhodamine were purchased from Molecular Probes. Polyclonal antibodies against the cytoplasmic domain of rat TGN38 were obtained from Keith Stanley (Heart Research Institute, Sydney, Australia) or from George Banting (University of Bristol, Bristol, United Kingdom) . Polyclonal antibodies against a conserved peptide sequence (TQMNDNRHGTRC) in the furin enzymatic site were obtained from Yukio Ikehara (Fukuoka University, Fukuoka, Japan) . Polyclonal antibodies against the CI-MPR were obtained from Peter Lobel (Robert Wood Johnson Medical School, Piscataway, NJ) . Polyclonal antibodies against fluorescein were purchased from Molecular Probes. Fluorescently labeled polyclonal antibodies against rabbit immunoglobulins were purchased from Sigma Chemical Co., Jackson ImmunoResearch, or Pierce Chemical Co. For microscopy, cells were passaged onto poly- d -lysine–treated Number 1 coverslips affixed beneath holes cut into the bottoms of 35-mm Petri dishes. For incubations of live cells with antibodies or ligands, cells were treated as described previously , except as indicated. For indirect immunofluorescence labeling, fixed cells were permeabilized with 0.01% (wt/vol) saponin (Sigma Chemical Co.) in Medium 1 (150 mM NaCl, 20 mM Hepes, 1 mM CaCl 2 , 5 mM KCl, 1 mM MgCl 2 , pH 7.4) with 0.5% BSA (AB buffer). Antibodies were diluted into AB buffer for application to cells, and all washes were with AB buffer. Labeling with NBD-C 6 -ceramide was performed as described . Digital epifluorescence microscopy was performed as described previously . Confocal microscopy was performed using an MRC600 laser scanning unit (BioRad) and an Axiovert 35 microscope (Carl Zeiss) with a 63× 1.4 NA plan Apochromat objective (Zeiss) , or an LSM 510 laser scanning unit (Zeiss) and an Axiovert 100M inverted microscope (Zeiss) with a 63× 1.4 NA plan Apochromat objective (Zeiss) . Excitation on the MRC600 unit was with a 25-mW argon ion laser emitting at 488 nm and 514 nm, and emissions were collected using standard fluorescein and rhodamine filter sets. Excitation on the LSM 510 unit was with a 25-mW argon laser emitting at 488 nm, a 1.0-mW helium/neon laser emitting at 543 nm, and a 5.0-mW helium/neon laser emitting at 633 nm; emissions were collected using a 505–530-nm band pass filter to collect fluorescein and Alexa 488 emissions and a 585-nm long pass filter to collect rhodamine, Cy3, and DiI emissions. For Fig. 5 , Cy3 emissions were collected with a 560–615-nm band pass filter, and Cy5 emissions were collected with a 650-nm long pass filter. Typically, 0.5-μm vertical steps were used, with a vertical optical resolution of <1.0 μm. To determine the rate of Tac-furin externalization by accumulation of fluorescent antibodies, TRVb-1/Tac-furin cells cultured on coverslips were incubated with Cy3–anti-Tac IgG (3 μg/ml) in McCoy's 5A + 0.1% BSA for 5, 10, 15, 20, 30, 40, 50, 60, 75, or 90 min. Unbound antibody was removed by washing, and cells were fixed. Cy3 fluorescence was imaged by epifluorescence microscopy. Fluorescence power per cell was determined as described below. Fluorescence power was relatively uniform among all cells for each time point. Determination of the externalization rate by accumulation of radiolabeled antibodies, measurement of the level of surface expression of Tac-furin, and determination of the endocytic rate constant of Tac-furin were performed using procedures described previously . To determine the exit rate of antibody-labeled Tac-furin from the cell, TRVb-1/Tac-furin cells were incubated with FITC–anti-Tac for 60 min, followed by a 30-min chase in McCoy's/BSA. After this procedure, anti-Tac is mostly detected in the TGN . Cells were further incubated for 5, 10, 15, 20, 30, 40, 50, 60, 75, or 90 min in the presence of antifluorescein antibodies (10 μg/ml) in the chase medium and fixed. We have shown previously that antifluorescein antibodies are not internalized by fluid-phase pinocytosis sufficiently to cause significant intracellular quenching . Fluorescein fluorescence was imaged by epifluorescence microscopy. Fluorescence power per cell was determined as described below. Cells that did not receive antifluorescein exhibited insignificant loss of fluorescence over the chase period (data not shown). To determine if Tac-furin undergoes rapid endocytic recycling, TRVb-1/Tac-furin cells were incubated with FITC-anti-Tac for 10 min, then washed in medium and chased in the absence or presence of antifluorescein for 5, 10, 15, or 20 min. Cells were fixed, and fluorescein fluorescence was imaged as described above. Experiments examining the effects of nocodazole and wortmannin on protein transport were performed in parallel. For all steps, BSA was omitted from incubation media to prevent adsorption and deactivation of reagents, and all cells received 0.1% (vol/vol) DMSO to control for effects of the solvent. Nocodazole-treated cells were pretreated with nocodazole (33 μM) for 30 min at 4°C, then 30 min at 37°C. To maintain identical conditions of temperature and DMSO exposure, wortmannin-treated cells were incubated for 30 min at 4°C in the presence of 0.1% DMSO, then pretreated with wortmannin (100 nM) for 30 min at 37°C. Untreated cells were incubated for 30 min at 4°C and 30 min at 37°C in the presence of 0.1% DMSO. After pretreatments, cells were incubated with ligands for 15 min followed by a 45-min chase in the continuous presence of nocodazole, wortmannin, or DMSO alone, respectively. Processing of digitized images was performed using the MetaMorph image processing software package (Universal Imaging). All images were corrected for background fluorescence and crossover between channels. To quantify fluorescence power per cell , the background fluorescence value was subtracted from images, and the remaining fluorescence power in the field was summed and divided by the number of cells in the field; typically, 10 fields of ∼20 cells per field were analyzed for each data point in a single experiment. For quantitative microscopic analyses and 125 I-antibody experiments, data points were fit using the SigmaPlot software program (SPSS Inc.). To quantify the colocalization of internalized fluorescent anti-Tac and LDL over a time course, cells were imaged using the MRC600 confocal microscope. Using routines available in the MetaMorph software package, images from the green and red channels were thresholded to detect labeled objects above background fluorescence, then labeled endosomes were selected on the basis of size (between 10 and 50 square pixels). Double-labeled endosomes were identified by performing a logical AND operation with the endosomes detected in the green and red channels. Intensities of singly and doubly labeled endosomes were transferred to Microsoft Excel for statistical analyses. Tac-furin (TTF) was shown by Bonifacino and co-workers to cycle between the plasma membrane and the TGN, maintaining a steady-state enrichment in the TGN ; similar behavior of exogenously expressed epitope-tagged furin was described by Thomas and co-workers . We transfected TRVb-1 cells with the TTF form of Tac-furin and isolated clonal populations expressing the chimeric construct (referred to as TRVb-1/TTF cells). Results presented here were indistinguishable from those observed for two different clones and for a polyclonal population; images and data from a single clone are presented. At steady state, we observed Tac-furin predominantly in the TGN, where it colocalized with endogenous furin and with TGN38 . The immunolocalized Tac-furin was not seen in endosomes labeled with antibodies to the CI-MPR or with internalized dextrans , indicating that the expressed chimera is not detectable in endosomes at steady state. We observed that the overlap of Tac-furin and endogenous furin was incomplete . This may be due to the transport of a portion of endogenous furin into the degradative pathway . Alternatively, the two molecules may be partially distributed into distinct subdomains of the TGN . When TRVb-1/TTF cells were incubated with fluorescently labeled anti-Tac antibodies, the antibodies were internalized and transported over time to the TGN, which was identified using anti-TGN38 antibodies , using antibodies against furin, or using the fluorescent lipid analogue NBD-C 6 -ceramide (data not shown). Tac-furin was detected in the TGN after ∼30 min of internalization , becoming further enriched there after 60 min . These results demonstrate the transport of Tac-furin to the TGN in our system and confirm previous reports in other cell types. Internalized anti-Tac Fab fragments were transported identically to intact IgG (data not shown), demonstrating that our findings are not an artifact of protein aggregation by divalent IgG. The delivery of internalized anti-Tac to the TGN in TRVb-1/TTF cells allowed us to describe the kinetics of Tac-furin trafficking by incubating cells with anti-Tac under various conditions. To determine the rate of exit of Tac-furin from TRVb-1/TTF cells, the cells were incubated at 37°C with Cy3–anti-Tac, and the cells were imaged by epifluorescence microscopy. The accumulation of the Cy3–anti-Tac was then quantified in terms of fluorescence power per cell versus incubation time . Under these conditions, the rate of accumulation of anti-Tac is equal to the rate of appearance of unlabeled intracellular Tac-furin at the plasma membrane . We found that the antibody was accumulated by TRVb-1/TTF cells with first-order kinetics, with a half-time of ∼26 min ( k = 0.026 min −1 ). Alternatively, cells in a 24-well plate were incubated at 37°C for various times with 125 I–anti-Tac antibodies, and the cell-associated radioactivity was measured for each time point. We found that the cells accumulated anti-Tac with a half-time of ∼36 min ( k = 0.019 min −1 ) . The difference in the exit rate constants obtained from the two different methods is small compared with other parameters of Tac-furin trafficking (see below), and may be due to the differences in the experimental procedures. From the specific activity of the 125 I–anti-Tac, the asymptote of the accumulation curve, and the number of cells in each well, we calculate that ∼2 × 10 5 copies of Tac-furin are expressed per cell. Tac-furin and endogenous furin localize predominantly to the TGN in these cells , so it is unlikely that sorting or retention mechanisms are saturated at this expression level. To determine the steady-state surface expression of Tac-furin, TRVb-1/TTF cells were incubated with 125 I–anti-Tac antibody at 0°C to prevent internalization, and the bound counts were compared with the asymptote of the 37°C 125 I–anti-Tac accumulation curve. We estimate that 5% of Tac-furin is at the plasma membrane at steady state (data not shown). We also measured the internalization rate constant of Tac-furin in TRVb-1/TTF cells. As determined from the ratios of internal to surface antibody over a brief time course at 37°C, the protein is internalized with a rate constant of 0.36 min −1 , which is consistent with the presence of rapid internalization signals in the furin cytoplasmic domain . At steady state, the relative rates of internalization and externalization determine the relative amounts of protein in internal compartments and at the plasma membrane. The ratio of the measured rates of endocytosis (0.36 min −1 ) and externalization (0.019 min −1 ) of 125 I–anti-Tac is about 19, which agrees well with the estimated internal-to-surface ratio of Tac-furin (also about 19). This indicates that our kinetic data accurately describe the rates of trafficking of Tac-furin. To demonstrate that antibody labeling did not perturb the kinetics of Tac-furin trafficking, we measured the externalization rate by another method. TRVb-1/TTF cells were incubated for 60 min with FITC–anti-Tac, followed by a 30-min chase. At this time, the FITC–anti-Tac predominantly labeled the TGN . Subsequently, antifluorescein was applied to the medium, and the cells were incubated over a long time course to allow externalization of Tac-furin. Over this time period, the pericentriolar fluorescence signal decreased, indicating that the antibody was externalized from the TGN to the plasma membrane . Cells were then imaged by epifluorescence microscopy, and the fluorescein fluorescence power per cell was quantified for each time point . The fluorescence power declined in a monoexponential fashion ( k = 0.026 min −1 ), with a half-time of about 26 min indicating the rate of exit of FITC–anti-Tac–labeled Tac-furin from the cells. This rate is similar to the rate of externalization of unlabeled Tac-furin from cells , confirming that antibody labeling has not altered the kinetics of Tac-furin transport. Since the majority of the FITC–anti-Tac was externalized from the TGN under this procedure, the measured rate constant mainly reflects the rate of transport of Tac-furin from the TGN to the plasma membrane. The exact pathway and the rate of exit of Tac-furin from the TGN per se are not directly shown by these studies. The agreement of this rate constant with that for whole-cell antibody accumulation is consistent with the existence of a single major exit route for Tac-furin (see below). Also, the fluorescein quenching procedure measures only the kinetics of externalization of the cycling Tac-furin pool. Our results suggest either that the cycling and biosynthetic pools are externalized at the same rate or that the contribution of the biosynthetic pool to these kinetics is small. Finally, the transport of FITC–anti-Tac back to the plasma membrane demonstrates that the antibody remains associated with Tac-furin throughout its trafficking itinerary. Otherwise, dissociated FITC–anti-Tac would accumulate in lysosomes, which was not observed. The low level of residual fluorescence power observed in these experiments after a prolonged chase is probably due to autofluorescence and the incomplete quenching of fluorescein by antifluorescein antibodies, as little TGN or endosomal anti-Tac staining could be detected at the longest time points. The rapid internalization of Tac-furin at the plasma membrane and the relatively slow movement from the TGN to the plasma membrane account for the steady-state localization of the chimera. We next determined the route by which Tac-furin is transported to the TGN. To evaluate if Tac-furin, like Tac-TGN38, transits through the endocytic recycling pathway, TRVb-1/TTF cells were incubated for 5 min with fluorescently-labeled anti-Tac and transferrin, then fixed immediately or chased in the continuous presence of transferrin to label the recycling pathway . As reported previously , transferrin was detected primarily in the pericentriolar ERC and also in peripheral sorting endosomes. In contrast, Tac-furin was observed in an exclusively punctate distribution at early chase times , accumulating in the TGN after a 40-min chase . In the absence of a chase, the two molecules partially colocalized in sorting endosomes (data not shown), and the extent of colocalization declined rapidly with increasing chase times. At no time was Tac-furin prominent in the ERC, unlike Tac-TGN38 which is enriched in that organelle shortly after internalization . We also found that endocytosed Tac-furin does not recycle rapidly to the plasma membrane. TRVb-1/TTF cells were incubated briefly with anti-Tac conjugated to fluorescein (FITC–anti-Tac), then chased over a short time course in the absence or presence of antifluorescein antibodies in the medium. Cells were imaged by epifluorescence microscopy, and the fluorescence power per cell was determined for each chase time point . If Tac-furin is rapidly recycled, then the internalized FITC–anti-Tac should reappear at the plasma membrane, where its fluorescence would be quenched by the antifluorescein antibodies. Instead, we observed no loss of cell-associated fluorescence over the time of the chase. This finding again contrasts with the rapid recycling of the bulk of internalized Tac-TGN38, which was demonstrated using the same approach . The most common fate of an endocytosed protein that is not recycled is accumulation in endosomes that over time have matured and are segregated from the recycling pathway. This class of endosomes is defined as late endosomes . For example, internalized LDL dissociates from its receptor in sorting endosomes and then is transported to late endosomes , where it no longer colocalizes with internalized transferrin. Also, growth factor receptors such as the epidermal growth factor receptor are transported to late endosomes and lysosomes after binding to their corresponding ligands . A population of late endosomes is enriched in the CI-MPR . To begin to characterize the endosomal intermediate in Tac-furin trafficking, we incubated TRVb-1/TTF cells for 5 min with anti-Tac antibody and chased for various times. Fluorescent transferrin was applied during the last 5 min of the chase to label sorting endosomes and the ERC. After fixation, the cells were permeabilized and stained for the CI-MPR by indirect immunofluorescence . At all time points, anti-Tac exhibited a detectable but low degree of colocalization with the CI-MPR . In the absence of chase, internalized anti-Tac colocalized with transferrin in punctate sorting endosomes (data not shown). This colocalization was lost with a chase, as anti-Tac distributed into endosomes near the center of the cell at 20 min and then eventually accumulated in the TGN at 40 min . Since the anti-Tac–labeled endosomes observed at 20 min of chase do not contain transferrin, they are presumably matured endosomes that have departed the recycling pathway. The absence of Tac-furin from CI-MPR–enriched endosomes may be due to transport into a novel pathway distinct from the classical degradative pathway, or may be explained by the exit of Tac-furin from endosomes before they have become significantly enriched in CI-MPR. The existence of multiple classes of matured endosomes with distinct properties has been reported in other systems . To establish the point at which a molecule that follows the degradative pathway colocalizes with the CI-MPR, fluorescent dextrans were followed through a pulse-chase protocol over the same time course. The dextrans were not enriched in CI-MPR–positive endosomes until 40 min of chase , at which time anti-Tac had already accumulated in the TGN. Therefore, transport of lysosomally targeted cargo into CI-MPR–containing endosomes may occur after the time of endosome maturation. This would allow a molecule such as Tac-furin to enter newly formed late endosomes and then exit before extensive delivery of the CI-MPR into this compartment. To confirm that endocytosed Tac-furin enters late endosomes, TRVb-1/TTF cells were pulsed for 5 min with fluorescently labeled anti-Tac and LDL . After a 5-min chase, the two probes were detected in peripheral spots, colocalizing significantly although incompletely . The frequency of double-labeled endosomes remained high through about 15–20 min of chase . At these chase times, the majority of LDL is in late endosomes , segregated from recycling molecules such as transferrin. Therefore, the colocalization of anti-Tac with LDL demonstrates the transport of Tac-furin into late endosomes. After a longer chase interval , anti-Tac no longer colocalized with LDL and was instead found in a compartment resembling the TGN. When anti-Tac and LDL were applied to cells with consecutive pulses, such that one probe experienced a 20-min chase and the other was not allowed a chase, the two probes did not colocalize (data not shown). This result indicates that the overlap of anti-Tac and LDL illustrated in Fig. 6 is not in sorting endosomes that have not yet matured. Also, anti-Tac rapidly lost colocalization with transferrin-containing peripheral punctate sorting endosomes over this time course . Transport of internalized Tac-furin through late endosomes en route to the TGN was also demonstrated by colocalization in punctate structures with internalized dextrans over a similar time course (data not shown). The extent of colocalization of anti-Tac and LDL was quantified by two different computational methods. Endosomes labeled with LDL were selected, and the proportion that was also labeled with anti-Tac was quantified versus chase time. Also, the ratio of anti-Tac to LDL fluorescence was measured for each double-labeled endosome, and the distribution of these ratios was determined as a function of chase time. These analyses were performed on images similar to those in Fig. 6 , using two independent data sets. We found that the proportion of LDL-labeled endosomes containing anti-Tac declined sharply between 20 and 40 min of chase, coincident with the appearance of anti-Tac in the TGN . In contrast, the population of endosomes labeled with both probes exhibited no change in fluorescence power ratios during this same interval , although the size of that population decreased over time. The abrupt loss of double-labeled endosomes and the relative invariance of fluorescence power ratios suggest that anti-Tac may be sorted away from LDL by a highly concerted process, rather than by a more gradual or iterative mechanism; in this way, a double-labeled endosome would suddenly become singly labeled. We failed to detect an accumulation of endosomes labeled with anti-Tac and not with LDL, so it seems plausible that Tac-furin follows a relatively direct route from endosomes to the TGN. This event apparently precedes or coincides with the delivery of CI-MPR to late endosomes from the TGN, such that the two molecules mostly do not overlap. In addition to cytoplasmic domain sorting signals, the transmembrane domains of some membrane proteins have been shown to perform a sorting function. In particular, the transmembrane domain of TGN38 has been shown to play a role in the localization of that protein to the TGN . To determine if the transmembrane domain of furin influences its postendocytic trafficking, we expressed in TRVb-1 cells a construct encoding the ectodomain of Tac and the transmembrane and cytoplasmic domains of furin (TFF) . We found that this chimeric protein, like TTF, was internalized and entered late endosomes, bypassing the recycling pathway (data not shown). The internalized chimeras entered the TGN over a similar time course, and the rates of externalization from cells were almost identical for each protein ( k e = 0.023 min −1 for TFF, and 0.025 min −1 for TTF in a single experiment). Therefore, the transmembrane domain appears not to be a major sorting determinant of the endocytic transport of furin to the TGN. The result is consistent with previous findings in other systems . The transport of Tac-furin to the TGN via late endosomes is distinct from the pathway that is followed by Tac-TGN38. The point at which the trafficking of the two proteins diverges is the sorting endosome: Tac-furin is retained as the sorting endosome matures into a late endosome, whereas Tac-TGN38 is transported to the ERC. To show directly the divergent routes taken by these two chimeras, we performed a series of pulse-chase experiments under conditions that are known to selectively alter different properties of the endosomal system. First, we took advantage of the effects of the microtubule-disrupting compound, nocodazole. Nocodazole has been shown to inhibit endosome maturation but does not affect the kinetics of internalization or endocytic recycling . We verified these effects in TRVb-1 cells expressing Tac-furin (TTF) or Tac-TGN38 (data not shown). Therefore, treatment of cells with nocodazole provides a means to discriminate between transport to the TGN via late endosomes and transport via the recycling pathway. TRVb-1/Tac-TGN38 and TRVb-1/TTF cells were pretreated with 33 μM nocodazole or 0.1% DMSO alone as described in Materials and Methods. The cells were then incubated with fluorescently labeled anti-Tac antibodies, alone or in combination with fluorescent LDL for 15 min, followed by a 45-min chase; nocodazole or DMSO was present throughout this time course. After fixation, cells that received anti-Tac alone were stained with NBD-C 6 -ceramide to label the TGN. The distributions of the probes were determined by confocal microscopy. In TRVb-1/TTF cells treated with DMSO alone, internalized antibody colocalized well with NBD-C 6 -ceramide and had very little overlap with LDL, which is expected to label mainly late endosomes after this time course . However, nocodazole treatment resulted in a substantial redistribution of anti-Tac into LDL-containing structures, and overlap with NBD-C 6 -ceramide was greatly diminished . Note the dispersion of the NBD-C 6 -ceramide staining, which is a consequence of the fragmentation of the TGN caused by microtubule disassembly . Since nocodazole inhibits endosome maturation, we presume that anti-Tac and LDL label sorting endosomes under these conditions. In contrast, treatment of TRVb-1/Tac-TGN38 cells with nocodazole had little effect on the transport of internalized anti-Tac antibody to the TGN , and the antibody exhibited limited overlap with internalized LDL . These results support the proposal that Tac-furin is directed to the TGN by way of late endosomes, whereas Tac-TGN38 travels via the endocytic recycling pathway. A second reagent having relevant effects on vesicular transport is the phosphatidylinositol 3-OH kinase (PI3 kinase) inhibitor wortmannin. Wortmannin exerts numerous effects on endosomal trafficking, including the inhibition of early endosome fusion , the swelling of late endosomes and impaired transport of hydrolases to lysosomes from the TGN , acceleration of the rate of transferrin internalization , and a moderate slowing of endocytic recycling . However, wortmannin allows the delivery of internalized material to late endosomes . These effects were also observed in TRVb-1 cells expressing Tac-furin or Tac-TGN38 (data not shown). Given these findings, we evaluated whether or not wortmannin would affect the transport of Tac-TGN38 or Tac-furin to the TGN from the plasma membrane. TRVb-1/Tac-TGN38 and TRVb-1/TTF cells were pretreated with 100 nM wortmannin or 0.1% DMSO alone as described in Materials and Methods. The cells were then incubated with fluorescently labeled anti-Tac antibodies, alone or in combination with fluorescent LDL for 15 min, then chased for 45 min in the presence or absence of wortmannin. After fixation, cells that received anti-Tac alone were stained with NBD-C 6 -ceramide to label the TGN. Samples that were not subjected to a chase were also analyzed to verify the efficient internalization of the probes irrespective of wortmannin treatment (data not shown). After 45-min chase, anti-Tac mostly labeled the TGN in mock-treated cells, as observed previously (data not shown). The trafficking of Tac-furin was severely inhibited by wortmannin treatment. Under these conditions, internalized Tac-furin was delivered to the TGN only very inefficiently, instead remaining colocalized with LDL . Note the nebulous appearance of the anti-Tac and LDL labeling in Fig. 9A , Fig. C , and Fig. D ; this pattern is presumably due to the effects of wortmannin on the morphology of endosomes. The appearance of Tac-TGN38 in the TGN also was reduced upon treatment with the drug, presumably due to slower transit of the chimera through the endocytic recycling pathway. However, significant colocalization of Cy3–anti-Tac and NBD-C 6 -ceramide was apparent at the end of the 45-min chase , increasing with a more prolonged chase (data not shown). Before delivery to the TGN, Tac-TGN38 remained colocalized with transferrin, and at no point was overlap with internalized LDL observed (data not shown). Given the reported activities of wortmannin and our own observations, the most straightforward conclusion is that Tac-furin trafficking is blocked from late endosomes to the TGN. This may represent a direct dependence of this transport event on PI3-kinase activity, or may be an indirect consequence of inhibiting the delivery of transport factors to late endosomes. Irrespective of the precise point of action of wortmannin, the absence of accumulation of Tac-TGN38 in LDL-containing endosomes under these conditions underscores the different pathways that these chimeras take in the endosomal system. Over the past several years, numerous reports have described the transport of certain membrane-associated proteins between the endosomal system and the secretory pathway, specifically the TGN. However, the exact route by which proteins are delivered to the TGN usually has not been specified. This is significant since the transport pathway determines where protein sorting must take place and also may suggest the mechanism of sorting. We demonstrated recently the postendocytic transport of a chimeric transmembrane protein, Tac-TGN38, to the TGN via the endocytic recycling pathway . In the present study, we have shown that another chimeric protein which is localized to the TGN, Tac-furin, is delivered there from late endosomes and does not traverse the recycling pathway. Therefore, CHO cells have at least two distinct mechanisms for endocytic transport of membrane proteins to the TGN . Selective removal of Tac-TGN38 from the recycling pathway apparently occurs from the ERC. In contrast, Tac-furin is segregated from recycling proteins at an earlier step, the sorting endosome, allowing it to enter late endosomes. Furthermore, whereas Tac-TGN38 is removed from the recycling pathway and delivered to the TGN in an iterative manner, Tac-furin is segregated from late endosomes by a more efficient, single-pass mechanism, possibly in one concerted step. The steady-state localization of a protein is determined by the slowest step in its trafficking. Irrespective of the transport itineraries and sorting mechanisms involved, both Tac-furin and Tac-TGN38 are localized to the TGN predominantly through their relatively slow rates of exit from that compartment. This is likely reflected in the rates of transport of the proteins from internal sites to the plasma membrane, which are slower than all other kinetic steps measured for each chimera. In the case of Tac-furin, the kinetics of exit from the cell appears to be independent of the transmembrane domain. However, this may not be the case for Tac-TGN38 . Tac-TGN38 exits the cell significantly slower than does Tac-furin , implying a possible role of the TGN38 transmembrane domain in retention of the chimera in the TGN. The two distinct endosomal pathways, which diverge at the level of the sorting endosome, indicate at least two sorting steps that are involved in transport to the TGN. Tac-TGN38 traverses the recycling pathway that is followed by the bulk of internalized membrane proteins in CHO cells in the absence of specific targeting. From some point along this pathway, Tac-TGN38 is then diverted for delivery to the TGN. This process must require a specific property of Tac-TGN38, such as an amino acid motif or selective partitioning into membrane domains due to the protein's physical characteristics. The cytoplasmic domain sequence, SDYQRL, appears to fulfill at least part of this sorting function . However, the SDYQRL motif is not sufficient for delivery from the ERC to the TGN . Additionally, the similarity of the trafficking itinerary of the Shiga toxin B fragment, a soluble protein which binds to the glycolipid globotriaosylceramide , suggests that lipid-based sorting may also be critical along this pathway . We have not determined the point of departure of Tac-TGN38 from the recycling pathway. The most likely possibility is that it exits from the recycling compartment, although a direct pathway from sorting endosomes cannot be excluded at present. The itinerary followed by Tac-furin is now more completely described. This protein evades the endocytic recycling pathway, neither appearing in the ERC nor rapidly returning to the plasma membrane. Rather, Tac-furin transits from sorting endosomes to late endosomes, which must require high-fidelity, active sorting of Tac-furin at the sorting endosome. From late endosomes, Tac-furin is transported to the TGN, apparently before the accumulation of CI-MPR in late endosomes. This late endosome to TGN step may also require active sorting, or this may be the pathway taken by most membrane proteins that have accessed late endosomes, in the absence of a positive sorting signal. There is evidence that internalized membrane proteins such as the transferrin and LDL receptors may be transported from endosomes to the TGN at a low constitutive rate . However, Tac-furin is delivered to the TGN with much higher efficiency than those receptors. Also, the transport of the cation-dependent mannose 6-phosphate receptor to the TGN requires an amino acid sequence in its cytoplasmic domain, and the receptor is degraded in lysosomes if this signal is disrupted . Whether dependent on a positive signal or not, the kinetics of separation of Tac-furin from LDL indicate that the mechanism for delivering Tac-furin to the TGN must be able to segregate a membrane-associated molecule from a soluble molecule very efficiently. This mechanism must also allow the efficient transport of growth factor receptors from late endosomes to lysosomes and not to the TGN. A series of reports has demonstrated the importance of an acidic cluster in the furin cytoplasmic domain for its delivery to the TGN , and specifically, the phosphorylation of serine residues within this region appears to be a major regulator of furin endosomal transport , possibly directing furin into a rapid recycling pathway. Extensive and detailed analyses will be required to determine at which steps the acidic cluster, phosphorylation, and other putative sorting signals are required. The different endosomal pathways followed by Tac-furin and Tac-TGN38 must require different sorting mechanisms, as supported by studies using nocodazole and wortmannin. The inhibition of Tac-furin transport by nocodazole is readily explained, since the chimera is delivered via late endosomes, and entry of endocytosed proteins into late endosomes is blocked by nocodazole. The absence of an effect on Tac-TGN38 transport is consistent with the properties of the endocytic recycling pathway, although it would not necessarily be predicted that a route linking the recycling pathway and the TGN would also be independent of microtubules. Our data also suggest that transport from late endosomes to the TGN depends on PI3 kinase activity since wortmannin apparently causes internalized Tac-furin to accumulate in late endosomes rather than in the TGN. This may reflect a general requirement for PI3 kinase in late endosome to TGN trafficking, or alternatively a specific role of PI3 kinase in the pathway that transports furin. Analyses of the effects of wortmannin treatment on endosome-to-TGN trafficking in other systems have yielded conflicting results . An extensive series of studies by Thomas and co-workers has revealed possible roles for a number of proteins, including phosphofurin acidic cluster sorting protein 1 (PACS-1) , actin binding protein of 280 kD , and protein phosphatase 2A , at various stages of furin transport to modulate either delivery to or retention in the TGN. Specifically, PACS-1 interacts with the furin acidic domain. PACS-1 also interacts with the MPR/IGF-II receptor but not with the primate TGN38 homologue, TGN46 , further supporting a role for PACS-1 in endosomal sorting. This interaction requires the phosphorylation of the furin cytoplasmic domain, which has also been shown to be a modulator of the intracellular distribution of furin . The TGN38 cytoplasmic domain can also be phosphorylated in vitro with effects on its protein interactions , and like furin the endocytic transport of TGN38 may be regulated by phosphorylation. However, the factors that are responsible for TGN38 sorting after internalization have not been conclusively identified. These may include as yet unidentified coat proteins and also may involve Rab11 and/or other regulatory GTPases. Careful examination of each step in endosomal transport will be necessary to assign these various species to their respective sites of action. | Study | biomedical | en | 0.999997 |
10469726 | Heterotypic gap junctions formed by two closely related connexins (Cx), 1 Cx32 and Cx26, display marked but different asymmetries in the transjunctional voltage (V j ) dependence of initial and steady state conductances . The reduction in the steady state conductance of Cx32/Cx26 heterotypic junctions that occurs only when the Cx26 side of the junction is depolarized relative to the Cx32 side is a consequence of the opposite gating polarity of a V j gate found in each of the two apposed hemichannels that form the intercellular junction. Cx26 hemichannels close upon the application of relatively positive transjunctional potentials, whereas Cx32 hemichannels close upon the application of relatively negative transjunctional potentials . Thus, in Cx32/Cx26 junctions one or both gates are closed by adequate positivity on the Cx26 side and neither is closed by positivity on the Cx32 side. Less is known about the mechanism underlying the V j dependence of initial conductance in the heterotypic Cx32/Cx26 junction. The demonstration that the rectification of single channel currents of the heterotypic Cx32/Cx26 junction parallels the voltage dependence of initial conductance indicates that the underlying mechanism involves changes in ion fluxes through the open channel rather than gating. Although considerably less steep, the rectification of initial currents observed in the heterotypic Cx32/Cx26 junction qualitatively resembles that of a rectifying electrical synapse present in the abdominal nerve cord of the crayfish and one found between giant fibers and motorneurons in hatchetfish that is formed by gap junction channels . In these electrical synapses, depolarizations but not hyperpolarizations of the presynaptic fiber produce virtually instantaneous changes in the potential of the postsynaptic fiber. Voltage clamp studies of the crayfish rectifying electrical synapse demonstrated that junctional currents elicited by presynaptic depolarization reach steady state within 800 μs and rectify extremely steeply, increasing e -fold per 4–5 mV of applied voltage . In their original report, Furshpan and Potter 1959 hypothesized that the junctional membrane behaved as an “electrical rectifier” or diode rather than a simple electrical resistor. The separation of fixed positive and negative charges across the junctional membrane resulting from the pairing of hemichannels with opposite charges at their channel entry could form a diode (p-n junction). Mauro 1962 and Finkelstein 1963 have examined the electrical characteristics of p-n junctions formed by fixed charged membranes. The steepness of the current–voltage (I–V) relations attainable by p-n junctions provides an attractive model for the generation of steeply rectifying electrical synapses. However, Jaslove and Brink 1986 provided evidence that the crayfish rectifying synapse contains a voltage-dependent gate, as kinetic components (with a time constant of ∼7.5 ms) could be resolved upon cooling the preparation to 9.5°C. They hypothesized that the steep rectification results from a structurally asymmetric junction, in which one hemichannel contains a “fast” voltage-dependent gate that closes upon depolarization of the presynaptic element. However, a substantial fast kinetic component remained in the cooled preparation. This result suggests that an additional process, unrelated to voltage-dependent gating, may also be involved. Giaume et al. 1987 also suggested that the crayfish synapse was structurally asymmetric and suggested that the steep rectification was a consequence of a highly voltage-dependent probability of opening of one of the hemichannels that forms the rectifying synapse. The connexins forming rectifying electrical synapses in vertebrates and invertebrates have not yet been identified and indeed the invertebrate gap junctions are formed by proteins encoded by another gene family with no primary sequence homology to the vertebrate connexin gene family . Although the rectification observed for Cx32/Cx26 heterotypic junctions is much less steep than that observed in rectifying synapses, the elucidation of the molecular determinants underlying the rectification of initial conductance in these channels may shed light on the mechanism governing the rectification of electrical synapses. The results of the molecular genetic studies described in this paper demonstrate that charged amino acid residues located in the amino terminus and first extracellular loop are major determinants of the rectification of the heterotypic Cx32/Cx26 channel. The rectifying I–V relation of the heterotypic channel results from a structural asymmetry arising from differences in the number and position of fixed charges present in the Cx32 and Cx26 hemichannels. The different I–V relations of homotypic channels can be explained by the different symmetrical charge distributions that result from homotypic pairings. Furthermore, we show that homotypic and heterotypic channels can enter substates that rectify. The nonlinearity of the I–V relations of these substates is likely to result from the increased electrostatic effect of charged residues present in the amino terminus of one hemichannel that arises from a conformational change associated with V j -dependent gating. This finding suggests that the change in conductance resulting from V j gating may correspond to a narrowing of the entry of the connexin hemichannel near the cytoplasmic surface. We suggest that the rectification of single channel currents like that observed in the fully open state and substates of gap junction channels is responsible for the steep and rapid rectification observed in some electrical synapses. Chimeras of Cx26 and Cx32 were constructed by the procedure described by Rubin et al. 1992a or, when possible, by using restriction enzyme sites that are conserved among connexins and chimeric constructs. In this study, we define for Cx26: the NH 2 terminus (NT) as containing amino acids 1–22 inclusively, the first transmembrane domain (TM1) amino acids 23–40, the first extracellular loop (E1) amino acids 41–75, the second transmembrane domain (TM2) amino acids 76–93, the cytoplasmic loop (CL) amino acids 94–131, the third transmembrane domain (TM3) amino acids 132–151, the second extracellular loop (E2) amino acids 152–189, the fourth transmembrane domain (TM4) amino acids 190–209, and the COOH terminus (CT) amino acids 210–226. Cx32 has one fewer amino acid in the CL domain than does Cx26. Consequently, CL of Cx32 consists of amino acids 94–130, TM3 amino acids 131–150, E2 amino acids 151–188, TM4 amino acids 189–208, and CT amino acids 209–283. In our notation, a chimera designated as Cx32*Cx26E1 has the sequence of the E1 domain of Cx32 replaced with that of Cx26. In a chimera designated as Cx32*Cx26(NT 1–11 + CL), the first 11 amino acids of the NH 2 terminus of Cx32 are replaced with the first 11 amino acids of the NH 2 terminus of Cx26 and the CL domain of Cx32 is replaced with the CL domain of Cx26. A chimera designated as Cx32*Cx26(CL–CT) would have the entire region spanning the cytoplasmic loop through the COOH terminus of Cx32 (amino acids 94–283) replaced with the equivalent region of Cx26 (amino acids 94–226). Site-directed point mutations were constructed using oligonucleotide primers and the polymerase chain reaction. All chimeric constructs and point mutations were cloned into the plasmid vector, pGEM-7zf (+) (Promega Corp.) and sequenced in entirety. RNA was transcribed in vitro from linearized plasmid templates as described in Rubin et al. 1992a . For oocyte expression of cloned connexins, approximately 50 ηl of 1 ηg/ηl RNA was coinjected with 0.3 ρmol/ηl of an antisense phosphorothioate oligonucleotide complimentary to Xenopus Cx38. This antisense oligonucleotide blocks all endogenous coupling between oocyte pairs attributable to Cx38 within 72 h . Oocytes were devitellinized and paired 12–24 h after RNA injection. Expression of junctional currents usually developed within 4 h of pairing. Only cells expressing <5 μS junctional conductance were employed to minimize the effects of access resistance on voltage dependence . Recordings were obtained with a dual voltage clamp with glass electrodes containing 1 M KCL solutions. Pipette resistance was ∼5 MΩ. Coupled oocytes had equal resting membrane potentials that varied between −30 and −60 mV. Cells were voltage clamped to their resting potential (0 mV transjunctional voltage) and a family of junctional currents for each cell pair was generated by applying transjunctional voltages of ±5–125 mV in an ascending series of 5-mV increments. Total pulse length was 20 s with a 90-s interpulse interval. Currents were digitized at two rates, first at 256 Hz for 2 s, and then at 28 Hz for the remainder of the trace. This allowed for greater accuracy in the measurement of initial currents while not requiring the collection of an excessive number of points during the extended time necessary for junctional currents to reach steady state. Each transjunctional voltage step was preceded by a 10-mV prepulse of short duration (100–500 ms) that was used to normalize junctional currents within a family of current traces. In heterotypic junctions, current traces were scaled independently to prepulses of appropriate polarity of V j since the prepulse amplitude elicited by a hyperpolarizing prepulse would not be the same as for a depolarizing prepulse administered to the same cell. Initial and steady state currents were determined by extrapolation of exponential fits to t = 0 and t = ∞. Initial ( g 0 ), steady state ( g ∞ ), and residual ( g min ) conductances were normalized to the value of initial conductance, g 0 at V j = 0 to obtain G 0 , G ∞ , and G min . Additional details are provided in Rubin et al. 1992a ,Rubin et al. 1992b and Verselis et al. 1994 . The coding regions of wild-type and mutant connexins were cloned into pCI-neo (CMV promoter) vector (Promega Corp.) and transfected into the mouse Neuro-2a cell line and a second Neuro-2a cell line that expressed green fluorescent protein (GFP). Heterotypic channels were formed by mixing cell lines expressing different connexins, in which one member of the cell pair could be identified by the presence of GFP. In some cases, GFP expression was linked to that of a given connexin by the presence of an internal ribosomal entry site (IRES) sequence provided by H.-S. Shin (POSTECH, Pohang, South Korea). Transfected cell lines expressing exogenous connexins but not Cx45, which forms endogenous channels in Neuro-2a cell lines, were selected for single channel analysis. Cx45 channels are easily distinguished by their single channel conductance (∼30 pS slope conductance), lack of substantial residual conductance states, and steep voltage dependence. Single channel and macroscopic (10–20 channels) records of gap junctions channels were obtained using double whole cell patch recordings as described in Oh et al. 1997 . Cells were held at 0 mV, their resting potential in the recording solutions used. Unless otherwise specified, data were acquired using pCLAMP 6.0 software, filtered at 1 kHz with a four-pole low pass Bessel filter and digitized at 5 kHz using Axopatch 200B integrating patch amplifiers and a Digidata 1200A interface (Axon Instruments). Pipette resistances were 5–10 MΩ. All points histograms were constructed with a bin size of 0.05 pA and fit to a Gaussian using Origin 4.0 software (Microcal Software Inc.). The Poisson-Nernst-Plank (PNP) model used is that of Chen and Eisenberg 1993 . Computer programs used to solve the PNP equations were downloaded from http://144.74.27.66/pnp.html. Additional details are provided in the discussion . Conductance–voltage relations and representative current traces of homotypic Cx32, Cx26, and heterotypic Cx32/Cx26 gap junctions expressed in pairs of Xenopus oocytes are shown in Fig. 1 . Both the initial and steady state conductance–voltage relations of homotypic gap junctions formed by Cx32 are symmetric about V j = 0 . Initial conductance (▾) is maximal at V j = 0 and reduced by ∼30% at large transjunctional voltages. At large transjunctional voltages, the steady state conductance–voltage relation decreases to a minimal conductance, G min , of ∼0.3. In Cx26 homotypic junctions , initial conductance displays some dependence on the absolute voltage difference between the inside and outside of the cells (termed V m or V i–o ), detected as asymmetry between the effects of depolarizing one cell and hyperpolarizing the other to the same extent . Junctional currents relax slowly for either polarity of V j and conductance decreases to a minimum at higher transjunctional voltages, corresponding to a G min of ∼0.2 at positive transjunctional voltages. There is a small asymmetry in the steady state conductance–voltage relation depending on whether one cell is hyperpolarized or depolarized. As reported previously , heterotypic junctions formed by Cx32 and Cx26 are characterized by an asymmetric conductance–voltage relation that depends only on V j . The rectification of initial currents results in an increase in conductance when the cell expressing Cx26 is made relatively positive and a decrease in conductance when this cell is made relatively negative. Typically, initial conductance is approximately three times greater when the Cx26-expressing cell is stepped to V j = +120 mV than when it is stepped to V j = −120 mV. The steady state conductance normalized to the initial conductance decreases only when the Cx26 side of the heterotypic junction is stepped to positive transjunctional potentials exceeding 40 mV. G min /G 0 is ∼0.2. A record of a homotypic rat Cx32 single channel between Neuro-2a cells is shown in Fig. 2 . The single channel properties of rat Cx32 are similar to those that we have reported for human Cx32 . The primary sequence of rat and human Cx32 differ by only four amino acid residues. In the segments of the single channel records shown in Fig. 2A and Fig. B , V j steps of ±60 mV were applied from a holding potential of 0 mV by polarizing one member of the cell pair at the times denoted by the numeral 1 in the figure. The changes in current, recorded from the unstepped cell, correspond to a junctional conductance of ∼68 pS in both cases, which we ascribe to the conductance of the fully open channel. The boxed portion of the record shown in Fig. 2 A illustrates the presence of three different subconductances in rat Cx32 homotypic junctions: ∼26 pS (S1), ∼19 pS (S2), and ∼10 pS (S3), determined from the all points histogram shown in Fig. 2 D. We believe that these subconductances correspond to at least three distinct subconductance states of rat Cx32 homotypic channels . The average conductance, determined from an all-points histogram of the entire trace (data not shown), is ∼20 pS, a value that corresponds to a 75% reduction in the conductance of the fully open state. This value is comparable to the G min /G 0 of 0.3 observed in macroscopic recordings of Cx32. This suggests that the voltage-dependent entry of the channel into one of several possible subconductance states is a major determinant of the residual conductance observed macroscopically at large transjunctional voltages . Cx32 channels can also display gating that involves a complex series of transitions between fully open and fully closed states . Although not demonstrated in this record, complete closure of the rat Cx32 channel by these transitions can occur from either the fully open state or the subconductance states. This gating is similar in appearance to the slow gating during block by CO 2 and recovery , the ionic currents expressed during channel formation, termed “docking or formation currents” by Bukauskas et al. 1995b , and to the transitions between fully closed and open states in Cx46 hemichannels, termed “loop gating” by Trexler et al. 1996 . Trexler et al. 1996 suggested that “loop gating” involves a series of conformational changes involving the extracellular loops of the Cx46 hemichannel. In contrast, V j -dependent gating involves conformational changes that result in the entry of the channel into one of several possible discrete subconductance states. Bukauskas and Weingart 1994 have noted the similarity of the complex transitions observed in vertebrate intercellular channels to V m gating observed in insect gap junctions. The partial closures of the channel observed between positions 1 and 2 in Fig. 2 B likely correspond to loop gating transitions that did not result in complete channel closure. The record also is suggestive of additional complexities in V j -dependent gating. Homotypic rat Cx32 channels appear to be able to enter into either long- or short-lived subconductance states. For example, at the beginning of the trace shown in Fig. 2 A, the channel flickered extensively, often entering a residual conductance state for <100 ms (exemplified by the transitions at position 2). In contrast, at position 3, the channel entered a 19-pS state that lasted 30 s and, later in the record, the channel entered a 10-pS state from which it had not exited when the V j step was terminated 20 s later. In other records, we have observed that a Cx32 homotypic channel can remain in a residual conductance state for several minutes (data not shown). The presence of short- and long-lived subconductance states is also evident in the records shown in Fig. 2 E and 3. Fig. 2 E shows that the I–V relation of the fully open state of a single Cx32 channel is nearly linear, although currents do decrease slightly, by ∼5%, for either polarity of large transjunctional voltages. A similar deviation from linearity is also observed in the conductance–voltage relations of initial currents of Cx32 recorded macroscopically in Neuro-2a cell lines that are expressing 15–20 intercellular channels . Fig. 3 illustrates the gating and I–V relations of Cx32 subconductance states as well as the fully open state. When the channel resided in the fully open state, the application of a 500-ms voltage ramp from −100 to + 100 mV elicited a junctional current that only slightly deviates from linearity. However, when the channel resided in a long-lived subconductance state, the application of the same voltage ramp resulted in a nonlinear change in junctional current . In this case, chord conductance decreased approximately twofold as the V j ramp became more positive (from 2.4 pA at −100 mV to 1.2 pA at +100 mV). The return of junctional current to the same level (2.4 pA) as measured before the application of the voltage ramp makes it unlikely that the nonlinearity of junctional currents resulted from gating transitions between different subconductance states that could not be resolved in the record. Furthermore, reversing the time course of the voltage ramp (from +100 to −100 mV) while the channel resided in the same subconductance state reversed the time course of the rectification; junctional current now increased (from 1.2 to 2.0 pA) as the voltage ramp became more negative (not shown). Based on the negative V j gating polarity of hemichannels formed by Cx32 , it is likely that the negative voltage step caused the closure of the hemichannel in the stepped cell. Fig. 3 D illustrates the rectification observed in another cell pair using a similar test paradigm but starting from an initial voltage step of +100 mV and applying a voltage ramp of shorter duration (100 ms). Unlike the previous example, the application of a positive voltage to the stepped cell is expected to cause closure of the V j gate in the hemichannel present in the unstepped member of the cell pair. In this case, junctional currents decreased nonlinearly as the voltage was ramped from +100 to −100 mV. The difference in the direction of rectification in these two cases is a consequence of the closure of opposite hemichannels. In the former case , it is likely that the V j gate in the hemichannel in the stepped cell closed, whereas in the second case , it is likely that the V j gate in the hemichannel in the unstepped cell closed. In other instances, we have observed that the I–V relations for other Cx32 substates do not rectify appreciably (data not shown). Thus, it appears that Cx32 hemichannels may adopt several different closed conformations. Although we have not examined the I–V relations of all substates extensively, the available data suggest that the longer lasting substates rectify. The linearity of the I–V relation of the fully open Cx32 channel differs from expectation based on the 30% reduction in initial conductance observed for Cx32 homotypic junctions formed between pairs of Xenopus oocytes. Differential modification of the protein subunits or the presence of ancillary proteins in one of the two expression systems is unlikely to cause the different behavior of the initial currents, because initial currents in Cx26/Cx32 heterotypic junctions are almost identical in oocytes and transfected Neuro-2a cell lines . A possible explanation, described in the discussion , is that the rectification observed in oocytes arises from the difference in the intracellular ion composition of the two experimental systems. The concentration of permeable anions should be less in oocyte cell pairs than in Neuro-2a cells dialyzed with CsCl patch recording solutions. A record of a single homotypic Cx26 channel between Neuro-2a cells is shown in Fig. 4 A. Initially, the cells appeared to be uncoupled, as the application of a +60-mV voltage step to one member of the cell pair did not change the current measured in the unstepped cell. The channel opened subsequently via a series of complex, poorly resolved events that resemble “loop gating” or “docking/formation currents.” In the segment of the trace shown in Fig. 4 B, , the channel opened and closed by a similar series of complex gating events while a V j of −60 mV was applied. In the record shown, the conductance of the fully open channel is 130 pS. In other records, we have observed the conductance of the fully open Cx26 channel to be ∼150 pS at ±60 mV. There was a single V j -dependent gating event corresponding to a 104-pS transition that resulted in a 26-pS residual conductance state (substate). The residual conductance, ∼20% of the fully open state, is in agreement with the G min /G 0 of ∼0.2 that is observed in macroscopic recordings. The relatively few gating transitions observed at ±60 mV is consistent with that expected, given a V 1/2 of ∼90 mV determined from the macroscopic conductance–voltage relation shown in Fig. 1 B. The channel shows more gating at higher transjunctional voltages (±120 mV). It is likely that homotypic Cx26 channels may also enter into one or more substates with residual conductances lying between 10 and 30 pS, as shown in the current trace and all points histogram . Cx26 junctional currents characteristically appear noisier when the channel resides in a high conductance state than when it resides in either a subconductance or the fully closed state. This suggests the existence of multiple open states whose average conductance is ∼130–150 pS. The I–V relation of the open state of Cx26 homotypic channels is linear . In contrast to the expression of Cx26 in oocytes, there does not appear to be any dependence on V m in Cx26-transfected cell lines, as the I–V relation is symmetric at V j = 0 over a ±120-mV ramp. The insensitivity of Cx26 hemichannels to V j (V 1/2 = ±90 mV) and the rapid opening of Cx26 hemichannels upon stepping to smaller transjunctional voltages (not shown) precluded the examination of the I–V relation of Cx26 substates. A record of a heterotypic Cx32/Cx26 channel between Neuro-2a cells is shown in Fig. 5 . Both cells were initially held at 0 mV. At position 1 in the trace shown in Fig. 5 A, the cell expressing Cx32 was stepped to −80 mV, eliciting a junctional current corresponding to a conductance of 120 pS . At position 2, the channel underwent an ∼95-pS transition, which resulted in an ∼25-pS subconductance state. The trace presented in Fig. 5 B and the corresponding all points histogram demonstrates the existence of at least three additional substates of ∼20, 30, and 40 pS in the heterotypic channel. We ascribe these transitions to subconductance states resulting from V j -dependent gating that could have occurred if either the V j gate in the Cx32 or the Cx26 hemichannel or both gates had closed. Recall that the polarity of V j -dependent gating is opposite in the two hemichannels with Cx32 closing for relatively negative V j at its cytoplasmic face and with Cx26 closing for relatively positive V j . The rectification of ionic currents through the fully open heterotypic channel is shown in Fig. 5A and Fig. F . In Fig. 5 A, the voltage was stepped from −80 to +80 mV at position 3. The conductance of the fully open channel was ∼2.5-fold greater at −80 (120 pS) than at +80 (50 pS) mV, . Fig. 5 F shows the I–V relation obtained by applying a ±120-mV ramp to the cell pair expressing Cx26. In this record, single channel currents rectify approximately threefold over the ±120 mV range. Rectification of the same polarity was also observed in a substate of the heterotypic channels. In Fig. 5 E, position 1, the voltage was stepped briefly from −100 to 0 mV, and then to +100 mV, but in this case the polarity of the V j step was reversed while the channel resided in a 25-pS substate. The channel remained in this substate briefly before transiting to the fully open state. The conductance of the substate at +100 mV was 9.5 pS, compared with 25 pS at −100 mV, corresponding to a rectification of ∼2.5-fold. The degree and polarity of rectification of this substate is similar to that observed for the rectification of the fully open state in this record (125–45 pS or 2.8-fold at comparable voltages). We observed discrete gating transitions (V j dependent) from the fully open state when the Cx26-containing cell was depolarized or the Cx32-expressing cell was hyperpolarized. This result is consistent with the observed relaxation of steady state currents in macroscopic recordings of Cx32/Cx26 heterotypic junctions. On occasion, we observe complete channel closure via “loop gating” during the application of either polarity of V j in this heterotypic junction (data not shown), which supports the hypothesis that this gating occurs by a mechanism distinct from V j -dependent gating. If the “loop gate” is weakly voltage dependent, then this observation could explain the slight difference in the initial and steady state conductance–voltage relations that is observed in macroscopic records of some heterotypic junctions at polarities of transjunctional voltage that should not close the V j gate. For example, there is a decrease in steady state junctional conductance at negative V j in the conductance–voltage relations in Fig. 6A and Fig. B . As a means of establishing the mechanism underlying the rectification of junctional currents, we sought to localize the molecular determinants of the process. Our approach can be summarized by the following question: What amino acid residues in Cx26 and Cx32 must be exchanged to interconvert their I–V relations in heterotypic as well as homotypic junctions? The difference in the gating polarity of the steady state V j dependence of Cx32 and Cx26 hemichannels is due to a single amino acid difference in the sequence of the NH 2 terminus of these two connexins . The replacement of the neutral asparagine residue (N2) normally present in Cx32 with the negatively charged aspartate residue (D2) found in wild-type Cx26 reverses the V j gating polarity of Cx32 hemichannels. Similarly, the reciprocal substitution (Cx26D2N) reverses the gating polarity of Cx26, from closure on relatively positive V j to closure on relatively negative V j . We proposed that amino acid residues at this position form a vestibule for the channel pore by virtue of the creation of a reverse turn in the NH 2 terminus that is initiated by a conserved glycine residue at position 12 . Thus, it is likely that a negative (D2 in Cx26) and a positive (associated with the NH 2 -terminal Met residue in both Cx32 and Cx26) charge reside near the cytoplasmic entry of the hemichannels formed by these two connexins. Our previous work also demonstrated that charge substitutions of amino acid residues located at the border of the first transmembrane domain and first extracellular loop may also reverse gating polarity. Consequently, we first examined the role of the amino terminus and first extracellular loop in the voltage dependence of initial currents. The results we present in the following sections show that charged amino acid residues in the amino terminus of Cx26 and Cx32 and in the extracellular loop of Cx26 play a major role. Fig. 6 A illustrates that the initial currents of the heterotypic Cx32/Cx32N2D junctions rectify similarly to those observed in wild-type Cx32/Cx26 heterotypic junctions when expressed in pairs of Xenopus oocytes. The effect appears to be due to the electrostatic contribution of the N2D mutation, as the voltage dependence of the initial conductance of Cx32N2E is similar to that of Cx32N2D , and substitutions with neutral amino acids Cx32N2Q and Cx32N2A have little or no effect on the voltage dependence of initial conductance (data not shown). These results suggest that charged amino acid residues in the amino terminus of Cx32 play a substantial role in the rectification of initial currents. However, studies of chimeras in which the first 11 amino acids of Cx26 were substituted for those of Cx32 suggest that residues in other domains may also play a role. For example, the chimera Cx32* Cx26(NT 1–11 ), which results in the substitution of seven amino acid residues in Cx32, including N2D, did not express junctional currents when paired homo- or heterotypically with Cx26 or Cx32. This result was unexpected as the reciprocal domain substitution, Cx26* Cx32(NT 1–11 ), forms functional channels [not shown, see also Verselis et al., 1994, where Cx26*Cx32(NT 1–11 ) is designated as Cx26*32NT-V13]. Expression of junctional currents can be restored if the Cx32*Cx26(NT 1–11 ) chimera also contains the cytoplasmic loop of Cx26; i.e., Cx32*Cx26(NT 1–11 +CL). This result suggests that the amino terminus and cytoplasmic loop of Cx26 may interact directly and that this interaction may be required for the expression of junctional currents. The conductance–voltage relation of the heterotypic Cx32/Cx32*Cx26(NT 1–11 +CL) junction is shown in Fig. 6 C. Notably, the initial conductance of this junction rectifies more steeply (five- vs. threefold over ±120 mV) than does the initial conductance of Cx32/Cx26 junctions . Other Cx32 chimeric junctions, which contain the cytoplasmic loop of Cx26; Cx32*Cx26(CL–CT)/Cx26 and Cx32* Cx26CL/Cx26 (not shown) also display increased steepness in initial conductance. The behavior of the heterotypic junction Cx26* Cx32(CL–CT)/Cx26, in which the CL through CT domain of Cx26 is replaced by that of Cx32, is interesting as the initial conductance increases at larger transjunctional voltages of either polarity of V j . This change in initial conductance corresponds to a super-linear I–V relation. Also, the gating polarity of the Cx26*Cx32(CL–CT) hemichannel appears to be reversed as steady state conductance only decreases when the Cx26 side of the junction is relatively positive or the Cx26*Cx32(CL–CT) side of the junction is relatively negative. This result is surprising as both Cx26 and Cx26*Cx32(CL–CT) hemichannels have a negatively charged residue at the second position (D2). We suggest that the chimera has a different conformation that effectively reduces the electrostatic contribution of the D2 residue to the voltage sensor of the Cx26* Cx32(CL–CT) hemichannel. We did not observe the expression of junctional currents in homotypic pairings of this hemichannel. The voltage dependence of initial currents in homotypic junctions formed by Cx32*Cx26(NT 1–11 +CL), and Cx32N2E is substantially greater than that observed in Cx32 homotypic junctions. Initial conductance declines by ∼50% over ±120 mV j in both Cx32N2E and Cx32*Cx26(NT 1–11 +CL) homotypic junctions. In addition, the shape of the initial conductance–voltage relations of these homotypic junctions differs markedly from that of Cx32 homotypic junctions, declining at small as well as large transjunctional voltages. The initial conductance of heterotypic junctions Cx32N2D/Cx26, Cx32N2E/Cx26, and Cx32*Cx26(NT 1–11 +CL)/Cx26 decreases for either polarity of applied V j , but substantially more when the Cx26 side of the junction is relatively positive. The conductance–voltage relation of Cx32N2D/Cx26 exemplifies the behavior of these junctions. The single channel I–V relations in Neuro-2a cells of the fully open Cx32*Cx26(NT 1–11 +CL) channel in homo- and heterotypic pairings with Cx32 and Cx26 are shown in Fig. 7 . In the case of the homotypic channel, Cx32*Cx26(NT 1–11 +CL), the I–V relation of the fully open channel is sigmoidal. Currents decline by ∼35–40% at ±120 mV from that predicted by a linear fit of the current trace at ±20 mV . The I–V relation of the fully open Cx32*Cx26(NT 1–11 +CL)/Cx26 channel is asymmetric, decreasing more when the Cx26 side of the channel is relatively positive than when it is relatively negative . Fig. 7 C illustrates that junctional currents rectify when the Cx32/Cx32*Cx26(NT 1–11 +CL) heterotypic channel resides in the fully open state; increasing ∼4.5-fold (±120 mV) when the Cx32 cell is made relatively negative (note that there are two channels in this record). This rectification is greater than that observed for wild-type Cx32/Cx26 heterotypic junctions . In all cases, the degree and direction of rectification of Cx32*Cx26(NT 1–11 +CL) channels in Neuro-2a cells is reasonably well predicted by the macroscopic behavior of initial currents of this junction when they are expressed in pairs of Xenopus oocytes. There is similar correspondence between the single channel I–V relation and the macroscopic conductance–voltage relation of the Cx32N2E homotypic junction (data not shown). Fig. 7 D shows the I–V relation of a Cx32*Cx26 (NT 1–11 +CL)/Cx26 channel substate that was obtained by applying a −120- to +120-mV ramp to the Cx26 side of the channel. The chord conductance of this substate decreased nonlinearly as the Cx26 side of the junction became more positive. Given the positive gating polarity of both Cx26 and Cx32*26(NT 1–11 +CL) hemichannels, this substate most likely resulted from the closure of the Cx32*Cx26(NT 1–11 +CL) hemichannel as the channel entered the substate when the Cx32*Cx26 (NT 1–11 +CL) cell was relatively positive. In the case of the homotypic Cx32*Cx26(NT 1–11 +CL) channel illustrated in Fig. 7 E, single channel currents rectified when the hemichannel in the unstepped cell entered a substate in response to the initial positive polarity of the voltage ramp that was applied to stepped cell (ramped from +120 to −120 mV). This would favor the closure of the V j gate in the stepped cell as this cell would be relatively positive. As in Fig. 7 D, chord conductance decreased nonlinearly through the substate when the voltage of the stepped cell became more negative. As noted, the substitution of a single negative charge in the amino terminus of Cx32 is sufficient to make both the initial and steady state conductance–voltage relations of heterotypic mutant/Cx32 junctions resemble that of the Cx32/Cx26 heterotypic junction. However, none of the initial conductance–voltage relations of homotypic Cx32N2D, Cx32N2E, or Cx32* Cx26(NT 1–11 +CL) junctions resemble that of homotypic junctions formed by Cx26 . Also, the initial conductance–voltage relation of junctions formed by pairing these mutant hemichannels heterotypically with Cx26 is markedly asymmetric . These results indicate that the molecular determinants of initial conductance differ in Cx26 and Cx32 channels. When the Cx26 side of the heterotypic Cx26D2N/Cx26 junction is relatively positive, initial conductance increases as shown in Fig. 8 A. Qualitatively, the voltage dependence of initial conductance is similar, but less steep than is observed in the Cx32/Cx26 junction. This result is consistent with the interpretation that the D2 residue in Cx26 plays a role in the process underlying the open channel rectification of Cx32/Cx26. However, the initial conductance of the Cx32/Cx26D2N junction does not resemble that observed in Cx32 homotypic junctions as would be expected if the Cx26D2N mutation had converted Cx26 into Cx32. Nor does the initial conductance resemble that of junctions exemplified by Cx32N2D/Cx26 junctions , as would be expected if Cx32 and Cx26 made equal contributions to the rectification of initial conductance. In fact the initial conductance of the Cx32/Cx26D2N junction rectifies more than that observed in the Cx26D2N/Cx26 junction and, unlike the Cx32/Cx26 junction, conductance increases when the Cx32 side is relatively negative. The results obtained with the Cx26D2N mutation must be interpreted cautiously, as the mutation causes a substantial shift in the steady state conductance–voltage relation. A simple implication of this result is that there is a higher probability that the mutant hemichannel resides in a substate at V j = 0. Therefore, it is likely that currents passing through one or more substates make a substantial contribution to the initial currents measured macroscopically in Xenopus oocytes when the experimental paradigm described in materials and methods is employed. A large shift in the steady state conductance–voltage relation of the Cx26D2N hemichannel towards positivity explains the failure to observe junctional currents in the homotypic Cx26D2N junctions. The conductance–voltage relation inferred for Cx26D2N hemichannels from Cx26D2N/Cx26 junctions predicts that junctional conductance of homotypic Cx26D2N junctions would be substantially reduced at all transjunctional voltages. We have not yet examined the single channel currents of Cx26D2N mutations. The conductance–voltage relation of Cx26 chimeras containing the NH 2 terminus and first transmembrane domains of Cx32, Cx26*Cx32(NT+TM1), do not display large shifts in their steady state conductance–voltage relations. Consequently, they provide a means of examining the effect of neutralization of the negative charge of the D2 residue on the voltage dependence of initial conductance. Fig. 8 C illustrates that the initial conductance of the heterotypic junction, Cx26*Cx32(NT+TM1)/Cx26, is similar to that of Cx32/Cx26. This result suggests that the Cx26 chimera has adopted Cx32 properties. However, the initial conductance of Cx26*Cx32(NT+TM1)/Cx32 junction is markedly asymmetric , decreasing more when the Cx32 hemichannel is stepped to positive transjunctional voltages. The substitution of either N2D or the first eight amino acids of Cx26 into the amino terminus of the Cx26*32(NT+TM1) chimera results in hemichannels, Cx26*Cx32(N2D+NT+TM1), and Cx26* Cx32(NT 9–22 +TM1), whose properties are indistinguishable from those of wild-type Cx26. The conductance–voltage relations of Cx26*Cx32(NT 9–22 +TM1)/Cx26 and Cx26*Cx32(NT 9–22 +TM1)/Cx32 are shown in Fig. 8E and Fig. F ). These results indicate that amino acids downstream of TM1 in addition to the charged aspartate residue (D2) are responsible for the linear initial conductance–voltage relation of Cx26 homotypic junctions. Our previous work indicated that the E1 domain of Cx26 was likely to be involved in the voltage dependence of initial currents, because a chimera in which the first extracellular loop of Cx32 was replaced with the E1 domain of Cx26 to form Cx32*Cx26E1 demonstrated an appreciable asymmetry in the conductance–voltage relation of initial currents when paired with Cx32 . The voltage dependence of initial currents in the Cx32*Cx26E1/Cx32 heterotypic junction is very similar to that of the Cx26*Cx32(NT+TM1)/Cx32 heterotypic junction . In both cases, initial conductance declines more when the Cx32 hemichannel is stepped to positive potentials. The initial conductance–voltage relations of Cx32*Cx26E1 homotypic and Cx32*Cx26E1/Cx26 junctions do not differ substantially from wild-type Cx32 homotypic and Cx32/Cx26 heterotypic junctions when expressed in pairs of Xenopus oocytes. As for the Cx32 wild-type homotypic junction, the I–V relation of the fully open Cx32*Cx26E1 channel does not correspond to the macroscopic data obtained in pairs of Xenopus oocytes. When Cx32* Cx26E1 is expressed in Neuro-2a cell lines, the I–V relation of the fully open channel is linear . The changes in voltage dependence of the Cx26E1 substitution into Cx32 are likely to involve amino acid residues located at the TM1/E1 border as the double mutation, Cx26(K41E,E42S) (previously termed Cx26* ES) displays a steeper initial conductance–voltage relation than does Cx32/Cx26 when it is paired with Cx32 and some initial conductance reduction at higher transjunctional voltages when this chimera is paired homotypically . The effects appear to be localized to residue E42 in Cx26 as the initial conductance–voltage relation of the point mutation, Cx32S42E, in heterotypic pairings with Cx32 is almost identical to those observed with the chimeras Cx32*Cx26E1 and Cx26* Cx32(NT+TM1) . Accordingly, the asymmetry in the conductance–voltage relation of the Cx32N2D/Cx26 heterotypic junction could be explained by the presence of the E42 residue in the Cx26 hemichannel and its absence in the Cx32N2D hemichannel. Thus, the presence of negative charge at residue 42 appears to be an important determinant of the voltage dependence of initial conductance. The reciprocal chimera, Cx26*Cx32E1, displays a large shift in the steady state conductance–voltage relation in heterotypic pairings with Cx32, as illustrated in Fig. 9 F. Although initial conductance increases in this heterotypic junction with positivity on the Cx26*Cx32E1 side, the large shift in the steady state conductance–voltage relation makes it likely that substates of the Cx26*Cx32E1 hemichannel make a substantial contribution to the initial conductance determined macroscopically and this possibility complicates any quantitative interpretation. We did not observe the expression of junctional currents in Cx26*Cx32E1 homotypic pairings, an observation that may be explained by a negative shift in the V 1/2 of both hemichannels conductance–voltage relations. Reciprocal substitutions of the second extracellular loop of Cx26 and Cx32 have no effect on the voltage dependence of steady state or initial conductance in homo- and heterotypic pairings . The initial conductance of homotypic junctions of Cx32 chimeras, which contain one or more of the four transmembrane domains of Cx26 (Cx32*Cx26TM1, Cx32*Cx26TM2, Cx32*Cx26TM3, and Cx32*Cx26TM4) are similar to homotypic junctions formed by Cx32, and the heterotypic junction Cx32/Cx26*Cx32(TM4-CT) behaves like wild-type Cx32/Cx26 (data not shown). Also, a truncation of the Cx32 carboxyl terminus after TM4 (at residue 248) has little effect on the voltage dependence of initial conductance in homo- and heterotypic pairings with Cx32 and Cx26 (data not shown). Although the substitution of the TM1 domain does not cause marked changes in the transjunctional voltage dependence of initial conductance, it does alter the steady state conductance–voltage relation (data not shown). This result suggests that some conformational changes have occurred. These data indicate that substitutions involving the second extracellular loop, the four transmembrane domains and the carboxyl terminus have little or no effect on the voltage dependence of initial conductance observed in junctions formed by Cx26 and Cx32. However, as described above, changing amino acids positioned in the amino terminus and first extracellular loop appear to have pronounced effects. Although the cytoplasmic loop alters the voltage dependence of initial conductance, it is not clear if this is the result of a direct electrostatic effect or an indirect effect caused by changes in the conformation of the amino terminus. Based on the single channel data presented in this paper, it is apparent that the voltage dependence of initial conductance observed in the heterotypic Cx32/Cx26 channel arises as a consequence of the rectification of ionic currents through open channels rather than by conformational changes associated with voltage-dependent gating. It is remarkable that the I–V relations of both Cx32 and Cx26 homotypic channels expressed in Neuro-2a cell lines are essentially linear. Evidently, the union of Cx32 and Cx26 hemichannels results in an asymmetry in the structure of the heterotypic channel that produces the rectifying current–voltage relation. The union of like hemichannels to form a complete intercellular channel always results in symmetric, although not necessarily linear, I–V relations. For example, the I–V relation of homotypic Cx32*Cx26(NT 1–11 +CL) channels is sigmoidal . Asymmetric conductance–voltage relations may also result if the channels display dependence on the absolute voltage difference between the inside and outside of the cells (termed V i-o or V m ). However, this form of voltage dependence does not underlie the open channel I–V relations described in this study. Our previous results have indicated that charged amino acid residues, positive (M1) and negative (D2) in Cx26 and positive only (M1) in Cx32, are responsible for the difference in the V j -dependent gating polarity of Cx26 and Cx32 hemichannels. We proposed that these residues are located near the cytoplasmic entrance of the channel pore based on their ability to sense transjunctional voltage and the accepted membrane topology of gap junctions. These charges may account for the slight anion selectivity of homotypic Cx32 channels and the slight cation selectivity of Cx26 homotypic channels . The data presented in this paper indicate that these same charged amino acid residues are also involved in the mechanism underlying the voltage dependence of initial conductance. The observations that the substitutions of the neutral second amino acid residue of Cx32 with negatively charged amino acid residues alter the initial conductance–voltage relation and that neutral amino acid substitutions do not change initial conductance are consistent with an electrostatic effect. The increased steepness of the rectification observed in the Cx32/Cx32* Cx26(NT 1–11 +CL) junction may arise from an interaction between the CL and NT domains that alters the position of the negatively charged residue located in the amino terminus of this chimera. Alternatively, it may reflect an electrostatic effect of charged residues that differ in the CL domain in Cx32 and Cx26. Of the 38 amino acids present in the cytoplasmic loop of Cx26, 50% are charged, 12 positive and 7 negative. Of the 37 amino acid residues in the CL domain of Cx32, 6 are positively and 5 are negatively charged. Nine charges (five positive and four negative) are conserved between the two connexins. Consequently, it is difficult to assess whether the effect of the cytoplasmic loop on open channel rectification is a direct effect of specific charged amino acid residues, or if this domain exerts its effect indirectly by changing the conformation of the NH 2 terminus. It is apparent that the substitution of a negative charge into NH 2 terminus of Cx32 does not recreate the properties of Cx26 homotypic junctions, since the I–V relations of Cx32*Cx26(NT 1–11 +CL) and Cx32N2E homotypic channels are sigmoidal . Therefore, other charged residues must also influence ionic flux in Cx26 channels. The asymmetry in the voltage dependence of initial currents observed in the Cx32*Cx26E1/Cx32 junction indicates that the E1 domain of Cx26 is also involved. The negatively charged E42 residue in Cx26 is the best candidate, as the initial conductance–voltage relation of the point mutation Cx32S42E paired heterotypically with Cx32 is almost identical to that of Cx32*Cx26E1/Cx32 . The view that E42 plays a role in the expression of the voltage dependence of initial conductance is further supported by the increased steepness of the voltage dependence of initial conductance observed in the Cx32/Cx26(K41E,E42S) junction . We have not ruled out the possibility that other charged amino acid residues in E1 play a role. The initial conductances of chimeric channels containing the second extracellular loop (E2), the four transmembrane domains (TM1, TM2, TM3, and TM4), and the carboxyl terminus differ little from wild type. If any charged residues in these domains are involved in the voltage dependence of initial conductance, they are likely to be conserved in Cx26 and Cx32. The results presented in this paper indicate that charged amino acid residues located in the amino terminus and first extracellular loop play a major role in shaping the I–V relations of channels formed by Cx26 and Cx32. In the following sections, we examine whether these charges can account for the I–V relations of wild-type and mutant channels presented in this paper by using the electrodiffusive model of Chen and Eisenberg 1993 that numerically solves the PNP equations in one dimension. The PNP model of Chen and Eisenberg 1993 is attractive in that it promises to predict the fluxes and current passing through a channel in all experimental conditions once the distribution of permanent (fixed) charge on an ion channel is known . The theory has been used successfully to describe permeation through a large diameter (∼8 Å) synthetic ion channel (LS channel) using only four adjustable parameters; the diffusion coefficients of two ions in the channel pore and the distribution and density of fixed charge at the ends and central region of the channel . The availability of a large data set, obtained using 15 different ionic solutions, allowed reliable estimates of these parameters and may have provided a unique solution to the question of charge distribution and ionic mobility in the synthetic channel. However, the approach may provide limited structural information on the LS channel since Chen et al. 1997 state that it is likely that more than one atomic structure would give the same average parameters. In this study, we adopt a different strategy since we have very limited ionic data for any given channel (at best two ionic conditions for Cx32 channels), but we have information describing how the I–V relations are changed by substitutions of charged amino acid residues in different regions of Cx32 and Cx26 in several different pairing combinations. We seek to determine whether we can use our molecular studies to derive a set of charge distributions that when incorporated into the PNP model will lead to I–V relations comparable with those observed in homotypic and heterotypic pairings of wild-type and mutant hemichannels. In our use of PNP theory, we vary only the magnitude, the position, and the width of fixed charges that we infer from our molecular studies. We use a fixed pore geometry of 7 Å in radius and 120 Å in length. The cation and anion mobilities are set to values of the aqueous bulk solution mobilities of Cs and Cl (2.06 and 2.03 · 10 −5 cm 2 s −1 ), the solutions used in single channel experiments that were performed. The ion concentrations were 150 mM in the symmetric salt case, and 15 and 150 mM in the case of 1:10 salt gradients. The dielectric constant for the channel pore and the membrane were fixed at 80 and 4, respectively (the default values of the PNP computer program). The I–V relations obtained were not very sensitive to changes in the value of the dielectric constant assigned to the channel pore in the 150-mM salt case using fixed charges > ±1 e . The PNP model appears to be quite sensitive to reductions in the dielectric constant of the pore when ionic concentrations and the values assigned to fixed charges are both reduced . This observation most likely reflects the “charge screening” property of the PNP model. The input file for the pore surface charge was generated using the PNP Windows computer program of Stephen Traynelis' group (Emory University, Atlanta, GA) using the program's defaults (sigmoid, smooth charge points = 7). In the case where more than two charges are modeled, the charge input file was assembled in overlapping pairwise combinations using Microcal Origin software and saved as PAS or TXT files in Programmers Notepad. Output files (I–V relations, anion and cation fluxes, and chord conductances) were generated using the PNP computer program of Dr. Duan P. Chen (Rush Medical College, Chicago, IL) using the unmodified Poisson Equation. The Fortran version of the computer program was compiled in C ++ by Brady Trexler (Albert Einstein College of Medicine). We restrict our use of the PNP model to channels for which we have single channel records. We did not attempt to use any fitting algorithms, as we feel that this approach is premature in the absence of a larger data set that includes a variety of ionic conditions. The availability of additional ionic data would be helpful in distinguishing among alternative charge distribution models, but such data are lacking at present, and additional ionic data are not easily obtained for intercellular channels expressed in cells. Initially, we explored a PNP model of Cx32 with a charge step of +6 e located at either end of the open channel, with the rationale that each of the six connexin subunits that form a hemichannel would contribute one positive charge. The I–V relation of a channel with this charge distribution that resulted with the PNP model was quite similar to the I–V relation observed for Cx32 homotypic channels in symmetric 150-mM CsCl solutions (deviating from linearity by ∼5% at ±120 mV, not illustrated). However, although not unexpectedly, the PNP-modeled channel was almost ideally anion selective in 15 mM:150 mM CsCl solutions. Virtually all the current was carried by the anion over the entire voltage range explored in the model (±120 mV) and the predicted reversal potential was approximately −58 mV in the 1:10 salt gradient of this ionic strength. A reversal potential approximating that obtained for Cx32 homotypic channels could be obtained by incorporating a baseline charge of −1.5 e . The baseline charge in the computer model could be interpreted as a smearing of a negative charge along the length of the pore surface, perhaps reflecting the negative charge contribution of backbone carbonyls or the negative charge of side chains of pore-lining amino acids. Amino acids such as serine, which are believed to carry partial negative charges , may also be involved. The PNP model employed does not consider the effects of fixed charges on the distribution of ions outside the pore. Consequently, we could not consider the extent to which fixed charges near the channel entry would alter the local ionic environment outside the channel pore and influence the ionic selectivity of the channel by this mechanism. The incorporation of a baseline charge resulted in a significant deviation from linearity in the I–V relation of the modeled channel with all symmetric salt conditions explored (10–500 mM). Consequently, we explored the I–V relations of channels in which the magnitude of the charge step was reduced. Using a charge step of +1 e , we were able to find several intra-pore charge distributions that when used as inputs to the PNP model resulted in I–V relations that closely resembled the I–V relation observed for Cx32 homotypic channels in the 1:10 salt gradient employed . The charge distribution shown in Fig. 10 C gives the I–V relation shown in Fig. 10 D in symmetric salt case. The PNP model is expected to change the shape of the I–V relation as a function of ion concentrations (charge screening). Consistent with this expectation, the I–V relation shown in Fig. 10 D (150 mM) becomes more sigmoidal at an ion concentration of 10 mM (not illustrated) and substantially less but still noticeably sigmoidal at ion concentrations of 500 mM (not illustrated). It is tempting to speculate that reduced charge screening due to a markedly lower concentration of permeant anions may contribute to the greater nonlinearity of Cx32 channels when they are examined in CsCl-dialyzed cells compared with undialyzed Xenopus oocytes. Although the shapes of the I–V relations observed correspond closely to those obtained with the PNP model with the charge distribution and parameters used, the predicted single channel conductance is substantially greater than observed (227 vs. 70 pS at ±60 mV). This problem may be solved by reducing the diffusion coefficients of the ions in the pore, as done by Chen et al. 1997 in the synthetic channel. If the mobility of both the cation and anion are reduced to 0.7 · 10 −5 cm 2 s −1 from the initial values of 2.06 and 2.03 · 10 −5 cm 2 s −1 , the single channel conductance closely approximates that observed (70 pS at ±60 mV). The reductions in ionic mobilities we employ are much less than those used by Chen et al. 1997 in the PNP model of the synthetic LS channel. In that study, the mobility of K + was reduced 10-fold while that of Cl − , 100-fold. The difference perhaps reflects the substantially smaller radius of the LS channel (4 vs. 7 Å). The reduction in ionic mobilities does not alter the shape of the I–V relation of Cx32 (not illustrated). The contribution of friction in the channel pore to ion permeation has been discussed recently by Laio and Torre 1999 . The results of the molecular analysis indicate that the I–V relation of Cx26 channels is likely to be determined by external positive and negative charges and an internal negative charge. Following these considerations, we modeled the charge distribution of Cx26 as illustrated in Fig. 10 E. The charge associated with the D2 residue was set to −2 e , the positive charge in the amino terminus set to +1 e , and the internal charge (E42) was set to −1 e as this residue may be located at some distance from the channel lining. We justify the increased magnitude of the D2 charge by considering that negative charge substitutions into the amino terminus of Cx32 appear to dominate the effect of the fixed positive charge in polarity reversal of V j dependence. We also incorporated a baseline charge of +0.25 e to make the Cx26 channel slightly cation selective in 150 mM:15 mM CsCl gradients in consideration of the results reported by Veenstra 1996 . The sign of the baseline charge used here is opposite that for Cx32, and we cannot invoke a comparable physical interpretation consistent with that invoked for Cx32. It is possible that the difference in ion selectivity of Cx32 and Cx26 channels is influenced by other charged residues located within the channel and that the incorporation of the baseline charge mimics their effect in both Cx26 and Cx32. Alternatively, the difference in selectivity may be due to the effects of surface charges that change the local ionic environment near the channel entry. As the PNP model employed does not consider the effects of fixed charge outside the ion pore, we could only approximate the weak cation selectivity of the Cx26 channel by incorporating a small positive baseline charge. The I–V relation resulting from the application of the PNP model with this charge distribution model is linear , like that observed for Cx26 homotypic channels, but the single-channel conductance is somewhat larger than observed (220 compared with 150 pS). The incorporation of the baseline charge does not alter the linearity of the I–-V relation of the Cx26 homotypic channel. An alternative charge distribution model also results in a linear I–V relation and a slightly cation selective channel (not shown), but in this case the predicted single channel conductance is identical to that observed (150 pS, not shown). We modeled the charge distribution of the heterotypic Cx26/Cx32 channel by merging the charge distribution of half the Cx26 channel shown in Fig. 10 E (i.e., covering the electrical distance from 0.0 to 0.5) with that of Cx32, shown in Fig. 10 C (covering the electrical distance from 0.5 to 1.0). The charge distribution at the point of contact was recalculated by using the Windows PNP program of Traynelis to remove the sharp discontinuity that would otherwise result. The I–V relation resulting from the PNP model is almost identical to that observed for single Cx26/Cx32 channels, with current increasing approximately threefold when the Cx26 side of the channel is made relatively positive (−12.4 pA at −120 mV to 36.2 pA at 120 mV). The single channel conductance of the modeled channel can be made identical to that observed by reducing ion mobilities to 0.85 · 10 −5 cm 2 s −1 . The charge distribution model formed by combining the right half of the charge distribution shown in Fig. 10 C with the left side of the charge distribution shown in Fig. 10 H also resulted in an I–V relation that resembled that of Cx32/Cx26 heterotypic channels, rectifying 2.7-fold at ±120 mV (increasing when the Cx26 side of the channel is positive). In the model of the Cx32*Cx26(NT 1–11 +CL) channel, the charge profile of Cx26 shown in Fig. 11 A was modified by removing the central charge (−1 e ) associated with E42 in Cx26 and moving the external charges outward (the charge step was initiated at an electrical distance of 0.025 rather than 0.1), but the magnitude of the external charges was not altered. The change in position of these charges was required to produce a charge distribution model that could replicate the experimentally observed I–V relations in all pairing combinations involving the Cx32*Cx26(NT 1–11 +CL) hemichannel. The baseline charge was increased by 0.25 e to 0.5 e to maintain the expected moderate cation selectivity of this channel (E rev = −10 mV in 1:10 salt gradient, not shown). The charge profiles for Cx26/Cx32*Cx26(NT 1–11 +CL) and Cx32*Cx26(NT 1–11 + CL)/Cx32 are shown in Fig. 11B and Fig. C , respectively. The I–V relations given by the PNP model for the homotypic Cx32*Cx26(NT 1–11 +CL) channel, and heterotypic Cx26/Cx32*Cx26(NT 1–11 +CL) and Cx32* Cx26(NT 1–11 +CL)/Cx32 channels closely resemble the observed I–V relations . The small difference in the modeled and observed single channel conductances could be further reduced by decreasing the mobilities of ions in the PNP model (1.4 · 10 −5 cm 2 s −1 for the homotypic channel, 1.2 · 10 −5 cm 2 s −1 for the heterotypic Cx26 channel, and 0.9 · 10 −5 cm 2 s −1 for the heterotypic Cx32 channel). The I–V relations of homotypic junctions formed by Cx32N2E and Cx32N2D and their heterotypic junctions with Cx32 and Cx26 can be explained using charge distribution models similar to those employed to explain the behavior of the Cx32*Cx26(NT 1–11 +CL) channels (not shown). However, using half of the charge distribution model shown in Fig. 10 H for Cx26 to form a model of Cx26/Cx32*Cx26(NT 1–11 +CL) channel produced a nearly symmetric sigmoidal I–V relation (not shown) that did not correspond to the I–V relation observed experimentally. As we were not able to find an alternate charge distribution model for Cx32*Cx26(NT 1–11 +CL) that would provide the observed I–V relation, we believe that the Cx26 charge distribution model shown in Fig. 10 H is unlikely to be correct. As detailed above, there is good qualitative agreement between the I–V relations obtained with the PNP model using the charge distributions inferred from the molecular studies and those observed in this study. However, the linearity of the I–V relation of the Cx32*Cx26E1 homotypic channel cannot be explained by charge distribution models using only the identified charges analyzed so far. We were surprised to be unable to obtain a linear I–V relation for Cx32*Cx26E1 using the PNP model with the identified external and internal charges (M1 and E42). The I–V relation of a channel containing only the central charges (E42) is linear (not shown), while the I–V relation of a channel containing an external charge at either end can be linearized by moving the charges centrally (not shown) in the PNP analysis. Yet all models that considered both charges together were characterized by substantially sigmoidal I–V relations (not shown). A nearly linear I–V relation could be obtained by the PNP model if one incorporated a partial negative charge near the ends of the charge distribution model for Cx32*Cx26E1. The conserved tryptophan residue at the third amino acid position (W3) is a reasonable candidate for the formation of this negative charge if one proposes that the tryptophan residue adopts a conformation such that the negative component of the electrostatic surface potential of the aromatic ring of the tryptophan residue is oriented parallel to the channel pore. We did not consider the positive component of the electrostatic surface potential. The aromatic ring of the tryptophan residue has a permanent quadrupole moment and it is believed that its electrostatic effect is important in cation–π interactions . Favorable electrostatics between an ion and the indole ring of tryptophan have been reported to facilitate the cation conductance of gramicidin A channels . Kumpf and Dougherty 1993 have proposed that cation–π interactions at conserved aromatic amino acids may be responsible for the ion selectivity of potassium channels. The charge distribution used and the resulting I–V relation of this class of model after the application of the PNP model are illustrated in Fig. 12A and Fig. D . However, the incorporation of a negative charge at the W3 position alters the predicted I–V relation of Cx32 homotypic channels and Cx26/Cx32 heterotypic channels. The I–V relation of Cx32 becomes linear and notably the predicted reversal potential in a 1:10 salt gradient is close to that observed without the incorporation of a baseline charge (not shown). Also, the predicted I–V relation of Cx26/Cx32 rectifies much less, approximately twofold over ±120 mV (not shown). Interestingly, a channel with the charge distribution shown in Fig. 12 B has a predicted I–V relation that closely resembles wild-type homotypic Cx32 channels . In this case, the magnitude of the charge at W3 was reduced to −0.25 e, and a central charge of −0.5 e was introduced. It is possible that this charge corresponds to residue E41 in Cx32 and that the charge profile shown in Fig. 12 B more closely approximates that of Cx32 than the one shown in Fig. 10 C. Consequently, we explored the effect of incorporating the negative charges associated with residues W3 and E41 in a model of the heterotypic Cx26/Cx32 channel. The modeled charge distribution is shown in Fig. 12 C. The resulting I–V relation closely resembles that of Cx26/Cx32, rectifying 2.8-fold (−14.3 pA at −120 mV/40.7 pA at +120 mV). The single channel conductance of the modeled channel is three times greater than that actually observed, but can be adjusted by reducing the intrapore ionic mobilities of both the anion and cation to 0.7 · 10 −5 cm 2 s −1 from the bulk solution mobilities of Cs and Cl. The I–V relation of the resulting channel is shown in Fig. 12 F. The incorporation of a negative charge (−0.5 e ) associated with E41 into the charge distribution models of Cx32*Cx26(NT 1–11 +CL) channels does not substantially alter the resulting I–V relations obtained with the PNP model. The effect of this charge on the I–V relation obtained for the Cx26/Cx32*Cx26(NT 1–11 +CL) channel is illustrated in Fig. 13 . We did not attempt to apply PNP theory to model the behavior of the remaining junctions for which we have no single-channel data. We expect that the I–V relations that can be inferred from the macroscopic conductance–voltage relations could be predicted by the charge distribution models we present for Cx32, Cx26, Cx32*Cx26(NT 1 − 11 +CL) and Cx32*Cx26E1 with only minor modifications. The only notable exception is the superlinear I–V relation that would correspond to the macroscopic conductance–voltage relation of the Cx26* Cx32(CL–CT)/Cx26 heterotypic junction . The apparent reversal of the V j gating polarity of the Cx26*Cx32(CL–CT) hemichannel suggests that a conformational change has taken place that effectively removes the D2 residue from the transjunctional field. In barrier models, superlinear I–V relations are easily explained by the presence of a dominant central barrier. We did not find a reasonable way to obtain a superlinear I–V relation with the PNP model, although superlinear I–V relations have been obtained with the PNP2 model . Superlinearity can be obtained with the PNP model we use by placing a widely dispersed charge in the center of the channel and reducing the mobility of the oppositely charged ion by several orders of magnitude to 10 −9 cm 2 s −1 . Further analyses of this interesting junction await confirming single-channel data. The preceding sections illustrate how the results of the molecular analyses could be used in conjunction with the PNP model of Chen et al. 1997 to derive a consistent set of charge-distribution models for hemichannels formed by Cx32, Cx26, and NH 2 -terminal charge substitutions of Cx32. For the most part, the inferred charge distribution models reasonably reproduced the observed single-channel I–V relations of homotypic and heterotypic channels formed by these connexins when used as input parameters of the PNP model. The only notable exception was the I–V relation of the homotypic Cx32*Cx26E1 channel. The charge distribution that was inferred for this channel, containing an internal negative charge contributed by the E42 residue of Cx26 in addition to the external positive charge associated with residue (M1), did not reproduce the observed linear I–V relation when it was used as an input to the PNP model. Exploration of other charge distributions with the PNP model indicated a potential solution; the involvement of a partial negative charge (possibly reflecting the negative component of electrostatic surface potential of the indole ring of the W3 residue). Explorations of other charge distributions with the PNP model also suggested that the negative charge of the Cx32E41 residue may play a role in determining the I–V relations of the channels examined. Additional molecular studies are required to confirm the hypothesized role of these charged residues in shaping the I–V relations of Cx32 and Cx26 channels. It is important to stress that the set of charge distribution models provided above are probably not unique, in that there are likely to be other sets of charge distribution models that may also consistently describe the available set of experimentally observed I–V relations. This statement is not meant to imply that all charge distribution models that were examined in the current study could satisfactorily account for the entire experimental data set. For example, the class of charge distribution model for Cx26 illustrated in Fig. 10 H did not give rise to the experimentally observed I–V relation for the Cx26/Cx32*Cx26(NT 1–11 +CL) channel, although it worked reasonably well in reproducing the I–V relations of Cx26 homotypic and Cx32/Cx26 heterotypic junctions. We were unable to find an alternate charge distribution of Cx32*Cx26(NT 1–11 +CL) that could remedy this discrepancy and continue to adequately describe the behavior of other channels. Similarly, the model appeared to be quite sensitive to changes in the position of charges located near the entry of the Cx32*Cx26 (NT 1–11 +CL) hemichannel. As described above, the charges in this region had to be moved outward relative to those used in the charge distribution model of Cx26 to satisfactorily reproduce the I–V relations of homotypic and heterotypic channels observed for this chimera. The PNP model appears to be sensitive to changes in the position of charges in the class of charge distribution model exemplified by the Cx32* Cx26(NT 1–11 +CL) chimera, but, as described below, less sensitive to other changes in charge distribution. We did not attempt to rigorously explore the sensitivity of the PNP model to alterations in the input parameters. As stated previously, the PNP model does not appear to be sensitive to changes in the assigned values for dielectric constant of the membrane and pore, but this insensitivity may be related to the ionic conditions and charge distributions used in our study. In some cases, the model did not appear to be very sensitive to rather substantial changes in charge distribution. This is illustrated in Fig. 13A–C , where two different charge distribution models of the Cx26/Cx32*Cx26(NT 1–11 +CL) channel result in very similar I–V relations with the PNP model in the symmetric 150-mM salt case. The two charge distribution models differ only by the presence of an internal negative charge (−0.5 e ), which we propose to correspond to residue E41 in Cx32 (see above). However, the two charge distribution models can in principle be discriminated in other pairing configurations or by varying the ionic concentration parameter used in the PNP model. For example, the I–V relations obtained for the two different models of Cx32*Cx26 (NT 1–11 +CL) differ somewhat when they are used to form homotypic channels . Also, the I–V relations (and predicted reversal potential) of the two different heterotypic Cx26/Cx32*Cx26(NT 1–11 +CL) channels differ slightly when asymmetric salts (15 mM inside:150 mM outside) are used . The incorporation of the negative charge shifts the reversal potential from −10 to 0 mV. Greater differences in the I–V relations are obtained with the PNP model in the symmetric salt case when the concentration of ions is reduced to 15 mM from 150 mM . Although it is not clear whether the differences in the I–V relations shown in Fig. 13 can be resolved experimentally, it seems at least theoretically possible that the PNP model is able to discriminate among different charge distribution models when the model's input parameters are varied. The results of our molecular studies in conjunction with PNP theory have enabled us to identify at least one internally consistent set of charge distributions that can explain the observed rectification of several hetero- and homotypic channels. This finding supports the view that charged amino acid residues in the NT and E1 domain of Cx32 and Cx26 are important molecular determinants of electrical rectification. The questions, if the charge distributions models derived here are unique and if the PNP model can provide a “unique” solution in all cases needs to be addressed further, but this aim is computationally and experimentally intensive and beyond the scope of the current paper. We cannot legitimately apply the PNP model to explain the rectification observed in substates of Cx32 and Cx32*Cx26(NT 1–11 +CL) hemichannels as the available model considers the surface charge distribution of channels of right circular cylindrical geometry. While the changes in conformation associated with V j gating to substates may not substantially change the magnitude of surface charge, it is likely that reductions in pore radius would increase the electrostatic effect of charges located in constricted regions. We illustrate this by considering the charge distribution models shown in Fig. 14 . If one increases the magnitude of the external charges in the simple charge distribution models of Cx32 and Cx32*Cx26(NT 1–11 +CL) to mimic an increased electrostatic effect of these charges, then the I–V relations obtained rectify in the same direction as observed . When the Cx32 homotypic channel enters a substate in response to negative V j , currents decrease as V j becomes more positive . In homotypic Cx32*Cx26(NT 1–11 +CL) channels, current increases as the cell containing the hemichannel rendering in a substate becomes positive . Note that Cx32 and Cx32*Cx26(NT 1–11 +CL) hemichannels close for opposite polarities of transjunctional voltage. Nonner and Eisenberg 1998 have considered the problem of nonuniform channel geometry. A comparable approach may allow a more appropriate consideration of the observed substate rectification. The results obtained from the application of the PNP model can provide a mechanistic explanation for the generation of steeply rectifying electrical synapses originally reported by Furshpan and Potter 1959 . The model illustrates how the separation of fixed positive and negative charges across the junctional membrane could result in a steeply rectifying electrical synapse . In this context, our results subsume the diode hypothesis (i.e., the formation of a p-n junction by the separation of fixed positive and negative charges) that was originally proposed by Furshpan and Potter 1959 . The steep rectification attainable by p-n junctions was also demonstrated by Mauro 1962 and Finkelstein 1963 using Nernst-Plank equations with the electroneutrality condition. Jaslove and Brink 1986 suggested that voltage-dependent gating may also contribute to the steepness of the rectification observed in the crayfish electrical synapse. They and Giaume et al. 1987 have postulated that the crayfish electrical synapse is asymmetric; i.e., composed of two different hemichannels, one of which contains a fast voltage-dependent gate. It is possible that both the crayfish and hatchetfish electrical synapses are formed by a heterotypic junction in which the steady state conductance–voltage relation of one hemichannel is shifted to smaller transjunctional potentials, comparable with the inferred steady state conductance–voltage relation of the Cx26D2N hemichannel in the heterotypic Cx32/Cx26D2N junction . The open probability of such a channel would be close to zero at V j = 0 and increase upon depolarization of the presynaptic cell. This model would explain the low conductance of electrical synapses when coupled neurons have (or are held at) similar resting membrane potentials . Depolarization of the presynaptic cell (or hyperpolarization of the postsynaptic cell) would increase the open probability of this hemichannel, and increase junctional conductance. Hyperpolarization of the presynaptic cell (or depolarization of the postsynaptic cell) would favor the closed configuration. In this model, the presynaptic cell would contain a V j gate like that of Cx32, which closes on relative negativity. Voltage-dependent gating is unlikely to be the sole determinant of the electrical rectification observed in electrical synapses because steady state conductance at these synapses is reached very rapidly within 800 μs . None of the cloned connexins that have been examined demonstrate sufficiently rapid kinetics. For example, Bukauskas et al. 1995b reported that the mean time to reopen a closed Cx40 channel is 9.5 ms when the polarity of a transjunctional voltage step is reversed from +50 to −50 mV. But this is still much too slow to account for the properties of the crayfish electrical synapse. In preliminary studies, we have measured the mean time to reopen a closed Cx32 channel to be much slower, ∼50–75 ms, when the polarity of a transjunctional voltage step is reversed from +100 to −100 mV (Oh, S., V.K. Verselis, and T.A. Bargiello, unpublished observations). The rectification of gap junction open states and substates described in this study provides a mechanism that would allow for a nearly instantaneous increase in conductance in response to presynaptic depolarization. Electrodiffusive models (PNP theory and Nernst-Plank with electroneutrality assumption) predict steeply rectifying I–V relations for channels in which positive and negative charges are located at opposite ends of an intracellular channel (p-n junction). Thus, the steepness of the I–V relations attainable by p-n junctions can adequately explain the observed behavior of electrical synapses and obviates the need to invoke voltage gating. Recently, several fish connexins as well as an unrelated family of invertebrate proteins encoding gap junctions have been cloned , and it should soon be possible to identify the connexins that form rectifying electrical synapses and to precisely define the mechanism responsible for their steep rectification. | Study | biomedical | en | 0.999997 |
10469727 | The large conductance Ca 2+ -activated K + (BK Ca ) channel has a multi-ion pore , like many other potassium channels . Neyton and Miller 1988b reached the conclusion that the pore of BK Ca channels can accommodate several K + ions and that the K + sites are of high affinity. In particular, they functionally characterized a high affinity K + binding site facing the external solution; this site was revealed by the observation that increasing external [K + ] slows down the rate of Ba 2+ exit from the channel . It is easy to interpret this observation by assuming that there is a K + binding site located externally to the blocking site. When the channel is blocked by Ba 2+ , the outer K + site is in equilibrium with the external [K + ] so that, at very low external [K + ], the site remains empty most of the time and Ba 2+ can exit toward the external side more easily than to the internal side. This external K + site was dubbed the “lock-in” site. In this study, we have determined the dissociation constant for K + at the lock-in site with increased accuracy by lowering the external [K + ] below the contamination level with a crown ether that binds K + with high affinity. The value we obtained for the dissociation constant, 2.7 μM, indicates that K + binding is approximately fivefold stronger than reported by Neyton and Miller 1988a . These results suggest that BK Ca channels bind K + as tightly as Ca 2+ channels bind Ca 2+ ions . In this study, we also found that the mean Ba 2+ blocked time is affected by tetraethylammonium (TEA + ). The stabilizing effect of TEA + on Ba 2+ block is mainly due to a “trapping” of K + in the lock-in site. The large conductance Ca 2+ -activated K + channel has a high degree of identity in the pore region with voltage-dependent K + channels. The crystal structure of a K + channel from bacteria was recently elucidated . It revealed that the bacterial K + channel could contain three K + ions in its conduction pathway. One K + ion was detected in a large water-filled cavity at the center of the pore near the cytoplasmic end of the selectivity filter. The other two were located at opposite ends of the selectivity filter, stabilized by backbone carbonyl groups. The TEA + binding site, which is located outside the selectivity filter, is made by a ring of four tyrosines near the extracellular end of the pore. Our results imply that TEA, K + , and Ba 2+ ions can coexist in the BK Ca channel pore and set molecular constraints on the location of the lock-in and the Ba 2+ sites. A picture that is consistent with our results and the potassium channel crystal structure is one in which the lock-in site corresponds to the K + site located on the extracellular side of the selectivity filter, and Ba 2+ binds to a site on the internal side of the selectivity filter. Despite the similarity with voltage-dependent K + channels, BK Ca channels do not show external K + -dependent phenomena such as C-type inactivation or the loss of functional channels after removal of K + ions from both sides of the membrane . Actually, it is possible to record BK Ca channel activity for periods of hours without a hint of inactivation . A possible explanation for the stability of channels in the virtual absence of K + is their avidity for K + ions. In other words, the affinity of the channel for K + is so high that the low [K + ] present in nominally “K + -free” solutions (≈4 μM) is sufficient to saturate the relevant K + binding site(s) in the pore. To test this hypothesis, we have lowered the external [K + ] below the K + -contamination level using a crown ether that chelates K + with high affinity. Our results show that when channels are exposed to external solutions containing less than 4 μM, K + channel electrical activity suddenly ceases, a result that is consistent with our hypothesis. All measurements were performed on planar bilayers with a single BK Ca channel inserted. Since depolarizing voltages and cytoplasmic Ca 2+ activates BK Ca channels, the “internal” side of the membrane was defined according to the voltage and Ca 2+ dependence of the channel. Accordingly, the physiological voltage convention is used throughout, with the external side of the channel defined as zero voltage. Bilayers were cast from an 8:2 mixture of 1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE) and 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) in decane. Lipids were obtained from Avanti Polar Lipids. Bilayers were formed in 0.01 M 3-[ N -morpholino]propane-sulfonic acid- N -methyl d -glucamine (MOPS-NMDG), pH 7. Concentrated KCl and CaCl 2 were added to the internal solution to a final concentration of 0.1 M and 125 μM, respectively. The internal [Ca 2+ ] used fully activates the BK Ca channel from skeletal muscle . In some experiments Ba 2+ (75–200 nM) was added to the internal solution to increase the probability of Ba 2+ blockade events. Rat skeletal muscle was used to prepare membrane vesicles containing BK Ca channels as previously described . Membrane vesicles were added very close to the bilayer and, once a channel incorporated, concentrated MOPS-NMDG, pH 7, and EGTA-NMDG were added to the extracellular side to a final concentration of 0.11 M and 400 μM, respectively. Single channel currents were recorded at 0 mV. Single-channel recordings were acquired using a custom-made current-to-voltage converter amplifier connected to the solution through agar bridges made with ultrapure NaCl (Alfa Aesar). Continuous single-channel current records (3–30 min) were filtered at 400 Hz and digitized at 500 μs/point. Open and closed events were identified using a discriminator located at 50% of the open-channel current. Dwell-time histograms were logarithmically binned and fitted to a sum of exponential probability functions with Pclamp 6 software (Axon Instrument, Inc.). Closed dwell-time histograms were fitted to the sum of two exponential functions. The slow component is a Ba 2+ block previously described in detail . The mean Ba 2+ blocked times were measured in the range of 2 × 10 −8 to 10 −2 M K + . Data were grouped in decades of K + concentration and the average of the logarithms of mean blocked time and the average of the logarithm of the K + concentrations ± SD were used in Fig. 2 . The mean Ba 2+ -blocked data obtained at various external [K + ] were described using 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*}{\mathrm{{\tau}}}_{{\mathrm{0}}-{\mathrm{Ba}}}= \left \left({1}/{ \left \left[{k_{{\mathrm{in}}}+k_{{\mathrm{ext}}}}/{ \left \left(1+{ \left \left[K\right] \right }/{K_{{\mathrm{d}}}^{K}}\right) \right }\right] \right }\right) \right {\mathrm{,}}\end{equation*}\end{document} where k in is the dissociation rate constant toward the internal side and k ext is the dissociation rate constant toward the external side of the channel when the lock-in site is empty, and K d K is the dissociation constant for K + from the channel containing a K + and a Ba 2+ simultaneously. We used a nonlinear least-square fit procedure to find the values of k in , k ext , and K d K , where the statistical weight of each point was the number of observations on each decade . Determination of the free K + concentration in solutions containing low K + and crown ether requires knowledge of the [K + ] of “K + -free” solutions and the dissociation constant of the K + -crown ether in the presence of 0.11 M MOPS-NMDG. The [K + ] was determined using an ion-specific electrode that is linear in the [K + ] range between 1 μM and 1 M. The average K + contamination of the MOPS-NMDG solutions used in the present study was 4.4 μM. The contaminating [K + ] of the stock of MOPS-NMDG and EGTA-NMDG solutions was also determined by atomic absorption spectrophotometry. A crown ether (a gift from Dr. Jacques Neyton, Laboratoire de Neurobiologie, Ecole Normale Supérieure, Paris, France), (+)-18-Crown-6-tetracarboxylic acid (18C6TA) from Merck, was used to chelate the contaminating external K + and contaminating Ba 2+ in the internal solution. The 18C6TA:cation stoichiometry is 1:1 . The crown ether binds K + , Ca 2+ , and Ba 2+ with dissociation constants of 3.3 × 10 −6 , 10 −8 , and 1.6 × 10 −10 M, respectively . The dissociation constant of the K-crown ether complex in the presence of 0.11 M MOPS-NMDG was 6.3 × 10 −6 M. This value was obtained using an ion-specific electrode to measure the free [K + ] in solutions of known concentrations of total K + and crown ether. The dissociation constant of the Ba-crown ether complex in the presence of 150 mM KCl was considered to be 1.6 × 10 -10 M . Fig. 1 shows K + currents from single BK Ca channel recordings with 70 nM internal [Ba 2+ ] and different external K + concentrations along with the corresponding closed dwell time histograms. Three different features are evident from the figure. (a) There is a slow internal Ba 2+ block described previously . Low concentrations (∼70 nM) of internal Ba 2+ induce long-lived nonconducting intervals separated by “bursts” of channel activity . At the largest external [K + ] used, the open probability inside a burst is close to 1. Vergara and Latorre 1983 showed that each of these long-lived blocked events represents the binding of a single Ba 2+ ion to the channel, and they presented strong evidence that the site of Ba 2+ binding is located within the conduction pore. (b) There is an increase in the number of fast closing events with decreasing external [K + ], clearly revealed by an increase in the size of the fast component of the closed dwell-time distributions . This increase in the number of fast closing events has not been described before and, as discussed below, may be due to a change in channel kinetics or to a fast Ba 2+ blockade. (c) A long-lasting closed state appears at very low external [K + ] . Fig. 1 , right, shows that the distribution of dwell times in the closed state is multiexponential. Note that mean block times of the slow component became shorter and the number of events increased as the external [K + ] was decreased. Upon decreasing the external [K + ] from 24 to 0.09 μM, the mean Ba 2+ blocked time decreased from 660 to 50 ms. Fig. 2 shows a fit to the τ o-Ba -[K + ] ext data using . The best fit was obtained with k ext = 7.6 ± 1.7 s −1 , k in = 0.11 ± 0.02 s −1 , and K d K = 2.7 ± 0.4 μM. The value of K d K found indicates that BK Ca channels bind K + fivefold tighter than previously thought . Our value of k ext was determined at 0 mV applied voltage. Neyton and Miller 1988a found that in a solution containing “0” external K + (<5 μM) and 150 mM Na + , k ext increased e-fold with every 27-mV depolarization. At 50 mV, in the presence of external 150 mM NMDG and contaminating K + , they measured a k ext = 20 s −1 . Using the expression k ext (V) = k ext (0)exp(V/27), we find that if k ext (0) is 7.6, then k ext (50) is 48 s −1 , which is 2.4× larger than the value found by Neyton and Miller 1988a . The larger value we predict is due to the reduction in the background contaminating [K + ] by the addition of the crown ether to the external solution. K d K is also voltage dependent and Neyton and Miller 1988b showed that the external lock-in site senses 18% of the voltage drop measured from the outside. Using the expression K d K (V) = K d K (0)exp(− z δ e V/ kT ) with z δ = 0.18 and a K d K (0) = 2.7 μM, we find that K d K (50) = 3.9, a value fivefold lower than the K d K of 19 μM determined by Neyton and Miller 1988a . Hence, by examining a wider range of external [K + ]s and using 18C6TA, we have been able to determine k ext and K d K with more precision. The fast component of the closed dwell-time distribution was also modified by external [K + ]. As in the case of the slow Ba 2+ block, the number of events increased as the external [K + ] concentration was reduced . However, in contrast to the slow component, the mean fast blocked time of the fast component of the closed time histogram is almost unchanged by a 10-fold reduction in the external [K + ]. Is the difference in slow and fast dwell-time dependence on [K + ] due to a modification of channel gating proper or is it the manifestation of a Ba 2+ flickering block? . To answer this question, we added crown ether to the internal side of the channel to a final concentration of 225 μM. This experimental maneuver decreases the internal [Ba 2+ ] from 70 to 5 nM, decreases the number of fast closed events by 30%, and increases P o by 18% (from 0.6 to 0.73). The number of fast block events per unit open time, N B , is predicted to be 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_{{\mathrm{B}}}=k_{{\mathrm{on}}} \left \left[{\mathrm{Ba}}^{2+}\right] \right {\mathrm{P}}_{o}{\mathrm{,}}\end{equation*}\end{document} where k on is the association rate constant for Ba 2+ binding, and P o the probability of opening. Therefore, a 14-fold decrease in [Ba 2+ ], considering the P o s before and after the addition of Ba 2+ , should induce a 90% decrease in N B. The theoretically expected decrease in N B after lowering [Ba 2+ ] int is much more pronounced than the one found experimentally. This analysis suggests that upon diminishing [K + ] ext , the increase in N B is only partly due to a Ba 2+ flickering block and that the reduction in external K + also induces the appearance of fast closed events. Occupancy of the outer mouth of the pore of Shaker K + channels by K + slows the rate of C-type inactivation . The site at which K + directly slows down inactivation appears to be a high-affinity binding site involved in the ionic selectivity mechanism . In fact, this site appears to be located in the neighborhood or in the channel selectivity filter, and Kiss et al. 1999 have argued that the selectivity filter itself is the inactivation gate. Since the selectivity filter is highly conserved among different K + channels, it is pertinent to ask why an inactivated state has not been previously observed in the BK Ca channel even at very low [K + ]. The single-channel current recorded at 0.09 μM K + in Fig. 1 shows a closed state of very long duration. Fig. 3 A shows that the channel enters this nonconducting state of very long duration when the [K + ] ext is reduced from the contaminating level to 0.01 μM by the addition of crown ether to the external solution . After spending several minutes in the quiescent state, normal channel activity was recovered by adding K + to the external side to a final concentration of 10 μM . The recovery of channel activity after a drastic reduction in external [K + ] occurred in 15 of 22 trials. It appears then that the BK Ca channel conductance collapses at external [K + ]s much lower than those necessary to arrest other K + channels. Lithium, Na + , Rb + , Cs + , and NH 4 + were also tested for their abilities to recover the BK Ca channel from its long-lasting nonconductive state. Rubidium (20 mM), Cs + (20 mM), and NH 4 + (3.5–50 mM) were able to recover the channel from the nonconducting state. Fig. 3 B shows an example of recovery from the quiescent state when NH 4 + is added to the external solution to a final concentration of 10 mM. Sodium (20–40 mM) and Li + (20–100 mM) were not able to recover channel activity, suggesting that only permeant cations are able to recover the channel from the conformation it adopts at very low [K + ]. In Shaker K + channels, one specific amino acid location in the pore-forming region (position 449) is crucial in determining sensitivity to external TEA + . An aromatic residue at the 449 position is a requirement for high affinity TEA + blockade and Heginbotham and MacKinnon 1992 showed that a bracelet of pore-lining tyrosines forms the high affinity TEA + receptor. BK Ca channels are also blocked by TEA + and they show a high affinity for this quaternary ammonium ion . In BK Ca channels, there is a tyrosine residue at a position corresponding to the TEA + -sensitive position in Shaker K + channels . We reasoned that since a TEA + binding site in BK Ca channels is structurally well defined , it would be of interest to see whether or not TEA + behaves as a lock-in ion like external K + . Since we expected TEA + to increase Ba 2+ mean blocked time, we reduced [K + ] ext to begin the experiment with short mean Ba 2+ block time. The crown ether concentration was adjusted to decrease the potassium concentration from the basal level down to values where the channels would not enter into the long lasting nonconducting state. Furthermore, TEA + seems to protect the channel from falling into the long lasting closed state since we observed stable channel activity with [K + ] ext as low as 0.007 μM. In the experiment shown in Fig. 4 , we reduced the external [K + ] concentration from 6 to 0.03 μM by adding 0.9 mM crown ether to the external solution. The figure shows the effect of external TEA + on the nonconducting dwell times induced by the presence of internal Ba 2+ . TEA + reduces the open channel current and also increases the duration of the closed dwell times. In the absence of TEA + , the measured mean block time was 160 ms; after increasing the external TEA + to 900 μM, the mean block time increased to 1,700 ms. Surprisingly, if external [K + ] is reduced from 0.06 to 0.007 μM by the addition of crown ether, in the presence of external TEA + , the mean block time is decreased . Fig. 5 , middle and bottom, shows that channel conductance is not affected by the addition of crown ether. Therefore, the complexing agent does not affect TEA + concentration. Fig. 6 shows that the effect of TEA + on Ba 2+ block strongly depends on external K + concentration. In the presence of 130 μM external K + , 2 mM TEA + brings the mean Ba 2+ blocked time to ∼20 s. Therefore, the blocked time is even longer than the maximum value expected for Ba 2+ leaving toward the internal side of the channel in the presence of high external [K + ]. However, in the presence of 0.04 μM K + , the same TEA + concentration induces a mean Ba 2+ blocked time of only 2 s. The ability of external TEA + to slow down Ba 2+ dissociation cannot be reconciled with the idea that TEA + and K + compete for the same binding site in the channel or with the simple picture of ion–ion repulsion within the pore. In both cases, it is expected that TEA + should behave less effective as a lock-in ion in the presence of K + . To interpret our results quantitatively, we propose the model illustrated in Fig. 7 . The channel is viewed as having three sites: a Ba 2+ -blocking site, a K + -binding site located externally to the blocking site, and the external TEA + site. As shown by Neyton and Miller 1988a and documented in Fig. 2 , at very low [K + ] and in the absence of external TEA, Ba 2+ can dissociate and exit to the external solution with a rate ( k ext ) much greater than the exit rate toward the internal side ( k in ) . The results shown in Fig. 2 were interpreted in terms of an increase in the occupancy of an external K + site as the external [K + ] was increased . The model proposes that TEA + can bind to the singly or doubly occupied channel. Therefore, TEA + can trap K + inside the pore and the blocking Ba 2+ ion must then either dissociate to the internal solution, or wait for the TEA + and K + sites to become empty. Assuming that the unblock–block reactions are slow compared with the K + and TEA + binding reactions, the model presented in Fig. 7 predicts the following relation for the mean Ba 2+ blocked time, τ ο-Βα : 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*} \left {\mathrm{{\tau}}}_{0-B{\mathrm{{\alpha}}}}= \left \left[k_{{\mathrm{in}}}P_{{\mathrm{Ba}}-{\mathrm{K}}-{\mathrm{S3}}}+k_{{\mathrm{in}} \left \left(K,{\mathrm{TEA}}\right) \right }P_{{\mathrm{Ba}}-{\mathrm{K}}-{\mathrm{TEA}}} \right +k_{{\mathrm{in}} \left \left({\mathrm{TEA}}\right) \right }P_{{\mathrm{Ba}}-{\mathrm{S}}2-{\mathrm{TEA}}}+ \left \left(k_{{\mathrm{ext}}}+k_{{\mathrm{in}}}\right) \right P_{{\mathrm{Ba}}-{\mathrm{S}}2-{\mathrm{S3}}}\right] \right ^{-1}{\mathrm{,}}\end{equation*}\end{document} where P Ba , P Ba-K , P Ba-TEA , and P Ba-K-TEA are the probabilities of finding the channel occupied by Ba 2+ only, by Ba 2+ and K + , by Ba 2+ and TEA + , or by Ba 2+ , K + , and TEA + , respectively. These probabilities are given by the following relationships: 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*}\begin{matrix}P_{{\mathrm{Ba}}-{\mathrm{S}}2-{\mathrm{S3}}}= \left 1+ \left \left({ \left \left[{\mathrm{K}}^{+}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}}}^{{\mathrm{K}}}\right) \right \right \\ \left \left \left(1+{ \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}1}}^{{\mathrm{TEA}}}\right) \right +{ \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}2}}^{{\mathrm{TEA}}} \right ^{-1}\end{matrix}\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*}P_{{\mathrm{Ba}}-{\mathrm{K}}-{\mathrm{S3}}}={ \left \left[{\mathrm{K}}^{+}\right] \right }/{ \left \left \left[{\mathrm{K}}^{+}\right] \right +{\mathit{K}}_{{\mathrm{d}}}^{{\mathrm{K}}} \left \left(1+{ \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}2}}^{{\mathrm{TEA}}}\right) \right + \left \left[{\mathrm{K}}^{+}\right] \right { \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}2}}^{{\mathrm{TEA}}} \right }\end{equation*}\end{document} 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*}P_{{\mathrm{Ba}}-{\mathrm{S}}2-{\mathrm{TEA}}}={ \left \left[{\mathrm{TEA}}\right] \right }/{ \left \left \left[{\mathrm{TEA}}\right] \right +{\mathit{K}}_{{\mathrm{d}}2}^{{\mathrm{TEA}}} \left \left(1+{ \left \left[{\mathrm{K}}^{+}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}}}^{{\mathrm{K}}}\right) \right \left \left(1+{ \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}1}^{{\mathrm{TEA}}}}\right) \right \right }\end{equation*}\end{document} 4d \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{Ba}}-{\mathrm{K}}-{\mathrm{TEA}}}={ \left \left[{\mathrm{TEA}}\right] \right }/{ \left \left \left[{\mathrm{TEA}}\right] \right +K_{{\mathrm{d}}1}^{{\mathrm{TEA}}} \left \left(1+{ \left \left[{\mathrm{K}}^{+}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}}}^{{\mathrm{K}}}\right) \right \left \left(1+{ \left \left[{\mathrm{TEA}}\right] \right }/{{\mathit{K}}_{{\mathrm{d}}2}^{{\mathrm{TEA}}}}\right) \right \right }{\mathrm{,}}\end{equation*}\end{document} where K d K is the dissociation constant for K + , K d1 TEA is the dissociation constant for TEA + from the triply occupied state, and K d2 TEA is the dissociation constant for TEA + from the doubly occupied state. There are five different rate constants for Ba 2+ exit: k ext is the rate of exit to the extracellular side when the channel is occupied only by a Ba 2+ ion, k in is the rate constant of exit to the intracellular side when the channel is occupied only by a Ba 2+ ion, and k in(K) , k in(TEA) , k in(K, TEA) are the rate constants of exit toward the extracellular side when the channel is occupied by Ba 2+ and K + , by Ba 2+ and TEA + , or by Ba 2+ , K + , and TEA + , respectively. The model accommodates rate constants for Ba 2+ exit toward the internal side that are different in the absence and presence of TEA + . Experimentally, we found that when the quaternary ammonium ion and K + are present in the external solution, k in(K,TEA) is approximately twofold slower than in the absence of TEA. On the other hand, the fit to the data with the model shown in Fig. 7 indicates that in the absence of K + , the rate constant for Ba 2+ exit, k in(TEA) , is approximately four times larger than k in(K,TEA) . We have tested our model by comparing the measured mean Ba 2+ blocked times at different [K + ] and [TEA + ] from 26 different single-channel membranes with the calculated mean blocked times obtained using . The model proposed in Fig. 7 describes the data rather well . The model is unable to predict the experimental results if triple occupancy is not allowed ( K d1 TEA = ∞) or if TEA + is unable to bind the channel unless it is occupied by K + ( K d2 TEA = ∞) . Fig. 8 B shows that if triple occupancy is not allowed, the model predicts an attenuated effect of TEA + on the mean Ba 2+ blocked time relative to that found experimentally. On the other hand, Fig. 8 C illustrates that if TEA + can only bind to the Ba 2+ -occupied channel in a triple occupancy configuration (when the lock-in site for K + is full), then the model fails to account for the data obtained at very low [K + ]. The best correlation between model generated and experimental values of the mean Ba 2+ block time was obtained when TEA + binding was allowed in any configuration of the model with rate constants of K d1 TEA = 180 μM and K d2 TEA = 67 μM. It is very interesting that the ratio between these two dissociation constants (2.5) reveals a K + –TEA + repulsion of ∼0.6 kcal/mol. This very low repulsion energy implies that the bound TEA + ion is essentially shielded from the K + ion occupying the external lock-in site. The kinetics of block by TEA + are rapid, operating in the time scale of microseconds ; therefore, the TEA + blocking events are too fast to be directly observed since they are filtered by the measuring electronics. Therefore, TEA + appears to reduce the observed channel current . Because of this effect, the ratio between the average single-channel current value in the absence of TEA + , i o , and its value in the presence of the quaternary ammonium ion, 〈 i 〉, is a measure of the channel occupancy by TEA + at its blocking site: 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*}{i_{0}}/{{\ll}i{\gg}}= \left \left(1+{ \left \left[{\mathrm{TEA}}^{+}\right] \right }/{K_{{\mathrm{d}}}^{{\mathrm{TEA}}}}\right) \right {\mathrm{.}}\end{equation*}\end{document} Since in this case TEA + blocks a channel containing only K + ions in its conduction machinery, it is pertinent to ask whether the dissociation constant for TEA + , K d TEA , is similar to that obtained from its effect on the mean Ba 2+ block time. Fig. 9 illustrates the dependence of the channel current on TEA + concentration at 0 mV and in different [K + ] (each symbol represents a different [K + ]). There is a linear relationship that is well described by with a K d TEA = 106 μM . Notice that the fit to is reasonably good for all the [K + ] tested, indicating that there is not a single hint of competition between K + and TEA + for a site(s). Moreover, the value of K d TEA + οβταιν∈δ ισ ω∈ρυ σιμιλαρ το τηατ οβταιν∈δ βυ Ωιλλαρρο∈λ ∈τ αλ> ≲+′°°≳ ≲+−″ μM) under symmetrical 100-mM KCl conditions. The crystal structure of the K + channel pore from Streptomyces lividans revealed two binding sites for potassium in the selectivity filter that are ∼0.75-nm apart . In this channel, like in the BK Ca channel, the TEA + binding site is comprised of four tyrosines located externally to the outer K + binding site . Since TEA + can trap K + ions inside BK Ca channels, we assigned lock-in site to the outer K + binding site described by Doyle et al. 1998 . Since the crystal radius of Ba 2+ is similar to the crystal radius of K + , Ba 2+ probably occupies the inner site. A third site was identified at the pore center in a large cavity . An ion is stabilized in this central position by the aqueous environment and by α helical structures pointing their partial negative charge toward the cavity where the ion is located. We hypothesize that the Ba 2+ flickering block originates from Ba 2+ entering and leaving the pore from the pore cavity. In the case of potassium channels, it is clear that permeating ions within the conduction pathway affect some of the structural changes associated with gating and C-type inactivation . Squid axon K + channels become nonfunctional when K + is removed from the internal and external solutions, but these channels can survive in an internal K + -free solution if external K + , Cs + , NH 4 + , or Rb + ions are present . Shaker K + channels in K + -free solutions become irreversibly nonconducting, but only after opening. This long-lasting state can be entered only if there is inward gating charge movement . Shaker K + channels can be protected against the deleterious effects of the absence of K + by mutations that remove C-type inactivation. On the other hand, some mammalian delayed rectifier K + channels remain stable after removal of internal and external K + and become permeable to Na + ions . The data presented here shows that occupancy of a very high affinity site for K + , most likely the lock-in site, controls ion permeation in the BK Ca channel. Emptying the channel of K + ions could lead to the equivalent of the C-type inactivation or to the K + conductance collapse phenomena described for other K + channels. When the lock-in site is empty, the channel clearly undergoes structural changes that lead finally to the long-lasting inactivated state. These changes are probably triggered by electrostatic repulsion of the carbonyl groups, which makes the selectivity filter atoms move apart. Fig. 2 shows that the K d K for the lock-in site is 2.7 μM, which corresponds to an energy well of −13 kT. Considering that this value of K d K is for the double occupied [K + –Ba 2+ ] channel, this energy is an upper limit that indicates that the binding of K + to BK Ca channels as tight as the binding of Ca 2+ to Ca 2+ channels . On the other hand, the ratio of the rate constants k ext / k in is 100 and this implies that Ba 2+ must jump an energy barrier 2.8 kcal/mol larger when leaving the channel toward the internal side. Although we do not know the details of the molecular mechanism that governs C-type inactivation, it is known that external TEA + , K + , and other monovalent cations inhibit it. Point mutations in Shaker K + channels have also shown that the rate of C-type inactivation and the K + permeability properties can be altered simultaneously . The general explanation of this phenomenon is that occupancy of a site by K + or other permeant cations hinders the C-type inactivation conformational change, probably a collapse of the selectivity filter . We have demonstrated here that TEA + binds to the Ba 2+ -blocked channel when the lock-in site is occupied by K + , and prevents K + from leaving this site. This turns TEA + into an ion that can protect the channel from C-type inactivation, by binding to a site different from the typical lock-in site. Considering that K + protects BK Ca channels from entering into a very stable nonconducting state, how is it that the BK Ca channel is not protected by the internal K + ions flowing through it? Our results suggest that Ba 2+ cuts off potassium flow to the external K + site. During a Ba 2+ block at very low [K + ] ext , the channel has two possibilities when the lock-in site is empty: (a) it can enter a long lasting nonconducting state leaving the Ba 2+ trapped inside or (b) Ba 2+ can occupy the lock-in site and exit the channel toward the external side, making the channel enter into a burst of activity. A similar effect of extracellular K + and internal blockade has been shown in Shaker K + channels . In this case, a hydrophobic TEA + analogue applied internally hindered the potassium flow to a site in the pore and thereby greatly increased the rate of C-type inactivation. Internal TEA + also prevents the refilling of the pore by K + in the case of the potassium channel of the squid axon . In the absence of external K + , this produces an irreversible decrease of the K + current. The effect of external K + on the ability of TEA + to lock Ba 2+ into the channel was explained using a model in which Ba 2+ , K + , and TEA + can simultaneously occupy the channel. The analysis of our results demonstrated that TEA + binding to the Ba 2+ -blocked channel is essentially the same whether or not a K + ion is bound and that the binding constant is not very different from the one obtained measuring the current amplitude in the presence of different [TEA + ] and [K + ]. This result implies that there is little electrostatic repulsion between the K + in the external lock-in site and the TEA + bound to its external receptor. The crystal structure of the K + channel from Streptomyces lividans showed that the distance separating the K + ion located in the external site of the selectivity filter and the TEA + ion is 0.8 nm . Given this distance, the expected electrostatic repulsion is 41 kcal/mol if a nonpolarizable medium separates the two ions, or 2 kcal/mol if a medium with a dielectric constant of 20 separated the ions. The fact that the expected repulsion between these two ions is not detected by our experiments can only be explained if the K + ion in the lock-in site is shielded from the TEA + . It is possible that the ring of aspartates located in position 295 in m Slo supplies this shielding. | Study | biomedical | en | 0.999998 |
10469728 | Cyclic nucleotide–gated (CNG) 1 channels are partially homologous to Shaker -type voltage-gated K + channels . They have six membrane-spanning segments and contain sequence motifs resembling the voltage-sensor (S4) and the pore-forming segment (P loop), which connects the S5 and S6 transmembrane domains . However, CNG channels typically require cyclic nucleotide to open, are only weakly voltage dependent in the presence of cyclic nucleotide, and are equally permeable to sodium and potassium ions . A model of the pore loop topology is a necessary step towards understanding gating and permeation in CNG channels. The substituted cysteine-accessibility method has been widely used for studying the pore of voltage-dependent channels. In brief, amino-acid residues within the P loop are replaced in turn with cysteines; the accessibility of each mutated residue to different thiol-specific reagents is then tested. For this purpose, methanethiosulfonate (MTS) compounds have often been used. Strong current inhibition by MTS reagents is usually considered to be an indication that the tested cysteine lines the pore lumen. The main assumption is that charged, polar reagents react predominantly with thiols in the water-accessible surface , the pattern of accessibility (from intra- or extracellular surfaces) gives information about pore topology. In particular, 2-aminoethyl MTS (MTSEA) covalently links an ethylammonium group to the thiol of cysteines . Sun et al. 1996 found that several residues within the P region of CNG channels are accessible to MTSEA from both sides of the plasma membrane, in contrast with results obtained for voltage-gated potassium channels . This unexpected result suggested a model of the pore in which the P loops belonging to the four channel subunits are parallel to the membrane surface. In this way, many amino acid residues should be accessible to MTS compounds from both sides of the plasma membrane. In addition, some residues are differentially accessible in the open and closed states, an indication that, during gating, the P region should change its conformation considerably. MTSEA, however, although unable to permeate the open channel as a charged amine , readily crosses the lipid bilayer, probably because the uncharged amine is partially soluble into the plasma membrane . Thus, the accessibility to MTSEA of several P loop residues from the two sides of the membrane could be due to MTSEA permeation through the plasma membrane instead of being a consequence of the CNG channel topology. It is therefore important to assess the accessibility of some impermeant MTS derivatives, such as [2-(trimethylammonium)ethyl]MTS . The P region of the α subunit of the CNG channel from bovine rod is composed of the amino-acid residues R345 to S371 . In the absence of atomic-resolution structure, the accessibility pattern of the different residues inside the P region to different toxins, ions, and chemical probes is useful to propose structural models of the pore. Unfortunately, no specific toxins are known for CNG channels. Moreover, a large fraction of the cysteine mutants in the P region of CNG channels are not functional . In particular, no data are available on the accessibility of residues between Y8 and L14. The lack of experimental information about the structure of long stretches of amino acid residues is a serious impediment to the development of quantitative molecular modeling of permeation and gating and to the performance of molecular dynamics simulations . It also prevents a thorough comparison between the pore structures of K + channels and CNG channels. This paper has two purposes. First, to analyze the effects of MTSEA and MTSET applied to the inner and outer surface of patches containing different cysteine mutants in the P region of rod CNG channel. This allowed us to refine the assessment of the relative position of pore residues. Second, to test the accessibility of three residues not studied so far: two mutants within the Y8–L14 segment, namely W9C and L12C, and mutant P21C. Our results indicate a different accessibility for MTSEA and MTSET and suggest that the accessibility of several residues to MTSEA from both sides of the plasma membrane is caused by MTSEA permeation through the lipid bilayer. MTSET effects suggest that the distinction between residues outwardly or inwardly accessible is clear cut, in the P loop of CNG channels, with an overall topology reminiscent of that of Kv2.1 voltage-dependent K + channels . Wild-type and mutant channels were expressed in Xenopus laevis oocytes (purchased from H. Kahler, Institute fur Entwicklungsbiologie, Hamburg, Germany). Oocytes were extracted from frogs anesthetized with 0.2% tricaine methanesulfonate and treated as previously described . After injection, the eggs were incubated at 18°C in Barth's solution containing (mM): 88 NaCl, 1 KCl, 0.82 MgSO 4 , 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 2.4 NaHCO 3 , 5 TRIS-HCl, pH 7.4, supplemented with 50 μg/ml gentamicin sulphate. Currents were measured 2–5 d after mRNA injection, at room temperature (20–22°C). During the experiments, oocytes were maintained in Ringer solution containing (mM): 110 NaCl, 2.5 KCl, 1 CaCl 2 , 1.6 MgCl 2 , 10 HEPES, pH 7.4. The P region of the α subunit of the bovine rod CNG channel , spanning the amino acid residues R1–S27 , was investigated using SCAM. The α subunit clone was mutated using the QuikChange™ Site-Directed Mutagenesis kit (Stratagene Inc.). All mutant RNAs were sequenced completely with the DNA sequencer LI-COR . RNAs for wild-type (WT) and mutant channels were synthesized in vitro by using the mCAP™ RNA Capping kit (Stratagene Inc.). Functional channels were observed for mutants K2C, V4C, S6C, L7C, W9C, L12C, T15C, T16C, I17C, T20C, P21C, P22C, V24C, and S27C, with and without the background mutation C505T. No cGMP-activated current was measurable from mutants Y3C, Y5C, Y8C, S10C, T11C, L14C, E19C, or P23C. Some single-channel activity was found for mutants T13C and G18C, but the open probability was too low to allow reliable accessibility tests (Becchetti, A., and K. Gamel, manuscript submitted for publication). We did not study residues R1, R25, or D26. Currents from inside- and outside-out patches were recorded with a patch-clamp amplifier (Axopatch 200B; Axon Instruments Inc.). Borosilicate glass pipettes (Brand GmbH) had resistances of 2–5 MΩ, in symmetrical NaCl solutions. Patch currents were low-pass filtered at 10 kHz and stored on PCM\VCR. During the analysis, single-channel traces were low-pass filtered again at 2 kHz and sampled at 5 kHz (pClamp6 hardware and software; Axon Instruments). Macroscopic currents measured at constant membrane potential were digitized from PCM/VCR at 50 Hz. Currents for current–voltage relations were low-pass filtered at 1 kHz and acquired on-line (at 5 kHz) with pClamp6. The perfusion system was as previously described . Currents were analyzed with pClamp6 or SigmaPlot (Jandel Scientific). Data are usually given as mean ± SEM. Pipette contained (mM): 110 NaCl, 10 HEPES, 0.2 EDTA (standard solution, buffered with tetramethylammonium hydroxide, pH 7.6). Inside-out patches were perfused with the same solution supplemented, when necessary, with 0.5 mM cGMP and/or the appropriate MTS compound (2.5 mM). Current gated by cGMP was the difference between the currents in the presence and absence of cGMP. After allowing current and baseline stabilization, by applying cGMP several times, the MTS reagent was applied for 2–3 min. We followed the MTS effect continuously, at −40 mV, either in the presence (open state) or absence (closed state) of cGMP . After washout, we measured the residual current for comparison with the initial current. At least once for each tested mutant, we also applied consecutive 200-ms voltage steps between −100 and +100 mV , to study the current–voltage (I/V) relations before, during, and after MTS application. I/V relations potentially provide more information about mutant channel behavior during gating. When the current inhibition was irreversible, as expected for block due to covalent reaction of MTS with substituted cysteines, we perfused the inside face of the patch with Ringer solution. This procedure always activated oocyte's calcium-dependent chloride channels and reassured us that the absence of a cGMP-activated current was not caused by the formation of a vesicle preventing the perfusion solution from reaching channels contained within the membrane patch. In the absence of MTS compounds, all mutants (except I17C) showed stable currents for at least 10 min (data not shown). The cGMP-activated current was measured from outside-out patches as the difference between the currents in the absence and presence of 5 mM external MgCl 2 . Pipette (internal side) contained our standard solution supplemented with 0.5 mM cGMP, whereas MgCl 2 was added to the bath standard solution. Mg 2+ ions completely block cGMP-gated currents from the extracellular side, at negative membrane potentials . The effect of 5 mM MgCl 2 on the basal leak current (i.e., in the absence of cGMP) was negligible (data not shown). MTS compounds were applied for 2–3 min in the bath. The time course of their effect was usually followed at −40 mV. As for the intracellular experiments, whenever possible I/V relations were obtained before and after the effect of MTSET. In this case, however, membrane potential was stepped from −100 to +40 mV because the Mg 2+ block is voltage dependent and complete only up to about +40 mV . We carried out inside-out experiments with MTSET in the pipette and, after measuring the initial current immediately after excision, followed the current kinetics at −40 mV by sampling the residual current with brief applications of cGMP . In these experiments, MTSET was constantly present on the outer side of the patch, from the moment of seal formation. Therefore, in mutants accessible from the outer side of the plasma membrane, we could follow the inhibition time course only when the procedure of seal formation and patch excision took ∼20–30 s. Otherwise, no cGMP-gated current was measurable in the presence of MTSET, even in patches excised from oocytes expressing high levels of CNG current . MTS reagents were purchased from Toronto Research Chemicals Inc. They were always dissolved in the appropriate solution at 2.5 mM before the experiment, and applied to the patch within ∼30 min. A relatively high concentration of MTS compounds is necessary when performing SCAM studies , because the time course of the hydrolysis of thiosulfonates in saline solution at pH 7.6 is not known with precision . This time course, however, is similar for MTSEA and MTSET . Furthermore, control experiments performed on mutants sensitive to MTS compounds, like T20C and P22C, showed that in our experimental conditions MTSEA and MTSET were still completely effective 30 min after dissolvement (data not shown). All other chemicals were from Sigma Chemical Co. In this paper, we describe results obtained with SCAM applied to the α subunit of the bovine rod channel . When expressed in Xenopus laevis oocytes, WT channels form homomultimers with the main properties of native channels . First, we show the effect of MTS compounds on WT channels . Then we examine the accessibility of the different cysteine mutants to MTSEA and MTSET, from the outer and inner surface of excised membrane patches. Experiments were done at saturating concentrations of cGMP , in symmetrical NaCl, and in the absence of divalent cations. We will mostly show data obtained with MTSET, since our results with MTSEA agree with those reported by Sun et al. 1996 . MTSET and MTSEA (not shown) produced a voltage-dependent block of WT channels, when applied to the patch inner side, in the presence of cGMP (i.e., when the channels were open). This block resembled the one caused by other organic cations . Since the channel inhibition at positive membrane potentials was reversible, the interaction of MTS's with the open channel does not involve any covalent reaction with endogenous cysteines. On the contrary, when MTSET or MTSEA (not shown) were applied in the absence of cGMP (i.e., when the channels were closed), the subsequent perfusion of cGMP, after MTS reagent had been washed out, revealed a partial (∼50%) irreversible current inhibition, independent of membrane potential. Sun et al. 1996 showed that MTSEA's irreversible block is removed by substituting the cysteine in position 505, within the cGMP-binding region, with a threonine. The same result applies to MTSET, which had no effect on C505T mutant channels either in the presence or absence of cGMP . Therefore, when studying the MTS effect on CNG channels from the cytoplasmic side in the closed state, it is necessary to use cysteine mutants containing the supplementary mutation C505T. Fig. 2B and Fig. D , also exemplifies our experimental procedure. Unless otherwise indicated, the MTS effect is shown for patches maintained at −40 mV. Usually, cGMP was applied two to three times before MTS application. The appropriate MTS reagent was then applied for 2–3 min. After washout, cGMP was applied again for comparison with the initial current (see methods ). The properties of cysteine mutants in the pore region were described in a previous paper . In brief, no major difference was found between cysteine mutants and WT channels, in the affinity to cGMP and the selectivity to monovalent alkali cations. However, several cysteine mutants had altered gating compared with WT channels. Some of these effects will be commented upon below. The supplementary mutation C505T did not cause any effect in addition to preventing irreversible block by MTS compounds (data not shown). Neither MTSEA nor MTSET had any effect on mutants K2C and S6C . cGMP-activated currents from mutant V4C were not affected by MTSET application to the inner side of the plasma membrane, in either the closed or open state . On the contrary, MTSET strongly inhibited cGMP-gated currents in mutant V4C, when applied to the outer side of membrane patches, both in the presence and absence of cGMP . These results suggest that V4 is outwardly accessible and possibly located in the outer pore vestibule. The segment formed by residues L7–L14 is very sensitive to cysteine mutation , leading to mutant channels with a reduced maximal open probability and an altered gating. After RNA injection, functional channels were only measurable from L7C, W9C, and L12C mutants. cGMP-activated currents from L7C mutant were scarcely different from those observed in WT channels. They were not affected by external MTSET . However, MTSET application to the inner side of membrane patches produced a partial but reproducible block: 60% (±9.9%, n = 4) in the closed state, and 25% (±3.6%, n = 5) in the open state . These results show that L7 residue is accessible from the internal side of the plasma membrane. The difference between MTSET effect in the open and closed state is statistically significant ( P < 0.01; Student's t test) and suggests that this residue changes its location during gating. Mutant channels W9C and L12C had altered gating with outwardly rectifying I/V relations. In particular, W9C currents were consistently smaller than currents from the other mutant channels, due to low single-channel open probability at all membrane potentials . External application of MTSET, in the presence of cGMP, had no effect on either mutant channel . On the other hand, W9C mutant was inhibited, while L12C was potentiated by internal MTSET . These data indicate that cysteines introduced in positions 9 and 12 are accessible from the inner side of the plasma membrane. The MTSET effect on the macroscopic W9C and L12C mutant currents could be due to an alteration of the single channel conductance and/or channel gating. To distinguish between these two possibilities, we applied MTSET to patches containing a limited number of CNG channels. Fig. 5 A illustrates current recordings before (top) and after (bottom) MTSET application from a patch (representative of five experiments) containing at least two W9C mutant channels, at −80 mV. After treatment, the CNG channel activity decreased, and double openings appeared very rarely. The single-channel current amplitude before and after application of MTSET was ∼2.1 pA, as shown in the amplitude histograms. Thus, MTSET did not alter the single-channel conductance of mutant W9C, but decreased its open probability. Fig. 5 B illustrates a similar experiment (representative of three) performed with mutant channel L12C. In this case, the higher frequency of channel fluctuations made it difficult to resolve a clear peak in the amplitude histogram, corresponding to the open level . However, no major change in the single-channel conductance was apparent from current traces and amplitude histogram, before and after (bottom) MTSET application, whereas the analysis of the amplitude histogram indicated a slight increase in the open probability after treatment, consistent with the small potentiation observed in macroscopic currents at the steady state (data not shown). These data suggest that cysteines introduced in position 9 and possibly also in position 12 are inwardly accessible to MTSET. Mutant channels T15C and T16C had similar properties ; therefore, only data from T16C mutant will be presented. The cGMP-gated currents were strongly and rapidly potentiated by internal application of MTSET (and MTSEA, not shown), in the presence and absence of cGMP . This potentiation varied from 50 to 200% and was almost absent when MTSET was applied from the outer side . When MTSET was applied in the presence of cGMP, it was possible to follow the time course of its effect. After the initial fast potentiation, we observed a slow partial irreversible current inhibition reaching the steady state ∼2 min after addition . The partial inhibition with respect to the maximal current, measured shortly after MTSET addition, was 55% ± 8%. Fig. 10 (below) shows only the net steady state potentiation; i.e., the potentiation of the current measured after MTSET washout with respect to the current before treatment. It should be noted that the I/V relations of T16C mutant exhibited a weak inward rectification, and current traces revealed a time dependency in the development of the steady state current . These features persisted after the MTSET effect . Moreover, we followed the potentiation in a patch containing a low number of channels . Before MTSET application, single-channel recording at −60 mV in the presence of 0.5 mM cGMP exhibited only two distinct current levels, corresponding to the closed state and to an open state of 2.3 pA. After MTSET application, the open state became more populated and it was often possible to observe current openings of 4.6 pA, corresponding to the simultaneous opening of two channels. Thus, MTSET produced no major change in channel conductance, and the potentiation appears to be due to an increased open probability. A similar potentiation was caused by MTSET application to the inner side of membrane patches containing T15C mutant channels. However, in this case, no inhibition followed the rapid initial potentiation (not shown). The potentiation produced by MTSET is a consequence of the low open probability of T15C and T16C channels, even in saturating cGMP . Furthermore, we investigated whether, in T15C, a partial inhibition produced by MTSET was masked by the strong potentiation. To assess this point, we perfused the inner side of patches containing T15C channels with our standard solution supplemented with 10 μM Ni 2+ to activate maximally the channels within the patch . After potentiation by nickel, MTSET did not produce any supplementary effect (data not shown). On the other hand, MTSET application to the outer face of the plasma membrane produced negligible effects on both T15C and T16C mutants . These results suggest that residues in position 15 and 16 are not accessible from the extracellular side, and that T16C is accessible to MTSET from the inner side of the plasma membrane and may be partially involved in channel gating. Contrary to what was observed in all other cysteine mutants in the CNG channel pore, cGMP-activated currents recorded from the mutant channel I17C rapidly decayed in inside-out patches. Current did not recover even when patches were maintained several minutes in the absence of cGMP (data not shown). The current lifetime was prolonged by adding the reducing agent dithiothreitol to the medium bathing the inner side of the plasma membrane . These results are reminiscent of those obtained in Na + channels, when mutants containing two substituted cysteines in the pore region are expressed in Xenopus oocytes . As proposed for sodium channels, a possible explanation for these results is that cysteines in close proximity form disulfide bridges. In this view, cysteines in position 17 of neighboring subunits should be in closer contiguity than cysteines in the other functional mutants. The half-time ( t 1/2 ) of the I17C current decay was 45.8 ± 5.2 s in the presence of 0.5 mM cGMP and 80.1 ± 12.1 s in the absence of cGMP . The difference is statistically significant (0.01 < P < 0.05, Student's t test). Application of MTS compounds to the inner side of membrane patches strongly reduced the t 1/2 in the presence of cGMP, the average value being 20.4 ± 2.5 s . The presence of MTS compounds also reduced the data scatter . These results argue that residue in position 17 was inwardly accessible to our probe. We propose that I17 is located near the narrowest section of the channel pore, at its inward side (see discussion ). The faster decay in the presence of cGMP suggests also that I17 residues of different subunits are closer to each other in the open state. Glutamate in position 19 is known to be accessible to both intra- and extracellular cations and is believed to be close to the narrowest section of the channel pore . When E19 was mutated to a cysteine, no functional channels were observed, thus the accessibility of this residue cannot be studied with SCAM. Therefore, it is particularly important to analyze the accessibility of residues in positions 20–22. Both MTS reagents, when applied outwardly, strongly blocked T20C currents, both in the closed and open state . However, MTSEA and MTSET gave different results when applied to T20C channels from the inner side of the plasma membrane. MTSEA inhibited T20C current in both the open and closed state , whereas MTSET was not effective in either condition . Similar results were found for P22C channels. In the latter however, at variance with WT and the other cysteine mutants, the reversible MTSET block was also present at negative membrane potentials. Furthermore, the inhibition of this mutant channel required a several-minute washout to be completely reversed. This suggests that the affinity of the channel pore for MTSET is increased in P22C mutant. As shown in Fig. 9 , Fig. 10 , and Fig. 12 , MTSEA applied to the inner and outer sides of membrane patches irreversibly inhibited T20C and P22C channels, while MTSET was only effective when applied to the external side. This suggests that the internal MTSEA inhibition, in these cases, is due to permeation of this compound through the lipid bilayer . To verify this possibility, we applied MTSEA to the inner side of inside-out patches containing either T20C or P22C, when the patch pipette contained 10 mM cysteine, thereby used as a thiol scavenger on the outer side of inside-out patches . The presence of cysteine in the pipette solution did not affect the cGMP-gated current appreciably . In the presence of cysteine, the inhibition produced by MTSEA was always incomplete and partially reversible after washout , although washout needed to be prolonged for several minutes, indicating again that cysteine mutation in residues T20 and P22 somewhat alters the channel affinity for MTS compounds. In the presence of external cysteine, MTSEA produced 48% ± 8.6% block on T20C channels ( n = 6), and 23% ± 12% block on P22C channels ( n = 3), whereas the inhibition in the absence of cysteine was always complete and irreversible even after 10–15-min washout for both mutants. In this case, we have pooled experiments in which MTSEA was applied in the presence and absence of cGMP, since no difference was found between the two conditions. Cysteine rescue of MTSEA internal block on T20C and P22C currents reinforces the conclusion that T20 and P22 are outwardly accessible. The effect of MTS compounds on mutants T20C and P22C was also studied at a single-channel level. Fig. 11 shows current traces from a patch probably containing two active P22C mutant channels, before (top) and after (bottom) MTSEA application to the inner side of the plasma membrane. Currents were recorded at −100 mV, in the presence of 1 mM cGMP. After treatment, no openings were observed and the amplitude histograms of current fluctuations in the presence and absence of cGMP became identical. This result is consistent with MTSEA causing a complete channel block and suggests that the residue in position 22 lines the permeation pathway. Similar results were obtained with T20C mutant (data not shown). Finally, inwardly applied MTSET had no effect on P21C currents, whereas it produced a small block when applied externally. Negligible effects were produced by MTSET on V24C and S27C mutant channels . We conclude that T20, P21, and P22 are outwardly accessible residues. The weak effect on P21C indicates that the side chain of this residue does not line the channel pore lumen, after cysteine mutation at least. The results presented in this paper show that several amino acid residues in the P loop of the rod CNG channel are differently accessible to MTSEA and MTSET. The accessibility map of the residues tested with MTSET is consistent with a topological structure of the pore region different from the one previously proposed , and more similar to that of voltage-dependent K + channels , which have a significant homology with CNG channels . Due to the experimental variability , we assumed that only average MTSET effects >20% inhibition or potentiation provided information on residue accessibility . Our results obtained with MTSEA (in the absence of cysteine on the trans side) are broadly in agreement with those reported by Sun et al. 1996 , but some differences have been found that merit a brief discussion. Our single-channel recordings show that mutant channels with a cysteine in the pore loop often had a low maximal open probability . Hence, the difference in MTS compounds accessibility in the “closed” and “open” states (i.e., in the absence or presence of a saturating cGMP concentration) must be interpreted with some caution, especially regarding the cysteine mutants within the segment W8-I17, which show the strongest alterations in the open probability. The cGMP-gated current rundown we observed when recording from I17C mutant channels was not reported by Sun et al. 1996 . A possible explanation for this discrepancy is that these authors have always applied MTSEA after current had reached the steady state, which would explain the very low I17C current amplitude they observed. Finally, the initial MTS-induced potentiation we observed in T15C and T16C mutant channels is larger than that described by Sun et al. 1996 . Again, if Sun et al. 1996 have plotted the steady state MTSEA effect, there is no contrast between our data and theirs; the slow incomplete block we have observed in T16C mutant, following the quick potentiation, agrees with the data reported by them. Despite this general agreement, our topological model for the pore, described in the last section below, differs from that of Sun et al. 1996 . The reason for this disagreement is that we base our model on the results we obtained with MTSET inwardly or outwardly applied and on the results we obtained with MTSEA applied inwardly, in the presence of cysteine on the external side (where MTSEA should react only with residues accessible from the inner side). The MTSEA and MTSET effects on cysteine mutant channels were different from one another. MTSEA targets several residues when applied from either side of the plasma membrane . In contrast, we found that residues V4C, T20C, P22C, and (to a lesser extent) P21C were only accessible to outwardly applied MTSET. Furthermore, cysteine on the outer side of the patch prevented MTSEA block from the inner side . We conclude that these differences arise because MTSEA, but not MTSET, is partly permeant through the plasma membrane as a charged amine , and propose that V4, T20, P21, and P22 are only accessible from the outer side of the membrane. Glutamate in position 19 is located at (or near) the narrowest region of the pore . Therefore, T20, P21, and P22 are likely to form the outer pore vestibule, in agreement with the strong current inhibition produced by external MTSET on T20C and P22C mutants. The block produced by MTSET applied to the outer side of V4C mutant channels suggests that V4, too, is an outwardly accessible residue . Data are consistent both with a location of V4 within the extracellular pore vestibule and with a possible crucial involvement of V4 in the gating process, not necessarily involving the channel vestibule. Current activated by cGMP from I17C mutant spontaneously decayed in inside-out patches . This decay is slowed down by inwardly applied dithiotreithol , suggesting that current rundown is due to the formation of disulfide bridges in excised patches. It is likely that the formation of disulfide bridges is prevented inside the intact oocyte, because of the reducing intracellular environment . As shown in Fig. 8 , the MTS compounds decreased the half time of current decay, when applied to the inner side of membrane patches. The time course of current rundown was slower in the absence of cGMP, suggesting that this residue may move slightly towards the pore axis during channel opening. These results suggest that I17C residue is inwardly accessible. In addition, when the adjacent residues G18 and E19 were replaced by a cysteine, no functional channels were observed. In this case also, the lack of expression may be due to the formation of disulfide bridges, since E19 residue is thought to line the channel pore. This residue is accessible to extracellular divalent cations and to monovalent cations applied to the inner side , and is a strong determinant of CNG channel permeation . Hence, we suggest that I17 residue faces the inner side of the plasma membrane and is located within the channel pore, and that G18 and E19 form the narrowest section of the pore itself. Within the segment L7-T16, only mutants L7C, W9C, L12C, T15C, and T16C produced functional channels in Xenopus oocytes. None of these mutants was affected by external MTSET, whereas all were sensitive to inward application of MTSET, though to different degrees. cGMP-gated currents from L7C and W9C were inhibited, indicating directly that residues L7C and W9C are accessible from the intracellular side. On the contrary, currents from L12C, T15C, and T16C were potentiated. It should be noted that, in all of these mutants, single-channel recording showed a decrease in open probability with respect to the WT channels in saturating cGMP . Therefore, we cannot exclude that cysteine substitution of these residues makes cGMP a partial agonist, analogous to what was found after mutating E19 . In this case, the potentiation exerted by intracellular MTSET may be caused by an aspecific interaction between the cationic MTSET and channel portions different from the substituted cysteine, as, for instance, the residue H420, responsible for cGMP-gated current potentiation, at submaximal cGMP concentrations . However, since intracellular MTSET had a partial irreversible inhibitory effect on T16C after the quick initial potentiation, and the residues L7, W9, and I17, which bracket the L12–T16 segment, were inhibited by intracellular treatment, we suggest that L12, T15, and T16 face the inner side of the plasma membrane. In light of the results presented and discussed in this manuscript, the model shown in Fig. 13 is proposed for the pore loop topology in CNG channels. Residues accessible to MTSET from the external side of the plasma membrane were colored in red, residues accessible from the internal side were colored in blue, and white residues were either not accessible to MTS compounds or were not studied because cysteine substitution on these positions did not yield functional channels. The arrows indicate suggested displacements occurring during channel opening. Residues I17, G18, and E19 form the narrowest portion of the pore, whereas residues T20, P21, P22, and P23 form the extracellular channel vestibule. The three prolines 21–23 may form a polyproline loop . The diameter of the extracellular vestibule lumen, lined by residues T20, P21, and P22, is likely to be wider than the diameter of the pore lumen at the level of the residues G18–E19 for two reasons. First, the large thiol reagent MTSET can readily reach all these residues. Second, in mutant channels T20C, P21C, and P22C, cysteines of neighboring subunits are not likely to become so close to each other to form disulfide bonds, leading to channel occlusion. In fact, macroscopic cGMP-gated currents from mutants T20C, P21C, and P22C, in symmetrical sodium and in saturating cGMP, usually have amplitudes comparable with those of WT currents, with no evidence of rundown . Residue G18 may cause a turn in the P loop so that only the adjacent I17 residues belonging to the different subunits are still sufficiently close to form disulfide bridges, when substituted with cysteines, whereas the following residues towards the amino terminal do not line the pore lumen. Residues in the segment from L7 to T16 are intracellular. In particular, residues L7 to T13 have a significant homology, with residues L66 to T72 forming the final portion of the pore outer helix in KcsA potassium channel . Therefore, residues L7 to T13 in CNG channels may form an alpha helix. Residues from L7 to V4 span the plasma membrane and residue V4 is extracellular, possibly being part of the outer pore vestibule. It should be recalled here that the residues that form the segment 321–339, towards the amino terminal with respect to V4, were shown to be extracellular by immunocytochemistry . Our model of the CNG channel pore differs from that of Sun et al. 1996 because we have not observed any substituted cysteines in the P loop to be accessible by MTSET from both sides of the plasma membrane. The difference in the two models reflects the fact that MTSEA can cross the lipid bilayer and thus react with residues inaccessible to MTSET (or MTSEA in the presence of cysteine on the trans side). The accessibility to MTSET of most of the tested residues was similar irrespective of whether MTSET was applied in the presence or absence of cGMP. In mutants whose open probabilities were 0.8 or larger, in saturating cGMP (as is also the case for WT channels), the presence or absence of cGMP was a tool to study the accessibility in the open or closed state, respectively. In mutants whose open probabilities were smaller , the distinction between accessibility in the open and closed state was not clear-cut, since even in the presence of saturating cGMP the channels remained in the closed state for a considerable fraction of the time. MTSET inhibition of L7C channels was slightly larger in the closed state, suggesting that, in the open state, L7 residue either moves towards the extracellular side of the plasma membrane or becomes less accessible. On the contrary, the accessibilities of residues T20, P21, and P22 were almost identical in the closed and open state, indicating that the outer vestibule does not undergo any large conformational rearrangement during gating. The faster rundown of cGMP-activated current in mutant I17C in the open state may be taken as an indication that residues I17 are closer to each other in the open than in the closed state. These results suggest, but do not prove, that the opening of the CNG channel is primarily mediated by a widening of the channel lumen near residues 18 and 19, which is accompanied by a movement of I17 residues towards the pore axis. This is in agreement with reports that residue E19 is accessible to internal tetracaine in the closed but not open configuration . The comparison between the SCAM data presented here and the pore structure of the voltage-dependent K + channels presents both similarities and differences. SCAM has been applied to the study of the pore loop topology of voltage-dependent Kv2.1 delayed rectifying channels from rat brain . In Kv2.1 channels, the MTSET effects identify two distinct pore loop segments whose residues are accessible from either the outer or the inner side of the plasma membrane. In particular, residues D378 to K382, corresponding to G18–P22 in CNG channels , were outwardly accessible . On the other hand, residues T370 to V374, corresponding to T13–I17 in CNG channels, were mostly accessible to MTSET applied inwardly . Furthermore, residue P361 in Kv2.1 channels, corresponding to V4 in CNG channels, was accessible to extracellular MTSET and Cd 2+ , whereas residues A362–T372, corresponding to Y5–T15 in CNG channels, were not affected by external MTSET and Cd 2+ . In contrast, in their studies of the Shaker K + channels, Lu and Miller 1995 , using Ag + , and Gross and MacKinnon 1996 , using MTSEA, found only outwardly accessible sites, with the periodicity of an α helix, within the segment D431–V438, consistent with the atomic resolution KcsA channel structure . These residues correspond to Y5–L12 in CNG channels , among which we found only inwardly accessible residues (L7, W9, and possibly L12). Therefore, K + and CNG channels share some structural homology in the pore region, but they also show significant differences in the first part of the P segment, even though, in this area, their amino acid sequences are very similar . | Study | biomedical | en | 0.999994 |
10469729 | Skeletal and cardiac muscle dihydropyridine receptors (DHPRs) 1 are highly homologous proteins that function both as voltage-gated L-type Ca 2+ channels (L-channels) and as links between sarcolemmal depolarization and the release of Ca 2+ from the sarcoplasmic reticulum (SR). Nonetheless, there are important differences between the two proteins. Cardiac L-channels activate 10-fold more rapidly upon depolarization and the resulting Ca 2+ influx triggers the intracellular release of Ca 2+ via ryanodine receptors (RyRs) located nearby in the SR . In contrast to this “cardiac-type” excitation–contraction (EC) coupling, it is generally accepted that activation of RyRs in skeletal muscle is coupled to conformational changes of the skeletal DHPR via a mechanical interaction that is independent of the entry of extracellular Ca 2+ . Studies of heterologously expressed Ca 2+ channels in dysgenic myotubes, which lack endogenous skeletal L-channels, are consistent with this difference in type of EC coupling: removal of external Ca 2+ , or addition of Cd 2+ and La 3+ to block Ca 2+ entry, abolishes EC coupling in dysgenic myotubes expressing cardiac L-channels , but not in dysgenic myotubes expressing skeletal L-channels . However, although there is little doubt that Ca 2+ -entry–induced Ca 2+ release is involved in cardiac EC coupling, recent work suggests that a mechanical coupling may occur between cardiac L-channels and RyRs when intracellular cAMP levels are elevated . In spite of these distinct functional properties, the selectivity of both skeletal and cardiac L-channels for Ca 2+ over monovalent cations appears to involve intrapore binding of Ca 2+ with high affinity . Mutational analysis of the cardiac L-channel indicates that this intrapore Ca 2+ binding is coordinated by a cage of four conserved glutamate residues residing in corresponding positions within each of the pore-lining regions of repeats I–IV of the α 1 subunit . In fact, a single mutation of the glutamate residue in repeat III to a lysine residue is sufficient to alter the cardiac L-channel such that it exhibits minimal divalent permeability, large monovalent permeability, and a >1,000-fold decrease in high affinity Ca 2+ block of monovalent currents . The effects of mutating pore region glutamates on the Ca 2+ permeability of skeletal L-channels have yet to be elucidated. Here, we have used expression in dysgenic myotubes to compare permeation and EC coupling in skeletal and cardiac L-channels after substitution of lysine for the repeat III glutamate. Compared with the wild-type channel, the mutant cardiac L-channel (CEIIIK) conducted small inward currents carried by Ca 2+ and large outward currents carried by monovalent cations. The mutant skeletal L-channel (SkEIIIK) conducted large outward currents, but no detectable inward Ca 2+ current. As a result of the greatly reduced Ca 2+ entry, CEIIIK lost the ability of wild-type cardiac L-channels to trigger the release of SR Ca 2+ in dysgenic myotubes. By contrast, SkEIIIK channels were able, despite the complete loss of Ca 2+ entry, to trigger the release of SR Ca 2+ with a voltage dependence similar to that of the wild type skeletal DHPR. The production of large outward currents by the SkEIIIK and CEIIIK channels allowed us to compare activation rates over a much broader range of voltages than wild type channels, which can only be compared for test potentials that are both sufficiently positive to cause activation and sufficiently negative to provide a significant driving force for inward Ca 2+ current. We found that the activation rate of both channels was very weakly voltage dependent and that the activation of SkEIIIK was >10-fold slower than that of CEIIIK, even at +100 mV. Skeletal L-channels are unusual in that channel activation is both slow and weakly voltage dependent, while channel deactivation is strongly voltage dependent. This behavior can be accounted for by a linear reaction scheme in which the rate-limiting transition has an asymmetric voltage dependence . Because activation is slow, this asymmetric transition would generate only a very small “ON” gating current (Q on ). However, because deactivation is fast at negative voltages, repolarization should produce a component of “OFF” gating current (Q off ) with a magnitude that is dependent on the extent of activation occurring during a preceding depolarization. An unambiguous test of this prediction is difficult for wild-type channels since any apparent increase in Q off might simply be due to the presence of incompletely blocked ionic tail current. However, SkEIIIK, which did not permit inward Ca 2+ current, provided a powerful tool for testing this prediction. A comparison of Q off for SkEIIIK after strong depolarizations of 20 or 200 ms showed that the longer depolarizations recruited additional gating current, which increased Q off by >20%. The size of the recruitable SkEIIIK Q off and the similarity of its voltage dependence to that of channel conductance indicates that the majority of skeletal muscle DHPRs not only undergo the voltage-dependent conformational changes that trigger SR Ca 2+ release, but also the voltage-dependent transitions that are required for L-channel opening. Primary cultures of myotubes were prepared from skeletal muscle of newborn dysgenic mice, as described previously . All experiments were performed 7–11 d after the initial plating of myoblasts and were carried out at room temperature (20–22°C). Numerical figures are presented in the text and figures as mean ± SEM. Data were taken to be statistically significant at P < 0.001. A glutamate-to-lysine mutation in the repeat III pore region of the skeletal muscle dihydropyridine receptor was constructed using PCR. Two initial PCR fragments were produced in separate but parallel reactions. Each initial fragment was generated by amplifying pCAC6 with an “outer” primer and a mutating primer. The final mutated fragment was obtained by mixing the two initial PCR products and amplification in a third reaction with the outer primers . The composition of the two mutating primers was, sense: 5′-GTGTCCACC TTTAAA GGATGGCCCCAG-3′ and antisense: 5′-CTGGGGCCATCC TTTAAA GGTGGACAC-3′, where underlining indicates the mutated region. The composition of the outer primers was, sense : 5′-GTCCGTGAGGAATCAGATCCTTGG-3′ and antisense : 5′-TCCACAGCAGCGTGCGCACGCCCT-3′. This mutagenesis strategy not only substituted a lysine codon (AAA) for the glutamate codon (GAG) in the pore region of repeat III, but also exchanged phenylalanine codons (TTT replaced TTC) so as to introduce a novel DraI restriction site (TTTAAA) that was used to screen clones for the mutation. Each PCR reaction contained template DNA, 100 pmol of each primer, 100 μM of each deoxyribonuclease triphosphate (GIBCO-BRL), 2 U Vent DNA polymerase, 10 μl polymerase buffer (10×), and sterilized water sufficient to bring the final volume to 100 μl. 35 amplification cycles were performed with a PCR thermal cycler (Perkin-Elmer Corp.); cycle 1, 95°C for 4 min, 60°C for 2 min, and 72°C for 2 min; cycles 2–35, 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. After the last cycle, an additional 5-min extension period at 72°C was used. This PCR mutagenesis strategy generated a 1.3-kB mutant fragment that was subsequently digested with XhoI and HincII to generate a 1.0-kB mutant fragment that was used to replace the corresponding fragment from the 2.7-kb XhoI–SacI fragment of pCAC6 subcloned into pBluescript (Stratagene Inc.). Finally, the 1.7-kB XhoI–DraIII fragment of the mutant subclone was used to replace the corresponding fragment from pCAC6 to generate SkEIIIK. The entire 1.0-kB XhoI–HincII region of SkEIIIK was ultimately sequenced using an ABI 377 automatic sequencer to verify sequence integrity. The cDNA encoding CEIIIK was a generous gift form Dr. William Sather. Both SkEIIIK and CEIIIK use the mammalian expression vector pKCRH2 . Approximately 1 wk after plating, myotubes were microinjected into a single nucleus with expression plasmid for either CEIIIK or SkEIIIK (200–500 ng/μl). Myotubes expressing SkEIIIK were identified by contraction in response to extracellular electrical stimulation (80 V, 10–30 ms). Myotubes expressing CEIIIK never exhibited electrically evoked contractions and were usually identified by coinjecting 20 ng/μl of a cDNA expression plasmid encoding an enhanced green fluorescent protein (GFP). In control experiments, coexpression of the GFP cDNA did not alter the function of expressed L-channels (data not shown). Coinjection of GFP cDNA was omitted when cells were to be used for measurement of intracellular Ca 2+ transients and the expression of CEIIIK was established on the basis of large, rapidly activating outward ionic currents. The whole cell variant of the patch clamp technique was used to measure ionic currents, intramembrane charge movements, and intracellular calcium transients. Pipettes were fabricated from borosilicate glass and had resistances of 1.8–2.2 MΩ when filled with the internal solution. Linear capacitative and leakage currents were determined by averaging the currents elicited by multiple (usually 10) 20-mV hyperpolarizing pulses from a holding potential of −80 mV. This control current was then scaled appropriately and used to correct test currents for linear components of capacitative and leakage currents. Electronic compensation was used to reduce the effective series resistance (usually to ∼1 MΩ) and the time constant for charging the linear cell capacitance to <0.5 ms. Cell capacitance was determined by integration of the capacity transient resulting from the control pulse and was used to normalize currents (pA/pF) or charge movements (nC/μF) obtained from different myotubes. Ionic currents were filtered at 2 kHz and digitized at 1 kHz. To measure macroscopic L-current in isolation, a 1-s prepulse to −30 mV followed by a 25-ms repolarization to −50 mV was administered before the test pulse (prepulse protocol) to inactivate T-type Ca 2+ currents. The activation phase of L-currents was fitted by the following exponential function: 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*}I \left \left(t\right) \right =I_{{\mathrm{{\infty}}}} \left \left[1-{\mathrm{exp}} \left \left({-t}/{{\mathrm{{\tau}}}_{{\mathrm{act}}}}\right) \right \right] \right {\mathrm{,}}\end{equation*}\end{document} where I ( t ) is the current at time t after the depolarization, I ∞ is the steady state current, and τ act is the time constant of activation. The voltage dependence of SkEIIIK L-channel activation was obtained by tail-current analysis. In brief, myotubes were stepped for 200 ms to test potentials (V) ranging from −50 to +80 mV; the instantaneous current ( I T ) was then measured immediately after stepping to +40 mV. These data were then normalized to I max (the maximal I T ) and fitted according to: 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*}{I_{{\mathrm{T}}} \left \left({\mathrm{V}}\right) \right }/{I_{{\mathrm{max}}}}={1}/{ \left 1+{\mathrm{exp}} \left \left[{ \left \left({\mathrm{V}}_{{\mathrm{G}}}-{\mathrm{V}}\right) \right }/{k_{{\mathrm{G}}}}\right] \right \right {\mathrm{,}}}\end{equation*}\end{document} where V G is the potential causing half-maximal activation of L-type conductance and k G is a slope parameter. For measurement of intramembrane charge movements, filtering was at 2 kHz (eight pole Bessel filter; Frequency Devices Inc.) and digitization was at 10 kHz. Voltage clamp command pulses were exponentially rounded with a time constant of 50–300 μs and the prepulse protocol (see above) was used to reduce the contribution of gating currents from sodium channels and T-type Ca 2+ channels. “OFF” transients of charge movement were measured for repolarization to −50 mV after test pulses to various test potentials (−50 to +60 mV, in 10-mV increments). The rectification of the mutant L-channels meant that influx of Ca 2+ ions was minimal for CEIIIK and nonexistent for SkEIIIK . However, since some dysgenic myotubes exhibit a small, endogenous, rapidly activating L-current, residual Ca 2+ channel ionic currents were blocked by the addition of 2.0 mM CdCl 2 + 0.2 mM LaCl 3 to the extracellular recording solution (see Solutions ). The integral of the OFF transient for each test potential (V) was normalized by the maximal value of Q off (Q max ) and fitted according 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*}{{\mathrm{Q}}}/{{\mathrm{Q}}_{{\mathrm{max}}}}={1}/{ \left 1+{\mathrm{exp}} \left \left[{ \left \left({\mathrm{V}}_{{\mathrm{Q}}}-{\mathrm{V}}\right) \right }/{k_{{\mathrm{Q}}}}\right] \right \right {\mathrm{,}}}\end{equation*}\end{document} where V Q is the potential causing movement of half the maximal charge, and k Q is a slope parameter. Changes in intracellular Ca 2+ were recorded with Fluo-3, as described previously . In brief, the salt form of the dye was added to the internal solution. After rupture of the cell membrane and entry into the whole-cell mode, a waiting period of >5 min was used to allow the dye to diffuse into the cell interior. A 75W xenon illuminator and a set of fluorescein filters were used to excite the dye present in a small rectangular region of the voltage-clamped myotube. A computer-controlled shutter was used to block illumination in the intervals between test pulses. Fluorescence emission was measured by means of a fluorometer apparatus (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA). The background fluorescence was measured for each myotube before membrane rupture and cancelled by analogue subtraction. Fluorescence data are expressed as Δ F / F b , where Δ F represents the change in fluorescence from baseline (Δ F = F − F b ) and F b represents the average myotube fluorescence immediately before depolarization. For measurements of macroscopic ionic and gating currents, the internal solution consisted of (mM): 140 Cs-aspartate, 10 Cs 2 -EGTA, 5 MgCl 2 , and 10 HEPES, pH 7.40 with CsOH. For the measurements of depolarization-induced Ca 2+ transients , the patch pipettes contained an internal solution composed of (mM): 110 Cs-aspartate, 5 MgATP, 22.8 EGTA, 2.66 CaCl 2 , 1.2 MgCl 2 , 0.2 K 5 Fluo-3 (Molecular Probes, Inc.), and 10 HEPES, pH 7.40 with CsOH. Ionic currents and intracellular Ca 2+ transients were recorded in an external solution containing (mM): 145 TEA-Cl, 10 CaCl 2 , 0.003 tetrodotoxin (TTX), and 10 HEPES, pH 7.40 with tetraethylammonium (TEA)-OH. Gating currents were recorded in an external solution containing (mM): 145 TEA-Cl, 8 CaCl 2 , 2 CdCl 2 , 0.2 LaCl 3 , 0.003 TTX, and 10 HEPES, pH 7.40 with TEA-OH. The dihydropyridine antagonist, (+)-PN 200-110, was kindly provided by Drs. E. Rossi and A. Lindenmann of Sandoz Ltd. (Basel, Switzerland). Fig. 1 shows ionic currents in dysgenic myotubes expressing SkEIIIK and CEIIIK, the skeletal and cardiac DHPRs, respectively, in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Under the recording conditions used, the wild-type skeletal and cardiac L-channels support inward Ca 2+ currents for test potentials ranging from about −20 (cardiac) or 0 (skeletal) mV to +60 mV or greater, with the currents reversing to outward at an extrapolated potential of more than +70 mV . As reported previously for expression in Xenopus oocytes , CEIIIK channels expressed in dysgenic myotubes had dramatically altered divalent permeability and mediated inward Ca 2+ currents for only a few potentials . At potentials greater than +20 mV, dysgenic myotubes expressing CEIIIK exhibited rapidly activating outward currents (presumably carried by Cs + , the predominant intracellular cation). Dysgenic myotubes expressing SkEIIIK L-channels did not exhibit inward currents at any potential, but did give rise to large, slowly activating outward currents at all potentials greater than +20 mV . The outward currents for both SkEIIIK and CEIIIK were blocked by a DHP antagonist, confirming that they were produced by expressed L-channels (at +100 mV, 1 μM PN 200-110 reduced the SkEIIIK current by 90.0 ± 3.6%, n = 3, and CEIIIK current by 93.5%, n = 2). Fig. 1 B illustrates peak current–voltage relationships determined from the cells illustrated in Fig. 1 A. Compared with wild-type cardiac channels, the reversal potential for CEIIIK was negatively shifted by ∼60 mV, indicative of a large change in the permeability of Ca 2+ relative to Cs + . The negative shift in reversal potential appeared to have been still larger for SkEIIIK since inward Ca 2+ currents were not elicited at any test potential, even though depolarizations >10 mV caused significant activation of SkEIIIK channels . Furthermore, when outward currents via SkEIIIK were elicited by strong depolarizations, subsequent repolarization to negative potentials failed to cause inward ionic tail currents (see below). Thus, the conserved glutamate in the pore region of repeat III appears to play a role in controlling Ca 2+ permeation through the skeletal L-channel, which is even more pivotal than its role in the cardiac L-channel. Like wild-type skeletal L-channels, the SkEIIIK channels activated slowly. At test potentials where both produce appreciable currents (e.g., +60 mV), the SkEIIIK and native skeletal L-channels both display slow activation (native: 52.4 ± 2.0 ms, n = 6; SkEIIIK: 40.8 ± 1.7 ms, n = 11). Thus, the identity of the permeating ion has little effect on activation and the EIIIK channels provide a convenient tool for extending the comparison of skeletal and cardiac activation to more positive potentials . Even at +100 mV, activation of SkEIIIK is more than 10-fold slower than that of CEIIIK (SkEIIIK: 30.7 ± 1.9 ms, n = 11; CEIIIK: 2.9 ± 0.5 ms, n = 7). Because activation of the skeletal L-channel is slow over a broad range of test potentials (+20 to +100 mV), the rate-limiting event for the transition of the channel from closed to open must be very weakly voltage dependent, as we have suggested previously from single channel analyses . Controversy remains as to whether Ca 2+ permeation and/or intrapore Ca 2+ binding are necessary for the ability of the cardiac and skeletal L-channels to mediate EC coupling. We reexamined this issue with the CEIIIK and SkEIIIK L-channels since they have a profoundly altered intrapore binding site for Ca 2+ . In initial experiments, contractions were observed in response to extracellular electrical stimulation (80 V, 10–30 ms) of dysgenic myotubes injected with cDNA for SkEIIIK but not in myotubes injected with CEIIIK cDNA. Thus, the substantial loss of Ca 2+ permeation caused by the EIIIK mutation appeared to have eliminated the ability of the cardiac L-channel, but not the skeletal L-channel, to mediate EC coupling. However, to determine whether CEIIIK supports Ca 2+ release too small to be detected by contractions, we also used whole-cell voltage clamping to measure membrane currents and intracellular Ca 2+ transients with patch pipettes containing the pentapotassium salt of Fluo-3 . Fig. 2 A illustrates Ca 2+ transients and membrane currents in dysgenic myotubes expressing either SkEIIIK (left) or CEIIIK (right). Large Ca 2+ transients were present in the myotube expressing SkEIIIK (left), but not in the one expressing CEIIIK, despite the presence of large ionic currents (right). Similar results were obtained in five myotubes expressing SkEIIIK, all of which displayed depolarization-evoked Ca 2+ transients, and five myotubes expressing CEIIIK, none of which displayed Ca 2+ transients. These data lend strong support to the notion that Ca 2+ permeation through skeletal muscle DHPRs is not required to trigger the release of Ca 2+ from the SR . The lack of SR Ca 2+ release in CEIIIK-expressing myotubes strongly argues against a Ca 2+ -independent interaction between the cardiac DHPR and the skeletal SR Ca 2+ release channel. Fig. 2 B plots both normalized conductance (G) and the amplitude of the Ca 2+ transient (Δ F / F ) as a function of test potential for SkEIIIK-expressing myotubes. As in myotubes expressing the wild-type skeletal DHPR , the conductance of SkEIIIK L-channels is positively shifted by ∼15 mV compared with that of Ca 2+ release. Moreover, the average voltage dependencies (i.e., averages of Boltzmann fits obtained from multiple experiments) of G (V G = 21.6 ± 2.3 mV, k G = 8.8 ± 0.4 mV, n = 16) and of Δ F / F (V F = 6.5 ± 3.2 mV, k F = 9.3 ± 0.7 mV, n = 5) for SkEIIIK L-channels are also similar to the values reported previously for dysgenic myotubes expressing the wild-type skeletal DHPR (V G = 24.0 mV, k G = 8.2 mV; V F = 6.6 mV, k F = 9.0 mV). The qualitative and quantitative similarity of Ca 2+ release caused by wild-type and SkEIIIK L-channels indicates that neither Ca 2+ permeation nor high affinity binding of Ca 2+ within the L-channel pore play an important role in skeletal-type EC coupling. In comparison with other voltage-gated Ca 2+ channels, skeletal L-channels have extremely slow (and relatively voltage-independent) activation kinetics. Despite this slow activation, repolarization to negative potentials causes rapid deactivation, a behavior that can be explained by a single transition, with an asymmetric voltage dependence causing it to be slow in the opening direction and fast in the closing direction . Accordingly, such a transition would produce only a small Q on but a significant Q off gating current. We tested this prediction by comparing Q off gating currents for SkEIIIK and CEIIIK. Fig. 3 illustrates representative Q off gating currents generated upon repolarization to −50 mV for dysgenic myotubes expressing either SkEIIIK (left) or CEIIIK (right). Data were obtained in the presence of 2.0 mM Cd 2+ and 0.2 mM La 3+ (which was required to block the small inward currents supported by CEIIIK). Q off is shown after either brief (20 ms, light traces) or long (200 ms, dark traces) depolarizing pulses to the indicated potentials. These two pulse durations were used because 20 ms is long enough to open cardiac L-channels fully and short enough to only marginally open skeletal L-channels, whereas 200 ms is sufficiently long to open both cardiac and skeletal L-channels. At potentials less than +10 mV, Q off was independent of the test pulse duration for both SkEIIIK and CEIIIK. However, at potentials that typically activate ionic current (greater than or equal to +10 mV), Q off was significantly larger only for SkEIIIK L-channels after the 200-ms test pulses. The additional Q off for SkEIIIK L-channels was manifested as a prolongation of the total gating current rather than simply as an increase in peak of the “OFF” gating current. Thus, the increase in SkEIIIK Q off caused by longer depolarizations is inconsistent with a contamination from an ionic tail current and presumably arises from an additional amount of gating current recruited during slow channel activation. In contrast to SkEIIIK, the magnitude of Q off for CEIIIK was not increased by the longer test pulse duration. Rather, a slight decrease in Q off was found for CEIIIK after the largest 200-ms depolarizations (to potentials greater than +30 mV), most likely owing to a small degree of inactivation during the 200-ms pulses. Fig. 4 illustrates the voltage dependence of Q off for SkEIIIK (A) and CEIIIK (B) after 20-ms test pulses (Q 20 ; •) and 200-ms test pules (Q 200 ; ▪), and the difference between the two (Q 200 –Q 20 ; ▴). For potentials >0 mV, Q off SkEIIIK was larger after 200-ms test pulses than after 20-ms test pulses. For CEIIIK, no such increase in Q off occurred after longer duration pulses. The normalized voltage dependence of Q 20 , Q 200 , Q 200 –Q 20 , and channel conductance for the SkEIIIK-expressing myotube shown in Fig. 3 are compared in Fig. 4 C. As reported previously for Q on , the voltage dependence of Q off after brief depolarizations (Q 20 ) was leftward shifted and shallower than that of channel conductance. However, the additional Q off recruited by long depolarizations of SkEIIIK L-channels (Q 200– Q 20 ; ▴) exhibited a voltage dependence nearly identical to that of channel conductance (○). In a total of four experiments, good agreement was found between the voltage dependence of conductance (V G = 24.2 ± 6.3 mV and k G = 7.5 ± 1.0 mV) and that of Q 200 –Q 20 (V Q = 31.4 ± 6.7 mV and k Q = 7.5 ± 0.7 mV). Fig. 4 D summarizes the results on maximal Q off obtained from multiple experiments. On average, the maximal value of Q off for SkEIIIK after 200-ms test pulses was significantly greater (121.4 ± 2.0%, n = 6, P < 0.001) than that after 20-ms test pulses. For CEIIIK, Q off after 200 ms was not significantly different from that after 20 ms ( P > 0.3). Additionally, for both SkEIIIK and CEIIIK, Q off after 20 ms had essentially the same magnitude as Q on (data not shown). Thus, the voltage and time dependence of the Q 200 –Q 20 gating charge, coupled with its presence only in the SkEIIIK channels, suggests that this extra gating charge underlies the rate-limiting transition of slow skeletal channel activation. Since contamination by inward ionic tail current would artifactually increase Q off , it was important to ensure that no such contamination was present under our recording conditions. As one approach, we attempted to reduce ionic currents by adding Cd 2+ and La 3+ to the external bath. At a test potential of +60 mV, these blockers reduced the outward current by 75.3 ± 2.6% ( n = 5) for SkEIIIK and 82.0 ± 2.7 ( n = 5) for CEIIIK. To determine the effectiveness of this block at more negative potentials, cells were depolarized to +60 mV for 200 ms to cause maximal activation of channels and then either maintained at +60 mV or repolarized to varying potentials. As shown for a SkEIIIK-expressing cell in Fig. 5 , the addition of Cd 2+ and La 3+ caused a large reduction at +60 mV, where the integrated current was entirely ionic, but only a small reduction at more negative potentials. The small effect of the blockers at negative potentials strengthens the conclusion that SkEIIIK has a very low permeability to Ca 2+ . Moreover, it seems likely that the block of this already small Ca 2+ permeability by Cd 2+ and La 3+ should have been sufficient to eliminate any ionic contamination of the Q off charge. In this paper, we have characterized the effects of mutational alteration of Ca 2+ permeability on the behavior of skeletal and cardiac L-channels expressed in dysgenic myotubes. Because there had been no previous descriptions of mutations affecting Ca 2+ permeability of skeletal L-channels, we chose to substitute lysine for glutamate in the repeat III pore region (EIIIK) since the corresponding mutation had already been shown to produce a dramatic alteration in the divalent selectivity of the cardiac L-channel expressed in Xenopus oocytes . Similarly, depolarization of dysgenic myotubes expressing the CEIIIK channel elicited inward Ca 2+ currents only for a few test potentials (<20 mV), while stronger depolarizations elicited outward currents. Thus, introduction of the EIIIK mutation into the cardiac L-channel caused a large (>60 mV) hyperpolarizing shift in reversal potential compared with wild-type cardiac L-channels under the same recording conditions . Unlike the CEIIIK channel, which retained a small Ca 2+ permeability, the SkEIIIK channel completely lost permeability to Ca 2+ as judged by the absence of inward ionic currents either during depolarizing steps or after repolarization to negative potentials . The greater effect of the EIIIK mutation on the selectivity of skeletal L-channels suggests functional differences between the geometric arrangement of the conserved pore region glutamates in skeletal and cardiac L-channels. To test this hypothesis, it will be necessary to carry out a more systematic analysis of mutations of the glutamates in each of the four repeats of the skeletal DHPR. Previous studies have used expression in dysgenic myotubes of skeletal, cardiac, and chimeric DHPRs to dissect mechanisms of EC coupling. With this approach, it was shown that substitution of skeletal sequence for all or part of the II–III loop of an otherwise entirely cardiac DHPR was sufficient to convert the reconstituted EC coupling from cardiac- to skeletal-type. Subsequently, a number of laboratories have tested whether peptides derived from the II–III loop can activate reconstituted RyRs and/or Ca 2+ release from SR vesicles. This approach has revealed that either the entire cardiac II–III loop or peptides corresponding to part of the loop are capable of causing activation of RyRs in these reduced systems. Furthermore, it has been reported that a skeletal-type EC coupling mechanism independent of Ca 2+ influx can contribute to SR Ca 2+ release in cardiac muscle cells, particularly when intracellular cAMP is elevated . Thus, it is important to be certain that the EC coupling mechanism truly differs when cardiac and skeletal DHPRs are expressed in dysgenic myotubes. This is particularly important since earlier assessments of cardiac-type coupling in injected dysgenic myotubes relied, in part, on the loss of Ca 2+ release after the addition of Cd 2+ to the external medium. However, in addition to blocking entry of external Ca 2+ , any Cd 2+ entering the cells could have interfered with the measurement of Ca 2+ by quenching the Ca 2+ -sensitive dye and/or have directly blocked RyRs . We have now shown that CEIIIK in dysgenic myotubes is unable to trigger the release of SR Ca 2+ , even in the absence of inorganic Ca 2+ channel blockers. Specifically, myotubes with robust CEIIIK expression never contracted in response to strong electrical stimuli and never exhibited depolarization-induced intracellular Ca 2+ transients. Thus, large Ca 2+ flux through cardiac L-channels appears to be required for reconstitution of cardiac-type SR Ca 2+ release in dysgenic myotubes. Moreover, the distinct behaviors of CEIIIK and SkEIIIK in triggering the release of SR Ca 2+ support the use of skeletal/cardiac DHPR chimeras for identifying regions important for skeletal-type EC coupling. However, since the RyR isoform in dysgenic myotubes (i.e., RyR1) differs from that of cardiac muscle (RyR2), our results do not rule out the possibility that under some conditions a skeletal-type mechanism of EC coupling may contribute to SR Ca 2+ release in native cardiac myocytes. Occupancy of a metal cation binding site, which is accessible from the extracellular space, has been reported to be required for the conformational changes of the skeletal L-channel that result in the activation of RyR1s . This site has a binding selectivity and a micromolar Ca 2+ affinity similar to that of the site governing ion permeability of L-channels , suggesting that this cation-binding site corresponds to the structures governing Ca 2+ selectivity of the skeletal L-channel. Our data demonstrate that SkEIIIK retains the ability of the wild-type channel to control the release of SR Ca 2+ in spite of a complete loss of Ca 2+ permeability. In fact, the voltage dependence of SR Ca 2+ release in dysgenic myotubes expressing SkEIIIK L-channels was nearly identical to that of the wild-type skeletal DHPR . Thus, the capability of the skeletal L-channel to mediate EC coupling does not depend on Ca 2+ permeation. Despite the lack of Ca 2+ permeation, it is possible that a low affinity site in the pore of SkEIIIK is appreciably occupied by Ca 2+ under our experimental conditions (10 mM external Ca 2+ ). Alternatively, the presence of large outward Cs + currents, which should have been blocked by binding of Ca 2+ , may indicate that there is very little residual binding of Ca 2+ within the SkEIIIK pore. If the latter is indeed the case, it would imply that the metal cation site described by Pizzaro et al. 1989 does not lie within the pore. However, a direct evaluation of the contribution of the pore to the metal binding site involved in EC coupling will require measurement of SkEIIIK monovalent ionic currents, intramembrane charge movements, and SR Ca 2+ release over a range of extracellular Ca 2+ concentrations. The results with SkEIIIK raise the obvious question of what function is played by the slowly activating L-type Ca 2+ current in skeletal muscle. Clearly, this current plays little or no role in contractions elicited by relatively brief depolarizations. Perhaps the Ca 2+ current becomes important for contraction only during tetanic stimulation , or has other functions such as the regulation of acetylcholine receptor gene expression or the metabolic stabilization of acetylcholine receptors . One approach for identifying these or other roles of the slow L-type Ca 2+ current would be to construct mice that carry a transgene for the SkEIIIK channel and lack the wild-type skeletal L-channel. As in the case for inward Ca 2+ currents via wild-type skeletal L-channels, outward currents carried by SkEIIIK L-channels also activate slowly. Moreover, because they are slow over a broad range of test potentials, the SkEIIIK L-currents extend previous conclusions that activation of skeletal L-channels is only very weakly voltage dependent . Nonetheless, steady state activation is strongly voltage dependent for wild-type and SkEIIIK channels , with Boltzmann fits of conductance–voltage (G–V) relationships yielding a value of k ≈ 8 mV, which corresponds to an effective valence of approximately three electronic charges. This effective valence represents ∼25% of the total gating charge expected for voltage-gated calcium channels . However, the G–V relationship for wild-type skeletal DHPRs expressed in dysgenic myotubes is rightward shifted ∼30 mV with respect to the Q on –V relationship . Thus, nearly all of the measured gating current moves at voltages more negative than those over which the conductance increase occurs. One possibility consistent with this result is that there may be two separate populations of DHPRs in skeletal muscle: one population related to EC coupling that produces nearly all of the measured charge movement and a second population that functions as Ca 2+ channels and produces negligible gating current. An alternative possibility is that all of the DHPRs may undergo gating transitions that lead to the activation of L-channels, but that one of these transitions moves appreciable charge (e.g., approximately three electronic charges) too slowly to be detected during brief depolarizations (as is typically used in measurements of Q on ). A simple linear kinetic scheme consistent with the second possibility above, as well as with measurements of unitary currents through skeletal L-channels is shown in Fig. 1 . In this model, rapid gating currents are produced by closed–closed transitions, which for simplicity are indicated by the single C 0 –C 1 transition. These gating transitions could be important for triggering release of Ca 2+ , since Ca 2+ release has a voltage dependence much closer to that of Q on than does L-channel conductance . In the model, the rate constant β must be large because single channel open times are brief , and slow activation of L-type conductance is a consequence of the C 1 –C 2 transition, with forward and reverse rate constants δ and ∈, respectively. The rate constant δ must be small and not strongly voltage dependent because activation remains slow for SkEIIIK even at +100 mV. By contrast, the rate constant ∈ must be strongly voltage dependent such that it is small at the depolarized potentials causing activation of current and large at negative voltages (e.g., −50 mV) causing deactivation. This behavior, which suggests that the energy barrier separating C 1 and C 2 is asymmetrically located with respect to the transmembrane potential drop, is required because activation is slow, whereas deactivation is rapid . An explicit prediction of this reaction scheme is that the component of gating charge that is associated with the C 1 –C 2 transition would move slowly outward during depolarization and rapidly inward upon repolarization. The rectification of SkEIIIK under our experimental conditions (TEA + and Ca 2+ as external cations) allowed a clear test of this prediction and revealed that additional Q off is recruited by depolarizations that are sufficient to activate ionic current. The voltage dependence of this additional charge is nearly identical to that of channel conductance, consistent with the hypothesis that this charge moves during a transition required for channel activation. Based on chimeras of the skeletal and cardiac L-channels, it was shown that the sequence of IS3 and the IS3–IS4 linker determines whether activation is fast or slow , perhaps by governing the rate of movement of the nearby voltage-sensing IS4 segment. Thus, it is tempting to speculate that repeat I produces the additional Q off that we found to be recruited by long depolarizations. If each of the four repeats contributed equivalent gating charge, then the long depolarizations should have caused a 33% (1/4 ÷ 3/4) increase in Q off , somewhat larger than the observed value of ∼20%. However, it seems likely that our measurement of the difference in Q off after 20- and 200-ms depolarizations underestimated the gating charge associated closely with channel activation, because 20-ms depolarizations cause some activation . In any case, the size of the recruitable Q off suggests that all DHPRs undergo the conformational change required for activation of L-type conductance. Neutralization of basic residues in the S4 segments would provide one test for the idea that this recruitable charge represents movements within IS4. | Study | biomedical | en | 0.999998 |
10469730 | The epithelia that line the stomach, bladder, renal collecting duct, and thick ascending limb of the nephron limit the dissipation of large proton, solute, NH 3 , and CO 2 gradients by creating and maintaining a barrier to diffusion . The means by which they do this is not entirely clear. However, the ability of certain substances to cross biological membranes correlates well with their oil/water partition coefficient; a relationship known as Overton's Rule . This early observation has been refined into the “solubility-diffusion” model, which states that for substances to cross a lipid membrane they must partition into or dissolve in the interfacial hydrocarbon region (adjacent to head groups), and then diffuse through the membrane before reemerging on the other side . Diffusion across the hydrocarbon interior is thought to occur via transport along self-propagating kinks or defects in the acyl chain packing. The rate of diffusion is therefore dependent on the thickness of the membrane, the length, saturation, and packing of the acyl chains, and the molecular volume and hydrophobicity of the solute . As predicted by this model, fluidity appears to be a major determinant in the rate of permeation of nonelectrolytes across lipid membranes . Indeed, there is compelling experimental evidence to suggest that reducing membrane fluidity is an important means by which epithelial cells can erect barriers to permeation. The exofacial leaflet of the apical membrane is able to maintain a lipid composition that is different from that of the cytoplasmic leaflet. This is accomplished in three ways: (a) asymmetric synthesis in the Golgi and vectorial delivery of specific lipids to the exofacial leaflet , (b) the presence of tight junctions that isolate the apical from the basolateral plasma membrane domains , and (c) the action of phospholipid flippases that can trans-orient phospholipids from one leaflet of the bilayer to the other in an energy-dependent process . The functional consequences of losing bilayer asymmetry were demonstrated when gastric apical vesicles were shown to have markedly lower water, proton, and nonelectrolyte permeabilities compared with the same membranes prepared from lipids quantitatively extracted from the vesicles and reconstituted into symmetric liposomes. . Membrane permeability to a number of substances is clearly dependent on fluidity, and cells appear to limit fluidity by creating asymmetric lipid membranes at their apical pole. However, for mainly technical reasons, there have been virtually no studies undertaken to model the effects of bilayer asymmetry on permeability in artificial membranes of known composition. We have previously shown by the use of two independent methods that rigidifying a single leaflet in a bilayer reduced water permeability . In the present studies, we rigidified and thereby reduced the fluidity of the outer leaflet of dipalmitoylphosphatidylcholine (DPPC) 1 liposomes with the rare earth metal praseodymium (Pr 3+ ) . The consequences of inducing bilayer asymmetry on the permeation of solutes, gases, and protons were investigated. Powdered DPPC was obtained from Avanti Polar Lipids and suspended by vortexing (at 25 mg/ml) in buffer appropriate for the permeability to be measured. Buffers used were as follows (mM): for solutes, 150 NaCl, 10 HEPES, 20 carboxyfluorescein (CF), pH 7.5; for NH 3 and protons, 150 NaCl, 30 KCl, 10 HEPES, 0.5 CF, pH 7.5; for CO 2 , 50 NaCl, 50 KCl, 20 HEPES, 0.5 CF, 0.5 mg/ml carbonic anhydrase, pH 7.4. Liposomes were prepared by probe sonication and after 90 min incubating on ice, extravesicular CF was removed by passing vesicles over a Sephadex G50 column (Sigma Chemical Co.). Vesicles were sized by quasi-elastic light scattering using a Nicomp model 270 submicron particle analyzer as described . Sonication conditions (intensity and duration) were chosen that reproducibly generated vesicles with a median diameter of 110 ± 20 nm . Permeability measurements were performed as described using a stopped-flow fluorimeter (SF.17 MV; Applied Photophysics) with a dead time of 0.7 ms. Experiments were performed either on the same day of vesicle manufacture or the next day. Liposome size was shown not to change significantly over 24 h. To perform solute permeability measurements, liposomes were incubated in buffer containing 200 mM solute (glycerol, urea, formamide, or acetamide) for 30 min before the experiment was commenced. Any residual extravesicular CF was quenched with anticarboxyfluorescein antibody, and then the liposomes were rapidly mixed with a solution of identical osmolality containing 100 mM solute, resulting in an extravesicular solute concentration of 150 mM. Osmolalities of all solutions were confirmed and adjusted, if necessary, by measuring freezing point depression on an Osmette A osmometer (Precision Instruments, Inc.). The applied concentration gradient results in solute efflux from liposomes followed by water efflux due to solvent drag. Vesicle shrinkage is monitored as a function of CF self-quenching. Fluorescence data from the stopped-flow fluorimeter from 6–10 individual determinations were averaged and fit to a single exponential curve using software supplied by Applied Photophysics. Solute flux across a membrane can be defined by the relation : 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*}J_{{\mathrm{z}}}={dz}/{dt}= \left \left(P_{{\mathrm{z}}}\right) \right \left \left(SA\right) \right \left \left({\mathrm{{\Delta}}}C\right) \right {\mathrm{,}}\end{equation*}\end{document} where J z is the flux and P z is the permeability of the permeant solute z , SA is the surface area of the vesicle, and Δ C is the concentration difference of the permeant solute between the inside and outside of the vesicle. If 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{V}}_{{\mathrm{rel}}}={{\mathrm{V}} \left \left(t\right) \right }/{{\mathrm{V}}_{0}}{\mathrm{,}}\end{equation*}\end{document} where V 0 is the initial volume of the vesicle and V rel and V( t ) are the relative and absolute volumes, respectively, at time t , then for our experimental conditions: 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*}{dz}/{dt}=500 \left \left({\mathrm{V}}_{{\mathrm{0}}}-{\mathrm{V}}_{{\mathrm{0}}}{\mathrm{V}}_{{\mathrm{rel}}}\right) \right \end{equation*}\end{document} and 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{{\Delta}}}C=900- \left \left({800}/{{\mathrm{V}}_{{\mathrm{rel}}}}\right) \right {\mathrm{;}}\end{equation*}\end{document} therefore, 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*}{d{\mathrm{V}}_{{\mathrm{rel}}}}/{dt}=P_{{\mathrm{z}}} \left \left({{\mathrm{SA}}}/{{\mathrm{V}}_{{\mathrm{0}}}}\right) \right \left \left({1}/{500}\right) \right \left \left[ \left \left({800}/{{\mathrm{V}}_{{\mathrm{rel}}}}\right) \right -900\right] \right {\mathrm{.}}\end{equation*}\end{document} By use of parameters from the single exponential curve fit to the data, P solute was solved using commercially available MathCad software . Proton permeabilities were measured using pH-dependent quenching of fluorescence as described . Stopped-flow experiments were performed in which the liposomes were pretreated with 1 μM valinomycin, and then rapidly mixed with an identical buffer acidified to pH 6.50. Buffer capacity was determined on an SLM-Aminco 500C spectrofluorimeter by adding 10 mM acetate (final concentration) to liposomes as described . Fluorescence data from the stopped-flow device were fit to a single exponential curve and fitting parameters were used to solve the following equation for P H+ : 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*}J_{{\mathrm{H+}}}= \left \left(P_{{\mathrm{H+}}}\right) \right \left \left(SA\right) \right \left \left({\mathrm{{\Delta}}}C\right) \right = \left \left({{\mathrm{{\Delta}pH}}}/{t}\right) \right \left \left(BCV\right) \right {\mathrm{,}}\end{equation*}\end{document} where J H+ is the flux of protons, Δ C is the initial difference in concentration of protons between the inside and outside of the vesicle, ΔpH is the change in pH when time equals τ, the time constant of the single exponential curve describing the initial change in fluorescence as a function of time, and BCV is the buffer capacity of an individual vesicle . NH 3 permeability was determined using stopped-flow fluorimetry by monitoring the pH-sensitive increase in fluorescence when vesicles equilibrated to pH 6.8 were rapidly mixed with the same buffer containing 20 mM NH 4 Cl as described . NH 3 in solution passes through the membrane and becomes protonated to NH 4 + in the vesicle interior. By combining values for the rate of change of intravesicular pH, the final intravesicular pH, and the buffer capacity (assessed in the same way as for proton permeability), P NH3 was calculated . CO 2 fluxes were determined by monitoring the pH-sensitive decrease in fluorescence when vesicles were mixed with a 100 mM NaHCO 3 /CO 2 , 20 mM HEPES, pH 7.4 buffer. CO 2 gas in the bicarbonate solution diffuses into the liposomes, whereupon it is converted to HCO 3 − and H + by the entrapped carbonic anhydrase. By combining values for the initial rate of change of fluorescence, the final pH, and the buffer capacity of the vesicles, P CO2 was calculated as described . Membrane fluidity measurements were performed by incubating DPPC liposomes in 1 mM DPH-HPC (Molecular Probes), and then measuring anisotropy using excitation/emission wavelengths of 360 nm/430 nm on a SPEX Fluorolog 1680 double spectrometer according to standard methods . A circulating water bath allowed precise control of the temperature of the cuvette chamber, and buffer temperatures were confirmed by electronic thermometer before measurement of anisotropy. Where indicated, PrCl 3 was added to a final concentration of 10 mM from a 1 M stock solution. For all comparisons, n = 4–6 liposome preparations. Groups were compared using unpaired t tests. P < 0.05 was considered significant. Before determining the effect of bilayer asymmetry on solute fluxes, it was necessary to ensure that the solute was not altering the ability of Pr 3+ to rigidify the outer leaflet of the liposome. We therefore examined the effect of Pr 3+ on anisotropy of the external leaflet of liposomes in the presence of 200 mM of solutes being examined. DPPC liposomes were incubated with the fluorescent phospholipid analogue and anisotropy probe, DPH-HPC [2-(3-(diphyenylhexatrienyl)propanyl)-1-hexadecanyl- sn -glycero-3-phosphocholine] to “label” the outer (exofacial) leaflet of the membrane for fluidity measurements . Liposomes incubated in buffer containing 200 mM permeant solute for 30 min had anisotropy measured as a function of temperature. Fig. 1 A shows the effect of increasing temperature on the anisotropy of the exofacial leaflet of the bilayer in the presence or absence of 10 mM Pr 3+ and in the presence of 200 mM glycerol. Between 24° and 34°C, there is little change in the anisotropy of the membrane and no difference between Pr 3+ and control liposomes. However, above 39°C, there is a sharp decrease in anisotropy that corresponds to a dramatic increase in leaflet fluidity at or near the phase-transition temperature (T c ) of the leaflet. In the presence of exofacially bound Pr 3+ , however, the acyl chains are rigidified and as a consequence the increase in fluidity occurs 2–3°C higher. Consistent with previous studies, this indicates that Pr 3+ binding has increased T c for the exofacial leaflet . Stopped-flow experiments in which CF and glycerol-loaded liposomes were rapidly exposed to a glycerol gradient defined the glycerol permeabilities of both native and Pr 3+ -treated liposomes . Upon exposure to the gradient, glycerol efflux from liposomes results in the formation of an immediate osmotic gradient for water. Water then exits the liposomes resulting in vesicle shrinkage and self-quenching of entrapped CF. The superposition of glycerol efflux from native and Pr 3+ -treated vesicles at 42°C reveals that the rate of shrinkage and hence the permeability is reduced when Pr 3+ is present in the exofacial leaflet. Combined glycerol permeabilities as a function of temperature are shown in Fig. 1 C. Solute permeability was found to increase dramatically above 40°C. With the addition of 10 mM Pr 3+ , glycerol permeabilities were significantly lower at temperatures above 40°C ( P < 0.05 where indicated by asterisks), thus demonstrating that rigidifying one leaflet of the bilayer was sufficient to reduce the overall permeability of the membrane to this nonelectrolyte. Glycerol (M r = 92.09) has a relatively large molecular volume. Therefore, we tested three other uncharged solutes of varying molecular weight to ascertain whether inducing bilayer asymmetry with Pr 3+ would result in reduced permeability for other nonelectrolytes. In panel A, Fig. 2 Fig. 3 Fig. 4 , it can be noted that addition of Pr 3+ to formamide (M r = 45.04), acetamide (M r = 59.07), and urea (M r = 60.06) equilibrated liposomes, respectively, resulted in a reduction in fluidity of the exofacial leaflet in a manner similar to what is observed in the presence of glycerol. Therefore, the nature of the solute did not alter the interaction with Pr 3+ or its effect on the membrane. Panel B, Fig. 2 Fig. 3 Fig. 4 , shows representative tracings of stopped-flow experiments carried out above 40°C in the presence or absence of Pr 3+ . These demonstrate a reduction in solute permeability as judged by the initial rate of shrinkage for each solute. Combined permeability data for each solute is shown as a function of temperature . Each shows a significantly lowered permeability above T c when fluidity is reduced by Pr 3+ . It is clear that reducing acyl chain fluidity in a single leaflet is sufficient to alter the permeability of the entire membrane to multiple small nonelectrolytes. Water and solutes appear to obey the solubility-diffusion model for permeation across a bilayer. However, less information is available on the mechanisms of permeation of gases and protons. Fig. 5 shows the results of proton permeability experiments on DPPC liposomes over a range of temperatures. Fig. 5 A shows two experiments in which liposomes were rapidly exposed to a pH gradient (pH 7.50 inside/7.06 outside). The pH-dependent quenching of entrapped CF illustrates the reduction in acidification rate at 48°C when Pr 3+ is present. Fig. 5 B shows H + permeability as a function of temperature. There is a noticeable increase in proton permeability above T c ; however, at 48°C it is only fourfold higher than baseline levels. This is compared with 129 ± 36-fold (SEM) increases at 48°C for the four small nonelectrolytes tested in this study. Therefore, dramatically increasing bilayer fluidity at temperatures above T c only results in modest increases in the ability of protons to cross the bilayer. Rigidification of the exofacial leaflet with Pr 3+ completely abolishes the phase-transition–induced permeability increase seen in native vesicles. These data argue that proton permeability is only weakly correlated with membrane fluidity. NH 3 and CO 2 diffuse rapidly across cell membranes, and NH 3 permeation is thought to occur by solubility diffusion. We examined the transport properties of both gases across DPPC liposomes and examined the influence of temperature and bilayer asymmetry on the process. Fig. 6A and Fig. B , shows the permeation of NH 3 into DPPC liposomes at 42°C in the presence and absence, respectively, of Pr 3+ . The rate of permeation is slower when the exofacial leaflet is rigidified . Of note is the extreme rapidity of this process, which is complete within 4 ms. The temperature dependence of NH 3 permeation is shown in Fig. 6 C. At 25°C, the permeability coefficient is ∼0.4 cm/s, compared with 4 × 10 −6 cm/s for formamide (the fastest of the solutes). Permeability rapidly increased above 40°C, indicating that fluidity is a major determinant in the rate of NH 3 permeation across phospholipid bilayers. At temperatures higher than 42°C, NH 3 permeation in response to the applied NH 3 gradient was complete within the dead time of the instrument (∼0.7 ms), and therefore unmeasurable. Rigidification of the exofacial leaflet resulted in a marked reduction in the permeability of the membrane to NH 3 at temperatures above T c . This suggests that NH 3 , like water and solutes, crosses biological membranes by a solubility-diffusion mechanism. A CO 2 permeability assay recently developed in our laboratory measures the acidification occurring within liposomes after exposure to a CO 2 gradient that is supplied in the form of a CO 2 /HCO 3 − solution . Upon diffusion across the bilayer, CO 2 is hydrated to carbonic acid, which subsequently dissociates to bicarbonate ion and a proton. The presence of entrapped carbonic anhydrase within the liposome ensures that the hydration reaction is not rate limiting, but that gas permeation is. When the CO 2 permeability of DPPC liposomes was measured, it was found to be independent of temperature . Fig. 7 A illustrates the equivalence of CO 2 -dependent acidification rates in liposomes at 25° and 45°C from stopped-flow experiments. Fig. 7 B shows the lack of effect of temperature on the calculated CO 2 permeability coefficients. This was in dramatic contrast to the response of NH 3 to elevated temperature and strongly implies that fluidity does not influence the ability of CO 2 to cross a model phospholipid bilayer. Even at temperatures well above T c , there was no effect on CO 2 permeability. This data suggests that in a biological context, lipid bilayer asymmetry or phospholipid composition play no role in either limiting or facilitating the transfer of CO 2 between body compartments. The rare earth metal Pr 3+ has been used as a nuclear magnetic resonance shift reagent to discriminate between the inner and outer choline methyl resonances of dimyristoylphosphatidylcholine liposomes . Addition of Pr 3+ to liposomes increased the gel to liquid-crystalline phase-transition temperature of just the outer leaflet by several degrees. Significantly, this implied that the coupling of both halves of a phospholipid bilayer is sufficiently weak that each leaflet can undergo a thermotropic phase-transition independently. Pr 3+ binding was shown to exert this effect by reducing the fluidity of the outer leaflet of DPPC liposomes . It appears likely that the Pr 3+ binds to multiple phosphate head groups, reducing their mobility and thereby the mobility of their attached hydrocarbon chains. Negrete et al. 1996b demonstrated that binding to the phospholipid head group stabilized and rigidified the leaflet, and this was reflected in a decrease in water permeability at temperatures above T c . In this study, we further exploited the hemi-bilayer rigidifying properties of Pr 3+ to examine the effect of inducing bilayer asymmetry on solute, proton, and gas fluxes in an effort to better understand their permeation properties. DPPC has been used extensively as a model lipid to explore the behavior of phospholipid bilayers and is therefore well characterized. The advantages of using DPPC in these experiments were the phosphorylcholine head groups necessary for Pr 3+ binding, and its high T c , which allows an exploration of permeant behavior in both the gel and liquid-crystal states. As most PCs have a very low T c and exist in cell membranes only in the liquid-crystal phase, they don't allow an exploration of membrane permeant behavior in asymmetric bilayers at temperatures above and below T c . In a series of solute flux experiments, we initially sought to determine whether Pr 3+ would reduce the fluidity of the outer leaflet in liposomes that were loaded with high concentrations of solute (200 mM). Panel A, Fig. 1 Fig. 2 Fig. 3 Fig. 4 , demonstrates that there was no observable difference on membrane fluidity in the presence or absence of Pr 3+ as measured by fluorescence polarization anisotropy between 24° and 35°C. The control liposomes exhibited a steep decrease in anisotropy when temperatures were raised higher than 39°C, which indicated dramatic increases in membrane fluidity as a result of the membrane phase transition from a gel to liquid-crystalline state. When Pr 3+ was added, the thermotropic phase-transition occurred 1°–3°C higher. This demonstrated that high concentrations of solute were not affecting Pr 3+ binding or its influence as a stabilizing reagent on the outer leaflet. The permeability of DPPC membranes to glycerol, formamide, acetamide, and urea were tested over a range of temperatures and the effect of membrane phase transition on permeability was striking, with permeabilities above baseline (i.e., 24°C) of 235-, 99-, 74-, and 106-fold for glycerol, formamide, acetamide, and urea, respectively. This is consistent with the high degree of disorder that prevails in the liquid-crystal state. Phase transition is associated with a conformational change in the acyl chains from a predominantly straight (trans) conformation to the gauche conformation, which occurs due to C–C bond rotation. This results in an expansion of the area occupied by the chains and a concomitant reduction in the thickness of the bilayer . At temperatures below 39°C, Pr 3+ had no influence on membrane permeability to any of the solutes. However, at 41°–42°C, there was a significantly decreased permeability due to outer leaflet rigidification. These data confirmed that membrane fluidity was a rate-limiting factor for nonelectrolyte permeability, and, more significantly, that reducing the fluidity of a single leaflet is sufficient to significantly retard the passage of small uncharged molecules. Each leaflet in the bilayer therefore appears to exert an independent resistance to the passage of solutes, with the overall permeability a function of the sum of the resistances exerted by two leaflets. This relationship has previously been found to apply to water permeability and may be expressed as: 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*}{1}/{P_{{\mathrm{ab}}}}={1}/{P_{{\mathrm{a}}}}+{1}/{P_{{\mathrm{b}}}}{\mathrm{,}}\end{equation*}\end{document} where P ab is the permeability of the membrane, P a is the permeability of leaflet a and P b is the permeability of leaflet b. To test whether this relationship accurately predicts the solute permeability behavior of Pr 3+ -rigidified liposomes, we calculated asymmetric bilayer permeabilities. At temperatures above T c , we know the temperatures at which anisotropies of native and Pr 3+ -treated liposomes are the same ; e.g., in Fig. 3 A the anisotropy of the Pr 3+ -treated leaflet at 44°C has the same value as the control leaflet at 41°C. For identical fluidities, we assume identical permeabilities and in this way derive a permeability value for a Pr 3+ -treated leaflet. From Fig. 3 C for acetamide, for example: \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*}\begin{matrix}P_{{\mathrm{acet.}}} \left \left[{\mathrm{DPPCbilayer}}\right] \right _{{\mathrm{41{^\circ}C}}}=1.266{\times}10^{-4}{\mathrm{cm/s}}{\Rightarrow}P_{{\mathrm{acet.}}} \left \left[{\mathrm{DPPCleaflet}}\right] \right _{{\mathrm{41{^\circ}C}}}=2.532{\times}10^{-4}{\mathrm{cm/s}}\\ =P_{{\mathrm{acet.}}} \left \left[{\mathrm{Pr}}^{{\mathrm{3+}}}-{\mathrm{DPPCleaflet}}\right] \right _{{\mathrm{44{^\circ}C}}}{\mathrm{.}}\end{matrix}\end{equation*}\end{document} We now calculate a DPPC leaflet permeability at 44°C from the experimentally determined value for control liposomes. \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{acet.}}} \left \left[{\mathrm{DPPCbilayer}}\right] \right _{44{\mathrm{{^\circ}C}}}=3.012{\times}10^{-4}{\mathrm{cm/s}}{\Rightarrow}P_{{\mathrm{acet.}}} \left \left[{\mathrm{DPPCleaflet}}\right] \right _{4{\mathrm{4{^\circ}C}}}=6.024{\times}10^{-4}{\mathrm{cm/s.}}\end{equation*}\end{document} Having now derived values for the acetamide permeability of a DPPC leaflet and a Pr 3+ -DPPC leaflet at 44°C, we can add their reciprocals to arrive at a predicted Pr 3+ -treated membrane permeability of 1.78 × 10 −4 cm/s. This compares favorably with the experimentally measured value of 2.12 ± 0.06 × 10 −4 cm/s. Predicted values compared with those measured for the other solutes were 1.08 × 10 −5 vs. 1.26 × 10 −5 cm/s for glycerol, 2.21 × 10 −4 vs. 2.78 × 10 −4 cm/s for formamide, and 8.58 × 10 −6 vs. 9.55 × 10 −6 cm/s for urea. This close concordance of measured and predicted permeabilities for asymmetric membranes strongly supports the model of leaflets offering independent resistances to solute permeation. Bilayer asymmetry is therefore a plausible mechanism by which epithelial cells may limit the permeation of solutes such as urea. The permeability of water, solutes, and NH 3 have been found to correlate strongly with membrane fluidity; however, proton permeability correlates only weakly . Although protons are ions, they traverse membranes at rates several orders of magnitude higher than alkali or halide ions . The fluidity dependence of H + permeability in DPPC liposomes can be seen in Fig. 5 B. In contrast to the massive increase in solute permeability upon phase transition of the liposomal membrane (approximately two orders of magnitude), H + permeability increased only fourfold. Rigidifying the outer leaflet eliminated that modest increase. Proton permeation has been postulated to occur by a pathway distinct from that of water and solutes. Two prevailing hypotheses as to the nature of that pathway are that protons can be shuttled from one side of the membrane to the other by virtue of hydrogen-bonded “water wires” embedded in the hydrocarbon . Alternatively, weak acids present as “contaminants” in the bilayer may act as proton carriers . According to the weak-acid hypothesis, protons cross the membrane in a non-ionic form (HA), and upon proton release the carrier must translocate back as an anion (A − ). This process may be driven either by voltage or pH gradients, but it is thought that A − translocation is the rate-limiting step. Our data confirms the weak dependence of proton transfer on fluidity; however, the effect of Pr 3+ in dramatically reducing permeability was unexpected and may not necessarily be due to its actions as a leaflet fluidity-reducing reagent. Other potential explanations are the possibility that externally bound Pr 3+ somehow blocks access to internal water molecules that constitute water wires, or that Pr 3+ is inhibiting access to, or freedom of movement of, the weak acid proton carrier. The experimental methodology employed doesn't allow us to discriminate between these possibilities, but the results do add to the growing body of evidence suggesting that protons cross phospholipid bilayers by a mechanism that is independent of the water and solute pathway. Mobility of the hydrocarbon chains does not appear to be as important. As such, bilayer asymmetry is unlikely to play a major role in providing a barrier to acid flux. Indeed, Bhaskar et al. 1992 have shown that gastric mucus may be the predominant barrier to proton flux in the stomach. NH 3 is a neutral lipophilic molecule that is freely diffusible across most cell membranes. Notable exceptions have been described, however, in the medullary thick ascending limb of Henle , colonic crypts , and rabbit urinary bladder epithelium . In all cases, the apical membrane was shown to be virtually impermeable to NH 3 , whilst the basolateral membrane presented no significant barrier to NH 3 transport. In the case of the thick ascending limb of Henle, low surface area of the apical membrane plays a critical role in its barrier properties. Our data clearly show that NH 3 permeability in DPPC liposomes is strongly influenced by membrane fluidity. Induction of a membrane asymmetry significantly restricts NH 3 permeability at temperatures above T c . This data confirms the nature of NH 3 flux that occurs by a solubility-diffusion mechanism and demonstrates the efficacy of reducing outer leaflet fluidity in reducing overall permeability to this gas. We conclude that an alteration to lipid structure in a single leaflet, such as is seen with the apical membranes of barrier epithelia, is sufficient to reduce NH 3 permeability. This implies that each leaflet offers an independent resistance to NH 3 flux. The system we have used to create bilayer asymmetry allows us to demonstrate the effect of a single leaflet rigidification on the permeation behavior of a range of biologically relevant molecules. These features are applicable to real cell membranes that, it should be noted, exist physiologically in the liquid-crystal rather than the gel state; i.e., in a state analogous to DPPC membranes at temperatures above 41°C. These results clearly demonstrate that epithelial cells with a requirement to restrict diffusional processes; e.g., in the thick ascending limb or collecting duct of the kidney, can do so by means of erecting apical membranes with asymmetric leaflet fluidities. Gastric glands, which contain parietal and chief cells, are the only epithelia described that possess a barrier to CO 2 permeation . In addition, since urine and possibly collecting duct P CO2 can reach 80 mmHg while blood P CO2 is 40 mmHg, it is highly likely that mammalian bladder also exhibits low permeability to CO 2 . CO 2 , like NH 3 , is readily permeable to most lipid membranes . However, because CO 2 permeates most membranes extremely rapidly, the mechanism by which it crosses membranes has not been studied. We recently developed a technique for measuring the CO 2 flux into liposomes and used it to examine the influence of membrane fluidity on CO 2 permeation. Unexpectedly, there was no change in permeability when liposomes underwent the gel to liquid-crystal phase transition at 40–41°C . This suggests that CO 2 transport across membranes is not governed by a solubility-diffusion mechanism. Prasad et al. 1998 demonstrated that liposomes of varying lipid composition with a range of membrane fluidities could substantially alter water permeability, while having no effect on CO 2 permeability. We conclude therefore that gastric gland cells and possibly the urinary bladder and renal cortex as well, maintain lumenal CO 2 gradients with respect to interstitium by mechanisms that do not primarily depend on apical membrane bilayer asymmetry and fluidity reduction in the exofacial leaflet. These results demonstrate that for molecules that permeate across membranes by a solubility-diffusion mechanism, reducing the fluidity of a single leaflet of the bilayer is sufficient to reduce permeability. This finding has implications for our understanding of permeation processes in that it allows us to treat the resistance to permeation offered by each leaflet as an independent parameter. Therefore, the permeability properties of the bilayer are not some amalgam or synergy of the activities of each leaflet, but are independent and additive in their own right. The bilayer can be considered, much like an electrical circuit, as a pair of resistors in series for the permeation of solutes, NH 3 and water . CO 2 permeability was shown not to occur by a solubility-diffusion pathway as its rate of passage across the liposomal membrane was completely independent of temperature and membrane fluidity. It is likely that these unusual properties are due to its molecular linearity and lack of any permanent dipole moment. Simon and Gutknecht 1980 demonstrated that CO 2 could dissolve into a number of organic solvents as well as into an egg lecithin bilayer with only small differences in the partition coefficient. Addition of cholesterol to the egg lecithin, which would have resulted in reduced membrane fluidity, had only a minor effect on the partition coefficient . Prasad et al. 1998 have recently shown that liposomes of widely varying fluidity have identical CO 2 flux rates. Therefore, neither solubility nor diffusion of CO 2 appears to be affected by lipid composition or lipid packing. To date, only the gastric gland has been directly shown to present a barrier to CO 2 diffusion ; however, the study did not investigate mechanisms for this remarkable property. Based on the results presented here, we conclude that it is unlikely to be the lipid composition of these cells that presents the barrier. In particular, our results suggest that proteins inserted in, or associated with, the membrane determine the membrane's permeability to CO 2 | Study | biomedical | en | 0.999996 |
10469731 | Inward-rectifier K + channels act as K + -selective diodes in the cell membrane . In a normal intracellular environment, when the extracellular K + concentration is raised to the same level as the intracellular concentration, inward K + current is much larger than the outward current. This property is commonly called inward rectification. Although the detailed mechanisms underlying rectification are still controversial , rectification in most inward-rectifier K + channels is induced by intracellular cations. It was first discovered that some inward-rectifier K + channels are blocked by intracellular Mg 2+ and that the extent of block depends on membrane voltage . More recently, inward-rectifier K + channels were also found to be blocked by intracellular polyamines in a voltage-dependent manner . Since both Mg 2+ and polyamines are cationic, the simplest explanation for the voltage dependence of channel blockade would be the Woodhull hypothesis , which proposes that the blocking ion site is located within the transmembrane electrical field. Therefore, to block the ion conduction pore, a blocking ion must travel across a portion of the electrical field, which results in the voltage dependence. Consequently, the degree of voltage dependence of the block, or rectification, is determined by the blocker's valence and the fraction of the electrical field that it needs to traverse to reach the blocking site. However, as early as a half century ago, it was observed that inward rectification is also sensitive to the concentration of extracellular K + . An increase in the concentration of extracellular K + anomalously increases the outward current rather than decreases it as one would predict from a simple change in the K + equilibrium potential. Furthermore, altering the concentration of extracellular K + causes a “parallel” shift of the current–voltage (I–V) 1 curve along the voltage axis. Thus, inward rectification does not simply depend on membrane voltage. Instead, on an empirical basis it appears to depend on the difference between the membrane voltage (V m ) and the equilibrium potential for K + (E K ); i.e., the driving force for K + (V m − E K ) . Subsequently, a seemingly related phenomenon was observed: the extent of channel blockade by intracellular tetraethylammonium (TEA) in a voltage-activated K + channel can be reduced by raising the concentration of extracellular K + ; this is commonly referred to as “trans knock-off of TEA by K + ” . Since then, the binding of most cationic K + channel blockers has been found to be sensitive to K + concentration on the trans side of the membrane . The knock-off effect has been suspected to be somehow related to rectification . Recently, not only the extent but also the voltage dependence of blockade of the ROMK1 inward-rectifier K + channel by both TEA and Mg 2+ were shown to depend on the concentration of extracellular K + . The discovery that the voltage dependence varies with the concentration of extracellular K + led to a further investigation of why the degree of rectification, or the voltage dependence of channel blockade, depends on both the membrane potential and the concentration of extracellular K + . In that study, the binding of extracellular K + to the pore was found to be voltage dependent, compatible with the K + -binding site being located halfway through the transmembrane electrical field. Since the binding of extracellular K + into the pore lowers the affinity of intracellular blocking ions such as TEA and Mg 2+ , the K + ion bound at the outer part of the pore and the blocking ion at the inner part are energetically coupled. Therefore, the voltage dependence of channel blockade must be determined by the movement of all energetically coupled ions in the electrical field. A variation in the concentration of extracellular K + will alter the saturation level of the external K + site ( K d ≈ 10 mM), and thus the degree of rectification. In other words, for a given intracellular ionic condition, the degree of rectification reflects the level of ion saturation at the external site. Thus far, it is unclear how much of the voltage dependence is due to the movement of permeant ions versus the movement of blocking ions in the electrical field. However, it is clear that a significant fraction of the voltage dependence results from the movement of permeant ions . Thus, the degree of rectification induced by a given intracellular blocking ion can be tuned by adjusting the concentration of extracellular permeant ion K + . To gain more insight into the role of permeant ions in determining the degree of rectification, we examined block of the ROMK1 channel by intracellular TEA when the extra- as well as the intracellular solutions contain various alkali metal ions. ROMK1 cDNA was cloned into the p-SPORT1 plasmid (GIBCO BRL) . RNA was synthesized using T7 polymerase (Promega Corp.) from Not1-linearized ROMK1 cDNA. Oocytes harvested from Xenopus laevis (Xenopus One) were incubated in a solution containing (mM) 82.5 NaCl, 2.5 KCl, 1.0 MgCl 2 , 5.0 HEPES, pH 7.6, and 2–4 mg/ml collagenase. The oocyte preparation was agitated using a platform shaker (80 rpm) for 60–90 min. It was then rinsed thoroughly and stored in a solution containing (mM): 96 NaCl, 2.5 KCl, 1.8 CaCl 2 , 1.0 MgCl 2 , 5 HEPES, pH 7.6, and 50 μg gentamicin. Defolliculated oocytes were selected and injected with RNA at least 2 and 16 h after collagenase treatment, respectively. All oocytes were stored in an incubator at 18°C. ROMK1 currents were recorded in the inside-out configuration from Xenopus oocytes (injected with ROMK1 cRNA) with an Axopatch 200B amplifier (Axon Instruments, Inc.). The recorded signal was filtered at 1 kHz and sampled at 5 kHz using an analogue-to-digital converter interfaced with a personal computer. pClamp6 software (Axon Instruments, Inc.) was used to control the amplifier and acquire the data. Macroscopic current–voltage curves were recorded as membrane voltage was linearly ramped (50 mV/s). Background leak current correction was carried out as previously described . Pipette solutions contained specified concentrations of alkali metal ions with (mM): 0.3 CaCl 2 , 1.0 MgCl 2 , and 10 HEPES, pH 7.6. Na + and N -methyl- d -glucamine (NMG + ) had nearly identical effects on all examined properties (see results ). Thus, when K + , Rb + , or Cs + concentrations were reduced, Na + was used to maintain the total concentration of alkali metal ions at 100 mM. The bath solutions contained the specified concentrations of TEA with (mM): 90 KCl, 5 K 2 EDTA, and 10 HEPES, pH 7.6. In some experiments, K + in the bath solution was replaced by Rb + . In the presence of 100 mM K + on both sides of the membrane and the absence of intracellular blocking ions, the I–V curve of the ROMK1 channel was remarkably linear . However, when extracellular K + was replaced by an equal concentration of Rb + , the channel conducted larger outward than inward current with a reversal potential of −5.3 ± 0.3 mV (mean ± SEM, n = 5) . As shown in Fig. 1C–E , no inward currents can be seen when extracellular K + was replaced by either Cs + , Na + , or NMG + . The reversal potentials were less than −60 mV in extracellular Cs + and less than −120 mV in extracellular Na + or NMG + . From these, we estimated the permeability ratios ( P K / P X ) using the Goldman-Hodgkin-Katz equation as P K / P Rb = 1.2, P K / P CS > 10, and P K / P Na and P K / P NMG > 100. Based on this empirical measure of ion selectivity, the channel has the same ion-selective sequence as other K + channels, K + ≈ Rb + > Cs + >> Na + , NMG + . Fig. 1 A shows the I–V curves recorded in symmetric 100 mM K + without or with intracellular TEA at the concentrations indicated. As shown previously, intracellular TEA causes the ROMK1 channel to conduct in an inwardly rectifying manner . This is because TEA blocks the channel in a voltage-dependent manner, manifested by the downward deflection of the I–V curves. Interestingly, when we replaced extracellular K + by other monovalent cations, we found that the I–V curves of the channel exhibit various degrees of curvature depending on the substituting ion species. As shown in Fig. 1 B, the curvature of the I–V curves appears to be, if anything, only slightly reduced when extracellular K + was replaced by Rb + . However, the curvature of the I–V curves was significantly reduced when K + was replaced by Cs + , and even further reduced when K + was replaced by Na + and NMG + . Thus, although inward rectification is induced by the binding of intracellular cations to the pore, the degree of rectification is at least in part determined by extracellular ions. In Fig. 2 , the fractions of unblocked current in the presence of 100 mM extracellular K + , Rb + , Cs + , or Na + were plotted against TEA concentration for several representative membrane voltages. Although the increment in membrane voltage between the adjacent curves is the same (20 mV) in all four plots, the distance between adjacent curves is not the same in the four cases. It decreases in the order of K + , Rb + , Cs + , and Na + , illustrating how voltage dependence of channel blockade by TEA varies with various extracellular alkali metal ions. The curves superimposed on the data are least-squares fits of an equation that assumes a 1:1 stoichiometry between the channel and TEA. From the fits, we determined the observed equilibrium dissociation constants for TEA ( TEA K obs ) in the presence of each of the four alkali metal ions. In Fig. 3 , we plotted TEA K obs determined in the presence of 100 mM of each of the four species of extracellular ions as a function of membrane voltage. The lines superimposed on the data represent least-squares fits of the Woodhull equation . From the Woodhull analysis we determined TEA K obs (0 mV) (the observed TEA equilibrium dissociation constant at 0 mV) and TEA ( z δ) obs (an empirical measure of the voltage dependence). The averages of TEA K obs (0 mV) and TEA ( z δ) obs determined in the presence of 100 mM extracellular K + , Rb + , Cs + , and Na + are presented in Fig. 4 . TEA K obs (0 mV) determined in K + was slightly smaller than that in Rb + , but larger than those in Cs + and Na + . Judged from TEA ( z δ) obs , the voltage dependence of channel blockade by TEA is similar in both K + and Rb + , although it may be slightly smaller in Rb + . However, the voltage dependence is significantly attenuated in Cs + and even more in Na + . The value of TEA ( z δ) obs in Na + is only about half what it is in K + . We next examined how channel blockade by TEA varies with the concentration of each ion species. When the concentrations of K + , Rb + , or Cs + were reduced, we used Na + as a substituting ion instead of the more commonly used NMG + , because in the present study extracellular Na + and NMG + behaved similarly and membrane patches tolerated Na + better. Both the I–V curves of the channel and its blockade by TEA were very similar in the presence of extracellular Na + or NMG + . Furthermore, TEA binds to the channel with nearly identical affinities in the presence of extracellular Na + or NMG + . Assuming NMG + does not bind at the external ion-binding site, we estimated the equilibrium dissociation constant of the site for Na + is in the molar range (see discussion ). Generally, the values of both TEA K obs (0 mV) and TEA ( z δ) obs increase with the concentration of extracellular alkali metal ions. In Fig. 5 , we plotted TEA K obs (0 mV) as a function of the concentration of all four alkali metal ions. In the presence of K + , Rb + , and Cs + , TEA K obs (0 mV) increases linearly with ion concentration. The plots for K + and Rb + are very similar, but the slope of the plot for Cs + is much smaller. In the case of Na + , the TEA K obs (0 mV) value is nearly the same in either 0 mM (i.e., 100 mM NMG + ) or 100 mM extracellular Na + . Furthermore, the value of TEA ( z δ) obs also increases with the concentration of alkali metal ions. For presentation purposes, the TEA ( z δ) obs data are presented in Fig. 7 (below). To gain insight into how intracellular permeating ions affect channel blockade by intracellular TEA, we examined how TEA-blocking behaviors would change if we replaced intracellular K + by Rb + . Fig. 6 A shows the I–V curves obtained in the presence of 100 mM intracellular Rb + and 100 mM extracellular K + . The inward current carried by K + is larger than the outward current carried by Rb + , and the currents reverse at approximately +10 mV. Addition of TEA to the intracellular solution significantly reduced the outward current. However, the curvature of the outward current induced by intracellular TEA is significantly smaller than that observed when 100 mM K + is present in both the intracellular and extracellular solutions . The I–V curves in Fig. 6 B were recorded in the presence of 100 mM Rb + on both sides of the membrane. Unlike the remarkably linear I–V curve in symmetric K + , the I–V curve in symmetric Rb + is nonlinear. The outward current is slightly smaller than the inward current, which reveals the asymmetric property of the channel. Addition of intracellular TEA also significantly reduced the outward current. Again, the TEA-induced curvature of the I–V curves obtained in the presence of intracellular Rb + is much less than that obtained in intracellular K + . Fig. 6C and Fig. D , shows the effects of reducing the concentration of extracellular permeant ions on channel blockade by intracellular TEA. All I–V curves in Fig. 6C and Fig. D , were recorded in the presence of 100 mM intracellular Rb + . The extracellular permeant ions were 20 mM K + and Rb + for C and D, respectively. The concentrations of intracellular TEA were as indicated. In the presence of 20 mM extracellular K + or Rb + , both the extent and the voltage dependence of channel blockade by TEA were very similar to those obtained in 100 mM corresponding extracellular ions . Fig. 7A and Fig. B , plots TEA K obs (0 mV) determined in the presence of 100 mM intracellular K + (open symbols) or 100 mM intracellular Rb + (closed symbols) as a function of the concentration of extracellular K + and Rb + , respectively. Replacing intracellular K + by Rb + dramatically reduced the dependence of TEA K obs (0 mV) on the concentration of either extracellular ion. To further illustrate how different intracellular ions alter the voltage dependence of channel blockade, in Fig. 7C and Fig. D , we plotted the TEA ( z δ) obs values determined under various intra- and extracellular conditions. Fig. 7 C plots TEA ( z δ) obs values against the concentration of extracellular K + , while Fig. 7 D plots the values against the concentration of extracellular Rb + . In both plots, the data represented by open and closed symbols were obtained in intracellular K + and Rb + , respectively. The values of TEA ( z δ) obs increased with increasing concentrations of extracellular K + or Rb + , compatible with a scenario where the observed changes in the voltage dependence result from titrating an ion-binding site in the external part of the pore. When K + in the intracellular solution was replaced by Rb + , the value of TEA ( z δ) obs was significantly reduced at all tested concentrations of extracellular K + or Rb + . However, regardless of whether the intracellular ion was K + or Rb + , the TEA ( z δ) obs values converged to the same minimum at vanishing extracellular K + or Rb + concentrations. These observations show that replacing intracellular K + by Rb + dramatically reduces the ability of extracellular ions to alter the voltage dependence of channel blockade. In the present study, we found that both the extent and the voltage dependence of channel blockade by TEA depend on the type of alkali metal ions, including K + , Rb + , Cs + , and Na + , in the extracellular solution . Since the voltage dependence is determined by the movement of all energetically coupled ions in the transmembrane electrical field , these results can be understood if the external ion site has different affinities for these ions. To test this idea, we determined the affinities of the site for the four alkali metal ions by examining how TEA K obs (0 mV) varies with the concentration of each ion. As shown in Fig. 5 , TEA K obs (0 mV) increases linearly with not only the concentration of K + , as observed previously , but also the concentrations of Rb + and Cs + . Altering Na + concentration has very little effect on TEA K obs (0 mV). The linear dependence of TEA K obs (0 mV) on the concentration of extracellular ions argues that extracellular ions in effect compete with intracellular TEA for binding to the pore. However, the apparent competition undoubtedly does not result from the binding of extracellular permeant ions and intracellular TEA to the same site. Rather, the competition is most likely an indirect process. For example, the binding of K + at the external site may perturb the binding site for intracellular TEA, which in turn affects TEA binding, and vice versa. This scenario is in line with the finding that mutations at some residues around the permeant ion-binding sites in the narrow region of the voltage-activated K + channels can significantly affect the binding of intracellular TEA to the more internal region of the pore . Furthermore, as discussed below, we show here that ion binding at an internal site in the ROMK1 pore can dramatically alter ion binding at the external site . The diagram in Fig. 8 is a simplified version of the kinetic models that we previously used to interpret the dependence of TEA K obs (0 mV) on the concentration of extracellular K + . The dissociation constants ( TEA K and X K ) for the two competitors (TEA int and X ext ) are defined 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*}^{{\mathrm{TEA}}}{\mathit{K}}=\frac{ \left \left[{\mathrm{Ch}}\right] \right \left \left[{\mathrm{TEA}}_{{\mathrm{int}}}\right] \right }{ \left \left[{\mathrm{ChTEA}}_{{\mathrm{int}}}\right] \right }\end{equation*}\end{document} and 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{X}}}{\mathit{K}}=\frac{ \left \left[{\mathrm{Ch}}\right] \right \left \left[{\mathrm{X}}_{{\mathrm{ext}}}\right] \right }{ \left \left[{\mathrm{X}}_{{\mathrm{ext}}}{\mathrm{Ch}}\right] \right }{\mathrm{.}}\end{equation*}\end{document} The fraction of unblocked channel is 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{{\theta}}}=\frac{ \left \left[{\mathrm{Ch}}\right] \right + \left \left[{\mathrm{X}}_{{\mathrm{ext}}}{\mathrm{Ch}}\right] \right }{ \left \left[{\mathrm{Ch}}\right] \right + \left \left[{\mathrm{X}}_{{\mathrm{ext}}}{\mathrm{Ch}}\right] \right + \left \left[{\mathrm{ChTEA}}_{{\mathrm{int}}}\right] \right }{\mathrm{,}}\end{equation*}\end{document} where [X ext Ch], [ChTEA int ], and [Ch] are concentrations of the channels bound with an additional extracellular ion X, intracellular TEA, or neither of them. Combining yields: 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{{\theta}}}=\frac{1}{1+\displaystyle\frac{ \left \left[{\mathrm{TEA}}_{{\mathrm{int}}}\right] \right }{^{{\mathrm{TEA}}}{\mathit{K}}_{{\mathrm{obs}}}}}{\mathrm{,}}\end{equation*}\end{document} where 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*}^{{\mathrm{TEA}}}K_{{\mathrm{obs}}}=\frac{^{{\mathrm{TEA}}}K}{^{{\mathrm{X}}}K} \left \left[{\mathrm{X}}_{{\mathrm{ext}}}\right] \right +^{{\mathrm{TEA}}}K{\mathrm{.}}\end{equation*}\end{document} Undoubtedly, the state diagram in Fig. 8 is an over simplification for the interaction between extracellular permeant ions and intracellular TEA. Nevertheless, it provides a simple and instructive way for considering the interaction between the extracellular permeant ions and intracellular TEA. In fact, derived based on this simple state diagram is the same as one we previously derived based on a more complex model . According to , we determined the equilibrium dissociation constant for TEA at 0 mV and in zero extracellular K + [ TEA K (0 mV)] from the y intercept of the plots in Fig. 5 as 1.4 ± 0.3 mM. We also determined the equilibrium dissociation constants of the channel for the four ions from the ratio of the y intercept and slope of the corresponding plots (mM): K K = 13.2 ± 2.4 (mean ± SEM), Rb K = 15.6 ± 3.1, Cs K = 34.0 ± 3.5, and Na K = 1.4 M. Since the slope of plot D is minimal, a Na K value of 1.4 M may not be very precise. Nevertheless, it does indicate that the affinity of the site for Na + is much lower. The results of the analysis indicate that the external ion-binding site strongly selects K + over smaller Na + , but only minimally or modestly selects K + over larger Rb + or Cs + . The ion affinity sequence of the site (K + ≈ Rb + > Cs + > Na + ) correlates with the degree of voltage dependence of channel blockade by TEA in the presence of the corresponding alkali metal ions . To account for the dependence of TEA K obs on membrane voltage, we combined and the Woodhull 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*}K_{{\mathrm{d}}} \left \left({\mathrm{X\;mV}}\right) \right =K_{{\mathrm{d}}} \left \left(0\;{\mathrm{mV}}\right) \right e^{\frac{- \left \left(z{\mathrm{{\delta}}}\right) \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}{\mathrm{,}}\end{equation*}\end{document} which gives 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*}^{{\mathrm{TEA}}}K_{{\mathrm{obs}}} \left \left({\mathrm{X\;mV}}\right) \right =\frac{ \left \left[{\mathrm{X}}_{{\mathrm{ext}}}\right] \right ^{{\mathrm{TEA}}}K \left \left(0\;{\mathrm{mV}}\right) \right e^{\displaystyle\frac{-^{{\mathrm{TEA}}} \left \left(z{\mathrm{{\delta}}}\right) \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}}{^{{\mathrm{X}}}K \left \left(0\;{\mathrm{mV}}\right) \right e^{\displaystyle\frac{^{{\mathrm{X}}} \left \left(z{\mathrm{{\delta}}}\right) \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}}+^{{\mathrm{TEA}}}K \left \left(0\;{\mathrm{mV}}\right) \right e^{\frac{-^{{\mathrm{TEA}}} \left \left(z{\mathrm{{\delta}}}\right) \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}{\mathrm{.}}\end{equation*}\end{document} Rearranging , we obtain 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*}^{{\mathrm{TEA}}}K_{{\mathrm{obs}}} \left \left({\mathrm{X\;mV}}\right) \right =\frac{ \left \left[{\mathrm{X}}_{{\mathrm{ext}}}\right] \right ^{{\mathrm{TEA}}}K \left \left(0\;{\mathrm{mV}}\right) \right e^{\displaystyle\frac{- \left \left[^{{\mathrm{TEA}}} \left \left(z{\mathrm{{\delta}}}\right) \right +^{{\mathrm{X}}} \left \left(z{\mathrm{{\delta}}}\right) \right \right] \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}}{^{{\mathrm{X}}}K \left \left(0\;{\mathrm{mV}}\right) \right }+^{{\mathrm{TEA}}}K \left \left(0\;{\mathrm{mV}}\right) \right e^{\frac{-^{{\mathrm{TEA}}} \left \left(z{\mathrm{{\delta}}}\right) \right F{\mathrm{V}}_{{\mathrm{m}}}}{RT}}{\mathrm{.}}\end{equation*}\end{document} Quantity X ( z δ) in is related only to the binding and unbinding of extracellular permeant ions to the external site. However, TEA ( z δ) is related to the movement of TEA and possibly other permeant ions (excluding the one bound to the external site) in the electrical field along the pore . According to , voltage dependence of channel blockade by intracellular TEA is determined by the movement of TEA as well as permeant ions in the transmembrane electrical field. Since the external site has different affinity for the various alkali metal ions, the site is occupied to a different extent in the presence of various ions at a given finite concentration. This explains why various degrees of voltage dependence are observed in the presence of 100 mM of the four extracellular alkali metal ions. Although we exploited the apparent competition between extracellular alkali metal ions and intracellular TEA to determine K d values for the alkali metal ions, these K d values should characterize the interaction of alkali metal ions with the channel in the absence of TEA . Even so, the absolute K d values determined here do not necessarily reflect the intrinsic affinities of the site for the various alkali metal ions for the reason discussed below. To learn how intracellular permeant ions modify blocking ion–induced rectification, we compared TEA block in 100 mM intracellular K + versus Rb + . Regardless of whether K + or Rb + was in the extracellular solution, the effect on TEA K obs (0 mV) of changes in the concentration of extracellular permeating ions was much larger when the intracellular ion was K + than when it was Rb + . For example, when the concentration of extracellular K + was decreased from 100 mM to near zero, TEA K obs (0 mV) decreased by eightfold in intracellular K + , whereas it decreased by less than twofold in intracellular Rb + . A similar phenomenon was observed when the concentration of extracellular Rb + was reduced . Analyzing the data acquired in intracellular Rb + using , we obtained K K = 155 ± 21 mM, Rb K = 159 ± 18 mM, and TEA K = 6.5 ± 0.4 mM (all at 0 mV). Thus, replacing intracellular K + with Rb + lowers channel affinity for extracellular K + and Rb + by ∼10-fold ( K K = 13 vs. 155 mM, Rb K = 16 vs. 159 mM), and channel affinity for intracellular TEA by ∼5-fold ( TEA K = 1.4 vs. 6.5 mM). The increase in TEA K accounts for the higher y intercepts of the plots corresponding to intracellular Rb + in Fig. 7A and Fig. B , while the larger changes in both K K and Rb K than in TEA K account for the shallower slopes of the plots corresponding to intracellular Rb + . These findings suggest that K + and Rb + interact quite differently with an internal ion-binding site, despite the fact that the external site has similar affinities for K + and Rb + ( K K = 13 mM vs. Rb K = 16 mM). Since the binding of intracellular Rb + reduces the affinity of the external site for both K + and Rb + , predicts that in the presence of subsaturating concentrations of extracellular permeating ions, the voltage dependence of channel blockade should be less in intracellular Rb + than in K + . Also, regardless of the types of permeating ions present in the intracellular solution, the voltage dependence should be the same when the concentration of extracellular permeating ions is zero, because in this case only the second term in applies. These predictions are consistent with what was observed . Therefore, the reduction in the voltage dependence due to replacing intracellular K + by Rb + can be explained by the resulting reduction in the affinity, and thus in the level of ion saturation, of the external ion-binding site. It is unclear thus far why the affinity of the channel for blocking ions, such as TEA, and the voltage dependence of channel blockade are much less sensitive to the concentration of extracellular K + in the voltage-activated K + channels than in the inward-rectifier K + channels . Here, we found that in the presence of intracellular Rb + , the affinity of the ROMK1 channel for extracellular permeating ions is significantly reduced. Consequently, in the presence of intracellular Rb + , both the affinity of the channel for TEA and the voltage dependence of its block by TEA are much less sensitive to the concentration of extracellular permeating ions, a property reminiscent of voltage-activated K + channels. This finding makes one wonder whether the relatively low extracellular ion sensitivity of TEA block in the voltage-activated K + channels may in part result from a lower affinity of the channels for extracellular ions under comparable conditions. Interestingly, the single channel conductance of the ROMK1 inward-rectifier K + channel is half-maximal at 10 mM K + , whereas the half-maximal single-channel conductance of the Shaker voltage-activated K + channel is not reached until the concentration of K + is 300 mM . We showed here that the external ion site in the ROMK1 channel interacts selectively with alkali metal ions with an affinity sequence K + ≈ Rb + > Cs + >> Na + . This sequence is similar to that previously determined in a Ca 2+ -activated K + channel by examining the interaction between K + and Ba 2+ ions , although the absolute K d values for all four ions determined in the present study are generally much larger than those determined in the quoted studies . (As already discussed, the affinity of a given ion binding site in the pore depends on the presence of other ions in the pore.) Our data show that the external site in the ROMK1 channel strongly selects against smaller Na + , but only modestly selects among the larger ions. Furthermore, we found that the binding of intracellular K + versus Rb + to an internal site has dramatically different effects on how the pore interacts with other ions. This finding argues that this internal site may be more selective among the larger ions. The idea that both internal and external sites in a K + pore are selective has previously been used by Neyton and Miller 1988a , Neyton and Miller 1988b to account for the fact that both intra- and extracellular permeating ions selectively interact with blocking Ba 2+ in a Ca 2+ -activated K + channel. This view is supported by a recent crystallographic study on KcsA, a bacterial K + channel, showing that two permeating ions can simultaneously reside in the narrow region of the pore, and that the locations of the binding sites are the same for various alkali metal ions . A multi-ion theory, often used to explain ion selectivity in K + channels , was originally proposed to explain ion selectivity in the voltage-activated Ca 2+ channels . The theory hypothesizes that there are two (or more) K + -selective ion-binding sites in the narrow region of the pore. K + ions can bind to either site with high affinity, which accounts for the K + selectivity of the pore. To account for the high throughput rate of the channel, the binding of K + to the second site is hypothesized to dramatically lower the affinity of both sites for K + due to electrostatic repulsion between the bound K + ions. We found here that the external site in the ROMK1 channel has very different affinities for extracellular permeating ions depending on whether the internal site is exposed to K + or Rb + , which provides experimental evidence for the hypothesized permeant ion interactions in the pore. This finding also argues that the interactions between permeating ions in the pore are not merely electrostatic. Conceivably, binding of ions of different sizes at one site (e.g., the internal site in this case) in the pore can induce different degrees of structural “deformation” at a second site (e.g., the external site) elsewhere in the pore by propagating the binding energy along the pore-lining protein elements. Since the interactions between ions and the narrow part of the pore almost certainly are ion–dipole interactions, they should be highly sensitive to a change in the distance between permeating ions and the dipole-generating atoms, such as carbonyl oxygen, in the channel protein . Thus, even a small change in the distance between a permeating ion and the dipole-generating atoms in the channel protein, induced by the binding of another ion elsewhere, could dramatically alter their interactions. In summary, the external ion site in the ROMK1 pore binds various alkali metal ions (Na + , K + , Rb + , and Cs + ) with different affinities, which can in turn be altered by the binding of various permeating ions at the internal site through a nonelectrostatic mechanism. Consequently, the saturation level of the external ion site depends on the ion species on both sides of the membrane. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, various degrees of rectification are observed with various combinations of extra- and intracellular ions. Although both the external and internal ion sites in the ROMK1 pore appear to be ion selective, they likely have different ion selectivity: the external site selects strongly against smaller Na + but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger ions K + and Rb + . | Other | biomedical | en | 0.999996 |
10469733 | Neurotransmitter transporters, many of which are of great medical importance , couple the “uphill” transport of neurotransmitters into cells to the “downhill” movement of Na + . Transport is often coupled to transmembrane movements of other ions, such as Cl − , K + , and/or protons . Two distinct gene families for plasmalemmal neurotransmitter transporters are identified, the family of Na + /Cl − -dependent transporters for γ-aminobutyric acid (GABA), 1 glycine, serotonin, and catecholamines and the family of excitatory amino acid transporters . GABA transport could be studied in native membranes, and a transport stoichiometry of 1GABA:2Na + :1Cl − was suggested . The GABA transporter, GAT1, was the first member of its family to be cloned , and initial findings on GAT1 expressed in Xenopus oocytes were consistent with the proposed stoichiometry. Stoichiometric transport of substrates is usually explained by the “alternating access” hypothesis , whereby conformational changes allow substrates to bind alternatively on one membrane side or the other, and to be transported across the membrane . However, recent studies have led several authors to question the validity of the alternating access model for GAT1: some findings suggest the existence of “leak modes” of the transporter (e.g., uncoupled current in the absence of GABA) that result in major departures from the accepted stoichiometry . Additionally, some GABA uptake studies, in combination with current measurements, suggest much larger Na + movements than predicted by a 1GABA:2Na + :1Cl − stoichiometry . Furthermore, channel-like behaviors have been identified as current noise in some recordings and as probable “single channel” currents under certain conditions in other recordings . Possibly, the channel-like, nonstoichiometric behaviors identified for GAT1 and other neurotransmitter transporters, including a Cl − conductance in glutamate transporters , represent important clues needed to understand the physical basis of neurotransmitter transport . To address these issues, we have characterized GAT1 function with the Xenopus oocyte expression system, using giant membrane patches to record GAT1 currents . For these studies, the ability to control substrate concentrations on both membrane sides and to voltage clamp with microsecond resolution are major advantages of the giant patch methods . In this article, we describe problems of measuring GAT1 currents in oocyte membrane and our lack of evidence for GABA-uncoupled currents. We characterize steady state GAT1-mediated currents with an emphasis on the reverse (outward) current and cis–trans substrate interactions for both transport directions. After analyzing our results in relation to the proposed “channel-like” cotransport models, we conclude that GAT1 operates by the alternating access principle. Thus, a major goal of our work has been to develop an alternating-access reaction scheme that can account for GAT1 function in Xenopus oocyte membranes . The reaction cartoons, shown in Fig. 1 , are intended to orient the reader to our overall goal; namely, to account for the dependencies of GAT1 function on all three substrates on both membrane sides, to account for voltage dependencies of transport, and to account for transporter kinetics. Fig. 1 A depicts groups of reactions thought to occur in the forward transport mode of GAT1 (i.e., moving GABA into cells) on the basis of previous work. Perhaps the most important detail is that a slow reaction can occlude one Na + ion from the extracellular side in the absence of GABA and chloride . This reaction is electrogenic and appears to move about one charge per transporter cycle through the membrane electric field. It is therefore marked with a large lightning symbol in Fig. 1 . As further indicated, the reaction is relatively slow (τ = 15–200 ms), and it probably rate-limits inward current in the middle potential range, where the slope of the current–voltage relations of the transport current is quite steep. Evidently, GABA and chloride can bind only after Na + has been occluded by the transporter, and when they do so, all three substrates are transported to the cytoplasmic side via a faster reaction that is nearly electroneutral. At large negative potentials where the “slow” charge-moving reaction is accelerated, this “fast” translocation reaction probably becomes rate limiting; this would explain the observed current saturation with hyperpolarization . It is known from previous giant patch studies that distinct fast reactions (τ < 1 ms) can occur in GAT1 in the absence of substrates on both membrane sides . Labeled “fast” and associated with a small lightning symbol in Fig. 1 A, these reactions are blocked by the presence of cytoplasmic Cl − , and they might represent a reorientation of the empty transporter sites to the outside to allow rebinding of Na + . Presumably, these reactions never become rate limiting for GABA transport. The results described in this article for the reverse transport mode (i.e., GABA transport from inside to outside) give rise to the reaction perspectives summarized in Fig. 1 B. In the absence of extracellular substrates, the reverse transport cycle appears to be rate limited by a weakly voltage-dependent reaction under most conditions. According to our analysis, the underlying reaction must involve the occlusion and/or translocation of substrates from the cytoplasmic side. One possible scheme is shown in Fig. 1 B, where one Cl − and one Na + are occluded in a slow step, and all other reactions in the reverse cycle are much faster. These include (a) the binding of a second cytoplasmic Na + and GABA, (b) electroneutral substrate transport and release to the extracellular side , (c) the electrogenic deocclusion of Na + (fast, with a large lightning symbol), and (d) the reorientation of empty binding sites that allows cytoplasmic Cl − and Na + to bind again. We point out that the results presented in this article alone do not justify these interpretations, and that the interpretations alone do not allow formulation of a reaction scheme for GAT1. Thus, in an accompanying article , we describe new kinetic findings on non–steady state GAT1 function that are critical to our interpretations. And we describe in another article the development of an alternating access model that can account for most details of GAT1 function in the Xenopus oocyte membrane . GAT1 cRNA was in vitro transcribed from pBSAMVGAT1 (gift of S. Mager, California Institute of Technology, Pasadena, CA) with T7 polymerase and injected into Xenopus oocytes, which were isolated and maintained according to Zühlke et al. 1995 . Transport currents and capacitance were recorded 4–7 d after injection in excised giant patches. In the absence of extracellular Na + , outward GAT1 transport current was 15–200 pA at 0 mV, depending on patch size and expression level, in the presence of 20 mM cytoplasmic GABA and 120 mM cytoplasmic NaCl. Giant inside-out oocyte membrane patches (6–12 pF; seal resistance > 1 GΩ) were obtained as previously described . Methods for pipette perfusion and concentration jumps are also detailed elsewhere . Unless indicated otherwise, transport currents were recorded at 32°C. Membrane currents were recorded using an Axopatch 200 patch clamp amplifier (Axon Instruments). Voltage protocols and data acquisition were executed with Digidata 1200 (Axon Instruments), using our own software. Membrane capacitance was measured according to Lu et al. 1995 , using a Princeton lock-in amplifier . Data analysis and curve fitting used Origin5.0 (Microcal Software, Inc.). Many experiments described in this and the next article were technically demanding. The results presented are from single experiments that we judge to be reliable. The results were verified in multiple observations, and, usually, two or more additional experiments with very similar results were obtained. However, patches sometimes became unstable before all data points in a protocol were acquired, and we judge that results from patches with low GAT1 expression are in general less reliable than results from patches with high expression. For these reasons, we do not include statistical analysis. Current–voltage relations for the outward GAT1 currents, presented in this article, are described well by a simple exponential (Boltzmann) function of the form, A · e q · 0.5 · Em · F / RT , where A is a scalar, Em is the membrane potential, F / RT has its usual meaning, and q is the equivalent charge. According to Eyring rate theory, and assuming an energy barrier midway through the membrane electrical field, the “equivalent charge” is the amount of charge that would have to move through the entire membrane field to account for the voltage dependence. This is “equivalent” to a proportionally larger charge amount that would move through a proportionally smaller fraction of membrane field to account for the same voltage dependence. RT/F was approximated as 26.5 mV. The standard bath solution contained (mM): 0–20 GABA, 120 NaCl, 0.5 magnesium sulfamate, 20 tetraethylammonium-OH, 10 EGTA, and 20 HEPES, pH 7.0. Unless noted otherwise, the standard pipette solution contained (mM): 20 N -methylglucamine (NMG) 1 -Cl, 100 NMG–2-( N -morpholino)ethanesulfonic acid (MES), 2 magnesium sulfamate, 4 calcium sulfamate, 0.02 ouabain, and 20 HEPES, pH 7.0. Equimolar NMG was substituted for Na + , and MES was substituted for Cl − . Giant membrane patches from control oocytes exhibited no GABA-activated currents (data not shown). Fig. 2 A shows the typical GABA-activated currents recorded in giant membrane patches from oocytes expressing GAT1. Outward current is activated when GABA (20 mM) is added to a cytoplasmic solution containing 120 mM NaCl, whereby the extracellular (pipette) solution contains 20 mM Cl − and no GABA or Na + . Inward current is activated in the absence of cytoplasmic GABA, Na + , and Cl − when GABA (0.2 mM) is added via pipette perfusion with a pipette solution that contains 100 mM NaCl. Inward currents at 0 mV were typically manyfold smaller than outward currents at 0 mV. In stable patches, the GABA-induced currents showed no change in magnitude for over 30 min. Fig. 2 B illustrates our standard protocol for studying the voltage dependence of steady state transport current. When the membrane current reached a new steady value after solution changes, the voltage protocol described in Fig. 2 B (middle) was applied. This is the cause of the current spikes a–d in Fig. 2 A. Typically, we used cumulative membrane voltage steps of 30 mV magnitude from 0 to –150 mV, up to +90 mV, and back to 0 mV. The step durations were just long enough so that pre–steady state transients of the inward GAT1 current decayed for the most part during each step. Fig. 2 B (top) shows the membrane current response during (a) and after (b) the application of cytoplasmic GABA, and the difference (a–b) is shown below. The same procedure was used to obtain membrane current responses before (c) and during (d) extracellular GABA application by pipette perfusion. The subtracted record (d–c) reveals pre–steady state current transients that are faster at negative potentials. To obtain the steady state current–voltage (I–V) relation of the subtracted current, the median current magnitude of the last 3 ms of each voltage step is plotted against membrane voltage . The I–V relations are monotonic. There is little or no hysteresis in the outward I–V relation, and the moderate hysteresis present in the inward I–V relation is expected from the slow time-dependent processes in the forward transport cycle . Fig. 3 shows that outward transport currents can be defined by two different means with very similar results. The extracellular solution contains Na + (120 mM) but no GABA to test for GAT1-mediated inward current in the absence of GABA. Definition of transport current by applying and removing 20 mM GABA from the cytoplasmic side; (□) the definition by applying NO-711, a high-affinity GABA uptake inhibitor, from the cytoplasmic side in the presence of 20 mM cytoplasmic GABA. The results are fitted by the Boltzmann equation, given in materials and methods , and the equivalent charge is 0.63. This slope is two-times larger than in the absence of Na + o . Pipette perfusion experiments that define the effect of Na + o in I–V's in a single experiment are described in Fig. 6 A of a companion article . If GAT1 mediated an uncoupled Na + current, these I–V relations would cross the zero current line and display an inward current component. That is not the case. As shown further in Fig. 3 (•), the presence of NO-711 (0.13 mM) on the cytoplasmic side completely blocks GABA-activated current. In re-lated experiments, we used NO-711 to further test whether GAT1 mediates ionic currents in the absence of GABA. In patches expressing GAT1, current responses to membrane voltage changes before and after applying NO-711 were compared, and no NO-711–inhibited current was clearly detected. Protocols used to define GAT1 charge movements provide one example. We point out that our ability to resolve small Na + currents is limited by the Xenopus oocyte Na + conductance. This conductance is slowly activated by depolarization beyond −20 mV in patches . It is large in many oocyte batches. Although it decreases continuously over 10–20 min after patch excision, a residual conductance is persistent. Outward I–V relations were also defined by addition and removal of cytoplasmic Cl − or Na + , leaving the other substrate concentrations constant (not shown). Results for Cl − , using MES as the Cl − substitute, were virtually identical to those with GABA subtraction. This indicates that, in the absence of cytoplasmic Ca 2+ , the Cl − conductance of the oocyte membrane patches is very small. Results for Na + , using NMG as the Na + substitute, were also very similar to those with GABA subtraction, provided that GAT1 expression was high and the endogenous Na + conductance of the oocyte patch had run down. In general, however, the Na + conductance of the oocyte membrane complicates results obtained with Na + subtraction. In our experience, activation of outward GAT1 transport current in oocyte patches requires the simultaneous presence of GABA, Na + , and Cl − on the cytoplasmic side. Activation by each substrate in the presence of cosubstrates is demonstrated in Fig. 4 . The results were obtained using a fast solution switch device whereby the pipette tip is moved in <3 ms through the interface between two solution streams by a computer-controlled manipulator . We present results from a patch with relatively low GAT1 expression since these results illustrate our reasons for defining GAT1 current with GABA application and removal in subsequent experiments. Dotted lines in Fig. 4 indicate the zero current level. Fig. 4 A shows the current record for applying and removing 20 mM cytoplasmic GABA in the presence of 120 mM NaCl. The outward current activates to a plateau in ∼30 ms on applying GABA i , and the current returns to baseline with an S-shaped time course over ∼200 ms. The patch has a small “leak current” (<1 pA) in the presence of 120 mM NaCl i . Due to the rise of the membrane into the pipette tip (∼50-μm diameter), the time courses of current activation and deactivation are, with good certainty, determined by the diffusion of GABA up to and away from the membrane, respectively. The rate of activation is relatively fast because the transporter binding sites become saturated with GABA i before diffusion equilibrium is reached. The time course of deactivation is slow because the concentration of GABA i at the membrane must decrease substantially before binding sites are freed of GABA i . Fig. 4 B shows the record obtained on applying 120 mM Cl − i , instead of 120 mM MES − i , in the presence of 20 mM GABA i and 120 mM Na + i . Current activation is just as fast as with GABA i because the half-maximal Cl − i concentration is only ∼10 mM at 0 mV . The current relaxation on removal of Cl − i is, however, biphasic. The fast phase of relaxation is probably an artifact caused by the liquid junction potential of these two solutions (∼8 mV), and this will affect the driving force for both leak current and transport current. Thus, Cl − i definition of GAT1 current will have significant artifactual components, particularly with low transport activities, high Cl − i concentrations, and at extreme potentials. Fig. 4 C shows the typical result obtained for application of 120 mM Na + i in the presence of 20 mM GABA i and 120 mM Cl − i , using NMG + as the Na + substitute. In this case, removal of Na + i causes a small inward background Na + current (<5 pA at 0 mV), which probably reflects Na + -mediated leak current through the patch seal. The activation of GAT1 transport current by Na + i application is somewhat slower than by GABA i and Cl − i . This is because 120 mM Na + i does not “super-saturate” binding sites. The current deactivates faster than with the other substrates because the Na + i dependence of the current is sigmoidal . From these results, we conclude that GAT1 transport current is best defined with GABA application and removal, and GABA definition is used in subsequent results. In experiments similar to those just described, we tested extensively whether the GAT1 transporter undergoes substrate- and time-dependent activity changes via “secondary modulation” or “inactivation” reactions, analogous to reactions identified for cardiac Na + –Ca 2+ exchange . In short, we have found no evidence for such behaviors, on either long or short time scales, down to the time scale of the slow extracellular Na + -dependent GAT1 charge movements (∼20 ms) described in the next article . Using pipette perfusion, we also tested whether inward or outward GAT1 currents could be enhanced by the presence of substrates or monovalent ions on the opposite membrane side, as reported for GAT1 currents recorded in stably transfected HEK293 cells . We did not observe any trans-activation effect. In contrast to the evident tight substrate-current coupling in the activation of reverse (outward) transport current in oocyte patches, forward transport current can be partially activated by extracellular GABA in the nominal absence of extracellular Cl − . Fig. 5 shows records from a single patch: (a) the outward current activated by 20 mM GABA i with 120 mM NaCl on both membrane sides. (b) The NO-711 i -sensitive inward current with 0.4 mM GABA o , 120 mM Na + o , no Cl − o , and no cytoplasmic substrates. (c) The NO-711 i -sensitive inward current with 0.4 mM GABA o , 120 mM NaCl o , and no cytoplasmic substrates. The Cl − o -independent inward current is more significant at negative membrane potentials (at –120 mV, ∼40% of that with 120 mM Cl − o ). These results are comparable with results in intact oocytes . To further probe substrate coupling by the GAT1 transporter, we measured reversal potentials with various extracellular-to-cytoplasmic substrate ratios and compared them with the expected reversal potentials, V rev , for 1GABA:2Na + :1Cl − stoichiometry: \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_{{\mathrm{rev}}}=\frac{R{\cdot}T}{F}{\cdot}{\mathrm{ln}} \left \frac{ \left \left[{\mathrm{GABA}}_{{\mathrm{o}}}\right] \right }{ \left \left[{\mathrm{GABA}}_{{\mathrm{i}}}\right] \right }{\cdot} \left \left(\frac{ \left \left[{\mathrm{Na}}^{{\mathrm{+}}}_{{\mathrm{o}}}\right] \right }{ \left \left[{\mathrm{Na}}^{{\mathrm{+}}}_{{\mathrm{i}}}\right] \right }\right) \right ^{{\mathrm{2}}}{\cdot}\frac{ \left \left[{\mathrm{Cl}}^{-}_{{\mathrm{o}}}\right] \right }{ \left \left[{\mathrm{Cl}}^{-}_{{\mathrm{i}}}\right] \right } \right {\mathrm{,}}\end{equation*}\end{document} where R , T , and F have their usual meanings. For these measurements, I–V relations were determined with all substrates on both membrane sides, before and after extracellular application of the high-affinity organic GAT1 inhibitor NO-711 via pipette perfusion. We note that cytoplasmic NO-711 did not block effectively the GAT1-mediated currents under these conditions, presumably because GABA binding sites are occupied on both membrane sides. Fig. 6 A shows a typical GAT1 I–V relation with a measured V rev of 45 mV. With the substrate concentrations employed , the predicted value is 97 mV. Fig. 6 B shows results obtained with a variety of substrate concentrations. The scatter of experimental results is rather large in the repeated measurements, and this reflects the fact that GAT1 current amplitudes in these conditions are rather small. With 120 mM NaCl and 2 mM GABA on both sides, for example, we could not define any GAT1 current. Nevertheless, all of the measured V rev 's, summarized in Fig. 6 B, scatter along the theoretical line without systematic deviation. Thus, within our experimental limitations, we find no contradiction to the idea that GAT1 transport is a well-coupled process. Since activation of outward GAT1 current with GABA involves changing solution osmolarity by up to 20 mosmol/liter, we tested whether osmotic strength and/or transmembrane osmotic gradients result in any alteration of GAT1 current. We observed no effects of osmolarity changes using dextrose, aspartate, glutamate, or polyethylene glycol (not shown). In a related issue, Loo et al. 1996 have reported that GAT1 transports water in a manner similar to the brush border Na + /glucose cotransporter, where >200 water molecules appear to be directly coupled to the transport of each sugar molecule. We therefore tested the effect of changing water concentration on GAT1 transport. There was no clear effect on the outward GAT1 transport current when 30% of water molecules were replaced by low molecular weight polyethylene glycol. For some ion channels, it is established that ions permeate in a single-file fashion along multiple binding sites . When two ion species can permeate a channel, permeation of one ion species can sweep the other ion species through the channel against its electrochemical gradient. This mechanism allows a cotransport function to be established in an ion/substrate “hopping” model when it is assumed that a substrate such as GABA can permeate through a Na + -selective channel . Specifically, the “multisubstrate single-file” model assumes that Na + and a substrate “hop” through a pore with three sites that can bind either the substrate or Na + . The model permits flux-coupling without generating large Na + leak currents in the absence of substrate, and it can recreate a number of results on GAT1 function. We now compare its function with an “alternating access” model that “simultaneously transports two substrates . Fig. 7 illustrates predictions for trans- and cis-substrate interactions in the two models (A and D) that we consider fundamental. Predictions for the channel-like model are shown in Fig. 7B and Fig. C ; those for the alternating access model are shown in E and F. We simulated the channel model with our own software using the published parameter values , and we checked that our implementation accurately recreated all published steady state simulations. Also, we checked that the model did not contradict thermodynamic constraints under a wide range of conditions. An inherent feature of the channel-like model is that binding of a substrate on one side of the membrane tends to be prevented by substrate flux from the opposite side. Thus, addition of Na + to the trans side decreases the apparent affinity for GABA i to activate transport from the cis side . On the other hand, substrates on the same (cis) membrane side compete for entrance into the pore. Thus, reduction of [Na + ] i results in an increase in the apparent affinity for GABA i . We use the simple cotransport model in Fig. 7 D to illustrate the equivalent predictions for an alternating access scheme. In this simulation, the cotransporter binds its substrates X and Y instantaneously and independently. The presence of a trans substrate (e.g., X o ) reduces the rate of the “empty carrier translocation” or “return” step (1) in the transport cycle, thereby limiting the rate of transport from the cis side. When the return step is slow, the apparent affinity for cis substrates (e.g., Y i ) is higher. The fractional increase of affinity for cis substrates should be proportional to the fractional decrease of maximal transport induced by addition of the trans substrate. The predictions for X = Na + and Y = GABA are shown in Fig. 7 E, together with the corresponding data from a pipette perfusion experiment. The simulated GABA i dependencies (solid lines) are scaled to the measured magnitudes of outward GAT1 currents with (○) or without (•) 100 mM extracellular Na + at 0 mV. In the presence of high [Na + ] o , the maximal GABA i -induced current is decreased, and the apparent affinity of GAT1 for GABA i is increased. The half-maximum GABA i concentrations are 0.90 and 0.52 mM, respectively, in the absence and presence of 100 mM Na + o . Clearly, the data agrees well with the simple alternating access transport model, but not the “channel model” . Fig. 7 F shows predictions for cis-substrate interactions by the same alternating access model , together with the corresponding experimental data. With a rate-limiting empty carrier translocation step in the reverse transport cycle, outward transport current activated by one substrate (e.g., Y i or GABA i ) becomes smaller if the concentration of its cis cosubstrate (e.g., X i or Cl − i ) is lowered. This reduction in current amplitude is “overcome” by a sufficiently high concentration of Y i , and a reduction of the cis-cosubstrate concentration results in a decreased apparent affinity for the substrate. This is the case here because the maximal transport rate is determined by the rate of the return step , rather than the translocation steps (2) from the cis side. From other observations, we expect that the empty carrier translocation step in GAT1 becomes rate limiting during reverse transport only in the presence of high extracellular Na + . As shown by the data in Fig. 7 F with 120 mM extracellular NaCl, reduction of [Cl − ] i from 120 to 30 mM shifts the half-maximal concentration for GABA i from 0.3 to 2.7 mM, while the maximal transport current is decreased by only 25%. This pattern is well simulated by the model in Fig. 7 D . In contrast, the channel model predicts that reduction of a cis cosubstrate (i.e., Na + i ) increases the apparent affinity for GABA i since GABA i competes with Na + i for entrance to the pore. Thus, both cis–trans and trans–trans substrate interactions of GAT1 are predicted by alternating access models of cotransport, and they contradict the channel-like model of cotransport. Up to now, we could not envision any modifications of the channel model that would predict the substrate dependencies of the alternating-access model. Also, we point out that our results are consistent with substrates being transported simultaneously, rather than sequentially, as concluded previously by Jauch and Läuger 1986 for a Na + –alanine cotransporter. We have tested whether extracellular GABA at concentrations up to 1 mM affects the outward current in the absence of extracellular Na + , and no clear effect was observed. For extracellular Cl − , we have detected only a small inhibition of outward current (∼20%) with 120 mM versus nominally Cl − free extracellular solution in the absence of extracellular Na + . To gain insight into how substrate interactions with GAT1 are affected by membrane potential, we first measured the I–V relations of outward GAT1 current over a wide concentration range for one substrate, while leaving the other substrates at fixed high (“saturating”) concentrations. A data set for each GAT1 substrate could then be replotted as a series of concentration–current relations at different membrane potentials, and the maximal current amplitude ( I max ) and half-maximum concentration ( K 1/2 ) for that substrate could be obtained by fitting the concentration–current relations by a Hill 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*}I=I_{{\mathrm{max}}}{\cdot}\frac{ \left \left[S\right] \right ^{n_{{\mathrm{H}}}}}{ \left \left[S\right] \right ^{n_{{\mathrm{H}}}}+K_{{1}/{2}}^{n_{{\mathrm{H}}}}}{\mathrm{,}}\end{equation*}\end{document} where I is the outward transport current magnitude, [ S ] is the substrate concentration, and n H is the Hill coefficient. The corresponding substrate dependencies are shown in Fig. 8 , whereby the GABA i , Na + i , and Cl − i dependencies of GAT1 outward current at 0 mV are given in A–C, respectively. The corresponding voltage dependencies of I max , K 1/2 , and n H , are shown in Fig. 8D–F , respectively. The I max –V relations are fit by the Boltzmann equation given in materials and methods . In each case, the equivalent charge is close to 0.3. Only small changes of I–V shapes were detected with changes of substrate concentrations: I–V ' s become slightly less steep with low GABA concentrations and slightly steeper at very low [Na + ] i . The fitted n H and K 1/2 are both relatively voltage independent . At membrane potentials between −30 and +90 mV, where measurements are most reliable, n H s are 1.2–1.4 for Na + i and 0.9–1.1 for Cl − i . n H for the GABA dependencies was always close to 1, and it was therefore fixed at 1 for this presentation. Over the same range of −30 to +90 mV, the K 1/2 s are 2.2–2.8 mM for GABA i , 53–83 mM for Na + i , and12.1–13.6 mM for Cl − i . In the more negative potential range there is a trend for the K 1/2 's for Na + i and Cl − i to increase. Fig. 9 describes how GAT1 substrates interact from the cytoplasmic side in the activation of outward transport current. Since the effects of voltage were minor in these interactions, we present only the data at 0 mV. Our protocol was to maintain a high concentration of one of the three substrates, and then systematically examine the effects of lowering one of the other two substrates on the concentration dependence of the third substrate. The transport current in all cases is defined by application and removal of cytoplasmic GABA with otherwise fixed substrate concentrations. In all results, the pipette solution contains 20 mM Cl − , no Na + , and no GABA. The solid lines presented with the data points in Fig. 9 represent fits by the functional model we developed for GAT1, which is detailed in another article . Fig. 9 A shows the cytoplasmic GABA dependencies of transport current. The data points were fit by the Hill equation with n H = 1 to obtain the corresponding I max 's and K 1/2 's (not shown). When [Na + ] i was lowered from 120 to 30 mM with 120 mM cytoplasmic Cl − , the I max decreased from 155.3 to 30.7 pA, while the K 1/2 remained unchanged . Similar effects were obtained when reducing cytoplasmic Cl − with 120 mM cytoplasmic Na + (bottom graph). The I max is 98.4 pA and the K 1/2 is 2.0 mM at 60 mM Cl − i ; at 3 mM Cl − i the I max and K 1/2 values are 69.4 pA and 2.9 mM, respectively. Fig. 9 B shows the cytoplasmic Na + dependencies of the transport current. The top graph shows the effect of changing cytoplasmic GABA from 20 to 2 mM with 120 mM cytoplasmic Cl − : the n H obtained from fit by the Hill equation (not shown) increased from 1.2 to 2.1, the K 1/2 for Na + i changed only slightly, and the I max decreased by 40%. The bottom graph shows the effect of reducing Cl − i from 50 to 5 mM with 12 mM GABA: the K 1/2 for Na + i increased by twofold (123 vs. 58 mM), while the n H and the I max changed only slightly (80 vs. 73 pA). Fig. 9 C shows the cytoplasmic Cl − dependencies of the transport current. These results were fit by the Hill equation with n H = 1 (not shown). The I max is reduced from 59 pA at 100 mM Na + i to 9.8 pA at 20 mM Na + i , while the K 1/2 is increased from 4 to 12 mM. Reduction of [GABA] i also decreases the I max for Cl − (239 pA at 20 mM GABA i vs. 155 pA at 2 mM GABA i ), but the K 1/2 increases by only 25% at low [Na + ] i (12 mM at 20 mM GABA i vs. 15 mM at 2 mM GABA i ). The substrate concentration dependencies presented in the previous section are predicted precisely by a binding scheme for GAT1 cytoplasmic substrates , where two Na + i and one Cl − i bind sequentially (Cl − i –Na + i –Na + i ) and GABA i binding is independent of Cl − i and Na + i . In this binding scheme, reduction of [Cl − ] i will be overcome by high [Na + ] i , and GABA i will not affect the apparent affinities of the other cosubstrate. Also, this binding order is mostly consistent with our capacitance data, which indicate that Cl − i interacts with the empty transport in the absence of other cosubstrates . However, there are three further predictions: (a) cytoplasmic GABA should inhibit the inward GAT1 current because it can bind from the cytoplasmic side in the absence of cosubstrates; (b) the apparent affinity for Cl − i binding, when measured as a capacitance change, should increase in the presence of high [Na + ] i ; and (c) inhibition of inward current by Cl − i should be enhanced by Na + i , just as lowering [Na + ] i decreases the maximal outward current activated by Cl − i . These predictions were tested in the following experiments. As described previously, inward transport current can be repeatedly activated in the patch using the pipette perfusion technique . Fig. 10A and Fig. B , present I–V relations of the inward transport current defined by 0.2 mM extracellular GABA in the presence of 120 mM extracellular NaCl. The activation of inward transport current does not require any cytoplasmic ions, nor is inward current enhanced by cytoplasmic ions. As shown in Fig. 10 A, the presence of high cytoplasmic Na + alone (120 mM) has a weak inhibitory effect, and GABA alone (20 mM) has almost no effect. A 25–50% inhibition of the inward current is achieved with the combined application of Na + i and GABA i . In contrast, cytoplasmic Cl − strongly inhibits the inward transport current. The inhibition by Cl − is monotonic, and its concentration dependence at 0 mV is shown in Fig. 10 C. These data points are from additional measurements in the same patch, and the solid line represents a fit by the 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*}I=\frac{I_{{\mathrm{max}}}}{1+\displaystyle\frac{ \left \left[Cl^{-}\right] \right _{i}}{K_{{1}/{2}}}}{\mathrm{,}}\end{equation*}\end{document} where I max is the current magnitude without Cl − i , and K 1/2 is the [Cl − ] i at which inward current is reduced by 50%. Inward currents were defined by subtraction of records with and without extracellular GABA. In the presence of 120 mM cytoplasmic Cl − , >90% of the inward current is inhibited. The K 1/2 from the fit is 12.4 mM. We also examined the kinetics of inhibition by performing automated concentration jumps, and we resolved no slow components of inhibition that would not be explained by diffusion of Cl − i to and away from the patch (not shown). In Fig. 11 , we have used the capacitance method to further analyze the interactions between GAT1 and its substrates. These results were obtained using 10 kHz/1 mV sinusoidal voltage perturbations. Fig. 11 A illustrates the membrane capacitance responses obtained when a patch is rapidly switched from high to 0 Cl − cytoplasmic solution. The magnitude of capacitance increase is larger when the cytoplasmic solution also contains GABA and Na + . Fig. 11 B shows the cytoplasmic Cl − dependence of the capacitance change under four conditions: with high GABA i and high Na + i , with high GABA i only, with high Na + i only, and with no GABA i or Na + i . GABA and Na + alone do not alter much the Cl − i -induced capacitance change. However, the simultaneous presence of cytoplasmic Na + and GABA results in two changes in the capacitance signal. First, the Cl − i -induced capacitance change is larger at all concentrations. Second, there is a fourfold decrease of the half-maximal Cl − i concentration. Together, the results described in Fig. 10 and Fig. 11 present definitive contradictions to the simple binding scheme for GAT1 cytoplasmic substrates discussed above. First, GABA i alone cannot inhibit the inward current or affect a change of capacitance in the absence of the other substrates. Second, Na + i does not strongly increase the apparent affinity for Cl − i . An adequate account of this data therefore requires a systematic simulation effort to model GAT1 function, and this is presented in an accompanying article . This study has demonstrated that GAT1-mediated transport currents behave largely as expected for a simple alternating-access cotransporter in the steady state. The Hill slopes of the substrate concentration dependencies and the measured reversal potentials are all consistent with the usual postulated stoichiometry of 1GABA:2Na + :1Cl − for GAT1. Admittedly, the reversal potential measurements show considerable scatter. However, the reverse GAT1 current absolutely requires the presence of GABA and both co-ions. Neither the reverse nor the forward current requires the presence of any trans ions, nor have we observed any trans-stimulation effects of substrates. Our conclusion, that GAT1 can function as a simple transporter with fixed stoichiometry, is similar to that of Jauch et al. 1986 for a Na + –alanine cotransporter that could be studied in pancreatic acinar cells with pipette perfusion. Our results are strikingly different from those of Cammack et al. 1994 , who employed whole-cell voltage clamp to study GAT1 transiently expressed in HEK293 cells. In their study, “ex-gated” (i.e., GABA o dependent) and “in-gated” (GABA i dependent) currents were differentiated; both required the presence of monovalent cations and/or anions on the trans-membrane side. In addition, the authors reported that GAT1 has modes of action that contradict the expectations for a tightly coupled “conventional transporter:” “leak” currents were observed in the absence of GABA, suggesting that real ion channels are formed by a small fraction of the expressed GAT1 transporters . These differences to our results might be due to different GAT1 functions in different expression systems ( Xenopus versus mammalian). Although our experience is limited, we have observed evidence for such expression system-dependent differences: in excised giant patches from HEK293 cells, transiently expressing GAT1, we found that the I–V of reverse GAT1 current was nearly voltage independent (data not presented). Two-electrode voltage clamp studies with Xenopus oocytes have previously provided much mechanistic insight into the forward transport mode, which mediates GABA uptake , and our studies now provide complementary data on the transporter's reverse mode. We can therefore outline several clear differences between the two modes of operation. (a) Although in the forward transport cycle Na + seems to bind to the empty transporter before Cl − and GABA at the extracellular side, in the reverse cycle the interaction of Cl − i with the transporter appears to be the first step. This is supported by the fact that only Cl − i was able to decrease capacitance of the empty carrier in a concentration-dependent manner and that only Cl − i strongly inhibits the forward transport current . (b) In the forward cycle, the rate-limiting step at 0 mV is a strongly voltage-dependent reaction; a voltage-independent step becomes rate limiting at large negative potentials. In contrast, the rate-limiting step of the reverse cycle is weakly voltage dependent, and it remains rate limiting under virtually all conditions; this explains the relative lack of shape changes of I–V relations. (c) The transporter has extremely asymmetrical affinities for extracellular and cytoplasmic GABA. The apparent affinity for extracellular GABA is more than two orders of magnitude higher than that for cytoplasmic GABA. The concentrations of GABA o that half-maximally activate inward GAT1 current range from ∼6 μM at −20 mV to ∼20 μM at −140 mV when extracellular Na + and Cl − are not limiting . Under comparable conditions, K 1/2 s for cytoplasmic GABA are in the millimolar range . (d) The apparent affinities for extracellular GABA and Na + depend on membrane voltage, being higher at more negative potentials. In contrast, the apparent affinities for cytoplasmic GABA, Na + , and Cl − do not vary much with membrane potential , which is expected if the reverse cycle is always rate limited by a weakly voltage-dependent step. The idea that cotransporters might operate in a channel-like fashion, without conformational changes, presupposes that a pore structure could allow hydrophilic molecules as large as GABA to bind specifically and permeate, while disallowing a nonspecific permeation of ions. We know of no clear evidence that this is physically possible, and we have presented two types of results for GAT1 that contradict the channel model . First, addition of extracellular Na + increases the apparent affinity for cytoplasmic GABA in parallel with a reduction of the maximum reverse GAT1 current. Second, reduction of cytoplasmic Cl − decreases the apparent affinity for GABA i in the presence of extracellular Na + , but not in its absence. In addition, the Cl − i -activated GAT1 capacitance changes become larger and saturate at lower Cl − i concentrations in the presence of both cosubstrates . This indicates that the binding of all three substrates enables further transporter reactions, presumably conformational changes. A final well-known property of alternating access models is that the presence of a substrate on one membrane side can stimulate transport of that substrate from the opposite side. This “self-exchange” of substrates is not predicted by the channel model, but it has been well documented for the GABA transporter . The interactions of cytoplasmic substrates in the activation of the reverse current are not straightforward. As noted above, it is evident that Cl − i can bind in the absence of the other substrates. Furthermore, reduction of Cl − i shifts the Na + i dependence of current to somewhat higher Na + i concentrations, as expected if Na + i binds after Cl − i . However, reduction of [Cl − i ] does not shift the dependence of current on GABA, indicating that GABA i probably binds independently from Cl − i . It is then perplexing that GABA i cannot inhibit the inward current. Furthermore, Na + i –GABA i interactions are also suggestive of independent binding sites; maximal currents are decreased when either cosubstrate concentration is reduced. That these complexities can be accounted for in the context of a simple transport model was highlighted in this article by including simulations of our tentative GAT1 model with the experimental data . Our observation that cytoplasmic Cl − inhibits potently GAT1 forward transport current raises multiple interesting points. First, since the physiological Cl − concentration inside the oocyte is probably 40–50 mM , more than threefold higher than our measured half-maximal inhibition . The inhibitory effect of Cl − i may partially explain why the GAT1 turnover rate appears to be several times higher in our patches, where [Cl − ] i is nominally zero, than estimated from whole cell experiments . In an accompanying paper , we provide evidence that cytoplasmic Cl − can indeed retard a slow charge moving reaction of the transport cycle. Second, it can be speculated that cytoplasmic Cl − changes might provide a mechanism for shaping GABA responses in neurons. For example, significant Cl − transients inside presynaptic GABA-anergic neurons may occur during and after inhibitory post-synaptic potential responses, thereby inhibiting and “deinhibiting” GABA transport. Moreover, it has been suggested that the cytoplasmic Cl − concentration is regulated in individual hippocampal neurons . | Study | biomedical | en | 0.999997 |
10469734 | If the transport of one neutral γ-aminobutyric acid (GABA) 1 molecule by the GAT1 transporter is coupled to the cotransport of two Na + ions and one Cl − ion , then one net positive charge must be moved in the direction of GABA in each transport cycle. In fact, a single partial reaction of the GAT1 transporter has been identified that would account for the expected movement of charge . This reaction occurs in the absence of GABA, it is slow (τ > 10 ms), and it is closely related to the binding and/or occlusion of extracellular Na + . We will refer to this reaction as “ Q slow .” Charge movements similar to Q slow occur in Na + –glucose cotransporters, but reactions that rearrange empty binding sites are thought to also generate large charge movements in those transporters . For GAT1, we have identified charge-moving reactions in the absence of substrates that are >1,000-fold faster than Q slow , but ∼10-fold smaller in magnitude . We will refer to these reactions, which were identified by capacitance measurements, as “ Q fast .” This article presents further kinetic and mechanistic studies of the GAT1 charge-moving reactions, as well as experimentation to define the kinetics of voltage-independent transport reactions in GAT1. These studies form the major experimental basis for a refined alternating access model of GAT1 function . The experimental methods and solutions used in this study were essentially the same as described in the previous article . Charge movements were measured as described previously . For all giant patch experiments, GAT1 expression levels in the oocytes were estimated by checking the magnitude of outward transport current activated by 20 mM cytoplasmic GABA in the presence of 120 mM cytoplasmic NaCl and the absence of extracellular GABA. Our first goal was to identify Q slow in giant excised oocyte patches and compare its operation to results of others using intact oocytes . For brevity, we describe this work without figures. First, we defined Q slow using the GAT1 inhibitor, SKF-89976A, applied from the extracellular side via pipette perfusion; current (or charge) records with inhibitor were subtracted from records without inhibitor. Second, we determined that very similar charge movements could be defined by subtracting current records in the absence of extracellular Na + (with substitution by N -methylglucamine) from records in the presence of extracellular Na + . This confirmed that Q slow is probably associated with extracellular Na + binding. Third, we determined that the GAT1 inhibitor, NO-711, in contrast to SKF-89976A, could be used from the cytoplasmic side to define Q slow , thereby eliminating the need to routinely perfuse pipettes. Fourth, we tested whether NO-711 might block additional slow charge movements taking place in the absence of extracellular Na + . We examined a voltage range from +200 to −200 mV, and all Na + -independent charge movements that we identified were >10× faster than Q slow . Inward GAT1 current and a fast GAT1 charge movement are suppressed by cytoplasmic Cl − , and, as described here, high cytoplasmic Cl − concentrations also suppress Q slow . Fig. 1 compares charge movements that are defined by the inhibitor, NO-711, with charge movements defined by cytoplasmic Cl − in the same patch. To directly visualize whether charge movements are reversible, and thereby whether any “leak currents” are blocked or induced by these interventions, we recorded charge movement per se, instead of membrane current, from the integrating patch clamp amplifier. The protocol employed was a series of 60-ms voltage pulses from a holding potential of −40 mV to potentials between −160 and +120 mV. The pipette contained 40 mM extracellular NaCl. Charge records in Fig. 1 A were obtained by subtracting signals in the presence of 0.13 mM cytoplasmic NO-711 from signals obtained in the absence of the inhibitor. Fig. 1 B shows the charge records defined by subtracting signals with 120 mM cytoplasmic Cl − from signals without Cl − , using methanesulfonate (MES − ) as substitute anion. The Cl − i -defined charge records were obtained before the NO-711 i –defined ones because effects of NO-711 reverse only slowly after its wash-out from the cytoplasmic side. The two sets of charge signals are similar, and they return nearly to initial values at the end of the observation period. However, the magnitudes of charge movements defined by inhibitor are larger, suggesting that Cl − i did not fully inhibit the underlying reactions. Note the small fast charge components, which appear as quasi-instantaneous charge jumps on return to −40 mV from 120 mV. These components might arise from electrogenic Na + binding, similar to signals described for the Na + , K + pump . However, from our results in the absence of extracellular Na + , to be described below, it is much more likely that these signals arise from empty GAT1 binding sites, rather than substrate-transporter interactions. Single exponential functions were fitted to the signals during the voltage pulse (“on” charge) and after returning potential to −40 mV (“off” charge). They are included as dotted lines that are mostly covered by the charge records. The magnitudes and rates of charge movements are plotted against pulse potential in Fig. 1C and Fig. D , respectively, for NO-711 i – and Cl − i -defined signals. Both charge–voltage ( Q –V) relations are well described by a Boltzmann equation . The Q –V relation for the inhibitor-defined charge has a slope of 0.94 and projects to 4.36 pC for the total charge moved. The Q –V relation for the Cl − i -defined charge has a shallower slope (0.74) and projects to a total charge of 2.93 pC. The inhibitor-defined Q –V (V 1/2 = −45 mV) is left-shifted from the Cl − i -defined one (V 1/2 = −35 mV). The rate–voltage ( k slow –V) relations are U shaped, and the minimum rates occur at approximately the midpoint of the corresponding Q –V relations. The solid lines represent a fit of the k slow –V relations by the sum of two exponential functions with opposite voltage dependence. The slope coefficients of these functions revealed an almost equal voltage dependence of the “forward” and “backward” reactions of the charge movement . We point out that we have observed clear variability of the midpoint of Q –V relations obtained from different batches of oocytes. Since excised patches tend to be unstable in the presence of high [Na + ] o and large negative potentials, our success rates were greater with patches in which the midpoint of Q slow occurred at less negative potentials. We considered three general mechanisms by which cytoplasmic Cl − may block Q slow . First, cytoplasmic Cl − might act through an inhibitory regulatory binding site (i.e., separate from transport binding sites). In this case, we would expect a biphasic (or complex) dependence of outward GAT1 current on cytoplasmic Cl − , and this was not the case . Second, GAT1 might be modulated by a slow autoinhibitory reaction that depends on Cl − i binding to transport sites, similar to the Na + i -dependent inactivation in the cardiac Na + /Ca 2+ exchanger . To test this, we performed rapid solution switches to examine the time courses with which inward and outward GAT1 currents change upon application and removal of cytoplasmic Cl − . All records revealed monotonic increases or decreases of current with time courses expected for ion diffusion up to and away from the membrane patch (30–400 ms). The third possibility, then, was that the effects of Cl − i are a direct consequence of its binding to transport sites. In an alternating access model, extracellular Na + and cytoplasmic Cl − binding could be linked by the conformational changes of the empty transporter that open binding sites alternatively to the extracellular or cytoplasmic sides. The minimum reactions involved are illustrated in a Reaction Scheme . Charge movement (★) occurs when a Na + is occluded into the transporter from the extracellular side (1). When the binding site is open, Na + o can dissociate (2), and the empty binding site can open alternatively to the extracellular or the cytoplasmic side (3). When the binding site is open to the cytoplasmic side, Cl − i can bind and dissociate (4). A saturating cytoplasmic Cl − concentration will disable the charge-moving reaction because transporters will all accumulate in the Cl − i -bound state. The actual effect of cytoplasmic Cl − , however, is to shift the Q slow –V relation to more negative potentials by decreasing the probability that Na + o can bind and be occluded. If the electrogenic reaction is slower than the other reactions, cytoplasmic Cl − will specifically decrease the rate of the forward charge movement (i.e., Na + occlusion). Based on model calculations, a 6–10-fold retardation would account for our ability to define charge movements by application and removal of cytoplasmic Cl − . As shown in Fig. 2 , our experimental results conform closely to these predictions. Current, rather than charge, was recorded to avoid signal drift during the long voltage pulses used in these protocols. As indicated in the lower part of Fig. 2 A, patches were held at 0 mV, and the on charge movement was recorded by applying 360-ms voltage pulses to potentials between +40 and −160 mV. Then the off charge movement was recorded by applying 80-ms test pulses to +80 mV, a potential positive enough so that all detectable charge moves in the backward direction. The charge movements in the absence and presence (bottom) of 120 mM cytoplasmic Cl − were defined by subtracting records taken before and after application of NO-711 i (0.13 mM). The on charge movements appear to be suppressed by Cl − i , as expected from Fig. 1 , while in fact they are taking place very slowly, such that the current is of small magnitude. The largest off current transients, recorded at +80 mV after voltage pulses to −160 mV, are nearly identical in magnitude and rate in the absence and presence of Cl − . Thus, the total charge that can move is unaffected by Cl − . The steady state voltage dependence of Q slow , shown in Fig. 2 B, was determined by integrating the current transients at +80 mV. The amount of charge moved is plotted against the “pretest” potential, and the Q –V relations with (□) and without (▪) 120 mM cytoplasmic Cl − are fitted by Boltzmann equations (smooth lines). In this case, we forced the charge magnitudes (−3.4 pC) and the slope coefficient (1.3) to be equal. The midpoint of the Q –V relation is −36 mV in the absence of cytoplasmic Cl − ; it is shifted to −96 mV in the presence of 120 mM cytoplasmic Cl − . This 60-mV shift corresponds to a 10-fold retardation of the forward charge movement, which indicates that ∼90% of binding sites are occupied by Cl − i at 120 mM. The Cl − i dissociation constant must therefore be ∼12 mM, and this is in good agreement with half-maximal Cl − i concentrations for activation of outward current and inhibition of inward current . Fig. 2 C shows the effects of lower concentrations of cytoplasmic Cl − on current transients at negative potentials. Charge movements were elicited by a 180-ms pulse from 0 to −150 mV with 0, 15, and 30 mM cytoplasmic Cl − . Then NO-711 i was applied, the protocols were repeated, and the corresponding current records with NO-711 i were subtracted from those without. The current transients are shown together with single exponential fits in Fig. 2 C. As predicted for a decrease of charge movement rate without a change of charge magnitude at −150 mV, the records obtained with different Cl − i concentrations “cross” each other. For clarity, we show the “crossover” of the corresponding exponential functions above the current records. The time constants are 12.1 ms in 0 Cl − i , 18.9 ms in 15 mM Cl − i , and 36.6 ms in 30 mM Cl − i . Another major prediction from Fig. 1 , described in Fig. 3 , is that rapid application of cytoplasmic Cl − should be able to release occluded Na + o from GAT1 and thereby generate an outward current transient. We first verified in control experiments that fast cytoplasmic Cl − switches, as described previously , could be carried out without generating significant current artifacts in patches from uninjected oocytes. To achieve this, the tips of the recording pipettes were thickly coated with Parafilm/mineral oil mix to reduce capacitance (not shown). When experiments were performed in GAT1-expressing patches, outward current transients were indeed obtained in the presence, but not the absence, of extracellular Na + . We mention that the presence of Cl − o in the pipette was also required to obtain signals at 0 mV. This is expected because extracellular Cl − shifts the Q slow –V relation to more positive potentials, presumably by facilitating occlusion of extracellular Na + . Fig. 3 presents typical results with 120 mM extracellular NaCl, whereby 120 mM cytoplasmic Cl − was rapidly applied and removed using MES − as the Cl − substitute. Fig. 3 A shows that, in the absence of NO-711 i (a), a 2-pA outward current develops within 40 ms upon application of 120 mM Cl − i , and the current decays over the next 150 ms. On removal of Cl − i , a smaller inward current develops over 100 ms, and it decays over 350 ms. The current transients are largely blocked in the presence of 0.13 mM NO-711 i (b). The subtraction record (a–b), presented below the concentration bar, shows that the magnitudes of on and off charge, defined by the areas under the current transients, are in good agreement. Fig. 3 B shows the voltage dependence of the Cl − i -induced charge movement from another experiment. The total charge moved (Q) was calculated as the area bound by the on current trace and the baseline current in the presence of Cl − i . GAT1 expression was only moderate in this patch, but the charge magnitude clearly diminishes at both positive and negative potentials (+70 and −80 mV). This is expected because at positive potentials there will be no occluded Na + to be released; at very negative potentials, the Na + -occluded state will be stabilized to such an extent that Na + cannot be released by 120 mM cytoplasmic Cl − . Thus, the results in Fig. 3 B reflect a simple shift of the charge–voltage relations by Cl − i , as already described in Fig. 2 . Fig. 3 C shows the use of voltage jumps combined with concentration jumps to analyze the relationships between Cl − i - and voltage-induced charge movements. The slow charge movements elicited by a voltage pulse (0 → +100 → −100 → 0 mV) before (a), during (b), and after (c) a cytoplasmic Cl − jump are shown in Fig. 3 C. After the slow, small (∼2 pA) outward current elicited by Cl − i has decayed (∼200 ms in this patch), the voltage-induced charge movement is suppressed. In this experiment, the charge movement remained suppressed 300 ms after Cl − i removal. This slow reversal is due to the fact that, to obtain large signals, patches employed were large and rose up substantially in the pipette tip. As shown in Fig. 3 C, d, from the same patch, the time courses of outward GAT1 current activation and deactivation were similarly asymmetrical. Activation of the current occurs within 20 ms, but deactivation is only partial even after 500 ms. As mentioned in the introduction , we have previously isolated fast charge-moving reactions that appear to occur in empty transporters. If binding of extracellular Na + and cytoplasmic Cl − are mutually exclusive, then Na + o occlusion should suppress or “immobilize” the fast charge movements. As described in Fig. 4 , we have tested this possibility by monitoring changes of the availability of fast charge moving reactions as capacitance changes. The results shown are with 120 mM extracellular NaCl, and changes of membrane capacitance were monitored via a 20-kHz sinusoidal perturbation of 1-mV amplitude. Membrane potential was held for 1 s at −150 mV, and then pulsed to 0 mV for 1 s, back to −150 mV for 600 ms, and finally returned to 0 mV. These protocols yielded essentially flat records in patches from uninjected oocytes (not shown). As shown in Fig. 4 A, membrane capacitance increases in patches with high GAT1 expression (∼200 fF) when membrane potential is depolarized from −150 to 0 mV. These slow capacitance changes require the presence of Na + o (not shown), their time courses are very similar to those of Q slow , and they are well described by single exponential functions. The time constant for the pulse from 0 to −150 mV increases from 52 ms in the absence of Cl − i , to 140 ms with 60 mM Cl − i , and to 203 ms with 120 mM Cl − i . The exponential functions are included as dotted lines. Fig. 4 B shows three superimposed capacitance traces for a voltage protocol in which the pulse duration at −150 mV was varied. These were recorded in the presence of 120 mM cytoplasmic Cl − . The “capacitance recovery” upon pulsing from −150 to 0 mV takes place somewhat more slowly after short pulses to −150 mV than longer pulses (2 and 3). However, in all three traces, the time constants for pulses from 0 to −150 mV remain >200 ms. From these results, it seems certain that our previous description of voltage-dependent capacitance changes for GAT1 did not reflect a steady state in the presence of 120 mM cytoplasmic Cl − . We stress at this point that the fast charge movements underlying the capacitance signals in Fig. 4 cannot arise solely from conformational changes of the empty transporter. The magnitude of the capacitance change induced by the voltage step is decreased only 20% by the presence of 120 mM Cl − i . From further work, illustrated in Fig. 5 and Fig. 6 , we will conclude that, in fact, fast charge moving reactions also occur in the GAT1 states with Cl − bound on the cytoplasmic side. Consistent with the observation in Fig. 4 , the magnitudes of those charge movements are only 20% smaller than the charge movements that occur in the empty transporter. That cytoplasmic Cl − does not suppress all fast charge-moving reactions in GAT1 was also apparent in capacitance responses to Cl − i : in the presence of 120 mM cytoplasmic Cl − , application of Na + and GABA to the cytoplasmic side induces a further decrease of capacitance when transport current is activated. Our working hypothesis, then, is that multiple charged groups of the transporter, located within the membrane field, can flex somewhat in response to changes of membrane potential. In some transporter configurations, especially the Na + -occluded state, fast charge-moving reactions may be hindered. However, it seems unlikely that a genuine “null state” for the fast charge movements exists. As described in Fig. 5 , the inhibitor, NO-711, decreases GAT1 capacitance, but does not abolish capacitance changes with changes of cytoplasmic [Cl − ]. Fig. 5 shows an example of simultaneous recording of membrane capacitance and charge. The pipette contains 40 mM Na + . The capacitance responses to 60-ms voltage pulses from +120 mV to more negative potentials in 40-mV steps are shown in Fig. 5 A. Upon hyperpolarizing, membrane capacitance declines in a biphasic fashion after artifactual “spikes.” The fast decline presumably reflects saturation of the underlying fast charge-moving reactions in either the on or off state at extremes of potential. The slow phase occurs with the time course of the slow charge movements, which are shown in Fig. 5 B as the subtracted charge records defined by Cl − i (left) and NO-711 i (right). With application of 120 mM cytoplasmic Cl − , capacitance declines at 0 mV by ∼100 pF. The capacitance responses to voltage steps are largely suppressed, as expected from previous results for relatively short (60 ms) voltage pulses. Application of NO-711 i in the absence of Cl − i causes capacitance to decrease by 415 pF, approximately four times larger than the decrease induced by 120 mM Cl − i . Then, in the presence of NO-711 i , application and removal of 120 mM cytoplasmic Cl − still induces capacitance changes that are about one-half as large as those without the inhibitor. From such results, it is evident that NO-711 i effectively silences some fast charge-moving reactions. However, even in the presence of NO-711 i Cl − can still bind and dissociate from the cytoplasmic side and can still suppress a fast charge-moving reaction. As just described, Cl − i can still suppress fast charge movements in the presence of NO-711. In addition to this complexity, we identified fast charge movements with altered kinetics in the presence of inhibitor (not shown). Thus, we attempted to study Q fast without inhibitors, and the most important result is shown in Fig. 6 . Q fast is defined in Fig. 6 A by treating signals in the presence of 120 mM Cl − i as pseudo “null signals,” which were subtracted from signals with 0 or 12 mM Cl − i . Fig. 6 A shows the typical charge signals obtained by 3-ms voltage pulses from 0 mV to voltages between +160 and −200 mV in 40-mV steps. The pipette solution contained 20 mM Cl − ; both the pipette and the bath solution were Na + free. The same protocols resulted in virtually blank records when they were carried out in patches from uninjected oocytes (not shown). Clearly, the Q fast signals resolved in this fashion are complex: the records from the 0 Cl − i subtraction (left) show a slow component in the positive potential range (time constant <1 ms). The 12 mM Cl − i subtraction records (right) reveal components whose time course is not well defined in the 3-ms pulse duration. The charge magnitude at the end of each voltage pulse in Fig. 6 A is plotted against pulse potential and fitted by a Boltzmann function in Fig. 6 B. The slope coefficients of the Boltzmann functions are 0.26 and 0.22 in the absence of Cl − i and with 12 mM Cl − i , respectively, the projected charge magnitudes are 81 and 43 fC, respectively, and the half-saturation points are 114 and 42 mV, respectively. The major complication in the signals presented in Fig. 6 A is that large “charge transients” occur upon changing voltage. These components cannot be artifacts because they were not found in patches from control oocytes (not shown). To better resolve the initial components, shorter voltage pulses (0.25 ms) were employed with 1 MHz voltage clamp resolution (i.e., using highly polished pipette tips; <20 kohm). Results shown in Fig. 6 C are the subtractions of records with 120 mM Cl − i from records without Cl − i , as in Fig. 6 A (left). To illustrate the time courses more clearly, two of the records (−40 → +160 mV and −40 → −200 mV) are shown alone in Fig. 6 C (bottom). Why does the subtraction give rise to transient charge signals (i.e., two charge components of opposite sign)? Based on previous results with capacitance measurements, our interpretation is that one fast charge-moving reaction is inhibited by Cl − i . It has a time constant of ∼13 μs at all potentials (i.e., a rate constant of ∼77,000 s −1 ). A still faster charge component, not resolved in time, is, however, activated by Cl − i . This reaction could reflect a flexing of charged binding sites, or of Cl − i within its binding sites, in the membrane field. In good agreement with the results of Fig. 4 and Fig. 5 , the slow component, which is inhibited by Cl − i , is ∼20% larger than the component activated by Cl − i . At this point, our kinetic analysis of GAT1 is limited to the slow binding/occlusion of extracellular Na + and to the Q fast reactions just described. No kinetic information is available about the actual substrate translocation reactions that must occur to allow coupled transport in an alternating access model. Accordingly, we tried extensively to isolate charge-moving reactions in the “fully loaded” transporter, via both voltage and concentration jumps. For example, we compared charge records in the presence of all substrates, at high concentrations, on both membrane sides, with records in which GAT1 inhibitors had been applied. To enhance the blocking action of inhibitors, we often removed GABA from the membrane side to which the inhibitor was applied. Nevertheless, no clear signal was identified (results not shown). We therefore conclude that substrate translocation reactions involving fully loaded transporters must take place in a nearly electroneutral fashion. Even though charge movements of the fully loaded transporter cannot be resolved, kinetic information about the substrate translocation reactions can be gained by indirect means. At large negative potentials, voltage-independent step(s) in the cycle become rate limiting in the forward transport mode . This coupling of voltage-dependent and –independent steps may be expected to give rise to pre–steady state current transients during voltage steps, and the magnitudes of transient versus steady state components can give accurate information about the rates of the voltage-independent steps. Fig. 7 presents current transients related to both forward and reverse GAT1 transport. Results for the outward GAT1 current (A), obtained in the absence extracellular Na + , were defined both as GABA i -induced current (solid lines) and as NO-711 i -inhibited current (dotted lines). For both subtraction procedures, transient current components in the outward current mode are fast and small in relation to the steady state current. The results are consistent with the outward current being rate limited by a single slow step with weak voltage dependence. In contrast, current transients associated with the inward transport mode are much more pronounced and have rates similar to those of the slow charge movements. Fig. 7 B shows typical results using 120 mM cytoplasmic Cl − (solid line) or 0.13 mM NO-711 i (dotted line) to define the current. Membrane current responds to the voltage step from 0 to −50 mV essentially in a step, without a pre–steady state transient. Thus, at −50 mV, the rate of the charge-moving step cannot be greater than the rates of other steps. The transient current components become larger with larger pulses (i.e., to −100 and −150 mV). At −150 mV, the current activated by hyperpolarization decays by ∼50% with a rate constant of ∼80 s −1 . This presumably reflects the major charge-moving step, which takes place faster with application of large voltages; the slowest electroneutral step, which presumably does not change in rate, must be taking place at ∼40 s −1 to account for the 50% decline of current. Finally, we used the rates of charge movements to estimate the turnover rate for the reverse GAT1 transport cycle. To do so, we measured the slow charge movements and the outward transport current in the same patch, both defined by cytoplasmic NO-711 in the presence of 120 mM extracellular NaCl . Both the Q slow –V and k slow –V relations in Fig. 8 B are similar to those described previously . As shown in Fig. 8 B, the Q –V relation for the slow charge movement has a slope of 0.99, a midpoint potential of −33.1 mV, and a Δ Q max of 2.6 pC. Given the patch size of 8 pF and an assumed specific membrane capacitance of 0.8 μF/cm 2 , the transporter density is ∼2,000/μm 2 . On this basis, the turnover rates in the presence of extracellular Na + can be estimated by the ratio of steady state current magnitude to Δ Q max , and they are plotted as a function of membrane potential in Fig. 8 B. The turnover rate increases from ∼3 s −1 at −120 mV to ∼60 s −1 at +120 mV. As expected, the turnover rate never exceeds the rate of the slow charge movement. Our studies of GAT1 charge movements suggest that extracellular and cytoplasmic substrate binding are mutually exclusive, as predicted for an alternating access model. With this perspective, we will first discuss the charge movements themselves, then their coupling, and finally GAT1 kinetics. Our work was greatly facilitated by the finding that the GABA uptake inhibitor NO-711, in contrast to SKF-89976A , eliminates both the steady state GAT1 transport current and the slow charge movements when applied from the cytoplasmic side. We have failed to define genuine “null states” for fast charge movements occurring in GAT1. Besides GAT1, several other Na + -dependent transporters display charge movements that are closely related to the binding of extracellular Na + —the question is how. The idea of an “ion well”, as sketched by Läuger 1991 , supposes that ions diffuse into their binding sites through a pore or a “channel-like” structure, along which there is a fall of membrane potential experienced by the ion. The major GAT1 charge movement is very slow (2–300 s −1 ), and the rates of charge movements are sensitive to temperature (Lu and Hilgemann, unpublished observations) and mutational changes . Thus, it seems much more likely that conformational changes, not diffusion of ions, underlie Q slow kinetics. The conformational changes could occur, in principle, either after or before the actual binding of Na + . Our simulations, described in the following article, incorporate both possibilities . For the Na/K pump, signals consistent with an ion well mechanism have been isolated for the weakly voltage-dependent binding of extracellular Na + at sites that probably can also bind K + . If a “deep ion well” for Na + exists, it must occur within a transitional pump state . In this case, ion binding will generate a fast charge movement whose magnitude is proportional to the fractional occupancy of the transitional state. We have attempted extensively to identify such charge movements for the Na/K pump in cardiac patches , for the Na/Ca exchanger in cardiac and oocyte patches , and now for the GAT1 cotransporter in oocyte patches. Up to now, we did not identify any signal that could reflect the existence of such a “deep ion well” in the literal sense of Peter Läuger's suggestion . It is noteworthy that the Q slow of GAT1 behaves differently from that of the Na/K pump. The rate of the Na/K pump charge movement decreases monotonically with depolarization to a plateau rate . In contrast, our results for GAT1 show a bell-shaped dependence of rate on voltage . In intact oocytes, the increase of rate with hyperpolarization is less pronounced , and our work suggests that the presence of cytoplasmic Cl − in oocytes could be a factor in this difference. We have developed our interpretation of the Q fast charge movements with the presentation of data. To reiterate briefly, charged residues of the GAT1 substrate binding sites might flex somewhat within the membrane field, and they would do so more easily when open to the cytoplasmic than the extracellular side. When a Cl − is bound on the cytoplasmic side, either the rate of one such reaction is increased or Cl − i itself can change position rapidly within its binding site. Thus, application of cytoplasmic Cl − suppresses one charge movement and gives rise to a faster one, such that subtractions of the charge records generate charge transients . Three different types of experiments indicate that the magnitude of the charge movement taking place in the presence of Cl − i is ∼20% smaller than that of the reaction taking place in the absence of Cl − i . One of the slower reactions in the absence of Cl − i might reflect opening and closing of the empty Cl − binding site, which would impart a weak voltage dependence on Cl − i binding. We have been able to identify such reactions, in spite of their small magnitude, only because they are very fast. Overall, the reactions underlying fast GAT1 charge movements are so weakly voltage dependent that they will not markedly influence the voltage dependence of GAT1 currents. While only Na + can clearly bind to GAT1 from the extracellular side in the absence of cosubstrates, only Cl − clearly interacts with GAT1 from the cytoplasmic side in the absence of cosubstrates. Four results suggest that extracellular Na + and cytoplasmic Cl − bind to the transporter in a mutually exclusive fashion. (a) High cytoplasmic Cl − concentrations appear to block the slow GAT1 charge movement . Binding of Cl − i retards the forward rate of the charge movement (i.e., Na + occlusion) and thereby shifts the charge movement to more negative potentials . (b) Rapid application of cytoplasmic Cl − induces a Na + o -dependent outward charge movement that is equivalent in magnitude to the backward charge reaction (i.e., Na + deocclusion) and that precludes the backward reaction in voltage pulse experiments . (c) Positive potential reduces the ability of Cl − i to induce Na + o -dependent outward current transients, as expected if Na + is already released; negative membrane voltage reduces the ability of Cl − i to induce Na + o -dependent outward current, as expected if the Na + -occluded state is stabilized . (d) The fast charge movements, which are suppressed by cytoplasmic Cl − , are also suppressed by the forward reaction (i.e., Na + occlusion) of the slow charge movement . Pre–steady state current transients induced by voltage pulses are slow and prominent during forward GAT1 operation, while they are fast and small during reverse transport operation . These results allow quite precise estimates of the rates of voltage-independent steps in GAT1 operation that are considered in the following article . The voltage-independent reaction that occurs after Q slow in the forward transport cycle, for example, must occur at ∼40 s −1 . Our estimation of turnover rates, from these and related measurements, depends on the assumption that GAT1 stoichiometry is quite constant. That uncoupled currents cannot be very large is indicated by the fact that steady state GAT1 current magnitudes never exceed current magnitudes that occur during reversible charge movements. The turnover rate for fully activated reverse transport at +120 mV is 60 s −1 , and this is approximately threefold slower than the rate of the slow charge movement at the same potential in the presence of extracellular Na + . In the following article , we describe how the major findings of this article, our previous findings, and findings of others from whole-oocyte studies can be reasonably well accounted for by a simple alternating access transport model. | Study | biomedical | en | 0.999997 |
10469735 | Recent success in determining the structures of several membrane transporters and ion channels has opened the way to a greatly improved understanding of membrane transport. Still, the answers to many questions about membrane transport, including structure–function issues, require improved functional studies. Our electrophysiological studies of the GAT1 cotransporter provide the first extensive data set on the cytoplasmic substrate dependencies of a neurotransmitter transporter. Also, we have obtained new information about the voltage dependencies and kinetics of GAT1 function, as well as cis–trans and cis–cis substrate interactions. This data, together with data from previous GAT1 studies, provides one of the most extensive data sets available for the function of any transporter. Thus, we can now construct models of cotransport function and test them in relation to the data set. Starting from the general principles and transport models outlined by Läuger 1987 , we have tried to formulate the simplest model that can account well for the available data. In addition to providing a framework for the molecular analysis of GAT1 function, the model may be useful to describe GAT1 function in higher-level simulations of synapse function. In this article, we describe the rationale for our model assumptions, the range of experimental observations that are accounted for, and limitations of the model in accounting for the available data on GAT1 function. A cartoon of our model is shown in Fig. 1 . Transporters exist primarily in two states, designated E in and E out , and the transitions between them (reactions 1–4) take place through transitional states, designated * E out and * E in . Within each state, substrate binding is assumed to be at equilibrium. In the E in state, binding sites are open to the cytoplasmic side, and they bind sequentially one Cl − ( K d , 3.7 mM) and two Na + ( K d s, 442 and 11.5 mM). When the E in binding sites are empty, a low affinity Na + binding site ( K d , 0.92 M) can open to the extracellular side (1a; 200 s −1 ), thereby forming the * E out state that cannot bind any substrate from the cytoplasmic side. When a Na + is bound to the * E out state, it can be occluded into the transporter (1b), thereby forming the stable E out state. This overall reaction (1; i.e., 1a + 1b) moves +1.1 equivalent charges through the membrane field from outside to inside. In the E out state, one Cl − ( K d , 8.2 mM) and one Na + ( K d , 10.1 mM) together with 1 γ-aminobutyric acid (GABA) 1 ( K d , 41 μM) can be bound from the extracellular side. The backward transition to the E in state (2; i.e., 2a + 2b; 2,000 s −1 ), which releases one Na + to the outside, occurs only when the E out binding sites are empty. When the E out binding sites for Na + and GABA are occupied, these two substrates can be translocated to the cytoplasmic side , regardless of whether extracellular Cl − is bound, thereby forming the transitional state, * E in . The GABA translocation reaction (3a) has a slight voltage dependence that opposes the overall forward transport process at negative potentials (−0.07 equivalent charges). In the transitional * E in state, GABA and Na + can dissociate to the cytoplasmic side. For the transition to the stable E in state (3b), an extracellular Cl − must be bound, and GABA must have dissociated to the cytoplasmic side. The bound extracellular Cl − is then translocated simultaneously with the release of the occluded Na + ion to the cytoplasmic side. The reverse reaction (4) to the E out state at first translocates one cytoplasmic Cl − and occludes one cytoplasmic Na + (4a; 42 s −1 ); thereafter, one cytoplasmic Na + and GABA are translocated (4b). The occlusion of Cl − and Na + (i.e., 4a) has a weak voltage dependence (+0.17 equivalent charges) that promotes the reverse transport cycle at positive potentials. In the E in state, charged residues of the binding sites can flex somewhat in the membrane field (−0.18 equivalent charges), which imparts weak voltage dependence on cytoplasmic Cl − binding. The model just described is the simplest Markovian transport model we have found to account for all significant features of GAT1 transport function in Xenopus oocyte membrane. To develop the model, we first constructed a database of all steady state and kinetic data that we judged to be reliable. Then, with the perspectives of the previous articles , we attempted to recreate the database, qualitatively and quantitatively, with various substrate binding schemes and assumptions about conformational changes. Most models were based on the alternating access principle, as it is independently supported by our data. Also, we assumed in most models that cytoplasmic Cl − binding and extracellular Na + binding are mutually exclusive . In a typical model-building cycle, a simple two- or three-state model was expanded to a model with many states to achieve an improved explanatory range. Then, it was probed how states could be merged without losing the improvement. For brevity, we omit discussion of “dilemmas” encountered during model development. Examples pointed out previously include the substrate binding scheme for the cytoplasmic side and the different kinetic behaviors of inward and outward current during voltage steps. To account for GAT1 kinetics, we find it essential to assume that only one Na + ion is occluded by the transporter during the E in → E out transition . Since the deocclusion reaction is blocked by binding of the second Na + ion from the extracellular side, reduction of [Na + ] o from 100 to a few millimolar causes a >100-fold acceleration of the electrogenic deocclusion reaction . Another key observation is that GABA transport remains possible in the nominal absence of Cl − i . Without violating a fixed transport stoichiometry, this will be explained by our assumption that one Na + and GABA are first translocated from outside to inside without cotransport of Cl − ; then Cl − transport takes place in a second step from a transitional state. Our fitting method was described previously . In brief, we used a generalized Newton method for solving simultaneous nonlinear equations to minimize the squared deviations of predicted (model) results from experimental results. The model parameters are improved one-at-a-time in a random order during each fitting cycle. In addition to the model-specific parameters, one scaling variable is fitted for each experiment. Individual experiments can be weighted such that deviations of model results from data points are roughly similar for the entire database. Although our model contains only the minimum parameters needed to simulate GAT1 cotransport, we often identified multiple least-squares minima with different initial estimates for the parameters. Since variation of certain rate constants and substrate affinities had nearly identical results on model function, we fixed two of the rate constants in the model fit described here. The model to be presented is a “pseudo–two-state model” because it has only two stable states. Other models that we explored included many different substrate binding schemes and the kinetic simulation of substrate binding reactions. Also, we carefully tested our treatment of transitional states by developing models in which the transitional states were simulated as stable states with rapid exit transitions. Results with the complex and simplified models were identical for the purposes of this article. For kinetic simulations of two- and three-state models, we used analytical solutions to integrate state transitions over time. For the more complex models, we usually used a stable implicit method to integrate the differential equations ( dy i / dt ) for the individual states (y 1…n ) over time with the integration interval , h : \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_{{\mathrm{ih}}}=y_{{\mathrm{i0}}}+h{\cdot}\frac{{dy_{{\mathrm{i}}}}/{dt}}{1+h{\cdot}{{\sum}}k}{\mathrm{,}}\end{equation*}\end{document} where y ih is the state value at the forward time point, y i0 is the state value at the backward time point, and ∑ k is the sum of rate constants leading into and away from the given state. The simulation programs were written in Pascal and C++ and compiled with Borland TurboPascal and Borland C++ Builder, respectively (Inprise). Variability in results from different groups of experiments and experimental methods is an important problem in our simulations. The two major cases are the cytoplasmic Cl − dependence of reverse GAT1 current and the voltage dependence of charge movements. Half-maximal Cl − i concentrations vary by a factor of about four, and the midpoint voltage of charge movements varies by at least 25 mV in experiments with different oocyte batches. A similar variability of charge movements was observed in whole-oocyte recordings (Dr. Sela Mager, personal communication). To demonstrate the kinetic behavior predicted by our model, we have simulated one experiment with 70 mM extracellular NaCl, rather than the 40 mM used in the experiment. We describe here the rationale for two implicit model assumptions. For some simulations, we carried out a kinetic simulation of all substrate reactions, similar to a cotransport model proposed by Sanders et al. 1984 . However, we assume instantaneous binding of all substrates in this simulation. It seems reasonable to assume that ion binding reactions with millimolar (or lower) affinities can take place in less than a microsecond and therefore will not influence the much slower reactions simulated here. The assumption of fast GABA binding is less reliable, and we thoroughly tested how kinetic simulation of GABA binding affects our model results. Our conclusion is that the potential influence is rather small, except for low [GABA] o , where GABA binding may limit inward GABA transport, especially at negative membrane potentials. The second assumption is related to the application of Eyring rate models to transporter function. We find it important to assume that the energy barrier in a charge-moving reaction can be located highly asymmetrically in the electrical field, such that a strong “partitioning” of the voltage dependence on forward or backward reaction rates can occur. To underscore this idea for transporters, we illustrate a simple transport reaction in Fig. 2 A. We assume that the transporter can exist in two stable states, one with open and one with closed binding sites. Transitions between these states will result in rearrangements of the membrane electrical field profile, whereby electrical current is generated when field moves across a charged residue. In this example, a negative binding site charge is assumed to reside within membrane field when binding sites are closed, and the opening reaction (α) moves the electrical field across the site. No “driving force” for binding site closure (β) is provided by the electrical field because the charge is outside of the field when the site is open. Thus, the reaction will be voltage dependent only in the opening direction, although the amount of charge that moves through electrical field is the same in both directions. A still more extreme asymmetry, which we allow in simulations, is that the valences of forward and reverse reactions can be of opposite sign. This is justified if the overall reaction simulated is thought of as two reactions through a transitional state; the different valences then correspond to two different reactions that can be simulated separately with identical results. We illustrate our simulation of electrogenic reactions for the case shown in Fig. 2 , assuming that one equivalent charge is moved. The opening rate coefficient ( k α ) is multiplied by e q α · E m 27 mV ,where “q α ” is the charge coefficient and RT / F is approximated as 27 mV. The closure rate coefficient is multiplied by e q β · E m 27 mV ,where q β = 0. Total charge moved in the reaction (i.e., one elementary charge) is the sum of the forward and reverse reaction coefficients ( q α + q β ). A major charge-moving reaction of Na + ,glucose transporters can be simulated as a very slow, electrogenic Na + binding reaction , and our database for GAT1 allows a similar treatment. Nevertheless, we favor the idea that transporter conformational changes underlie the movement of charge through field, and the cartoon in Fig. 2 B shows how we have modeled the major charge-moving reaction for GAT1. We assume that an extracellular Na + binding site of GAT1 becomes available in an unstable transitional state (* E ), meaning that the binding site has a strong tendency to close to either the unloaded state ( E 1) or to the loaded, Na + -occluded state ( E 2), via reactions labeled β and γ, respectively. The binding site opening rates, α and δ, are assumed to be voltage dependent, whereby reaction α moves a fixed negative charge out of membrane field and the Na + occlusion reaction (γ) moves the positively charged Na + into the electrical field. Assuming that the *E state never accumulates significantly, this scheme predicts simple monoexponential charge movements with rate constants determined by the rates, α and δ. Although the opening of empty binding sites (α) is a major source of charge movement, this reaction cannot be isolated in the absence of extracellular Na + . Modifications of this scheme to allow significant accumulation of the * E state predict slow charge movements in the absence of Na + , as predicted and measured for Na,glucose transporters and for proton-coupled peptide transporters . Since we do not observe slow charge movements without Na + o , nor do we observe fast Na + o -dependent charge components, we estimate that the * E out state can never be occupied by >2% of total transporters. At 0 mV, the opening rates used to simulate our data are 200 s −1 for 1a and 2,000 s −1 for 2a . To ensure low occupancy of the transitional state, binding site closure rates (1b and 2b) would be in the range of 20,000–200,000 s −1 . This is still much slower than the expected dissociation rate of Na + from a binding site with a dissociation constant of 1 M ( K no ). Designating the fractional occupancy of the binding site by Na + in Fig. 2 as f no , and the extracellular Na + concentration as N o , 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_{{\mathrm{no}}}={N_{{\mathrm{o}}}}/{ \left \left(N_{{\mathrm{o}}}+K_{{\mathrm{no}}}\right) \right }{\mathrm{.}}\end{equation*}\end{document} From rate theory, the state flux from E 1 to E 2 (φ 12 ) will be 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{{\phi}}}_{12}={E_{1}{\cdot}{\mathrm{{\alpha}}}{\cdot}{\mathrm{{\gamma}}}{\cdot}f_{{\mathrm{no}}}}/{ \left \left[{\mathrm{{\gamma}}}{\cdot}f_{{\mathrm{no}}}+{\mathrm{{\beta}}}{\cdot} \left \left(1-f_{{\mathrm{no}}}\right) \right \right] \right }{\mathrm{,}}\end{equation*}\end{document} and the state flux from E 2 to E 1 (Ν 21 ) is 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{{\phi}}}_{21}={E_{2}{\cdot}{\mathrm{{\delta}}}{\cdot}{\mathrm{{\beta}}}{\cdot} \left \left(1-f_{{\mathrm{no}}}\right) \right }/{ \left \left[{\mathrm{{\gamma}}}{\cdot}f_{{\mathrm{no}}}+{\mathrm{{\beta}}}{\cdot} \left \left(1-f_{{\mathrm{no}}}\right) \right \right] \right }{\mathrm{.}}\end{equation*}\end{document} These same expressions were derived by Dr. Vladislav Markin (University of Texas Southwestern Medical Center at Dallas) from the analytical solution of the corresponding three-state model. Since variation of the rates, γ and β, simply changes the apparent Na + o affinity, these rates can be eliminated from the model. Thus, 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{{\phi}}}_{12}=E_{1}{\cdot}{\mathrm{{\alpha}}}{\cdot}f_{{\mathrm{no}}}\end{equation*}\end{document} and 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*}{\mathrm{{\phi}}}_{21}=E_{1}{\cdot}{\mathrm{{\delta}}}{\cdot} \left \left(1-f_{{\mathrm{no}}}\right) \right \end{equation*}\end{document} whereby the rate coefficients, α and δ, will be voltage dependent. We point out one interesting feature of this scheme, which could be relevant to the kinetic function of other transporters. Because Na + binding in the transitional state inhibits the overall reaction that releases Na + to the outside, the “off” rate of the charge movement is accelerated by reducing extracellular Na + . This would not be the case for a simple ion binding/dissociation reaction. The mathematical description of our model contains 18 parameters. One of these is eliminated to enforce microscopic reversibility of rates and one to enforce charge conservation (i.e., movement of one total charge per transport cycle). We fixed the intrinsic rates of 1a and 2a to 200 and 2,000 s −1 , respectively , because the effects of varying these rates by two- to threefold could be fully compensated by changes of substrate binding affinities. Another parameter that is not varied is designated f x . This parameter determines the ratio of extracellular Cl − dissociation constants in the E out and the * E in states; it affects only the simulations shown in Fig. 7 and Fig. 9 . Thus, 13 parameters were adjusted by the fitting routine for the results presented. Designations of the rate coefficients ( k 1 , k 2 , k 3 , and k 4 ) correspond to the reaction numbers in Fig. 1 . Na + ions are designated N, Cl − ions are designated Cl, and GABA molecules are designated G. Cytoplasmic and extracellular Na + concentrations are designated ni and no , respectively; Cl − concentrations are designated ci and co , and GABA concentrations are designated gi and go . Dissociation constants for the extracellular side are designated Kno1 and Kno2 for the first and second Na + ion to bind, respectively, during forward transport. Kgabo and Kclo are the extracellular GABA and Cl − dissociation constants. For the cytoplasmic side, our designations are Kni1 and Kni2 for the first and second Na + ions to bind, respectively, during reverse transport. Kgabi and Kcli are the cytoplasmic GABA and Cl − dissociation constants. Each of the reactions simulated has an apparent valence, designated q1, q2, q3 , and q4 , according to the corresponding rate coefficients. Finally, we assume that the cytoplasmic Cl − binding site, while empty, undergoes a weakly voltage-dependent reaction that allows and disallows Cl − i binding. Its valence is designated q5 . The parameter values for the results presented were as follows: k 1 = 200 s −1 , k 2 = 2,000 s −1 , k 3 = 39.8 s −1 , k 4 = 42.0 s −1 , Kno 1 = 917 mM, Kno 2 = 10.1 mM, Kgabo = 41.0 μM, Kclo = 8.16 mM, Kni 1 = 442 mM, Kni 2 = 11.5 mM, Kgabi = 1.77 mM, Kcli = 3.66 mM, q 1 = 0.684, q 2 = 0.387, q 3 = −0.071, q 4 = 0.167, and q 5 = −0.167. Microscopic reversibility was enforced at each fitting cycle by forcing a correction factor on one parameter, such that 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*}{ \left \left(Kni1{\cdot}Kni2{\cdot}Kgabi{\cdot}Kcli{\cdot}k1{\cdot}k3\right) \right }/{ \left \left(Kno1{\cdot}Kno2{\cdot}Kgabo{\cdot}Kclo{\cdot}k2{\cdot}k4\right) \right }=1{\mathrm{.}}\end{equation*}\end{document} The factors, h1–h5 , modify the rates of voltage-dependent reactions. Those reactions that move positive charge in the outward direction are multiplied by their respective factor, and those moving positive charge in the inward direction are divided by their respective factor: 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*}h_{ \left \left(1{\mathrm{{\ldots}}}5\right) \right }=e^{q \left \left(1{\mathrm{{\ldots}}}5\right) \right {\cdot}{ \left \left(Em\right) \right }/{{\mathrm{27\;mV}}}}{\mathrm{.}}\end{equation*}\end{document} The sum of apparent valences, q (1…5), is 1, corresponding to one net charge moved per transport cycle, 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*}q1+q2+q3+q4+q5=1{\mathrm{.}}\end{equation*}\end{document} Using d 1– d 5, r 1– r 4, and Kgat as temporary variables, the two-state model is simulated as follows. f 0cn is the fraction of E in transporters whose Cl − /Na + binding sites are empty and are not available to bind Cl − i (i.e., closed by the fast voltage-dependent reaction related to q 5). f cn is the fraction of E in transporters whose cytoplasmic Cl − /Na + binding sites are occupied by one Cl − i and the first Na + i to bind in the reverse transport cycle with the Kni1 dissociation constant. Although two Na + ions can bind in the E in state, only the first site must be occupied for the transition to the * E in state: 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*}d1={{{1+h5+h5{\cdot}ci}/{Kcli+h5{\cdot}ci{\cdot}ni}}/{ \left \left(Kcli{\cdot}Kni1\right) \right +h5{\cdot}ci{\cdot}ni^{2}}}/{ \left \left(Kcli{\cdot}Kni1{\cdot}Kni2\right) \right }{\mathrm{,}}\end{equation*}\end{document} 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_{0{\mathrm{cn}}}={1}/{d1{\mathrm{,}}}\end{equation*}\end{document} and 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{nc}}}={ \left \left[{{h5{\cdot}ci{\cdot}ni}/{ \left \left(Kcli{\cdot}Kni1\right) \right +h5{\cdot}ci{\cdot}ni^{2}}}/{ \left \left(Kcli{\cdot}Kni1{\cdot}Kni2\right) \right }\right] \right }/{d1}{\mathrm{.}}\end{equation*}\end{document} Na + i and GABA i bind sequentially in the * E in transitional state, although these binding reactions can be treated as parallel reactions with no important changes. f 0g is the fraction of transporters in the transitional state whose GABA i binding sites are empty; f nag is the fraction of transitional transporters whose Na + i /GABA i sites are occupied by both Na + i and GABA i : 12 \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*}d2={{1+ni}/{Kni2+ni{\cdot}gi}}/{ \left \left(Kni2{\cdot}Kgabi\right) \right }{\mathrm{,}}\end{equation*}\end{document} 13 \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{0g}}}={ \left \left({1+ni}/{Kni2}\right) \right }/{d2}{\mathrm{,}}\end{equation*}\end{document} and 14 \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{nag}}}={{ni{\cdot}gi}/{ \left \left(Kni2{\cdot}Kgabi\right) \right }}/{d2{\mathrm{.}}}\end{equation*}\end{document} f 1no is the fraction of * E out transporters with a Na + bound, and f 0no is the fraction without Na + bound: 15 \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*}d3={1+no}/{Kno1}{\mathrm{,}}\end{equation*}\end{document} 16 \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{1no}}}={{no}/{Kno1}}/{d3{\mathrm{,}}}\end{equation*}\end{document} and 17 \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{0no}}}={1}/{d3{\mathrm{.}}}\end{equation*}\end{document} f fullo is the fraction of E out transporters occupied by Na + o and GABA o , whereby extracellular Na + ( no ) and GABA ( go ) bind sequentially. Again, these binding reactions can be treated as parallel reactions without important changes. f 0o is the fraction of E out transporters with empty Na + /GABA binding sites: 18 \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*}d4={{1+no}/{Kno2+no{\cdot}go}}/{ \left \left(Kno2{\cdot}Kgado\right) \right }{\mathrm{,}}\end{equation*}\end{document} 19 \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{fullo}}}={{go{\cdot}no}/{ \left \left(Kno2{\cdot}Kgabo\right) \right }}/{d4{\mathrm{,}}}\end{equation*}\end{document} and 20 \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{0o}}}={1}/{d4{\mathrm{.}}}\end{equation*}\end{document} f clo is the fraction of extracellularly-oriented Cl − binding sites which is occupied by Cl − : 21 \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{clo}}}={co}/{ \left \left(co+k_{{\mathrm{clo}}}\right) \right }{\mathrm{.}}\end{equation*}\end{document} The rate coefficients, k 1 to k 4 , are multiplied by the appropriate factors to calculate the rates of the E in → E out ( r 1 and r 4) and the E out → E in ( r 2 and r 3) transitions: 22 \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*}r1={k_{1}{\cdot}f_{0{\mathrm{cn}}}{\cdot}f_{1{\mathrm{no}}}}/{h1}\end{equation*}\end{document} and 23 \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*}r2=k_{2}{\cdot}f_{0{\mathrm{o}}}{\cdot} \left \left(1-f_{{\mathrm{clo}}}\right) \right {\cdot}f_{{\mathrm{0no}}}{\cdot}h2{\mathrm{.}}\end{equation*}\end{document} Calculation of the reaction rates, r 3 and r 4, is more complex because more substrates interact with the * E in state than the * E out state. These rates are modified by a denominator, h 6, derived analogously to that in and . The denominator is the sum of the factors that modify exit rates from the * E 2 state . The dissociation constant for Cl − in the E out state is multiplied by a factor, f x , to give the dissociation constant in the * E in state. Microscopic reversibility is maintained by modifying the E out → * E in transition rate in the absence of Cl − o by the same factor. The extracellular Cl − dependence of the overall reaction, r 3, is then 24 \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[{{co}/{ \left \left(co+k_{{\mathrm{clo}}}\right) \right +f_{{\mathrm{x}}}{\cdot}k_{{\mathrm{clo}}}}}/{ \left \left(co+k_{{\mathrm{clo}}}\right) \right }\right] \right {\cdot} \left \left[{co}/{ \left \left(co+k_{{\mathrm{clo}}}{\cdot}f_{{\mathrm{x}}}\right) \right }\right] \right {\mathrm{,}}\end{equation*}\end{document} which simplifies to f clo . f x was assigned a value of 0.2 for the simulations presented, and its variation from 0.1 to 0.3 is without significant consequence. The r 3 and r 4 rates are calculated as follows: 25 \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*}h6= \left \left[{ \left \left(co\right) \right }/{ \left \left(co+k_{{\mathrm{clo}}}{\cdot}f_{{\mathrm{x}}}\right) \right }\right] \right {\cdot}f_{{\mathrm{0gabi}}}+f_{{\mathrm{gabi}}}{\mathrm{,}}\end{equation*}\end{document} 26 \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*}r3={k_{3}{\cdot}f_{clo}{\cdot}f_{{\mathrm{fullo}}}{\cdot}f_{0{\mathrm{g}}}}/{ \left \left(h3{\cdot}h6\right) \right }{\mathrm{,}}\end{equation*}\end{document} and 27 \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*}r4={k_{4}{\cdot}f_{{\mathrm{nc}}}{\cdot}f_{{\mathrm{nag}}}{\cdot}h4}/{h6}{\mathrm{.}}\end{equation*}\end{document} The fractional occupancy of the E in and E out states, and the steady state transporter turnover rate ( Rgat ) are calculated as follows: 28 \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*}Kgat=r1+r2+r3+r4{\mathrm{,}}\end{equation*}\end{document} 29 \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*}E_{{\mathrm{out}}}={ \left \left(r1+r4\right) \right }/{Kgat}{\mathrm{,}}\end{equation*}\end{document} 30 \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*}E_{{\mathrm{in}}}={ \left \left(r2+r3\right) \right }/{Kgat{\mathrm{,}}}\end{equation*}\end{document} and 31 \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*}Rgat={ \left \left(r2{\cdot}r4-r1{\cdot}r3\right) \right }/{Kgat}{\mathrm{.}}\end{equation*}\end{document} As required by thermodynamics for a tightly coupled transport process, the complete equation system obeys the relationship, 32 \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(r1{\cdot}r3\right) \right }/{ \left \left(r2{\cdot}r4\right) \right }={ \left \left(go{\cdot}co{\cdot}no^{2}\right) \right {\cdot}e^{-{Em}/{{\mathrm{27\;mV}}}}}/{ \left \left(gi{\cdot}ci{\cdot}ni^{2}\right) \right }{\mathrm{.}}\end{equation*}\end{document} For non–steady state (kinetic) simulations, the E in state at time t , E in ( t ), is calculated from its value at time zero, E in (0), and steady state value, E in (∞): 33 \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*}E_{{\mathrm{in}}} \left \left(t\right) \right =E_{{\mathrm{in}}} \left \left(0\right) \right + \left \left[E_{{\mathrm{in}}} \left \left({\mathrm{{\infty}}}\right) \right -E_{{\mathrm{in}}} \left \left(0\right) \right \right] \right {\cdot} \left \left(1-e^{-t{\cdot}Kgat}\right) \right {\mathrm{.}}\end{equation*}\end{document} The charge moved per second by a single transporter is calculated as follows: 34 \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*}\begin{matrix}Igat=E_{{\mathrm{out}}}{\cdot} \left r2{\cdot} \left \left[q1+q2+ \left \left(1-f_{{\mathrm{0cn}}}\right) \right {\cdot}q5\right] \right \right \\ \left +r3{\cdot} \left \left(q3+q4-f_{{\mathrm{0cn}}}{\cdot}q5\right) \right \right \\ -E_{{\mathrm{in}}}{\cdot} \left r1{\cdot} \left \left[q1+q2+ \left \left(1-f_{{\mathrm{0cn}}}\right) \right {\cdot}q5\right] \right \right \\ \left +r4{\cdot} \left \left(q3+q4-f_{{\mathrm{0cn}}}{\cdot}q5\right) \right \right {\mathrm{.}}\end{matrix}\end{equation*}\end{document} This equation takes into account the model assumption that E in transporters undergo a fast (instantaneous) charge-moving reaction that enables Cl − i binding. Thus, for each transition that alters the E in occupancy, it is calculated how much charge is moved simultaneously by a shift of the E in distribution between the states with and without available Cl − i sites. We note that simulation results were nearly identical when the charge-moving reaction within the E in state was simulated kinetically, using a three-state model, with forward and backward rate constants of 77,000 s −1 , roughly as measured experimentally for Q fast . Charge signals are presented only for the case that GABA is absent on both membrane sides, so that the r 3 and r 4 rates are zero. With this limitation, the total transporter-associated charge ( Qgat ), which has moved through the membrane electrical field, relative to the E out state (i.e., with one occluded Na + o and no substrates bound), can be calculated: 35 \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*}Qgat=E_{{\mathrm{in}}}{\cdot} \left \left[q1+q2+ \left \left(1-f_{0{\mathrm{cn}}}\right) \right {\cdot}q5\right] \right {\mathrm{.}}\end{equation*}\end{document} Finally, the unidirectional GABA extrusion rate is calculated to relate model function to GABA radioisotope flux studies. The flux has two components: first, an outward GABA flux that occurs via the overall reaction 4 , and second, an exchange component that occurs when the E out sites undergo conformational changes to the * E in transitional state, and then return to the E out state without reaching the E in state. Thus, 36 \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{{\phi}}}_{{\mathrm{GABA}} \left \left({\mathrm{in}}{\rightarrow}{\mathrm{out}}\right) \right }={e1{\cdot}k4+ \left \left({e2{\cdot}k3}/{h3}\right) \right {\cdot} \left \left[f_{{\mathrm{clo}}}+ \left \left(1-f_{{\mathrm{clo}}}\right) \right {\cdot}f_{{\mathrm{x}}}\right] \right {\cdot}f_{{\mathrm{gabi}}}}/{h6{\mathrm{.}}}\end{equation*}\end{document} Results were nearly identical when the transitional state was simulated as a stable state with high exit rates, and GABA efflux was calculated as occupancy of that state times the transition rate to the E out state. Fig. 3 shows fully activated current–voltage relations predicted by the model, together with corresponding data from experiments. Here, and in subsequent figures with steady state model predictions, we plot the calculated transporter turnover rates (y axis), rather than membrane current magnitudes from experiments. The membrane currents (data points) are proportional to the simulated turnover rate (lines), and the current magnitudes are available for all data simulated from the relevant figures in the previous articles . Fig. 3 includes results for the fully activated outward current (120 mM NaCl + 20 mM GABA on the cytoplasmic side; 20 mM Cl − and no other substrates on the extracellular side) and for the fully activated inward current from the same patch (120 mM NaCl + 0.2 mM GABA on the extracellular side; no cytoplasmic substrates). Simulated results (dotted line) are also given for a “mixed” mode (reversing) condition with all substrates in equal concentrations on both sides (100 mM Cl − , 100 mM Na + , and 2 mM GABA). The mixed mode current reverses at 0 mV, as required by thermodynamics. The shapes of the forward and reverse current–voltage relations are predicted in detail by the model, as are their relative magnitudes. Also, the small magnitude of the mixed mode current corresponds to our experience, that we could not confidently define any GAT1-mediated current, steady or transient, with these high substrate concentrations on both sides. These simulations allow us to summarize concisely major features of the model's function: outward GAT1 current, in the absence of extracellular Na + , has weak voltage dependence that corresponds to the voltage dependence of substrate occlusion from the cytoplasmic side ( k 4 ). This step is rate limiting for reverse current because the deocclusion of Na + to the extracellular side ( k 2 ) is very fast in the absence of extracellular Na + . For the same reason, the ratio of E in to E out occupancy does not change when voltage changes or cytoplasmic substrate concentrations are changed over a substantial range (not shown). The maximum turnover rate for outward transport at 0 mV, simulated for results at 32°C, is ∼40 s −1 . The relative slope of the fully activated inward current is larger than that of the outward current. This slope is determined mostly by the valence of reaction 1a . The relatively slow rate of this process, even with 120 mM extracellular Na + , determines the 4.5-fold smaller magnitude of fully activated inward current, compared with outward current, at 0 mV. The inward current saturates with increasing hyperpolarization because the GABA translocation step becomes rate limiting. This saturation behavior is enhanced by our assumption that this step moves a small amount of negative charge from outside to inside (i.e., in opposite direction from Na + occlusion). In fact, the model predicts that negative slopes of the current–voltage relations should be found at more negative potentials. Also, the weak voltage dependence of the GABA translocation reaction contributes to a small voltage dependence of current activation by extracellular GABA . The maximum forward transport rate at 0 mV is ∼8 s −1 . As indicated with bar graphs in Fig. 3 , changes of membrane voltage in the inward current condition result in large changes in the fractional distribution of the E in and E out states. In contrast, voltage changes result in very little shift from the E in configuration in the outward current condition. As described previously , an alternating access model of cotransport function predicts that the presence of one substrate on the trans membrane side will increase the apparent affinity for a substrate on the cis side in proportion to its inhibition of cis-to-trans transport activity. Fig. 4 A shows the GABA dependence of the reverse GAT1 current at 0 mV with 120 mM cytoplasmic NaCl, with and without 120 mM extracellular Na + . The half-maximal GABA i concentration shifts from ∼1.5 to ∼0.8 mM in the presence of 120 mM extracellular Na + , which inhibits the current by ∼40%. The magnitudes of inhibition and the changes of concentration dependence are both predicted well by the model. Fig. 4 B shows the simulation result obtained for cis–cis substrate interaction, when reverse current is limited by the “return” step of the alternating access model (i.e., with NaCl in the pipette). In this case, reduction of the cytoplasmic cosubstrate concentration, [Cl − ] i , from 120 to 3 mM increases the half-maximal concentration of cytoplasmic GABA. The predicted effect is smaller than the experimental effect. As described at the end of results , this discrepancy is completely alleviated when the * E in state is simulated as a stable state that can accumulate significantly. Isotope flux studies of GABA–GABA exchange provide another important test of our model. In outside-out synaptic membrane vesicles, extracellular GABA promotes GABA efflux both in the presence and in the nominal absence of extracellular Cl − . This result is accounted for by our model because Na + and GABA can be translocated from outside to inside regardless of whether the parallel Cl − binding site is occupied. As noted in materials and methods , we have simulated this reaction so that it takes place fivefold faster when extracellular Cl − is bound than when Cl − is not bound. Fig. 5 A shows the relevant measurements of GABA efflux by Kanner et al. 1983 . With 100 mM extracellular NaCl, GABA efflux in the presence of all substrates on the cytoplasmic side is roughly doubled by the presence of 20 μM extracellular GABA, and this effect is similar when extracellular Cl − is ∼5 mM. Our simulation of GABA efflux in the presence of all cytoplasmic substrates is shown in Fig. 5 B. The cytoplasm was assumed to contain 40 mM NaCl and 20 mM GABA, the extracellular solution was assumed to contain 100 mM Na + , and membrane potential was assumed to be 0 mV. The GABA dependence of GABA efflux is shown with 100 and 5 mM extracellular Cl − . In both cases, GABA efflux is stimulated for increasing extracellular GABA concentrations. The maximum GABA efflux rate is larger with high than with low [Cl − ] o . The results are in reasonable qualitative agreement with the experimental data, particularly in light of the fact that membrane potential and cytoplasmic substrate concentrations are not controlled in the experiments. The model predictions for cytoplasmic substrate interactions in the activation of reverse GAT1 current were presented together with the relevant data . Here, we summarize the major features. (a) The GABA i dependence of the fully activated current shows almost no change when either cytoplasmic Na + or Cl − is reduced. This is because a time-dependent transition takes place between the binding of Cl − i and the first Na + i ion, and the binding of GABA i . Also, the second Na + i ion binds with such a high affinity that reduction of Na + to a few millimolar has no effect on the apparent GABA affinity. (b) There is almost no change of the apparent Cl − i affinity with reduction of cosubstrate concentrations. This is because the second Na + i and GABA i bind in a state that is temporally separated from that in which Cl − i binds. Also, it is important that one Na + i binds immediately after binding of Cl − i . This arrangement explains why Na + i does not inhibit the inward GAT1 current in the absence of Cl − i . (c) With [Cl − ] i reduction, there is a shift of the half-maximal Na + i concentration to higher values. This is accounted for by the assumed sequential Cl − → Na + → Na + binding order; the inhibitory effect of reducing the Cl − i concentration can be overcome by higher Na + i concentrations. Fig. 6 shows experimental and predicted current–voltage relations for the outward GAT1 current (20 mM Cl − o and no other extracellular substrates). Fig. 6 A shows the effect of adding 120 mM extracellular Na + via pipette perfusion. Inhibition is ∼75% at −120 mV, but only ∼10% at +90 mV. The strongly voltage-dependent deocclusion reaction becomes rate limiting for the reverse transport cycle at negative potentials in the presence of Na + o . Positive membrane potential relieves the inhibition because transporters are driven to accumulate in the E 1 state. Discrepancies between the experimental and predicted results are in the range of our experimental error. Fig. 6B–D , shows results in the absence of extracellular Na + . Fig. 6 B shows the effect of lowering the cytoplasmic Na + concentration from 120 to 20 mM on the outward current–voltage relation. The model predicts no significant change of the shape of the current–voltage relation; the measured current–voltage relation in low [Na + ] i is somewhat steeper than predicted. This could reflect a small voltage dependence of Na + i binding (not simulated), whose influence becomes more pronounced when Na + i concentrations are not saturating. Fig. 6 C shows the effect of lowering cytoplasmic GABA from 20 to 0.5 mM. In this case, the simulated current–voltage relation at the low GABA i concentration is somewhat steeper than experimental results. One possible explanation is that GABA i interaction (binding and/or occlusion) from the cytoplasmic side becomes rate limiting at low GABA i concentrations; the three-state simulation described at the end of results gives a more accurate account of this result. Fig. 6 D shows the effect of reducing cytoplasmic Cl − from 120 to 5 mM on the current–voltage relation. The discrepancy between predicted and observed results reflects an experimental variability of the apparent Cl − i affinity, as noted in materials and methods . The shapes of current–voltage relations are predicted with reasonable accuracy. Fig. 7 shows the substrate dependence of the inward GAT1 current in whole-oocytes at different membrane potentials. These data points have been replotted from Mager et al. 1993 , and they are simulated by assuming that intracellular Cl − and Na + concentrations are 50 and 12 mM, respectively. Fig. 7 A shows the extracellular Na + dependence at −60 and −140 mV with 0.2 mM GABA o and 100 mM Cl − o . The shapes of the Na + o dependencies are reasonably predicted. Saturation comes about with negative potentials because at high [Na + ] o the E out → * E in → E in transition becomes rate limiting. Fig. 7 B shows the Cl − o dependence of the inward current at −140 and −40 mV. The Cl − o dependence is biphasic. Approximately 50% of the current activates with very high affinity, and ∼50% with low affinity ( K d = 8 mM). The high-affinity component comes about because the overall E out → E in transition becomes very fast when [GABA] i is low. This, in turn, depends on our assumption that GABA i can be translocated from the extracellular side in the absence of Cl − o . The apparent affinity will be determined by the ratio of rates 3b to 4a , which in our simplified model is infinity. The effect of membrane potential and the overall Cl − o dependence are predicted accurately. Fig. 7 C shows the GABA o dependence of the inward current. When maximum current is strongly reduced by depolarization, there is a modest increase in the apparent GABA o affinity at less negative potentials. In current–voltage relations , this effect results in a more pronounced saturation of current with hyperpolarization when the GABA o concentration is low. Fig. 8 shows the inhibition of inward GAT1 current in giant patches by cytoplasmic substrates (0 mV; 120 mM extracellular NaCl and 0.2 mM extracellular GABA). Results in Fig. 8A–C , are for the individual substrates, Cl − i , Na + i , and GABA i , respectively. Cytoplasmic Cl − monotonically inhibits the inward current with half-inhibition at ∼15 mM . Cytoplasmic Na + and GABA, when applied individually, have almost no effect (B and C). The lack of effect of Na + i and GABA i relies on the assumption that the * E in state does not accumulate significantly during inward current. The complete lack of effect of GABA i , in the absence of Cl − i and Na + i , derives from the assumption that Na + i binding precedes GABA i binding in the * E in state. However, the results are only marginally different when binding of cytoplasmic Na + and GABA is simulated as parallel reactions (not shown). In the presence of 120 mM Na + i and the absence of Cl − i , GABA i inhibits the inward current with low affinity ; the predicted inhibition is ∼75% with 20 mM GABA i , while the inhibition obtained experimentally is ∼60%. For brevity, we do not show model results on the inhibition of outward GAT1 current by substrates applied to the extracellular side. The inhibitory effect of extracellular Na + on outward current was described in Fig. 6 A. In the absence of extracellular Na + , outward current is inhibited by only ∼10% when [Cl − ] o is increased from 0 to 120 mM in the model, and this is in close agreement with our experimental experience. The Cl − o inhibition is small because the E out state does not accumulate significantly in this condition. Extracellular GABA is without effect in the absence of extracellular Na + because GABA o binds after Na + o in the model. Fig. 9 shows the predicted and measured current–voltage relations of the inward GAT1 current in patches (A) and whole oocytes (B–D). Fig. 9 A shows the effect of Cl − i (0, 30, and 120 mM) on inward current in an oocyte patch. With high [Cl − ] i , inward currents lose their tendency to saturate at negative potentials. Fig. 9B–D , shows simulation results for whole-oocyte experiments, whereby we have assumed cytoplasmic Na + and Cl − to be 12 and 50 mM, respectively. Fig. 9 B shows the effect of reducing [Na + ] o from 96 to 29 mM. In the absence of Cl − i , the current–voltage relation would be shifted by ∼30 mV to more positive potentials. For the most part, the effect of reducing [Na + ] o is to shift the current–voltage relation to more negative potentials, and this is well predicted. Fig. 9 C shows the experimental effect of removing extracellular Cl − . We assume for this simulation that nominally Cl − -free solutions will still contain 1 μM Cl − . With this assumption, the simulated current–voltage (I–V) relations describe the experimental data accurately without violating a fixed transport stoichiometry. Removal of Cl − scales down the I–V relation and somewhat enhances the saturation with hyperpolarization. Fig. 9 D shows the effect of reducing extracellular GABA from 100 to 10 μM; saturation of I–V relations at negative potentials becomes more pronounced at low extracellular GABA. Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 describe model predictions for GAT1 kinetic function. Fig. 10 shows the charge movements predicted by the two-state model. These results are shifted by ∼25 mV from results shown subsequently under identical conditions. We suspect that this variability, already pointed out in materials and methods , reflects a variable regulatory process in the oocytes that influences GAT1 function. To demonstrate the kinetic behavior of the model in relation this data, therefore, we have used 70 instead of 40 mM NaCl o to simulate this single data set. The results are calibrated as charge moved ( e ) per single transporter. In agreement with experimental results, the simulated charge signals contain immediate charge jumps on changing potential from positive values to −40 mV. These jumps arise from the charge-moving reaction of the empty Cl − i binding sites ( q 5), which moves a total of −0.08 equivalent charges per transporter. Clearly, the kinetics of slow charge movements are simulated accurately by the model. Fig. 11A and Fig. B , shows the predicted and measured rate– and charge–voltage relations from another experiment with 40 and 60 mM extracellular Na + and Cl − , respectively. The shapes and positions of both the rate– and charge–voltage relations are predicted accurately. Fig. 11 C shows the predicted and measured effect of 120 mM cytoplasmic Cl − on charge–voltage relations in the presence of 90 mM extracellular NaCl. Qualitatively, the simulations are in good agreement with the experimental data. Since the results described next were performed at room temperature with intact oocytes, we describe here the effect of temperature on GAT1 currents. Fig. 12 shows the temperature dependence of both the inward and outward transport currents in oocyte patches. Increasing temperature from room temperature (23°C) to 32°C at 0 mV causes a 2.2-fold increase in both currents. Although we have not characterized the temperature dependence of charge movements in detail, we have observed rate changes for individual voltage pulses in this general range. To fit the charge movement rates determined in oocytes, it was essential to divide the predicted model rates by a somewhat larger factor of 3.6. This larger factor might reflect the loss of some inhibitory influence on GAT1 transport upon patch excision. Fig. 13 and Fig. 14 show results from whole-oocyte experiments. Fig. 13 shows the rates of slow charge movements with 96 mM Cl − o at different extracellular Na + concentrations (96, 58, 12, and 3 mM). These results are replotted from Mager et al. 1996 after converting time constants to rate constants. Reduction of [Na + ] o shifts the rate–voltage relations to more negative potentials, and this is predicted accurately. The measured charge movement rates with 96 mM Na + o increase somewhat less steeply with hyperpolarization than the simulated rates. This shifting of rates with changing [Na + ] o comes about because the E out → * E out transition (i.e., opening of binding sites from the loaded state) is strongly inhibited by Na + o binding at the second extracellular site. There is an additional acceleration at low [Na + ] o because the overall E out → E in transition is inhibited by Na + o binding to the transitional * E out state. Fig. 14 shows the voltage and Na + o dependencies of the slow charge movement in intact oocytes and in the model. Fig. 14 A presents the voltage dependence of charge moved at different extracellular Na + concentrations (12, 24, 48, 77, and 96 mM). Again, the results are replotted from Mager et al. 1996 and scaled to the magnitude of charge moved per transporter ( e ) in the simulation. The shifts of voltage dependence and the shapes of charge–voltage relations are predicted accurately, although the predicted shifts are somewhat larger than those observed experimentally. Fig. 14 B presents the concentration dependence of charge moved at −80 mV when different extracellular Na + concentrations are applied. The shape of the Na + o dependence of charge available at 0 mV is reasonably well predicted, but the apparent Na + o affinity is somewhat lower in the model than determined experimentally. An important interpretive point is that the sigmoidal shape of this relationship does not require that both Na + ions bind before they are occluded. Binding of the second Na + ion after the slow occlusion of the first Na + ion has the same effect, and this order of events is essential to explain the changes of charge movement rates with changes of Na + o concentration. Fig. 15 shows simulations of GAT1-mediated currents under the different conditions studied with voltage pulse protocols. The corresponding experimental results were not included in the simulation database, so these simulations provide a test of model constraint. The voltage protocol is shown below the results; membrane potential was stepped, in 40-mV increments, from 0 mV to different potentials, and then back to 0 mV. Fig. 15 A shows simulated outward transport current with 20 mM Cl − o , 0 mM Na + o , and 0 mM GABA o . With voltage pulses to positive potentials, pre–steady state transients are very small and fast, consistent with our experimental results. The relative lack of transients is due to the fact that the transport cycle is rate limited by a single step in the E in → * E in transition; the Na + deocclusion reaction ( E out → * E out ) takes place 10× faster. A predicted experimental result, which we have not tested, is that significant current transients should occur after pulsing to large negative potentials. Fig. 15 B shows simulated results for outward current in the presence of 120 mM extracellular NaCl. In this case, current transients at positive potentials are substantial. They come about because in this condition the relatively slow deocclusion of Na + o from the E out state allows transporters to accumulate in the Na + o -occluded E out state, which is subsequently released by voltage pulses to positive potentials. Fig. 15 C simulates the inward current condition (i.e., with all substrates on the extracellular side and none on the cytoplasmic side). Upon hyperpolarization to −120 mV, the inward current relaxes by ∼75%, and on returning to positive potentials, the “off” transients are smaller (i.e., they would integrate to a smaller total amount of charge moved). The model behaviors are in reasonable agreement with experimental results . Fig. 15 D simulates results with 120 mM NaCl o , and no other substrates—the same condition used to monitor slow GAT1 charge movements. The simulations reproduce in reasonable detail the rates of charge movements and their voltage dependence. Fig. 15 E shows simulation results for the “reversal” condition (6 mM Cl − , 120 mM Na + , and 2 mM GABA on the cytoplasmic side; 120 mM Na + , 40 mM Cl − , and 2 mM GABA on the extracellular side). Small steady state currents are generated, but there are essentially no pre–steady state transients. The major reason is that the transport reactions involving fully loaded transporters are nearly electroneutral. The simulation equations assume that empty transporters undergo a voltage-dependent reaction ( q 5). This reaction gives rise to a capacitance that decreases when cytoplasmic Cl − binds from the cytoplasmic side, but other details of the Q fast reactions are not represented. In particular, we know that charge-moving reactions still occur in the Cl − i -bound state. To simulate roughly results on capacitance, we assume that the entire E out state is a null state that contributes no capacitance. We assigned the fractions of the E in state with no substrates bound a relative capacitance of unity, and we assigned a relative capacitance of 0.93 to the fractions of the E in state that have at least one bound substrate. From our experimental data , the latter value would be 0.8 since charge movements in the presence of Cl − i are ∼20% smaller in magnitude than those without Cl − i . With these assumptions, Fig. 16 shows model predictions for the cytoplasmic Cl − dependence of capacitance changes in the absence of cosubstrates, in the presence of 120 mM cytoplasmic Na + , and in the presence of 120 mM cytoplasmic Na + and 20 mM GABA. The Cl − i dependence in the absence of cytoplasmic Na + is reproduced well. However, the model predicts that the Cl − i dependence shifts ∼2.5-fold to lower Cl − i concentrations in the presence of Na + i . This result was obtained in only one of four similar experiments. The following discrepancies between the two-state model predictions and corresponding experimental data cannot be explained by experimental variability and therefore appear fundamental. (a) In the simulation of cis–cis substrate interactions with extracellular Na + , reduction of cytoplasmic Cl − does not shift the GABA i dependence of outward current strongly enough to higher GABA i concentrations. (b) The measured current–voltage relations for outward current become relatively more shallow with reduction of GABA i . (c) The extracellular Na + dependence of inward current does not saturate strongly enough with increasing [Na + ] o at negative potentials . (d) In the simulations of capacitance results , the relative capacitance of the substrate-bound E in fractions must be assumed to be larger than measured experimentally. Also, the presence of Na + i shifts the Cl − i dependence of capacitance to lower Cl − i concentrations. All of these discrepancies were reduced significantly, or eliminated, in simulations that included kinetic simulation of the * E in state. The rate coefficients of reactions 3b and 4b were selected by the fitting routine such that the * E in state accumulated substantially during reverse GAT1 operation, while its occupancy remained negligible during forward GAT1 operation. Reaction 4b was assigned the voltage dependence of q 4, and for simplicity the reactions 4a and 3b were left voltage independent. With these assignments, all other simulation results remained at least as accurate as those presented for the two-state model. The fitted parameters were as follows: k 1 = 53.7 s −1 , k 2 = 1,642 s −1 , k 3 = 61.7 s −1 , k 4 = 365.8 s −1 , Kno1 = 237 mM, Kno2 = 7.4 mM, Kgabo = 68.3 μM, Kclo = 54.0 mM, Kni1 = 1,283 mM, Kni2 = 8.0 mM, Kgabi = 0.66 mM, Kcli = 5.18 mM, q 1 = 0.652, q 2 = 0.419, q 3 = −0.059, q 4 = 0.215, q 5 = −0.22, and f x = 0.3. The additional rate constants for reactions 3b and 4b were 1,767 and 52.2 s −1 , respectively. Our ability to simulate GAT1 function in oocyte membrane by a model with only two stable states lends confidence to our conclusion that GAT1 works by a simple alternating access mechanism. The model described, while simple, incorporates many detailed assumptions about substrate binding and the dependencies of state transitions thereon. It accounts for many aspects of GAT1 function that we could not explain before undertaking a comprehensive simulation effort. We recognize that our specific assumptions are ad hoc in nature. However, our central assumption is the fundamental principle of enzyme kinetics that substrate binding can either enable or disable individual enzymatic reactions. Also, we recognize that experimental variability limits confidence in the model parameters determined. Nevertheless, the biological variability of GAT1 function, which may reflect the influence of important regulatory processes, does not compromise the simulations presented in any obvious way. We have discussed our simulations for the most part with their presentation. Our simulations give us no new insight into the significance of uncoupled GAT1 currents, as reported for GAT1 expressed in HEK cells and in the presence of lithium in oocytes . Since we are confident of an alternating access model for GAT1, our bias is that uncoupled Na + fluxes, when present, represent failures of the transport mechanism that either do not occur physiologically or occur with such rarity that they play no important role in electrophysiology or ion homeostasis. An important related issue, which has received less attention in recent years, is the coupling of GABA transport with Cl − movements. Our model assumes tight 1:1 Cl − :GABA coupling during transport, and for the reverse GAT1 transport mode, 20 mM cytoplasmic GABA activates no current in the absence of cytoplasmic Cl − . Our model predicts that GABA o -induced inward currents can be significant at negative potentials with micromolar (or even submicromolar) concentrations of extracellular Cl − . These predictions evolved from our search for an explanation as to how GABA–GABA exchange in synaptic vesicles could be possible in the nominal absence of Cl − on the extracellular side . Since GABA/Na + translocation occurs to a transitional state, the next step involving Cl − translocation occurs with very high probability when cytoplasmic substrate concentrations are low. Thus, extracellular Cl − can be swept into the cytoplasm with very high apparent affinity as the transporter returns to the E in state. We stress that experimental evidence for this explanation is still lacking, and three other possibilities must be considered. (a) Extracellular Cl − contamination might be greater than we expect, both in the clefts of oocyte surface and in the pipette tip during our pipette perfusion experiments. (b) The Cl − substitutes employed in experiments might be transported at a slow rate in place of Cl − . (c) Genuine Na + /GABA cotransport may occur under Cl − o -free conditions via transporter reactions that do not occur in the presence of Cl − . This last possibility was suggested from recent isotope flux studies in Xenopus oocytes (Loo, D.D.F., S. Eskandari, and E.M. Wright, personal communication). These authors found that GABA uptake is well coupled with Cl − o uptake in the presence of Cl − o , but that Na + -dependent GABA uptake remains substantial at negative potentials in the absence of extracellular Cl − . Since the current-to-uptake ratio is not much changed in Cl − o -free solution, a 1 Na + /1 GABA uptake mode would explain the results. Finally, it is interesting to compare our model of GAT1 function with relevant models of other transporters. First, we predict that only one Na + is occluded in an energetically stable state in the GAT1 transporter. This is different from the Na + /K pump in which stable occluded states are formed with three bound Na + as well as two bound K + . Second, we are impressed that transitional states seem important to account for GAT1 function. This is how Na + occlusion from the outside can be tightly coupled with the empty carrier conformational change that alternates binding site access. Third, our general modeling scheme for the Na + o -dependent charge movements and their kinetics in GAT1 can probably be applied to Na + /glucose transporters, although there is no obvious sequence similarity between these transporters. In conclusion, our analysis of GAT1 function does not exclude cotransport coupling mechanisms other than the alternating access mechanism. Nevertheless, our analysis of GAT1 function clearly favors conservative interpretations. We have verified rigorously the alternating access model, established probable cytoplasmic and extracellular substrate binding schemes, identified probable sources of electrogenicity, and refined the kinetic analysis of others. Our model of GAT1 function should be useful in understanding GAT1 mutants that exhibit altered kinetics and charge movements . Our model predicts the existence of two parallel substrate binding sites in GAT1, rather than a single pore-like structure with single-file sites, and this structural implication will ultimately be verified or contradicted. The physiological significance of “nonconservative,” uncoupled modes of operation of GAT1 remains to be established. | Study | biomedical | en | 0.999997 |
10477545 | PTX was purchased from Sigma Chemical Co. and B-oligomer was from Calbiochem. The purity of B-oligomer was verified by gel electrophoresis analysis, which revealed complete lack of contamination with A-protomer. MIP-1β, RANTES, and SDF-1α were from PeproTech, and Fura-2/AM, Ro-31-8220, and Gö 6979 were from Calbiochem. T cell–enriched, monocyte-depleted cultures were established from PBMCs from HIV-1–negative donors by Ficoll-Hypaque gradient centrifugation and two rounds of adherence to plastic. Nonadherent cells were collected by centrifugation, resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, and stimulated with phytohemagglutinin (5 μg/ml) for 3 d. Cells were then washed and cultured for another 7 d in medium supplemented with 20 U/ml of recombinant IL-2. By that time, ∼84% of the cells were CD3 + , ∼40% were CD4 + , ∼30% were CCR5 + , ∼95% were CXCR4 + , ∼10% were CCR5 + CD4 + , and ∼35% were CXCR4 + CD4 + , as determined by flow cytometry. Five HIV-1 strains were used in this study: three R5 viruses (92US660, 92US657, and ADA) and two X4 strains (92UG21, a primary isolate, and LAI, a T cell line–adapted virus). Before infection, viral stocks were treated with 200 U/ml of RNase-free DNase for 1 h at room temperature to eliminate DNA contamination. Viral inoculae were adjusted according to reverse transcriptase activity to 6 × 10 4 cpm per 10 6 cells. After a 2-h adsorption, cells were washed and cultured in IL-2–supplemented medium. Cells were treated with B-oligomer (1 nM) for 10 min and then inoculated with heat-inactivated HIV-1 (2 × 10 6 cpm/ml of reverse transcriptase activity) or with 5 μg/ml of recombinant gp120 JR-FL (Progenics Pharmaceuticals) or gp120 LAI (Intracel). After 60 min at 37°C, cells were washed and fixed in 4% buffered formaldehyde. After washing, cells were incubated for 20 min in 1% FCS/PBS at room temperature, and 2 × 10 5 cells were incubated for 30 min at room temperature in 0.1 ml of 1% FCS/PBS with anti-CCR5 or anti-CXCR4 mAb (2D7 and 12G5, respectively, obtained from PharMingen; both antibodies neutralize HIV-1 infection), followed by a 30-min incubation with a secondary rhodamine-labeled anti–mouse IgG. After washing, cells were incubated with FITC-labeled anti-CD4 mAb (13B8.2, Immunotech; this antibody does not compete with gp120 for CD4 binding), then washed, spotted on slides, dried, and analyzed on an immunofluorescent imaging system using a dedicated software (MetaMorph; Universal Imaging Corporation). For each binding reaction, 10 6 cells resuspended in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl 2 , 5 mM MgCl 2 , and 0.5% BSA) were mixed with B-oligomer (1 nM), incubated for 10 min at 37°C, and then transferred to ice. 550 pM of 125 I-labeled MIP-1β (specific activity 2,200 Ci/mmol) was added to cells in the presence or absence of a 1,000-fold molar excess of unlabeled MIP-1β, or 0.5 μg/ml of gp120, and reactions were incubated at 4°C for 4 h on a horizontal shaker. After washing with binding buffer containing 0.5 M NaCl, cell-bound radioactivity was counted in a gamma-counter. The assay was performed as previously described 33 . In brief, 0.6 ml of Fura-2–loaded cells (5 × 10 6 cells/ml) was transferred to an acrylic cuvette and stimulated with B-oligomer (500 ng/sample), SDF-1α (100 ng/sample), or MIP-1β (500 ng/sample). Fluorescence emission at 340 and 380 nm was measured on a Perkin-Elmer Luminescence Spectrometer LS50B. Several previously published reports 16 17 18 19 20 demonstrated that activity of chemokine receptors as coreceptors for HIV-1 does not involve signaling via coupled G i proteins. A somewhat conflicting result has been reported recently 21 , demonstrating that entry of X4 HIV-1 strains into primary T cells correlated with actin-dependent cocapping of CD4 and CXCR4 receptors, thus implicating signaling in the process of HIV-1 entry. To better define the role of signaling from chemokine receptors in HIV-1 entry into primary T cells, we used PTX to specifically inactivate G i -like proteins that transduce signals from both CCR5 and CXCR4 13 , and measured HIV-1 entry by PCR, using primers LTR R/U5 specific for early products of reverse transcription 34 . The fragment of HIV-1 cDNA amplified by these primers is produced either within the virion or very early after virus–cell fusion, and thus reflects the efficiency of virus entry. Surprisingly, PTX inhibited entry of R5 HIV-1 strains 92US660 and ADA (not shown), but not of X4 strains LAI and 92UG21 (not shown). PTX is a complex protein composed of an active (A) and a binding (B) subunit, and certain T cell activities have been shown to be initiated by the B-oligomer of PTX independently of inactivation of G i -like proteins by the A-protomer 35 . We therefore tested whether activity of B-oligomer could account for the observed inhibitory effect of PTX on entry of R5 HIV-1. Similar to results observed with PTX, B-oligomer inhibited entry of R5 HIV-1 strains in a dose-dependent fashion, but not of X4 HIV-1 strains . Analysis of the B-oligomer preparation by SDS-PAGE confirmed the lack of A-protomer (not shown), and G i -mediated signaling was not impaired in B-oligomer–treated cells . We thus conclude that PTX blocks entry of R5 HIV-1 strains by a mechanism that is independent of G i protein inhibition. Interestingly, no inhibitory activity of PTX or B-oligomer was observed on entry of R5 HIV-1 into PM1 cells (not shown), consistent with recently reported results 36 . This result underscores the differences between the primary cells and T cell lines. To analyze the effect of B-oligomer on HIV-1 replication in long-term cultures, we measured virus production after in vitro infection of primary PBMCs from HIV-seronegative donor with R5 or X4 HIV-1 strains. As predicted from the entry studies , B-oligomer at 1 nM concentration inhibited replication of a primary R5 strain, 92US660 . Unexpectedly, a similar level of inhibition was also observed with primary and cell line–adapted X4 strains, entry of which was not affected by B-oligomer . This result is consistent with recently reported 37 inhibitory effect of PTX on replication in PBMCs of another X4 strain, RF, and indicates that, in addition to its effect on HIV-1 entry via the CCR5 receptor, B-oligomer exerts its anti-HIV activity via another, as yet unidentified, mechanism that works at the post-entry step of viral replication and does not depend on the identity of chemokine receptor used by the virus. Interestingly, this post-entry inhibitory effect of B-oligomer depended on the multiplicity of infection and was overcome when high virus inoculum was used (data not shown). Similar to results of entry studies, replication of neither R5 (92US660) nor X4 (LAI) strains was inhibited in PM1 cells . To further investigate the anti-HIV activity of B-oligomer, we analyzed its effect on replication of uncloned primary HIV-1 in cocultures of uninfected donor PBMCs with activated PBMCs from HIV-1–infected patients. The number of infected cells in the PBMCs from such patients is usually low 38 , so the measured virus output in cocultures is primarily a result of virus spread to new targets. A dose-dependent inhibition of virus replication by B-oligomer was observed . This effect was reproduced with PBMCs from three different patients (not shown); in all cases 1 nM of B-oligomer completely inhibited virus replication. To demonstrate that the inhibitory effect of B-oligomer was not due to its cytotoxic activity, we measured uptake of [ 3 H]thymidine and the change in cell numbers in long-term cultures. As shown in Fig. 2 E, no significant difference in thymidine uptake between B-oligomer–treated and untreated cultures was observed during the whole course of the experiment. In fact, treated cultures demonstrated higher proliferation rate , consistent with the well-known mitogenic effect of B-oligomer 24 . Therefore, B-oligomer inhibits replication of a wide variety of primary and cell line–adapted isolates. This result may explain a recently reported inhibitory effect of PTX on replication of SIV mac251 (which uses the CCR5 receptor) in vivo 39 . The inhibitory effect of B-oligomer is exerted at both the entry (for R5 strains) and post-entry (probably for both R5 and X4 viruses) steps of HIV-1 infection. In this work, we concentrated on the mechanisms of B-oligomer–mediated inactivation of CCR5. A possible mechanism by which B-oligomer could specifically inhibit entry of R5 but not X4 HIV-1 strains is by interfering with binding of the virus, either directly (by blocking the HIV-1 binding site on CCR5) or indirectly (by changing conformation of CCR5). To test this possibility, we performed binding studies with 125 I-labeled MIP-1β and gp120. Results in Fig. 3 A demonstrate that binding of MIP-1β to PBMCs was competed by recombinant gp120 of an R5 strain, HIV-1 JR-FL , but not by gp120 of an X4 strain, HIV-1 LAI . Although treatment of the cells with B-oligomer increased 125 I-labeled MIP-1β binding threefold, binding of this ligand was still effectively competed by gp120 JR-FL . To exclude the possibility that B-oligomer nonspecifically affected binding of CCR5 ligands, for instance by acting as a bridge between the ligand and the cell, analysis was also performed using cells treated for 10 min either with B-oligomer at 4°C (to block signaling from B-oligomer receptor), or with BSA at 37°C (to mimic the protein concentration in the experimental samples). Both treatments had no effect on 125 I-labeled MIP-1β binding, compared with untreated sample . Therefore, we conclude that the inhibitory activity of the B-oligomer on HIV-1 entry is a signal-mediated process that does not involve blocking of HIV-1 binding to its receptors. This result also suggests that binding of gp120 to CD4 and CCR5 is not sufficient to induce fusion of the viral and cellular membranes. A logical conclusion from the results presented above is that a post-binding step of virus entry was inhibited in B-oligomer–treated cells, and that this effect could be mediated by B-oligomer–induced alteration of an intracellular portion of the CCR5 molecule. As this part of the receptor is involved in interactions with coupled G proteins, we measured Ca 2+ mobilization in response to natural ligands of CCR5 and CXCR4, MIP-1β or RANTES, and SDF-1α, respectively, after pretreatment of the cells with B-oligomer. Treatment of the cells with B-oligomer blocked Ca 2+ flux initiated by MIP-1β and RANTES (not shown), but not by SDF-1α . Although pretreatment of the cells with MIP-1β desensitized response to B-oligomer , pretreatment with SDF-1α induced only partial desensitization . Of note, B-oligomer at the suboptimal concentration used in these experiments (2 nM) did not desensitize its own receptor (not shown). The use of higher concentrations of B-oligomer for desensitization studies was confounded by a very slow return of the Ca 2+ response to the baseline (>30 min), thus precluding analysis by Fura-2–based technique due to leaking of the dye from the cells. The pattern of Ca 2+ response to sequential treatment of cells with B-oligomer and MIP-1β is best described by the phenomenon of heterologous desensitization. Potentially, this desensitization is attributable to signaling from a B-oligomer receptor, similar to the heterologous desensitization of CXCR1 and CXCR2 chemokine receptors by signaling from opiate receptors 40 . Although we can not formally rule out the possibility that B-oligomer binds to CCR5, several considerations make this scenario unlikely. First, B-oligomer did not compete with MIP-1β or gp120 for binding to CCR5 . Second, B-oligomer did not induce downregulation of CCR5, as evidenced by flow cytometric analysis (not shown). Third, B-oligomer did not induce Ca 2+ flux (not shown) and did not affect HIV-1 replication in PM1 cells, supporting the notion that B-oligomer signals through its own receptor and this signaling is required for its anti-HIV activity. Treatment of T lymphocytes with PTX or B-oligomer has been reported to rapidly increase PKC activity 30 . To determine whether PKC was involved in the observed effects of B-oligomer on CCR5 function, we used two selective PKC inhibitors, Ro-31-8220 and Gö 6979. As shown in Fig. 5 , Ro-31-8220 reversed the inhibitory effect of B-oligomer on entry of R5 HIV-1 and on Ca 2+ flux induced by RANTES, another ligand for CCR5 . On its own, Ro-31-8220 did not significantly alter either of those activities of CCR5 . It also did not act upon entry of X4 HIV-1 or signaling by SDF-1α , which are mediated by CXCR4 and are not affected by B-oligomer. A similar result was observed with a different PKC inhibitor, Gö 6979 (not shown). Taken together, these results indicate that PKC is involved in signal transduction from B-oligomer (and PTX) receptor to CCR5. Our results thus far demonstrated that B-oligomer–treated cells did not signal after MIP-1β stimulation. We wished to demonstrate that signaling initiated by HIV-1 binding was also affected. Because our attempts to detect Ca 2+ flux 14 or Pyk2 phosphorylation 13 in primary cells after inoculation with HIV-1 or gp120 were unsuccessful, we decided to use receptor capping as an indicator of HIV-1–induced signaling. Inoculation of PBMC cultures with either R5 or X4 HIV-1 strains, or their corresponding gp120s ( Table ), induced a characteristic cocapping of chemokine receptors with CD4. The fact that we were able to detect capped CCR5 and CXCR4 using antibodies that compete with gp120 for chemokine receptor binding suggests that the observed signal came from free receptors that cocapped together with gp120-occupied receptors. Such interpretation is consistent with previously observed gp120-induced association of CD4 with several surface molecules, such as CD3, CD45RA, CD26, and HLA class I 41 42 , with RANTES-induced colocalization of CXCR4 and CD4 43 , and with cocapping of CCR2 and CCR5 in response to MCP-1 or RANTES 44 . Pretreatment of the cells with B-oligomer for 10 min prevented capping of HIV-1 receptors, but only when capping was induced by R5 HIV-1 . This result indicates that capping is mediated by HIV-1–induced signaling from chemokine receptors, rather than from CD4, and that treatment with B-oligomer blocks this signal. To further illustrate the role of signaling from chemokine receptors in capping, we analyzed the effect of B-oligomer on capping induced by SDF-1α and MIP-1β. This analysis was confounded by rapid downregulation of CXCR4 or CCR5 after stimulation with SDF-1α or MIP-1β, respectively. Nevertheless, results presented in Table demonstrate that B-oligomer blocked capping induced by MIP-1β, but not by SDF-1α. Similar to HIV-1– or gp120-induced polarization, the capping involved not only the ligand-specific receptor, but also other molecules, including CD4. This result suggests that signaling from chemokine receptors induces a major actin-dependent rearrangement of cellular membrane. Taken together, the results presented in this report suggest that the inhibitory effect of PTX and its B-oligomer on HIV-1 infection of primary cells is mediated through desensitization of CCR5. Exposure to B-oligomer causes T cells to lose signaling activity associated with binding of the natural CCR5 ligand, MIP-1β, and to fail to cap after binding of R5 HIV-1; however, ligand-binding activity is preserved. The role of receptor capping in HIV-1 infection of primary cells, and the nature of signals involved in the regulation of capping are now under investigation in our laboratory. It is clear that receptor capping is not required for binding of the virus, but it might be necessary for fusion with primary cells. In addition to the potential value of the B-oligomer as an anti-HIV agent, these studies may define new targets in the search for novel therapeutic approaches against HIV infection. | Study | biomedical | en | 0.999997 |
10477546 | TCR-α −/− mice with a background of C57BL/6 were obtained from The Jackson Laboratory. The mice were originally generated by a targeted disruption of TCR genes in embryonic stem cells 2 . They were housed in the Experimental Animal Facility at the Research Institute for Microbial Diseases, Osaka University, under specific pathogen-free conditions and received sterilized food and autoclaved distilled water ad libitum. In this study, a standard protocol was used for mAb in vivo treatment 10 . TCR-α −/− mice from 6 to 25 wk of age were intraperitoneally injected with rat anti–mouse IL-4 mAb (BVD4-1D11, 1 mg/mouse; PharMingen) or rat IgG2b (R35-38, 1 mg/mouse; PharMingen) as mock Ab in 250 μl of PBS once a week. These treatments did not produce any signs of serum sickness. Levels of IgA, IgG, and IgM Ab in serum and fecal extracts at 25 wk of age were examined by ELISA as described previously 14 . In brief, ELISA plates (Nunc) were coated with 100 μl of 5 μg/ml goat anti–mouse Ig (H+L) (Southern Biotechnology Associates) in PBS and were incubated for 16 h at 4°C. Serial twofold dilutions of sera or fecal extracts were added (100 μl/well). The fecal extracts were the supernatants obtained after fresh feces were mixed with PBS containing 0.5% sodium azide (100 mg wet weight of feces/ml) and then centrifuged 15 . After 2 h incubation at 37°C, unbound Abs were removed, and alkaline phosphatase–conjugated goat or rat anti–mouse α, γ, and μ chains (Southern Biotechnology Associates) were added. The plates were incubated at 37°C for 2 h and developed with p -nitrophenyl phosphate (1 mg/ml; Wako) in 10 mM diethanolamine buffer (pH 9.6). Ab concentrations were calculated from the standard curve by using purified mouse IgA, IgG, and IgM (Southern Biotechnology Associates). In addition, subclass–specific IgG Ab titers were also analyzed by using alkaline phosphatase–conjugated goat anti–mouse IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechnology Associates) as detection Ab. Further, murine IgG1, IgG2a, IgG2b, and IgG3 antibodies (Southern Biotechnology Associates) were used for the generation of a standard curve. Mice anesthetized with ketamine (Sigma Chemical Co.) were killed at 25 wk of age. The SPs and MLNs were aseptically removed, and single-cell suspensions were prepared by a standard mechanical procedure. Mononuclear cells from the LP of the colon were dissociated using type IV collagenase (Sigma Chemical Co.) to obtain single-cell preparations as described previously 16 . Total IgA, IgG, and IgM Ab–forming cells in SP, MLNs, and colonic LP were analyzed by an enzyme-linked immunospot (ELISPOT) assay as described previously 10 . Nitrocellulose microtiter plates (Millipore Co.) were coated with 100 μl of anti–mouse IgA, IgG, or IgM (Southern Biotechnology Associates) at a concentration of 5 μg/ml in PBS. For the detection of Ab-producing cells, alkaline phosphatase–conjugated anti–mouse IgA, IgG, or IgM (1 μg/ml; Southern Biotechnology Associates) was added and then visualized with a substrate. The substrate used was 5-bromo-4-chloro-3-indolyl phosphate (Wako)/nitroblue tetrazolium (Wako) in alkaline phosphatase buffer (100 mM Tris-HCl [pH 9.5] containing 100 mM NaCl and 5 mM MgCl 2 ). For the analysis of the profile of CD4 + ββ T cells by flow cytometry, PE-conjugated anti-CD4 (anti-L3T4; RM4-5) mAb and FITC-conjugated anti–TCR-β mAb (H57-597) were obtained from PharMingen. Single-cell suspensions of lymphocytes (10 6 /sample) were then stained with optimal concentrations of PE-conjugated anti-CD4 mAb and FITC-conjugated anti–TCR-β mAb. These samples were subjected to flow cytometric analysis by using a FACScan™ (Becton Dickinson). Data were analyzed by using CellQuest software (Becton Dickinson). For the analysis of the cytokine profile, CD4 + ββ T cells were purified by FACS Vantage™ (Becton Dickinson). Cytokine production by purified CD4 + ββ T cells from colonic LP was analyzed by modified cytokine-specific reverse-transcription (RT)-PCR as described 17 18 . To isolate RNA from the CD4 + ββ T cells purified by flow cytometry, TRIzol reagent (GIBCO BRL) was used. Purified RNA was reverse transcribed into cDNA using Superscript II reverse transcriptase (GIBCO BRL) and DIG DNA Labeling Mix ® (Boehringer Mannheim), which incorporates DIG-labeled dUTP every 20–25 nucleotides during reverse transcription. The DIG-labeled, synthesized cDNA and a series of diluted DIG-labeled control cDNA (Boehringer Mannheim) were dot blotted onto the nucleic acid transfer membrane (Amersham Pharmacia Biotech) and cross-linked by UV cross-linker (Spectronics). The membrane was subjected to blocking by 1% Blocking reagent ® (Boehringer Mannheim) in 0.15 M NaCl, 0.1 M maleic acid for 30 min, followed by an additional 30-min incubation with 7.5 U/liter of alkaline phosphatase–conjugated anti-DIG Abs (Boehringer Mannheim) in 1% Blocking reagent ® (Boehringer Mannheim). The membrane was then incubated with 1% chemiluminescent substrate for alkaline phosphatase, CDPstar™ (Tropix), in 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl 2 . The developed chemiluminescent signals on the membrane were exposed to an imaging screen for 18 h, and then characterized by an image analyzer (Molecular Imager ® system; Bio-Rad Laboratories). The images on the screen were extracted by a laser scanner, and the number of synthesized cDNA samples were quantitated using the image analyzer. PCR amplification of 10 ng of cDNA for each sample was performed with the GeneAmp PCR System 9700 (Perkin-Elmer Cetus). The cytokine-specific primers and amplification protocols used have been described previously 18 . The amplified products were separated by electrophoresis in 1.8% agarose gel and were visualized with ethidium bromide (1 μg/ml). Lymphocytes (10 6 /well) isolated from colonic LP of anti–IL-4 mAb– or mock Ab–treated mice were stimulated in vitro with precoated purified anti–mouse CD3∈ mAb (145-2C11, 10 μg/ml; PharMingen) or anti–mouse TCR-β (H57-597, 10 μg/ml; PharMingen) in 24-well microplates for 72 h. After the incubation period, the supernatants were collected to analyze Th1 (IFN-γ) and Th2 cytokine (IL-4 and IL-6) production. Cytokine synthesis in the culture supernatants was analyzed by using the Biotrak™ ELISA system (Amersham Pharmacia Biotech) according to the manufacturer's protocol. Tissue samples were fixed in 4% paraformaldehyde in PBS for 4 h, embedded in paraffin, and sectioned at a thickness of 5 μm. Sections were stained by the conventional hematoxylin and eosin staining method. The periodic acid–Schiff–alcian blue procedure was also performed in order to stain goblet cells 19 . Data were statistically analyzed by the Student's t test. As increased levels of Abs are one of the immunological features of TCR-α −/− mice with IBD 10 , we sought to determine and compare the levels of serum and fecal IgA, IgG, and IgM Abs in anti–IL-4 mAb– and mock Ab–treated TCR-α −/− mice at 25 wk of age by using ELISA. Serum as well as fecal Ab titers were increased in mock Ab–treated TCR-α −/− mice . The levels of Ab titers in these mice were comparable to those of untreated mice, as observed in previous reports 9 10 . However, the levels of IgA, IgG, and IgM Abs in serum and fecal extracts were significantly decreased in TCR-α −/− mice treated with anti–IL-4 mAb . When IgG subclass Ab titers of TCR-α −/− mice treated with anti–IL-4 mAb were examined by ELISA, levels of IgG1 and IgG2b were found to have decreased and those of IgG2a to have increased significantly . To further confirm the reduction of Ab production at the cellular base, mononuclear cells were isolated from systemic and mucosal tissues of TCR-α −/− mice treated with anti–IL-4 mAb and mock Ab for subsequent ELISPOT assay. The numbers of Ab-forming cells were increased in the systemic lymphoid (e.g., SP) as well as in mucosa-associated tissues (e.g., MLNs, colonic LP) of TCR-α −/− mice treated with mock Ab . On the other hand, numbers of IgA, IgG, and IgM Ab–forming cells from TCR-α −/− mice treated with anti–IL-4 mAb were significantly decreased both in the systemic lymphoid and mucosa-associated tissues . Since the administration of anti–IL-4 mAb inhibited Ab production in TCR-α −/− mice , we next used flow cytometry to assess the influence of mAb treatment on the development of CD4 + ββ T cells. A subset of CD4 + ββ T cells costained with PE-conjugated anti-CD4 mAb (RM4-5) and FITC-conjugated anti-TCR-β (H57-597) was detected in the mucosal and peripheral tissues of mock Ab–treated TCR-α −/− mice. Surprisingly, a similar frequency of CD4 + ββ T cells also developed in TCR-α −/− mice treated with anti–IL-4 mAb . Additionally, the number of total lymphocytes in colonic LP obtained by dissociation with collagenase showed no statistical change between the two groups of mice (Mock Ab, 4.4 ± 0.8 × 10 6 cells/mouse; and anti–IL-4 mAb, 4.0 ± 0.6 × 10 6 cells/mouse). Further, mice treated with anti–IL-4 mAb did not show obvious clinical signs of IBD (see section below). These findings show that anti–IL-4 mAb treatment did not influence the development of CD4 + ββ T cells. As the development of aberrant CD4 + ββ T cells was not affected by treatment with anti–IL-4 mAb , we next analyzed the cytokine profile of CD4 + ββ T cells isolated from TCR-α −/− mice treated with anti–IL-4 mAb or mock Ab. The CD4 + ββ T cells were isolated from the colonic LP of TCR-α −/− mice by FACS Vantage™, and the profile of Th1 and Th2 cytokine expression was examined by cytokine-specific RT-PCR. Our previous study showed that CD4 + ββ T cells could be considered to be of the Th2 phenotype, since a cytokine-specific ELISPOT assay showed that they produced IL-4 but not Th1 cytokines 10 . This pattern of Th2-type cytokine production was also confirmed by cytokine-specific RT-PCR, since CD4 + ββ T cells isolated from the colonic LP of mock Ab–treated TCR-α −/− mice expressed mRNA specific for IL-4, IL-5, IL-6, and IL-10, but not for Th1-type cytokines . Conversely, in the TCR-α −/− mice treated with anti–IL-4 mAb, the pattern of cytokine production was significantly changed; namely, specific messages for Th2-type cytokines such as IL-4, IL-5, and IL-10 were not detected, whereas those for Th1-type cytokines (e.g., IFN-γ) were upregulated . In addition, the analysis of CD4 + ββ T cells isolated from the MLNs of IL-4–specific mAb–treated mice resulted in the alteration of cytokine expression from a dominant Th2 to a Th1 type. The reduction of Th2-type cytokine production and the enhancement of Th1-type cytokine production in mice treated with anti–IL-4 mAb were further confirmed at the protein level through ELISA analysis of secreted Th1 and Th2 cytokines . Thus, in vitro stimulation of colonic lymphocytes isolated from anti–IL-4 mAb–treated mice with anti-CD3∈ or anti–TCR-β resulted in the decrease of Th2 cytokines (e.g., IL-4 and IL-6) and the increase of IFN-γ synthesis. In contrast, treatment with mock Ab resulted in high levels of Th2 cytokine production. These results demonstrate that a shift from a Th2- to Th1-type response was induced in anti–IL-4 mAb–treated TCR-α −/− mice. We next investigated the effect of anti–IL-4 mAb on the induction of IBD in the TCR-α −/− mice. Approximately 60% of TCR-α −/− mice treated with mock Ab or left untreated were observed to develop the morphological changes that have been reported previously to be typical of IBD, including anorectal prolapse, diarrhea, and the weight loss and hunched posture characteristic of wasting syndrome 2 10 . In contrast, the mice treated with anti–IL-4 mAb showed no significant weight loss . Histological examination of the intestinal LP of TCR-α −/− mice treated with anti–IL-4 mAb showed more elongation of epithelial villi and markedly less infiltration of inflammatory cells than in that of mice treated with mock Ab . Because a reduction in the number of goblet cells has also been reported in the colon of humans with IBD 20 , we stained tissue sections prepared from the colon of anti–IL-4 mAb– and mock Ab–treated TCR-α −/− mice with alcian blue in order to detect goblet cells. As in the colons of humans suffering from IBD, the number of goblet cells in colons of mock Ab–treated mice was reduced. In contrast, the number of goblet cells was almost normal in the colon of TCR-α −/− mice treated with anti–IL-4 mAb . These findings suggest that the inhibition of the Th2-type response by anti–IL-4 mAb treatment results in the prevention of mucosal inflammation in TCR-α −/− mice. The mucosal immune system consists of several immune components, including α/β and γ/δ T cells, IgA B cells, macrophages, dendritic cells, and epithelial cells, that form a molecular and cellular inter/intranet that provides a protective barrier against pathogens and environmental antigens in the gut 21 22 23 . Under normal conditions, the mucosal immune system also properly regulates the intestinal mucosa. However, in those suffering from IBD, the mucosal immune system, and more particularly the T cell–dependent regulatory system, is disrupted 1 3 . In TCR-α −/− mice, CD4 + ββ T cells are considered to play a key role in the induction of IBD 9 10 13 . The results of this study demonstrated two new and important points regarding the role of Th1- and Th2-type cells in the development of IBD. In all murine IBD models used, including specific gene-manipulated and hapten-induced mice, a common observation was that the enhancement of Th1-type activity was associated with disease development 24 25 26 . However, previous studies have posited that Th2-like responses are involved in the development of colon inflammation in human ulcerative colitis 27 28 29 30 31 . Our study directly demonstrates that CD4 + ββ T cells isolated from the inflamed colon possess characteristics of Th2-type cells based on their cytokine profile at the mRNA and protein levels . Further, it was shown that alteration of the cytokine profile from a Th2 to a Th1 type by treatment with anti–IL-4 mAb resulted in the prevention of colitis in TCR-α −/− mice . The decrease of IL-4–dependent IgG1 and IgG2b Ab responses further confirmed that anti–IL-4 mAb treatment inhibited Th2-type responses . These two important findings suggest that aberrant mucosal Th2-type cells are involved in the development of chronic inflammation in the intestinal tract. Two very recent studies support this conclusion by demonstrating that Th2-type CD4 + T cells play a major role in the development of hapten-induced murine colitis 32 33 . IL-4 initially received attention as an enhancer of DNA synthesis in mouse B lymphocytes 34 . Since then, extensive molecular and immunological investigations have established IL-4 as a biologically potent and essential cytokine for B cell development and responses, including those of IgE and IgG1 35 . Further, this cytokine has been shown to play an essential role in the generation of Th2-type from Th0-type cells 35 . Although IL-4 is considered to be a hallmark cytokine for Th2-type T cells, it should be noted that other immunologically competent cells such as mast cells, basophils, γ/δ T cells, and NK1.1 + T cells can produce this cytokine 35 36 37 38 39 40 . NK1.1 + CD4 + T cells have been shown to be a particularly important source of IL-4 37 . Histological analysis with alcian blue and flow cytometric analysis using mAb specific to NK1.1 detected only a small number of mast cells and NK1.1 + T cells in TCR-α −/− mice (data not shown). Although our previous and present results clearly demonstrate that CD4 + ββ T cells are the primary source for the production of IL-4 in TCR-α −/− mice with IBD, it is still possible that a minor population of NK1.1 + CD4 + T cells provides an alternative source of the cytokine. When mucosal γ/δ T cells were isolated from the intestinal tract of TCR-α −/− mice with IBD for the analysis of cytokine production, no specific message for IL-4 was detected (data not shown). Therefore, the population of CD4 + ββ T cells is most likely the major source of IL-4. In regard to the increased B cell responses in these diseased TCR-α −/− mice, Th2 cytokines produced by CD4 + ββ T cells are responsible for the activation of auto- and food antigen–specific IgG, IgE, and IgA Ab production pathways 10 . IL-4 is a well-known switch factor for IgE and IgG1 induction 35 . In addition to auto- and food antigen-specific IgG1 and IgE responses, IgA Ab responses were also upregulated in TCR-α −/− mice with IBD 10 . A similar pattern of response was also observed in TCR-α −/− mice treated with mock Ab. The increased B cell responses in TCR-α −/− mice with IBD could be explained by our present finding that these CD4 + ββ T cells were capable of producing IL-5, IL-6, and IL-10 in addition to IL-4 . These Th2-type cytokines further support the production of Abs, including those of the IgA isotype, in TCR-α −/− mice. In fact, studies using both murine and human experimental systems have shown that IL-5, IL-6, and IL-10 are IgA-enhancing cytokines 20 21 . When TCR-α −/− mice were treated with anti–IL-4 mAb, the levels of all the isotypes of Abs were significantly decreased in their serum and fecal extracts. Inasmuch as our results demonstrated that anti–IL-4 mAb treatment altered the cytokine profile of CD4 + ββ T cells from a Th2 to a Th1 type , it is most likely that an abrogation of Th2 cytokine synthesis resulted in the reduction of B cell development. It is not yet known whether Abs against auto- and food antigens play a pathological role, much less by what means they might do so. Several reports have provided evidence that the levels of autoantibodies (autoAbs, e.g., anti-goblet cell, antitropomyosin, and anticolon autoAbs) were increased in the colon of humans suffering from IBD and have suggested that these autoAbs are the causative pathogens for the destruction of mucosal tissues by Ab-dependent cell-mediated cytotoxicity 40 41 42 43 44 . Therefore, one possible mechanism for the prevention of IBD by anti–IL-4 mAb could be the reduction of autoAb-producing B cell responses. However, a separate study demonstrated that the disease process accelerated in double knockout mouse, which lacked both Ig μ and TCR α chains and which were generated by crossing TCR-α −/− and Ig-μ–deficient mice 45 . It also showed that, in some cases, the removal of B cells worsened the symptoms of colitis. The results of that study suggest that Abs play a protective rather than a pathological role in the development of mucosal inflammation. It remains to be examined whether aberrant B cells and their derived Abs are involved in the initiation of the inflammatory process in IBD. IL-4 has been shown to directly regulate intestinal epithelial cell functions. For example, IL-4 can regulate the growth of epithelial cells. It is also capable of disrupting the barrier function of the intestinal epithelium, of enhancing the adherence of neutrophils to the epithelia, and of upregulating transepithelial neutrophil migration 46 . Overproduction of IL-4 in the intestinal epithelium may disrupt immunological homeostasis between the mucosal immune system and environmental antigens, including gut lumen microorganisms, allowing recruitment of inflammatory cells for the initiation of disease. Therefore, a second possible mechanism for the prevention of colitis development by mAb treatment might be the direct inhibition of IL-4 effects on the intestinal epithelial cells. IL-4–specific mAb treatment directly inhibited the development of Th2-type aberrant CD4 + ββ T cells, which are thought to be directly involved in the induction of IBD. For example, Th2-dominated conditions such as those characteristic of IFN-γ −/− mice have been shown to be conducive to the development of a delayed-type hypersensitivity reaction 47 48 , and Th2-type cells are thought to contribute to the development of autoimmune diseases 49 50 . Finally, a recent study from another group provided direct evidence that IL-4 produced by Th2-type CD4 + ββ T cells was associated with the development of murine colitis by showing the reduction of disease incidence in mice of the TCR-α −/− and IL-4 −/− double knockout models 51 . In summary, this study has demonstrated that the pathological ability of CD4 + ββ T cells to induce IBD is inhibited by their alteration from a Th2 to a Th1 type. It has also shown that the treatment of TCR-α −/− mice with anti–IL-4 mAb resulted in the blockage of colitis formation. | Study | biomedical | en | 0.999997 |
10477547 | BALB/c mice were obtained from Charles River Japan. BALB/c background recombination activating gene (RAG)2 −/− mice were donated by Dr. M. Ito (Central Institute for Experimental Animals, Kanagawa, Japan). OVA 323–339 -specific I-A d –restricted TCR-Tg mice (DO11.10) maintained on the BALB/c background were donated by Dr. K.M. Murphy (Washington University School of Medicine, St. Louis, MO ). All the mice were female and were used at 5–6 wk of age. IL-12 was donated by Genetics Institute. Anti–IL-12 mAbs (C15.1 and C15.6) were a gift from Dr. G. Trinchieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA). PMA, brefeldin A, recombinant murine IL-4, and antiasialo GM1 Ab were purchased from Wako Pure Chemical Industries, Ltd. Anti–IL-4 mAb (11B11) was purchased from American Type Culture Collection. PE–anti-CD4 mAb, peridinine chlorophyll protein (PerCP)–anti-CD4 mAb, FITC–anti-CD45RB mAb, FITC–anti-CD8 mAb, purified anti-CD3 mAb, purified anti–very late antigen (VLA)-4, purified anti–intercellular adhesion molecule (ICAM)-1 mAb, and recombinant mouse IFN-γ and anti–IFN-γ mAb (R4-6A2) were purchased from PharMingen. Anti–LFA-1 mAb was produced by our established KBA hybridoma clone 26 . OVA 323–339 peptide was supplied by Dr. H. Tashiro (Fujiya Co. Ltd., Hadano, Japan). CD4 + CD45RB + naive T cells were isolated from nylon-passed spleen cells from DO11.10 TCR-Tg mice using cell sorting (FACS Vantage™; Becton Dickinson) as described previously 27 . Purified CD4 + CD45RB + cells were stimulated with 10 μg/ml OVA 323–339 peptide in the presence of mitomycin C–treated BALB/c spleen cells, 20 U/ml IL-12, 1 ng/ml IFN-γ, 50 μg/ml anti–IL-4 mAb, and 20 U/ml IL-2 for Th1 development. Th2 cells were induced from the same naive Th cells in the presence of 1 ng/ml IL-4, 50 μg/ml anti–IFN-γ mAb, 50 μg/ml anti–IL-12 mAbs, and 20 U/ml IL-2. At 48 h, cells were restimulated with OVA 323–339 under the same conditions, and used at 9–12 d of culture. Th1 or Th2 cells (2 × 10 6 cells/well) were cultured in 12-well plates and stimulated with 2 μg/ml of anti-CD3 mAb or 20 ng/ml of PMA for 1–2 h. The ability of Th1 or Th2 cells to form homotypic aggregation was determined by counting the number of cell aggregates under the microscope as described previously 28 . The blocking effect of mAbs was determined by adding 50 μg/ml of each mAb into the culture. IFN-γ or IL-4 activities of culture supernatants were measured using ELISA kits (Nycomed Amersham plc). The IFN-γ activity was determined using Biotrak IFN-γ ELISA kits , and IL-4 activity was determined using Biotrak IL-4 ELISA kits . The mean of triplicate samples was calculated. For the detection of cytoplasmic cytokine expression, cells stimulated with immobilized anti-CD3 mAb for 6 h in the presence of brefeldin A were first stained with PerCP–anti-CD4 mAb, fixed with 4% paraformaldehyde, and treated with permeabilizing solution (50 mM NaCl, 5 mM EDTA, 0.02% NaN 3 , 0.5% Triton X-100, pH 7.5), then the fixed cells were stained with PE-conjugated anti–IL-4 mAb and FITC-conjugated anti–IFN-γ for 45 min on ice. The percentage of cells expressing cytoplasmic IL-4 or IFN-γ was determined by flow cytometry (FACSCalibur™; Becton Dickinson). A20 B lymphoma cells were transfected with chicken OVA cDNA, which was donated by Dr. M.J. Bevan, Research Institute of Scripps Clinic, La Jolla, CA 29 . Transfectants were designated as A20-OVA tumor cells. The cytotoxicity mediated by Th1- or Th2-dominant cells was determined by 4-hr 51 Cr-release assays as described previously 30 . A20 parental cells, A20-OVA cells, or OVA 323–339 peptide–pulsed A20 cells were used as target cells. The cytotoxicity (as a percent) was calculated by the method described previously 30 . A20-OVA cells (2 × 10 6 ) were intradermally inoculated into BALB/c mice. When the tumor mass became palpable (6–8 mm), Th1 or Th2 cells (2 × 10 7 ) were intravenously transferred into the tumor-bearing mice. The antitumor activity mediated by the transferred cells was determined by measuring changes over time of the means of two perpendicular diameters of the tumor mass. The mean of six mice per group was indicated. In all experiments, tumor-free mice were followed for >90 d. Th1 and Th2 cells were induced from CD4 + CD45RB + naive Th cells isolated from DO11.10 TCR-Tg mice, which recognize OVA 323–339 peptide (OVA-pep) bound on I-A d molecules. Th1 cells were generated from naive Th cells by culture with OVA-pep in the presence of IL-2, IL-12, IFN-γ, and anti–IL-4 mAb, whereas Th2 cells were derived from naive Th cells by culture with OVA-pep in the presence of IL-2, IL-4, anti–IFN-γ, and anti–IL-12 mAb. As summarized in Fig. 1 , 99% of the cells cultured under Th1 conditions for 10 d consisted of CD4 + T cells, and ∼70% of the cells expressed intracellular IFN-γ but not IL-4 . In contrast, 99% of the cells cultured under Th2 conditions for 10 d consisted of CD4 + T cells, and ∼60% of the cells expressed intracellular IL-4 but not IFN-γ . To apply these OVA-specific Th1 and Th2 cells to tumor immunotherapy, we generated I-A d –positive A20-OVA tumor cells expressing OVA antigen by transfection with the OVA gene. The A20-OVA tumor cells secreted OVA protein into the culture supernatant, which was detectable by ELISA (data not shown). A20-OVA cells stimulated OVA-specific Th1 and Th2 cells to induce the production of IFN-γ and IL-4, respectively . Moreover, Th1 cells lysed A20-OVA tumor cells, although Th2 cells exhibited a negligible cytotoxicity . Th1 cells appeared to lyse A20-OVA tumor cells mainly mediated by perforin, but not by TNF-α and the Fas/FasL pathway, because inhibition of granular exocytosis 30 caused almost complete inhibition of the cytotoxicity, whereas this blocking effect was not demonstrated by anti–TNF-α mAb plus anti-FasL mAb (data not shown). In some cases, Th2 cells showed low but significant cytotoxicity against A20-OVA, but this appeared to be derived from the contamination of IFN-γ–producing Th1 or Th0 cells (data not shown), because IFN-γ–nonproducing pure Th2 cell populations exhibited negligible cytotoxic activity . The same stimulatory effect against Th1 and Th2 cells was also demonstrated by OVA-pep–pulsed A20 tumor cells, but not untreated A20 tumor cells . These results demonstrated that the OVA peptide fragment from A20-OVA bound to I-A d and was able to stimulate class II–restricted OVA 323–339 -specific Th1 and Th2 cells to trigger their immunological functions. To determine the precise role of Th1 and Th2 cells in antitumor immunity, we designed a novel adoptive tumor immunotherapy model using OVA-specific Th1 and Th2 cells in conjunction with A20-OVA tumor-bearing mice. Fig. 2 A shows the experimental set-up for these experiments. BALB/c mice were inoculated with 2 × 10 6 A20-OVA cells, and when the tumor mass became palpable (6–8 mm), 2 × 10 7 Th1 or Th2 cells were transferred into the tumor-bearing mice. As shown in Fig. 2 B, the established A20-OVA tumor mass was completely rejected by adoptive transfer of OVA-specific Th1 cells, but not nonspecific Th1 cells, which were induced from splenic Th cells from wild-type BALB/c mice by activation with anti-CD3 mAb under Th1-inducing conditions. However, the growth of parental A20 tumor cells was not inhibited by transfer of OVA-specific Th1 cells . These results clearly demonstrated that this adoptive tumor immunotherapy model allowed us to investigate the role of antigen-specific Th cells in antitumor immunity. As shown in Fig. 3 A, both Th1 and Th2 cells exhibited strong antitumor activity in vivo and completely eradicated the tumor mass after adoptive transfer. All the mice cured from the tumor by Th1 or Th2 cell transfer were free of tumor for >90 d (data not shown). These experiments were repeated >10 times, and identical results were obtained. For the complete cure of tumor-bearing mice, the transfer of >10 7 cells was required for both Th1 and Th2 cell therapy (data not shown). Although both Th1 and Th2 cells exhibited antitumor activity in vivo, totally distinct processes of tumor rejection appeared to be included. The typical pattern of tumor growth or tumor rejection in control, Th1-transferred, or Th2-transferred mice is shown in Fig. 3 B. Interestingly, the tumor mass of mice that received Th1 cells gradually changed into a small, white mass and completely disappeared 7–10 d after cell transfer. In contrast, in tumor-bearing mice that received Th2 cells, the tumor mass changed to a red color 7–10 d after cell transfer, and a strong tumor necrosis was observed. This clear difference suggested that Th1 and Th2 cells eradicate tumors using distinct immunological mechanisms. To understand these distinct antitumor mechanisms, we studied the tumor tissue by histological analysis . Although no significant lymphocyte infiltration was observed in control tumor tissue , a marked lymphocyte infiltration was present in tumor tissue of mice that received Th1 cells . However, in mice that received Th2 cells, a marked infiltration of inflammatory cells such as eosinophils and neutrophils was observed around the center of the tumor mass . These results suggested that both Th1 and Th2 cells exhibited a strong antitumor activity in vivo, but that these cells used distinct tumor rejection mechanisms. Fig. 5 A shows that intravenous injection of anti-CD4 mAb, anti-CD8 mAb, or anti–IFN-γ mAb completely inhibited the therapeutic effect of Th1-mediated adoptive immunotherapy. These results demonstrated that Th1 cells by themselves are not enough to induce complete tumor eradication and that the interaction between Th1 cells and CD8 + T cells through cytokines such as IFN-γ may be essential to induce successful tumor rejection. The requirement for CD8 + T cells in Th1-cell therapy was confirmed by demonstrating that the therapeutic effect of Th1 cell transfer was not induced in DO11.10 TCR-Tg mice that have I-A d –restricted OVA-reactive CD4 + T cells, but not CD8 + T cells . To further extend these findings, we carried out adoptive transfer experiments using tumor-bearing RAG2 −/− mice that lack T, NKT, and B cells. A20-OVA tumors were inoculated into BALB/c background RAG2 −/− mice, and when these tumors became palpable, the mice received OVA-specific Th1 or Th2 cells. As shown in Fig. 6 , the growth of A20-OVA tumors in RAG2 −/− mice was initially strongly inhibited by both Th1 and Th2 cells, but the tumor cells finally grew out in both types of mice, although regrown tumor cells expressed OVA antigen and could stimulate cytokine production of Th cells (data not shown). Thus, a single transfer of Th1 or Th2 cells by itself cannot completely cure tumor-bearing mice. These results strongly suggested that OVA-reactive CD8 + T cells, activated in wild-type BALB/c mice early after tumor inoculation, play an important role in Th1- and Th2-mediated adoptive immunotherapy. To further investigate the role of CD8 + T cells in Th1 and Th2 cell therapy, OVA-reactive CD8 + T cells (2 × 10 5 cells) obtained from A20-OVA–immunized mice were transferred into RAG2 −/− mice 7 d before the experiment. The small number (2 × 10 5 cells) of CD8 + T cells transferred into RAG2 −/− mice revealed marked expansion in vivo, and they made up >10% of spleen cells of RAG2 −/− mice 1 wk after the cell transfer (data not shown). The transfer of CD8 + T cells alone did not inhibit the growth of A20-OVA tumor cells . However, when A20-OVA–bearing RAG2 −/− mice first received CD8 + T cells from A20-OVA–immunized mice and then received OVA-specific Th1 or Th2 cells, all of the mice were completely cured from the tumor. A significant but incomplete tumor growth inhibition was also observed when unprimed CD8 + T cells were transferred into RAG2 −/− mice . These results clearly demonstrated the critical role for antigen-specific CD8 + T cells in complete tumor eradication induced by adoptive transfer of antigen-specific Th1 or Th2 cells. Next, we examined the therapeutic mechanisms of Th1 and Th2 cell therapy in terms of acquisition of immunological memory beneficial for CTL generation. Mice cured from tumors by Th1 and Th2 cell therapy were rechallenged with A20-OVA tumor cells. Fig. 7 A shows that both types of mice rejected A20-OVA tumors, but were unable to reject syngeneic Meth A tumor cells (data not shown). These results indicated that mice cured from A20-OVA tumors had acquired immunological memory. However, these memory cells, elicited after restimulation with mitomycin C–treated A20-OVA cells, produced totally different patterns of cytokines and differed in their cytotoxic potential . Spleen cells from mice cured by Th1 cell therapy produced IFN-γ and revealed high cytotoxicity against A20-OVA but low cytotoxicity against A20 tumor cells . In contrast, spleen cells from mice cured by Th2 cell therapy produced high levels of IL-4 but little IFN-γ , and had little cytotoxicity against A20-OVA . Thus, these findings strongly suggested that immunotherapy using adoptive transfer of Th1 cells appeared to be more effective than Th2 cells for inducing immunological memory suitable for the generation of CTL response. It was also demonstrated that some of the cured mice from A20-OVA by Th1 cell therapy, but not by Th2 cell therapy, showed resistance against rechallenged parental A20 tumor cells (data not shown). Finally, we examined the role of distinct cell adhesion molecules in Th1 and Th2 cell therapy. While investigating the expression and function of adhesion molecules involved in cell migration, we found that Th1 cells, in response to stimulation with anti-CD3 mAb and phorbol ester, show strong LFA-1/ICAM-1–dependent homotypic adhesion . However, no significant cell aggregation was observed for similarly activated Th2 cells . This homotypic cell–cell aggregation was strongly blocked by anti–LFA-1 or anti–ICAM-1 mAb, but not by control rat Ig or anti–VLA-4 mAb (data not shown). From these results, we concluded that Th1 and Th2 cells have distinct capacity to use LFA-1/ICAM-1 cell adhesion interactions. This differential ability to form homotypic cell–cell aggregates did not result from a defect in LFA-1 or ICAM-1 expression, because both Th1 and Th2 cells expressed high levels of LFA-1 and ICAM-1 at the cell surface . To investigate whether LFA-1/ICAM-1 interactions are important for the induction of antitumor activity in vivo, we examined the effect of anti–LFA-1 mAb administration on Th1 and Th2 cell therapy. Mice were inoculated with A20-OVA tumor cells, and when tumors became palpable, mice were treated by intravenous injection of anti–LFA-1 mAb (500 μg/mouse) twice at days −1 and 0 before adoptive transfer of Th1 or Th2 cells. As shown in Fig. 5 E, the therapeutic ability of Th1 cells was completely abrogated by anti–LFA-1 mAb administration. However, no significant inhibitory effect by anti–LFA-1 mAb was observed for Th2-mediated antitumor activity . These results indicated that Th1 and Th2 cells showed distinct requirements for LFA-1–dependent cell–cell interactions in vitro and in vivo: LFA-1–dependent cell migration appeared to be critical for Th1-cell therapy but not for Th2-cell therapy. Several lines of evidence have indirectly demonstrated a role for the Th1/Th2 balance in antitumor immunity. First, the cytokine IL-12, which stimulates Th1-dominant immunity in vivo, was shown to have strong in vivo antitumor activity against a variety of tumors, including primary tumors 18 19 31 32 . Second, in vivo neutralization of IFN-γ caused the inhibition of the antitumor effect of IL-12, suggesting that IFN-γ–producing Th1 cells may play an important role in tumor rejection. However, this conclusion is weakened by the observation that other T cells, including NKT cells and CD8 + T cells, can produce IFN-γ in response to IL-12, and that these cell types can activate Th1-dominant immunity 33 34 . Indeed, it was recently demonstrated that the antitumor activities of IL-12 are mediated, in large part, by NKT cells 34 . Moreover, the finding that Th2-derived cytokines (IL-4, IL-5, IL-6, IL-10) show antitumor activities in vivo that are as strong as the antitumor activities of Th1 cytokines 20 21 22 23 35 has made it difficult to conclude which cell type is the most effective for eliciting complete tumor regression in vivo. To address this issue, we designed a new adoptive tumor immunotherapy model using tumor antigen–specific Th1 or Th2 cells. As tumor cells, we used OVA gene–transfected tumor cells, and Th1 and Th2 cells were induced from mice transgenic for an OVA-specific TCR. Our results demonstrated that both Th1 and Th2 cells show potent antitumor activities in vivo . Interestingly, Th1 cells induced a marked lymphocyte infiltration into the tumor mass and eradicated the tumor mass via cellular immunity. In sharp contrast, Th2 cells induced inflammatory responses at the tumor site and induced tumor necrosis . The finding that Th2 cells, which can produce high levels of IL-4, induced the inflammation characterized by eosinophils and neutrophils is consistent with previous results with IL-4 gene–transfected tumors, which were rejected by inflammatory cells that included eosinophils and neutrophils 22 36 . However, it remains unclear how Th2 cells induce tumor necrosis. Another important difference between Th1 and Th2 cell therapy is that Th1 therapy was able to induce a strong immunological memory suitable for the generation of CTLs, whereas Th2 cells did not induce the immunological memory for CTL generation very well . Some of the cured mice from A20-OVA by Th1 cell therapy but not Th2 cell therapy showed resistance against rechallenged parental A20 tumor cells (data not shown), indicating that Th1 cell therapy might also be beneficial for the generation of CTLs, which recognize unknown tumor-rejection antigen expressed on parental A20 tumor. Based on our observation that in vivo administration of anti-CD4 mAb, anti-CD8 mAb, or anti–IFN-γ mAb blocked the therapeutic effect of Th1 cells against tumors , we suggest that transferred Th1 cells migrate into local tumor sites, produce IFN-γ, and facilitate the induction of antitumor CD8 + CTLs in vivo. The requirement for CD8 + T cells in Th1 cell therapy is also demonstrated by our finding that DO11.10 TCR-Tg mice that have OVA-reactive CD4 + T cells, but not CD8 + T cells, were unable to permanently clear the tumor by Th1 cell therapy . Direct evidence for the requirement for CD8 + T cells was demonstrated using adoptive transfer of CD8 + T cells into RAG2 −/− mice . This experiment also indicated that NKT cells are not involved in Th1- and Th2-induced antitumor activity in vivo because NKT cells are not differentiated in RAG2 −/− mice, which are deficient in NKT cells, mainstream T cells, and B cells. Th2 cells contained <2% of IFN-γ–producing cells , which might exhibit negligible cytotoxicity against A20-OVA . Indeed, in some cases, Th2 cells exhibited low but significant cytotoxicity against A20-OVA in parallel with the increased production of IFN-γ. However, even in such cases, the IFN-γ produced by contaminating cells (Th1 or Th2) appeared not to be involved in the triggering of antitumor activity of Th2 cells, because administration of anti–IFN-γ mAb caused no significant blocking of the Th2-mediated therapeutic effect (data not shown). In mice that were cured from A20-OVA tumors by Th2 therapy, A20-OVA–specific CTLs were not detected. These findings suggest that antigen-nonspecific CD8 + killer T cells are involved in Th2-mediated adoptive immunotherapy. Alternatively, CD8 + TC2 cells 33 induced by IL-4 may contribute to tumor eradication in Th2 cell therapy. Since the immunological memory in mice cured by Th2 cell transfer may be mediated by humoral immunity, we are currently investigating whether Th2 immunological memory can be transferred into normal mice by serum isolated from tumor-cured mice. The distinct antitumor immunity mediated by Th1 and Th2 cells may be due to the distinct cell adhesion interactions involved in the migration of these cells into tumor tissues across endothelia. Consistent with previous results 37 , we found that Th1 cells express higher levels of P-selectin ligands and produced higher amounts of chemokines compared with Th2 cells (data not shown). In addition, we found that Th1 cells exhibit strong LFA-1/ICAM-1–dependent cell–cell interactions , which are critical for lymphocyte activation, cell-mediated cytotoxicity, and transmigration of lymphocytes into inflammatory tissues 38 39 . In contrast, Th2 cells were defective in LFA-1/ICAM-1–mediated cell–cell interactions , but were able to interact with the extracellular matrix on endothelia through the integrin αVβ3 (data not shown). These results suggest that Th1 cells express adhesion molecules that facilitate transmigration into tumor tissues across the tumor vessels. Indeed, antitumor therapeutic activity of Th1 cells was completely blocked by administration of anti–LFA-1 mAb, whereas the activities of Th2 cells were not affected by anti–LFA-1 mAb injection . From these results, we speculate that, at the tumor local site, Th1 cells actively respond to tumor cells and produce cytokines, which recruit other effector cells such as CD8 + T cells, NKT, or NK cells into the tumor tissue. In contrast, Th2 cells, which are unable to enter tumor tissue because of a defect of adhesion mechanisms, may accumulate on the endothelial cells around the tumor mass and induce tumor necrosis via molecules such as TNF-α that damage tumor vessels 40 . However, we have recently demonstrated that in vivo administration of anti–IL-4, anti–IL-10, or anti–TNF-α was unable to block the tumor necrosis induced by Th2 cell therapy (data not shown). Therefore, unknown mechanisms appear to be involved in Th2-induced tumor necrosis. One possibility would be that Th2-derived cytokines activated other inflammatory cells and the products of these cells damage endothelial cells to induce tumor necrosis. This hypothesis is strongly supported by recent findings by Hung et al. 41 that CD4 + T cells play an important role in inducing antitumor activity in vivo through activation of eosinophils and macrophages that produce superoxide and nitric oxide. The finding that transfer of >10 7 Th1 or Th2 cells with CD8 + T cells is required for the complete rejection of tumor (data not shown) means that, at an early phase of tumor rejection, the bursting of a strong cytokine storm derived from Th1 or Th2 cells may be essential for overcoming a strong suppression in the tumor-bearing host and for induction of CD8 + CTL–mediated antitumor protective immunity in tumor-bearing mice. The present data demonstrate that Th1 and Th2 cells use distinct tumor eradication mechanisms. However, based on the following considerations, Th1 cells may be more suitable for adoptive tumor immunotherapy in the future: (a) Th1 cell therapy, but not Th2 cell therapy, induces strong immunological memory beneficial for CTL generation ; (b) Th2 cells produce high levels of IL-6, which can contribute to cachexia in late stage tumor-bearing hosts 42 ; and (c) in our experience, IFN-γ–producing Th1 cells are easily expanded from total spleen or peripheral blood cell populations, while it is hard to induce pure Th2 cells producing IL-4 but not IFN-γ from total spleen or peripheral blood cells in humans and mice (data not shown). In a previous report 43 , we demonstrated that culture of tumor-infiltrating lymphocytes (TILs) with IL-2 plus IL-12 results in a profound increase in the development of autologous tumor-reactive CTLs. Moreover, we showed that this protocol enhanced the generation of autologous tumor-reactive Th1-dominant cells (our unpublished data). Therefore, if we can develop a large scale culture system for the generation of autologous tumor-reactive Th1 cells from TILs or PBLs of tumor patients, the adoptive tumor immunotherapy using tumor-specific Th1-dominant cells may be possible. The cytokine IL-12 shows great promise for the development of tumor immunotherapy 18 19 31 32 . However, recent findings have demonstrated that IL-12 also has adverse effects owing to overstimulation of Th1-dominant immunity 44 . In terms of side effects, adoptive transfer of in vitro IL-12–activated Th1-dominant cells may minimize side effects, and could make the management of side effects easier. Thus far, IL-12 has been suggested for cytokine therapy and gene therapy of cancer. This paper further indicates that IL-12 may be a useful tool for application to a novel tumor immunotherapy protocol using the adoptive transfer of Th1-dominant T cells and/or CTLs (Th1 helper/killer therapy). | Study | biomedical | en | 0.999998 |
10477548 | LTα −/− mice (backcrossed to C57BL/6 mice for seven generations) and their wt littermates on a C57BL/6 background were bred under specific pathogen-free conditions as described 6 . LTβR −/− mice were provided by Dr. Klaus Pfeffer (Technical University of Munich, Germany) 8 . TCR −/− , BCR −/− , RAG-1 −/− , TNFR-I −/− , and TNF −/− mice as well as CD3∈-transgenic mice were purchased from The Jackson Laboratory. B6-Ly5.1 mice were purchased from Frederick Cancer Center, National Cancer Institute, Bethesda, Maryland. Animal care and use were in accordance with institutional guidelines. Splenic DCs were treated and collected basically according to the method developed by Inaba et al. 24 . In brief, spleen fragments were digested with 2 mg/ml of collagenase and 100 μg/ml DNase for 30 min at 37°C and then gently pipetted in the presence of 0.01 M EDTA for 1 min. Single-cell suspensions were stained and analyzed by two-color flow cytometry on a FACScan™ (Becton Dickinson). Biotinylated anti-CD11c and CD11b (Mac-1), FITC-conjugated anti–I-Ab, anti-CD11c, and anti-CD8α antibody were all obtained from PharMingen. Spleens were harvested, embedded in OCT compound (Miles-Yeda, Inc.), and frozen at −70°C. Frozen sections (6–10 μm thick) were fixed in cold acetone. Endogenous peroxidase was quenched with 0.2% H 2 O 2 in methanol. After washing in PBS, the sections were stained by first incubating with FITC-conjugated anti-B220 for B cells and biotinylated anti-CD11c for DCs (PharMingen) at 1:50–100 dilution. Horseradish peroxidase–conjugated rabbit anti–FITC (DAKO Corp.) and alkaline phosphatase–conjugated streptavidin (Vector Labs., Inc.) were added 1 h later. Color development for alkaline phosphatase and horseradish peroxidase was performed with an alkaline phosphatase reaction kit (Vector Labs., Inc.) and with 3,3′-diaminobenzidine (Sigma Chemical Co.). Anti-LTβ antibody and some aspects of the control LTβR–Ig fusion protein used in this study have been previously described 4 . The method for the generation of LTβR–Ig fusion protein was used as previously described with a minor modification 4 . In brief, cDNA encoding the extracellular domain of murine LTβR was isolated by RT-PCR using the sense primer (5′-AAAGGCCGCCATGGGCCT-3′) and the antisense primer (5′-TTAAGCTTCAGTAGCATTGCTCCTGGCT-3′) from mouse lung mRNA, digested by NcoI/HindIII, and then fused to an IL-3 leader sequence in p30242 vector. The fusion fragment was then subcloned into pX58 vector containing the IE-175 promoter and the Fc portion of human IgG1, which was then transfected into BHK/VP16 cells. The mouse LTβR–human Ig in culture supernatants was purified on a protein A column. No difference can be found between LTβR–human Ig in this preparation and a previous LTβR–Ig preparation in Chinese hamster ovary cells 4 . To block membrane LT activity in mice, the LTβR–Ig or anti-LTβ antibody (50–100 μg/injection) was given intraperitoneally, and the number of DCs was determined 10–14 d later by either flow cytometry or immunohistology. Bone marrow (BM) cell or splenocyte transfer was performed as previously described 12 . In brief, BM-derived DCs (BMDCs) from Ly5.1 mice were obtained by culturing BM cells with GM-CSF (5 ng/ml) and IL-4 (2 ng/ml) according to the procedure developed by Inaba et al. 25 . BMDCs (5 × 10 6 ) or splenocytes (5 × 10 7 ) were intravenously transferred into sublethally irradiated recipient mice (600 rads). Spleens and LN cells were collected for analysis within 24 h after transfer. As stimulating cells, splenocytes from wt or LTα −/− mice were isolated by gentle pressure through a cell strainer (Becton Dickinson), or spleen fragments were treated with collagenase as described earlier 24 . The stimulating cells were irradiated at 2,000 rads. The LN cells from BALB/c mice were collected by gentle pressure using a cell strainer and cultured in a petri dish for 2 h. The nonadherent LN cells were then harvested and used as the source of responding cells. The different amounts of stimulating cells as indicated and 4 × 10 5 responding cells were cocultured for 72 h, and [ 3 H]TdR at 1 μCi/ml was added during the last 18 h. TNF can promote the growth of DCs in vitro 15 16 . To assess the role of TNF in the development of DCs in vivo, splenocytes from TNF −/− and wt mice were stained for CD11c and MHC class II (I-A b ), and the number of DCs in the preparation was determined by flow cytometry. The total number of DCs in both types of mice was similar, suggesting that TNF is not essential for the development of DCs . Interestingly, the number of DCs in LTα −/− mice was greatly reduced, especially for the CD11c high class II high subset , suggesting a role for LTα in DC development. Soluble LTα and TNF-α are structurally related homotrimers (LTα 3 and TNF-α 3 ) that exhibit similar biological activities by binding to the defined TNFRs 1 , so TNFR −/− mice were used to determine the role of TNFR in DC development . However, the normal number of DCs in the spleens of TNFR −/− mice suggests that signaling via TNFR by either LTα 3 or TNF-α 3 is not essential for the presence of DCs in the spleen. CD11c + DC subsets preferentially migrate to distinct areas in the spleen 18 19 : myeloid DCs (CD8α − /CD11b + ) are mainly located in the marginal zones (MZs) of white pulp, whereas lymphoid DCs (CD8α + /CD11b − ) are preferentially located in the T cell zones of white pulp. To study whether LT or TNF preferentially regulates a subset of DCs, the distribution of DCs and B cells in the spleens of TNF −/− mice and LTα −/− mice was visualized histologically . Clusters of splenic DCs were readily observed in the T cell zone and MZ of wt and TNF −/− mice; however, only a few dispersed DCs were randomly present in the spleens of LTα −/− mice. The distribution pattern and number of DCs visualized in situ closely correlated to that measured by flow cytometry, which showed that both myeloid and lymphoid DCs were proportionally reduced in LTα −/− mice . Considering that myeloid and lymphoid DCs may be distinct populations of DC subsets 18 19 , it is interesting to notice that the presence of both subsets was regulated by LT. LTα −/− mice lack both soluble LTα 3 and membrane-associated LTα 1 β 2 , which bind to separate receptors, TNFR and LTβR, respectively 1 2 . As the number of DCs in TNFR-I −/− mice was similar to that in wt mice , it was possible that membrane LTα 1 β 2 , instead of soluble LTα 3 , was required for the presence of DCs in the spleen. To test this hypothesis, LTβR–Ig was used to block membrane LT activity in wt adult mice, which resulted in the absence of FDCs in 1 wk. Interestingly, the number of DCs but not lymphocytes in the spleens was markedly reduced 10 d after the administration of a single dose of LTβR–Ig . Moreover, the distribution pattern of the remaining DCs in the spleen was similar to that in LTα −/− mice. Expression of LT has been detected primarily in activated T, B, and NK cells 1 2 . However, the percentage of DCs in the spleen of TCR −/− BCR −/− CD3∈-transgenic mice or RAG-1 −/− mice is not obviously reduced (data not shown). In fact, the percentage of DCs in the splenocytes of RAG1 −/− mice is three- to fourfold higher than that of wt mice . This suggests that the development of DCs could be independent of LT expression on T and B cells. To rule out whether the DC development observed in RAG-1 −/− mice might be occurring via an LT-independent pathway, RAG-1 −/− mice were treated with LTβR–Ig for 10 d . A significant reduction of splenic DCs (60–90% reduction) was readily detected, demonstrating that LT-expressing cells other than T and B cells control the migration of DCs ( Table ). Although NK cells in RAG1 −/− mice were plausible candidates for regulating DC migration in an LT-dependent pathway, RAG-1 −/− mice depleted of NK cells (with 300 μg of PK136, an anti-NK1.1 antibody) did not exhibit reduced numbers of splenic DCs. Consistent with this data, no reduction of DCs was detected in CD3∈-transgenic mice lacking both NK and T cells. It is likely that cells other than T, B, and NK cells also express low levels of LT, regulating the migration of DCs. Murine LTβR–Ig may block ligands other than membrane LT. It has been shown that human LTβR–Ig can also bind to human LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a recently identified membrane-associated TNF family member 26 . The biological consequence of this binding is unclear. To exclude the potential effect of LIGHT, an anti–murine LTβ mAb, which specifically binds to the LTβ chain but not LIGHT, was administered to wt mice. Such treatment also resulted in a reduced number of DCs and their subsets similar to the effect of LTβR–Ig . Our data clearly indicate that LTα 1 β 2 is the ligand required for the presence of DCs in the spleen. As ligands from the TNF family can bind to more than one receptor, the number of splenic DCs in LTβR −/− mice was determined to directly address whether signaling via LTβR is required for the presence of DCs in lymphoid tissue. The number of DCs in these mice was also lower than in wt mice . Thus, the data strongly suggest that signaling via LTβR by membrane LT is essential for the presence of DCs in the spleen. Fewer DCs in the lymphoid tissues of mice lacking LT may be related to a reduction of DC progenitors in BM, impaired migration, or an accelerated removal of these cells. To test whether there was a deficiency in DC progenitors or the growth of DCs in LTα −/− mice, BM cells from either wt or LTα −/− mice were cultured by standard protocol using different doses of GM-CSF and IL-4 25 . The number of DC colonies and total number of DCs was comparable between wt and LTα −/− mice. In addition, the number of DC colonies from wt mice was not altered by coculture with LTβR–Ig (data not shown). Together, the data suggest that LT is not an essential survival factor or growth factor for DCs or their progenitors. It has recently been shown that LT and, to lesser degree, TNF stimulates stromal cells to release chemokines, which may determine the migration or segregation of T and B cells in the spleen 16 . It is possible that the migration of DCs into lymphoid tissues of LTα −/− mice is impaired due to the lack of LT-mediated chemokines for DCs. If the migration of DCs into lymphoid tissues is impaired in LTα −/− mice, the question would be where DCs accumulate in the absence of LT. If the BMDC development remains functional in the absence of LT, we would expect that the reduced number of DCs in lymphoid tissues in the absence of LT might be associated with an increased number of DCs in nonlymphoid tissues. Interestingly, there is an accumulation of lymphocytes around perivascular areas in lungs, liver, pancreas, submandibular glands, kidneys, and other tissues in LTα −/− , LTβ −/− , and LTβR −/− mice 7 8 9 . To test whether the number of DCs was also increased in nonlymphoid tissues, DCs in lungs were quantitated in wt and LTα −/− mice. In contrast to the reduced number of DCs in lymphoid tissues, the number of DCs in lungs of LTα −/− mice was much higher than in wt mice (10.5 ± 1.8 × 10 5 vs. 2.9 ± 1.3 × 10 5 ). This suggests that LT is required for the proper distribution of DCs. To directly study whether the migration of DCs into the spleen was impaired in LTα −/− mice, DCs expanded from the BM of Ly5.1 wt mice were transferred into LTα −/− and C57BL/6 mice (Ly5.2), respectively. The number of Ly5.1 DCs recovered from the spleens of wt mice was two- to fourfold higher than that from LTα −/− mice, although both groups received similar numbers of DCs from the same source . Ly5.1 + CD11c − donor cells, mainly macrophages, in both groups were roughly the same . As the number of splenic DCs in wt mice was not reduced within the first week after administration of a high dose of LTβR–Ig, it is unlikely that transfer of Ly5.1 DCs into LTα −/− mice leads to the premature death (<24 h) of these DCs. It is possible that the splenic environment in LTα −/− mice did not allow the efficient sequestration or migration of DCs. The splenic environment essential for the localization of DCs may include its architecture, the size and shape of white pulps, and cytokines, such as chemokines, produced from the spleen. Altered splenic architecture and smaller white pulp in LTα −/− mice are readily visualized defects that may structurally impair the migration of splenic DCs into the proper area. However, short-term blockage of membrane LT by LTβR–Ig in wt mice had no detectable impact on the architecture or size of white pulps, yet this treatment still prevented the effective migration of DCs into the T cell zone and B cell follicles . This suggests that altered architecture itself is not the primary cause of reduced migration of DCs into the spleens of LTα −/− mice. Interestingly, the altered T/B cell segregation correlated with the altered localization of DCs and with altered chemokine production in the absence of LT 16 . To study whether additional membrane LT can restore the localization of DCs in the spleens of LTα −/− mice, we transferred LT-expressing lymphocytes and DCs from wt mice into LTα −/− mice. The altered splenic architecture remained, but the number of CD11c + cells in LTα −/− recipients was comparable to that in wt recipients 10 d after transfer , again suggesting that the overall architectural defect in LTα −/− mice may not be the primary cause of reduced number DCs in the spleen. It appears that the microenvironment in the spleen required for the presence of DCs is rather flexible and can be altered in 1–2 wk. Interestingly, the timing of the reduction of DCs is also consistent with the maximum reduction of various chemokines in the spleen 1–2 wk after administration of LTβR–Ig 16 . Thus, the data suggest that the reduced number of DCs in LTα −/− mice may be due, at least in part, to the impaired migration of DCs that may be mediated through altered chemokine production. The nature of the LT-responsive stromal cells and the exact type of chemokines remains to be determined. BM transfer in long-term reconstitution provides a model to evaluate the role of LTα in determination of the splenic microenvironment that permits the migration of DCs. 6 wk after lethally irradiated LTα −/− mice were reconstituted with wt BM, DCs were restored to a level similar to that seen in irradiated wt mice reconstituted with wt BM . This suggests that the altered microenvironment that impairs the migration of DCs is not developmentally fixed and that LT-expressing BM cells could restore the migration of DCs. In contrast, when lethally irradiated wt mice were reconstituted with LTα −/− BM, the number of DCs in the spleen was reduced, as is seen in LTα −/− mice or LTβR–Ig-treated mice . Therefore, the LTα-mediated microenvironment that permits the migration of DCs is primarily determined and maintained by LT-expressing BM-derived cells. To examine whether reduced numbers of DCs in lymphoid tissues of LTα −/− mice could impair the overall function of DCs, the ability of DCs in LTα −/− mice to stimulate allogenic T cells was evaluated by mixed leukocyte reaction (MLR). Mechanically separated splenocytes from LTα −/− mice showed a decreased ability to stimulate allogenic T cells in a dose-dependent manner . To rule out the possibility that reduced antigen-presenting activity in the splenocytes of LTα −/− mice is associated with the failure to release DCs from altered architecture of the spleen using physical separation, spleen fragments from both LTα −/− mice and wt mice were subjected to collagenase digestion to release DCs. The collagenase-treated splenocytes from LTα −/− mice showed profound defects (four- to eightfold lower) in antigen-presenting activity compared with those from wt mice, especially when the total splenocytes was in the range of 0.2–1 × 10 5 cells . To exclude the impact from either of the developmental defects in LTα −/− mice, the splenocytes from LTβR–Ig-treated C57BL/6 mice were collected by mechanical pressure and used as stimulators. Severalfold reductions of radiation count were readily detected in the LTβR–Ig-pretreated group, as in the case of LTα −/− mice . In general, the lower MLR closely correlated with the lower number of DCs . The number of other potential APCs, such as B cells, in the spleens of LTα −/− mice or mice treated with LTβR–Ig appears to be comparable to that in wt mice. It was proposed a decade ago that DCs are the principal stimulators of MLR in the spleen 27 28 ; our results further support the proposal, as reduced numbers of DCs in LTα −/− mice could account for the impaired MLR. Our results have revealed that membrane LT and LTβR are the natural ligand–receptor pair essential for the presence of splenic DCs in vivo. LTα −/− mice exhibit reduced numbers of DCs in the spleen, whereas both TNF −/− and TNFR −/− mice show normal numbers of splenic DCs, suggesting that signaling via TNFR by either soluble LTα or TNF is not an essential pathway for the regulation of DCs in the spleen. The notion that membrane LT is an essential ligand for the presence of DCs in the spleen is further supported by the reduced number of DCs in the wt spleen after the administration of either LTβR–Ig or anti-LTβ mAb. The results also suggest that signaling via LTβR by membrane LT is required for the presence of DCs, as LTβR is the only identified receptor for membrane LT. Finally, the lower number of splenic DCs in LTβR −/− mice confirms our hypothesis. In terms of the regulation of development or migration of DCs in the spleen, an essential role of either soluble LTα 3 or TNF-α 3 has not been demonstrated. However, TNF-α 3 or LTα 3 can coordinate membrane LTα 1 β 2 in the development of lymphoid tissues 2 10 and also may play a minor role in the migration of DCs in some situations. Interestingly, recent studies reported that high levels of soluble LTα 3 were able to induce chemokines and adhesion molecules in vitro 29 . Ectopic expression of LTα 3 induces lymphocyte infiltration in nonlymphoid tissue, suggesting that the overexpression of LTα 3 may still play a role in the migration of some lymphoid cells 30 31 32 . Ectopic LT in LTα −/− (RIPLT.LTα −/− ) mice also restored some LN, but a decreased number of interdigitating DCs was apparent in the LN 31 . Therefore, proper expression of LT in the LN may also be required for the presence of DCs in the LN. The ineffective migration of DCs may account for the reduced number of DCs in the spleens of mice lacking membrane LT or its receptor: (a) compared with wt recipients, fewer donor DCs were present in the spleens of LTα −/− recipients; (b) a reduced number of DCs is not developmentally fixed and can be repaired by LT-expressing cells; (c) the timing of altered numbers of DCs is consistent with the altered expression of chemokines in the spleen; (d) no significant impairment of DC growth or reduced DC progenitors can be detected; and finally, (e) DCs accumulate in nonlymphoid tissues in both LTα −/− and LTβR −/− mice, strongly supporting our notion that the reduced number of DCs in the spleen is caused by impaired migration. Interestingly, fewer randomly distributed DCs in the spleens of LTBR–Ig-treated mice could still move to the T cell zone after intravenous injection of LPS, suggesting that fine positioning of DCs in the spleen could be regulated in an LT-independent fashion. A number of chemokines are constitutively secreted in the lymphoid organs in an LT-dependent fashion 16 . Altered distribution of T cells, B cells, and DCs in vivo may be regulated by some chemokines. Whether proper distribution of DCs and FDCs will facilitate T/B cell segregation remains to be determined. Although the expression of several chemokines has been found to be downregulated in the absence of LT, the exact chemokine that is essential for the migration of DCs has yet to be identified. Which chemokines are upregulated for directing DCs into nonlymphoid tissues in the absence of LT is completely unknown. Interestingly, the migration of most subsets of macrophages in the spleen is largely unchanged in the absence of LT , suggesting that the chemokines that regulate the distribution of DCs may be distinct from those that regulate the distribution of macrophages. It will be important to determine whether the differences in the migration patterns of macrophages and DCs may account for differences in their biological activities. In addition to the action of LT on stromal cells, it is also possible that direct signaling via LTβR on DCs by membrane LT is required for the migration of DCs in the spleen. Reduced numbers of DCs may account for reduced MLR, which is a DC-based T cell response. However, migration of DCs into lymphoid tissues for systemic immune responses may be more important for the generation of immune responses in vivo. In fact, after capturing antigens outside lymphoid tissues, DCs must migrate into lymphoid tissues to prime rare antigen-specific lymphocytes, which constantly recirculate through peripheral lymphoid tissues 18 19 . Regulation of the migration of DCs may provide an additional means to manipulate immune responses, T cell responses in particular. Consistent with that notion, we have found that inhibition of membrane LT has profound effects in several T cell–based disease models. For example, administration of LTβR–Ig reduced severity of colitis 17 , collagen-induced arthritis, and experimental autoimmune encephalitis (J.L. Browning, unpublished observation). Clearly, the membrane LT/LTβR system provides an interesting model to further study DC biology and DC-mediated diseases. | Study | biomedical | en | 0.999997 |
10477549 | The following three strains of mice were constructed from our BALB/c-based Tgic lines: AM14Vh/B6/lpr (H chain Tgics), AM14Vκ/B6lpr, and AM14Vκ/B6/lpr/IgH a (L chain Tgics). These were derived by continuous backcrossing to either B6/lpr (originally obtained from The Jackson Laboratory) or B6/lpr/IgH a mice 7 (a gift of Dr. Robert Eisenberg, University of Pennsylvania, Philadelphia, PA). B6/lpr and B6/lpr/IgH a mice were also maintained by intercrossing at Yale University. At each generation, Tgic mice were identified by PCR (see below) for breeding to the next generation. At BC1, mice were typed for homozygosity for the Fas lpr mutation by PCR 5 , which was confirmed at BC2. The IgH a genotype was also confirmed at BC1 by an allele-specific PCR assay for IgG2a a versus IgG2a b . From BC4 (97% B6 genes) and beyond, AM14Vh/B6/lpr mice were crossed with AM14Vl/B6/lpr mice to create Ag − double-Tgic controls (HL b mice) or to AM14Vh/B6/lpr/IgH a mice to create Ag + double-Tgic experimental mice (HL ab ). All other transgenotypes (H, H chain only; L, L chain only; and N, non-Tgic) were also obtained in these crosses and were analyzed as additional controls (see Results). Age-matched IgH b or IgH ab mice 40 that were wild type at the Fas locus were available on the BALB/c background and were analyzed as controls. All mice were housed in the same room in our specific pathogen–free barrier colony. PCR to genotype for H and L Tgs, IgH genotype, and the lpr mutation was performed as described 40 . PCR to genotype the IgG2a locus was performed as previously described 46 . The selection, preparation, and labeling of antibodies was as described 40 41 . Anti-CD3–biotin was obtained from PharMingen. These were performed essentially as described 41 with the following modifications. Spleens were harvested, weighed, and then divided, with a portion being quick-frozen in OCT for later immunohistochemical analysis. The other portion was weighed again and then processed into a single-cell suspension. Red cells were then lysed with ammonium chloride/Tris solution. Cell number was determined by counting in a hemocytometer, and a corrected total number of cells in the spleen was derived by considering the fraction by weight of the total spleen that was used to create the cell suspension. In most experiments, cells were preincubated with saturating concentrations of 2.4G2 (rat anti–mouse FcR) to block nonspecific binding. Stained cells were analyzed on a FACSCalibur™ (Becton Dickinson Immunocytometry Systems). When possible, live gating using propidium iodide was used to exclude dead cells. When four labeled mAbs were used, forward and side scatter profiles were used to exclude most dead cells and RBCs. At least 30,000 events were collected for two- and three-color analysis, and 50,000 events were collected for four-color analysis. The assays were performed as described 41 . Data were not distributed normally, mandating the use of Mann-Whitney nonparametric tests (two-tailed) to compare groups. Tests were performed using StatView 4.5 (Abacus Concepts, Inc.). Age-matched cohorts of AM14 Tgic mice were established on the B6/lpr (IgH b ) and B6/lpr/IgH a backgrounds. As these were generated by intercrossing H and L single-Tgic mice, all possible genotypes were created, among which H, HL, and N were extensively studied. In addition, similar age-matched cohorts were generated on the BALB/c (IgH a ) and congenic CB.17 (IgH b ) backgrounds. These strains were available as nonautoimmune, Fas-sufficient controls. Mice were allowed to age to 4–7 mo to allow spontaneous autoimmunity to develop, at which point they were killed and analyzed as described below. The number of total splenocytes was greater in the HL ab Tgics (which had the AM14 autoAg) compared with the corresponding HL b Tgic mice, which lacked the autoAg . Interestingly, a similar difference was observed in the H chain Tgic mice . This was not due to differences between B6/lpr and B6/lpr/IgH a per se, as no such difference was seen in the N controls ( Table ; P = 0.53). Notably, this difference was also not observed in the comparison between BALB/c and CB.17 HL Tgics of similar age ( P = 0.76). Percentages and numbers ( Table ) of AM14 Id + B cells were greater in the HL ab Tgic mice compared with HL b mice. Again, H chain Tgic mice were similar in this regard. Indeed, many H ab mice had substantial percentages of Id + B cells, whereas this was only rarely observed among H b mice. Such cells are detectable only at low frequency in young (reference 40) or old BALB/c or CB.17 H Tgics (not shown). AM14 Id + cells in H ab Tgics presumably represent cells with endogenous Vκ8 L chains that are identical to or resemble the germline-encoded Tgic Vκ8. This interpretation is supported by the fact that among LPS hybridomas isolated from BALB/c H chain Tgics, both of the Id + cell lines that were isolated had the same endogenously derived Vκ8 sequence as the Tg L chain (Shlomchik, M., unpublished data). If indeed these Id + cells in H-only mice reconstitute the RF specificity of the original AM14 HL pair, finding higher percentages in the H ab mice compared with the H b mice would be consistent with an active, autoAg-driven process in which rare RF B cells are substantially expanded. RF ELISpot data (see below) is also consistent with this interpretation; however, confirmation of it will require isolation of the cells from H ab mice and sequencing of their L chains. Larger numbers of AM14 Id + cells in mice that express the autoAg (i.e., when AM14 is an autoAb) suggests that spontaneous activation may be occurring in this setting. This is indeed the case, as demonstrated by expression of CD44 47 48 and by the presence of Id + ELISpots, which reflect differentiation into plasma cells. Fig. 2 shows CD44 expression in AM14 HL and H Tgics of both IgH a and IgH b allotypes. Many of the IgH a mice had a substantial fraction of CD44 hi /Id + cells, a phenotype only rarely observed in IgH b mice. These data are summarized over the entire cohort in Fig. 2 B as the ratio of CD44 hi /CD44 lo Id + cells. There is a statistically significant difference in this measure between the IgH a and IgH b mice of both HL and H genotypes. A similar comparison of IgH a and IgH b mice on the BALB/c background did not reveal any differences, suggesting that accumulation of spontaneously activated RF B cells is dependent on Fas deficiency. Another measure of activation is differentiation into high rate Ab-secreting cells, which we measured by ELISpot. Similar to the CD44 data, a substantial proportion of both HL ab and H ab mice had high numbers of Id + ELISpots . The median difference between IgH a and IgH b mice was 16-fold for HL Tgics and 407-fold for H Tgics ( P = 0.002). Again, there were no significant differences between BALB/c and CB.17 mice. To directly demonstrate this, on a subset of H chain mice, we measured RF ELISpots in both IgH a and IgH b B6/lpr mice. RF spots were much higher in most H ab than in H b mice ( P = 0.009). These findings directly demonstrate that in H a mice, concurrent with the expansion and differentiation of Id + cells, RF B cells are also expanded. In combination with our other observations, this suggests that most of the Id + B cells are RF B cells as well. To confirm the identification of AM14 Id + B cells and antibody-forming cells (AFCs) by FACS™ and ELISpot and to identify their locations in vivo, frozen sections of spleens from representative Fas-deficient animals (HL or H) were prepared and stained for CD3 and Id expression . Spleens from age-matched HL ab and HL b Fas-sufficient mice were similarly prepared as controls . Control mice of either allotype had normal splenic architecture, with most AM14 Id + cells in B cell follicles and few AFCs in the red pulp. In contrast, large numbers of cells staining darkly with the 4-44 anti-Id were detected in Fas-deficient IgH a mice of both HL and H genotypes . Such cells were prominent in the red pulp, a site known to harbor plasma cells early after immunization in normal mice 49 . These putative AFCs were more prominent in mice that also had large numbers of ELISpots (data not shown). In addition, such cells were also seen at the border of the T and B cell zones. In contrast to IgH a HL and H mice, darkly staining cells were rare to absent in all IgH b mice examined. In mice of both allotypes, cells staining moderately for the AM14 Id were seen scattered within the T cell zones, a site where B cells are rarely observed in normal mice . Others who have examined the splenic architecture of Fas-deficient mice have also noted the presence of B cells scattered in the T zones 50 51 . Our data suggest that such localization is not necessarily dependent on B cell receptor engagement, as it was equally seen in the presence or absence of Ag. Finally, residual B cell zones, located at the edges of the T zones, containing lightly staining Id + cells were noted in all IgH a mice. Some IgH b mice virtually lacked these B zones , whereas in others they appeared less well developed (not shown). This immunohistological phenotype may underlie the observation that overall, IgH b mice have fewer AM14 Id + B cells as determined by FACS™. B cells play a key role in the activation of T cells in MRL/lpr mice 5 52 . Many activated T cells in Fas-deficient mice express the B220 antigen 53 . This is an activation marker in normal mice 54 , and the weight of data suggests that in Fas-deficient mice, B220 + T cells represent accumulating cells that had been activated and, in the presence of Fas, would have been eliminated after activation. Thus, the frequency of such T cells is a measure of the extent of T cell activation. In the course of our analysis of the B6/lpr cohorts, we determined the percentages of B220 + T cells as demonstrated in Fig. 5 . The extent of T cell activation, as measured by B220 + T cell percentages, depends on whether the B cell autoAg is present. This was true in both the HL and H Tgics, but the IgH genotype did not have an effect on B220 + T cell numbers in N mice. Similar conclusions were reached based on B220 + T cell numbers (data not shown), which is not surprising because HL ab mice also have greater numbers of splenocytes than HL b mice ( Table ). The origin of autoreactive B cells has been a question of great interest: are these cells that aberrantly escape central tolerance, are abnormally rescued from an anergic state, or are derived from clonally ignorant B cells whose activation or cell death is improperly regulated? Although several of these pathways may operate—depending on the autoAg, B cell affinity, and stage of disease—it is important to demonstrate experimentally which pathways are possible and thereby have appropriate models for further study. Here, we show that RF B cells that are clonally ignorant in BALB/c mice are no longer ignorant but rather are stimulated in the presence of their autoAg in the B6/lpr genetic background. We were able to demonstrate both increased numbers of autoreactive B cells and differentiation of these B cells into AFCs in mice that were genetically capable of expressing the autoAg, compared with congenic mice that lacked the autoAg. The existence of low-affinity “natural” autoantibodies in normal individuals has been known for a long time 55 56 . These B cells are likely to be clonally ignorant, as suggested by the phenotype of AM14 B cells in normal mice. What has remained controversial is whether such B cells had any relevance or relationship to pathologic autoantibodies produced in systemic autoimmune disease 56 57 . Our results indicate that clonally ignorant cells can indeed be relevant precursors for pathologic autoantibodies. A second issue that our data addresses is the role of autoAg-stimulated versus nonspecific B cell activation in the induction of autoAbs. Several studies had shown that antibodies to a wide variety of antigens can be detected in autoimmune animals, suggesting that polyclonal activation was at work 58 59 60 . On the other hand, the oligoclonal nature of B cell hybridomas specific for IgG, DNA, nucleosomes, or Sm isolated from autoimmune animals suggested an antigen-driven process 11 13 14 15 16 17 18 19 . Notably, a nonrandom pattern of somatic mutations and the presence of certain mutations that increased the affinity for Ag suggested that Ag was playing a role in driving at least some autoimmune responses. These conflicting interpretations were based on indirect inferences. Moreover, neither argument could establish whether autoAg was required or was simply altering or exacerbating an underlying polyclonal process. The data in this report provide more direct evidence that autoAg is required for the activation of autoreactive B cells. In the presence of autoAg, substantial accumulations of RF plasma cells were observed in the spleens of Tgic mice compared with those mice that lacked the autoAg. Remarkably, only in the presence of the autoAg did H chain Tgic mice efficiently select rare endogenous L chains that reconstruct both the idiotype and RF specificity of the original HL pair that comprised AM14. This phenomenon provides an additional strong argument for the role of autoAg in driving RF B cells. These conclusions are based on direct ELISpot data and splenic histology; we did not systematically measure and do not present serum RF data that are at best an indirect measurement of B cell activation and differentiation. Serum RF data are widely gathered in humans, where such direct measurements as ELISpot are not feasible. In our system, serum RF levels would be influenced by Ag competition and increased clearance rates of immune complexes as well as by competition by IgG2a naturally occurring in serum. These confounding features would be present in IgH a mice but not in IgH b mice, precluding any meaningful comparison. Furthermore, it would be difficult in such assays to distinguish RF secreted by B cells expressing endogenous Ig genes. ELISpot and histology assays are not subject to these concerns. These observations raise the related issue of the identification of the true autoAg. It has been controversial whether the nominal Ags assayed in vitro as targets of autoAbs are actually those that drive autoreactive B cell clones in vivo 61 62 . Perhaps the strongest evidence in favor of nominal Ags as actual Ags came from a single study in which the clonality and specificity were simultaneously determined for a large number of hybridomas isolated from a single autoimmune mouse 63 . Nearly all of the expanded clones could be assigned to one of a relative few nominal autoAb specificities, again suggesting that the nominal antigens were driving most of the clonal expansion. As congenic strains were compared in this study, the only important difference between the two strains is almost certainly the IgH locus allotype and, in particular, the presence or absence of IgG2a a , which is the only ligand for AM14 that is encoded by the IgH locus (Shlomchik, M., unpublished data). Thus, IgG2a a is sufficient in vivo to drive B cell clonal expansion and to recapitulate the original expansion that must have occurred in the MRL/lpr mouse from which AM14 was isolated as a hybridoma. No other autoAg on the B6/lpr (IgH b ) background was capable of driving this activation. A surprising observation was that T cell activation, as reflected by the accumulation of B220 + T cells, was affected by the presence of the B cell Ag. This means that the extent of B cell activation of one particular clone was sufficient to affect the activation of polyclonal T cells. The mechanism for this is unclear but intriguing. It could be an indirect effect due to cytokines produced by B cells 64 65 66 67 . More likely, it is a direct effect of T–B cell collaboration. Recently, our group has shown that the absence of B cells affects T cell activation 5 . Sobel and colleagues also showed in mixed BM chimeras that Fas-deficient B cells are largely responsible for promoting T cell activation 52 and that cognate interactions are required for autoAb elicitation 68 . Our recent data 69 demonstrate that this effect on T cell activation is independent of secreted antibody, again arguing for a direct, cognate interaction. To explain the effect of the B cell Ag on T cell activation we observed here, one would have to suppose that AM14 B cells could activate a substantial enough fraction of T cells to make this pathway apparent by bulk FACS™ analysis. It is unlikely that there is a high frequency of T cells specific for self-IgG2a, the AM14 antigen. However, RF B cells can take up immune complexes and present a wide variety of autoAgs, provided they were complexed with self-IgG 70 . This is expected to be the case in B6/lpr mice, which produce a variety of IgG2a autoAbs, including antichromatin 68 . Indeed, this feature of RF B cells—the ability to present many autoAgs to T cells and thus garner T cell help—may explain why RF is a predominant specificity in Fas-deficient mice 63 as well as in several autoimmune diseases. It is worth noting that although the origin of B220 + T cells in lpr mice is unclear, most “double-negative” T cells arise from a CD8 + T cell precursor 71 72 . If the generation of B220 + T cells is related to the same process, then an effect of B cells on CD8 + T cells may be playing a role. In this regard, activated and memory phenotype CD8 + T cells fail to accumulate in MRL/lpr mice in the absence of B cells 5 . Further work will be required to expand on this unexpected observation and to identify the T cells promoted by RF B cells and the autoAgs that are recognized. Fas deficiency is the major determinant of autoAb production in B6/lpr mice 44 45 . Because of constraints and logistics of animal breeding as well as the desire to compare results to those previously obtained on the BALB/c background 40 41 , we have only been able to compare Fas-deficient mice on the B6 background to Fas-sufficient mice on the BALB/c background. Thus, we cannot formally rule out a role for background genes in the B6 strain. However, this seems very unlikely, as neither strain background is associated with autoimmunity or RF production and especially because many of the phenotypes we show—high numbers of RF ELISpots, activated B cells at the T–B interface and in the red pulp, and accumulation of B220 + T cells—are distinctive to the Fas-deficient phenotype. With this caveat in mind, our results bear on how Fas normally prevents autoAb production. Fas is expressed throughout B cell ontogeny, including in developing B cells, newly anergized B cells, newly activated B cells, germinal center (GC) B cells, and plasma cells 73 74 75 76 77 78 79 . In principle, Fas deficiency could be critical at multiple stages of B cell development and tolerance in promoting autoimmunity. Our data demonstrate that Fas deficiency need not act at early stages of tolerance (editing, deletion, and anergy) to promote autoreactivity. In the presence of functional Fas, AM14 B cells develop beyond these stages and are quiescent in the presence or absence of antigen. Fas must therefore play an important role in the regulation of RF B cells during or after autoAg-specific activation, because when Fas is deficient, these B cells expand, persist, and make autoAb, but only in the presence of autoAg. It is also possible that Fas is acting more indirectly, e.g., by causing increased IgG2a autoAg levels. In our case, this is doubtful, as the average serum IgG2a level in a small group of 4–5-mo-old HL ab mice is 40 ± 19 μg/ml (± 1 SD), levels that are similar to those in BALB/c mice. More work needs to be done to pinpoint at which stage(s) after the initial Ag activation event Fas is playing a role. It is notable in this regard that although Fas is expressed in GC B cells, GC reactions appear relatively normal in Fas-deficient mice 80 81 . We observed that RF Tgic B6/lpr mice accumulated large numbers of RF AFCs with few GCs (data not shown) and had a relative B lymphopenia. This raises the possibility that Fas on plasma cells is playing at least one critical role. Fas deficiency is likely not the cause of human systemic autoimmunity. However, given the central role of Fas/FasL and possibly other homologues in the TNFR/TNF family in immune system homeostasis, it does seem likely that subtle defects in apoptotic pathways could underlie human disease 82 83 . In this regard, pure Fas deficiency may be an excellent model for understanding the in vivo pathogenic mechanisms of such deficiencies. The overall impact of this idea will only be known when genes that promote lupus are identified 84 85 and the Fas and Fas-related signaling pathways are better elucidated. Previous studies had investigated how autoimmune-prone genetic backgrounds affected the regulation of B cells that would normally be tolerant rather than ignorant. In the case of mice that edited or deleted B cells specific for model self-antigens such as Class I or HEL, it was generally found that regulation was intact even in autoimmune-prone backgrounds 25 26 . These data suggested that Fas and Fas/MRL defects did not grossly impair central tolerance at this level for these autoAgs. Regulation of B cells specific for either HEL or DNA that are anergic in normal mice has also been investigated on the B6/lpr or MRL/lpr backgrounds. In the case of HEL, induction of anergy was essentially intact 25 . In related experiments, Rathmell et al. transferred anergic anti-HEL B cells along with activated CD4 + T cells and demonstrated that anergic cells are sensitive to elimination; this did not occur when the T cells were deficient in FasL, indicating a role for this pathway in the elimination of anergic cells when T cell help was also being delivered 86 . This remains a potential pathway by which autoreactive B cells could escape regulation, although unmanipulated Fas- or FasL-deficient mice did not show gross defects in self-tolerance, as discussed above. The situation is different in our model, as the B cells are not anergic and should be capable of rescue by surface Ig cross-linking 87 ; thus, the Fas pathway must function during additional regulatory steps (see below). The situation is more complex for anti-DNA B cells; their fate may depend on the specificity/affinity of the DNA-specific B cell. In normal mice, anti-ssDNA B cells appear anergic, albeit with a somewhat different phenotype from the anti-HEL anergic B cells 27 33 . Some dsDNA-specific B cells may be subject to receptor editing/deletion 28 29 30 88 , but others may persist in the periphery localized at the T–B interface, where they turn over rapidly 32 34 . In autoimmune MRL/lpr mice Tgic for the 3H9 H chain, which can generate a variety of anti-DNA depending on the endogenous L chain 89 , anti-DNA is seen in the serum and hybridomas secreting antinuclear Abs with homogenous nuclear staining are readily detected 31 39 . These autoAbs may arise through several pathways; they may result from loss of central tolerance, though recent detection of anti-dsDNA B cells in BALB/c spleen by Roark et al. 34 suggests that rescue from anergy may also be possible. Recent work by Weigert and colleagues on anti-ssDNA site-directed Tgic mice also suggests that rescue from anergy in MRL/lpr mice can occur 90 . As there cannot be an antigen-free anti-DNA Tgic, determination of the ontogeny of the autoreactive B cells and, in particular, proving that this is a specific process is less straightforward than in the RF system. In any case, the anti-DNA models represent a different scenario from ours in that the precursors of anti-DNA Abs are thought to escape from either deletion or anergy, whereas in the case of AM14, ignorant B cells are being positively selected by Ag. We have shown that B cells that would be ignored in a normal mouse are driven to activation, expansion, and secretion in an autoimmune-prone Fas-deficient mouse. This process is specific and requires the presence of autoAg as well as the Fas defect. Because this is the only autoAb Tgic system, to our knowledge, that demonstrates Ag-specific activation of defined, nontolerant mature B cells, it provides a unique opportunity to study the temporal and spatial course of the initiating events of Fas-deficient B cell autoimmunity and perhaps ultimately other scenarios of B cell autoimmunity. It may also shed light on the induction of autoreactive T cells and may be a system for their generation and study in vitro and in vivo. | Study | biomedical | en | 0.999996 |
10477550 | Nine metastatic melanoma patients, admitted to our Institute (Istituto Nazionale per lo Studio e la Cura dei Tumori) for surgery and chosen for expression of the HLA-A*0201 + allele as determined by single-stranded oligonucleotide probe–PCR typing 6 , were selected for this study. Characteristics of the patients are described in Table . At the time the PBLs were isolated for CTLp frequency determination, all patients had already developed lymph node metastases (stage III, AJCC). Further progression of disease occurred in seven out of nine patients after CTLp analysis ( Table ). Six out of nine patients died of disease between 1 and 16 mo after CTLp evaluation; the remaining three patients are still alive at 41 mo (patient 5, alive with disease) and 38 mo (patients 7 and 8, both without evidence of disease) after CTLp evaluation ( Table ). None of the patients enrolled in this study had been subjected to chemotherapy or any other therapy with immunosuppressive activity before isolation of the PBLs used for limiting dilution analysis (LDA; Table ). Tumor lines used in this study were isolated as previously described 6 from HLA-A*0201 + patients admitted to our Institute for surgical treatment of either primary or metastatic melanoma. Expression of Melan-A/Mart-1 antigen in these lines, as well as in fresh tumor cells isolated from some surgical specimens, was determined by intracellular fluorescence analysis on saponin-permeabilized cells followed by FACS™ analysis (Becton Dickinson) with the Melan-A/Mart-1–specific mAb M27C10 12 , a gift of Dr. F. Marincola (National Cancer Institute, Bethesda, MD). In addition, all lines were found positive by flow cytometry for HLA-A2, CD54, and lymphocyte function associate (LFA)-3, whereas none expressed CD80. Fresh tumor cells from surgical specimens were also characterized for HLA-A2 expression by flow cytometry after staining with mAb CR11.351 (anti–HLA-A2) 13 . Melan-A/Mart-1 27–35 (AAGIGILTV), influenza A (Flu) matrix 58–66 (GILGFVFTL), and tyrosinase 366–378 (YMNGTMSQV) peptides were used in this study 11 14 15 . All synthetic peptides were ≥95% pure (PRIMM srl; San Raffaele Biomedical Science Park, Milan, Italy). Stock solutions of peptides were set up in DMSO and kept at −20°C. The concentrations of Melan-A/Mart-1 27–35 (10 μg/ml) and Flu matrix 58–66 (5 μg/ml) peptides to load the TAP-deficient T2 cell line to be used in LDA assays were determined by the binding assay based on HLA-A2 stabilization and resulting in the same fluorescence ratio of 3.2 as previously described 16 . These peptide concentrations were also used to load other APCs used in the study. 5 × 10 6 T lymphocytes purified by nylon wool column after Ficoll separation were resuspended in 100 μl of PBS containing 0.5% autologous human serum and 0.6% acid citrate dextrose (Baxter Healthcare Ltd.). To two identical aliquots of this cell suspension, 20 μL of MACS ® CD45RA or MACS ® CD45RO Microbeads (Miltenyi Biotec) were added, mixed, and allowed to incubate at 4°C for 15 min. After incubation, the cells were washed by adding 5 ml of PBS followed by centrifugation at 800 rpm for 10 min at 4°C. The supernatant was discarded and the cell pellet resuspended in 1 ml of cold PBS. The separation column (MS type; Miltenyi Biotec) was primed by washing with 1 ml of cold PBA and placed in a magnetic field. The washed cell pellet, pretreated with either MACS ® CD45RA or MACS ® CD45RO microbeads, was applied to the prefilled columns. Cells expressing either CD45RA or CD45RO were retained in the columns, whereas the negative fraction (i.e., CD45RO + in columns loaded with CD45RA + -stained cells or vice versa) was eluted and used for the LDA assays. Purity of the two T cell subsets was assessed by flow cytometry and resulted in ≥98% in all instances. The HLA-A*0201 + TAP-deficient T2 cell line, an effective APC for the activation of even naive T cells and the generation of CTLs against peptides from self- and non-self proteins 17 , was used as APC for peptide presentation in all LDA assays. Lymphocytes isolated from peripheral blood of melanoma patients or HLA-A*0201 + healthy donors by Ficoll gradient centrifugation were used for determination of peptide-specific CTLp frequency after monocyte depletion. The LDA technique for determination of peptide-specific CTLp frequency was performed as described 6 16 . After 4 wk of culture, each of the replicate wells of all LDA cultures was split into two aliquots and tested against an empty or peptide-loaded HLA-A*0201 + LCL . Melan-A/Mart-1 27–35 or Flu matrix 58–66 peptides were used depending on the LDA sets. To evaluate the frequency of HLA-A2–restricted precursors, in some experiments the split well analysis was performed by testing lysis of an HLA-A*0201 + Melan-A/Mart-1 + melanoma that was or was not preincubated with an anti–HLA-A2 A28 mAb (CR11.351). The cytolytic assay was performed as described 6 16 . To increase the efficiency of the assay, all cytolytic tests, involving either 9742 LCL or melanoma cells as targets, were performed in the presence of 3 × 10 2 targets per well. The threshold of significant lysis, criteria to score a well as containing a peptide-specific CTLp, and data analysis for CTLp frequency determination have been described elsewhere 6 16 . As developed, this LDA assay cannot detect CTLp frequencies <1/200,000. This LDA technique was also used to evaluate frequency of CTL effectors in bulk T cell cultures. In these instances, T cells from bulk cultures were seeded in LDA sets and tested for specificity by the split well technique after 1 wk. After monocyte depletion, PBLs were cultured in 24-well plates (Costar Corp.) at 10 6 cells/ml in 2 ml of RPMI 1640 supplemented with 10% heat-inactivated human serum in the presence of 0.5 × 10 6 /ml irradiated, peptide-loaded T2 cells. Independent cultures were set up using T2, either empty or loaded with Melan-A/Mart-1 27–35 or tyrosinase 366–378 peptides. Low dose (10 U/ml) IL-2 was added on day 2 to all cultures. All cultures were then restimulated weekly with peptide-loaded or empty T2 cells. The resulting T cell lines were tested weekly from days 21–70 for specificity (by 51 Cr-release assays) on peptide-loaded or empty 9742 LCL cells. Specificity of these T cell lines was also tested on HLA-A*0201 + melanomas that did or did not express Melan-A/Mart-1. HLA-A2 restriction of melanoma lysis was checked by comparing lysis of melanomas that were or were not preincubated with mAb CR11.351. Induction of Melan-A/Mart-1 27–35 –specific CTLs was also carried out in two patients (patients 7 and 8) by coculture with peptide-loaded autologous dendritic cells (DCs) derived from CD34 + progenitors or monocytes. DCs were differentiated from purified CD34 + progenitors or monocytes as recently described 16 . CD34 + progenitors were initially mobilized by G-CSF treatment (a written informed consent was signed by patients for this treatment). Phenotype of DCs and monocytes was evaluated by flow cytometry with mAbs to CD1a (Coulter Immunology), CD14, HLA-DR, HLA-DQ (Becton Dickinson), CD40, CD80 (Serotec Ltd.), CD86 (Ancell), and CD54 (Immunotech). Control cultures from the same two patients were also set up with autologous fresh monocytes loaded with either Melan-A/Mart-1 27–35 or Flu matrix 58–66 peptides. All cultures, set up with autologous DCs or monocytes as APCs, were restimulated weekly with fresh, peptide-loaded monocytes. HLA–peptide tetrameric complexes were synthesized as described 18 . Staining of PBLs, TILs, and T cell lines was performed by incubating for 30 min in ice with PE-conjugated HLA-A*0201–Melan-A/Mart-1 26-35 tetramers. The modified Melan-A/Mart-1 26-35 peptide (ELAGIGILTV) used for refolding the HLA–peptide complex has been shown to increase the binding affinity for HLA-A*0201 without affecting CTL recognition 19 . Negative controls for tetramer staining included PBLs from HLA-A*0201–negative healthy donors and HLA-A2–restricted CTL clones directed to tyrosinase peptides 6 ; a Melan-A/Mart-1 27–35 –specific CTL clone, A83 16 , was used as positive control. Two-color fluorescence analysis was performed in some instances by staining T cell lines with FITC-conjugated anti-CD8 and PE-conjugated tetramers. At least 10 5 cells were analyzed for staining with PE-conjugated tetramers. Immunohistochemical analysis was performed on routinely formalin- or Bouin's-fixed and paraffin-embedded specimens. 4-μm-thick tissue sections were deparaffinized through graded series of ethanol passages and rehydrated in distilled water. Endogenous peroxidase was inhibited by a 30-min incubation in methanol containing 0.3% H 2 O 2 . To optimize immunodetection of Melan-A/Mart-1, gp100, and CD8, nonenzymatic antigen unmasking was performed by heating tissue sections at 95°C for 6 min in an autoclave in 5 mM citrate buffer, pH 6 20 . After cooling, sections were incubated with normal goat serum (1:50; DAKO Corp.) diluted in PBS containing 1% BSA for 30 min. Primary antibody incubation was performed overnight at 4°C with the following antibodies: M27C10 12 , anti–Melan-A/Mart-1 (1:50), HMB45, anti-gp100 (1:25; DAKO Corp.), and CD8 (1:20; DAKO Corp.). Staining with polyclonal antibody CD3 (DAKO Corp.) was performed after 0.1% trypsin treatment for 5 min as described for antigen unmasking. Sections were subsequently rinsed three times in PBS plus Triton X-100 and treated with biotinylated goat anti–mouse Ig (1:100; DAKO Corp.) or with biotinylated goat anti–rabbit Ig (1:200; DAKO Corp.) for CD3 staining. The slides were then covered with streptavidin–horseradish peroxidase (1:300; DAKO Corp.) for 30 min and finally visualized with the use of red 3-amino-9-ethylcarbazole (Sigma Chemical Co.) in 0.05 M acetate buffer containing 0.015% H 2 O 2 . Sections were then counterstained with hematoxylin and mounted with glycerine-gelatin 4. Tissue sections subjected to the same treatment but without incubation with primary antibody were used as negative controls. Positive controls were a reactive lymph node for CD3 and CD8 and a Melan-A/Mart-1 + human melanoma cell line grown in nude mice that was subsequently formalin fixed and paraffin embedded for Melan-A/Mart-1. Using LDA, we evaluated the CTLp frequency against a peptide from the melanocyte lineage–specific antigen Melan-A/Mart-1 in peripheral blood of nine HLA-A*0201 + metastatic melanoma patients. Two CTLp frequency groups were found ( Table ): a group (patients 1, 2, 6, and 9) with high frequency of CTLp to Melan-A/Mart-1 27–35 (between 1/1,400 and 1/2,225) and a group (patients 3, 4, 5, 7, and 8) with low frequency of CTLp (between 1/40,000 and <1/200,000) to the same antigen. In agreement with our previous results 6 21 , independent LDA assays performed using PBLs isolated from the same patient a few months apart indicated that the CTLp frequency remained in the same range in each of the two groups of patients (data not shown). Furthermore, staining with Melan-A/Mart-1–HLA-A*0201 tetrameric complexes indicated a frequency of peptide-specific T cells of 1/1,456 and 1/1,843 in PBLs of patients 6 and 9, respectively, in good agreement with CTLp frequency by LDA. By using lymphocytes from the same blood sample, CTLp analysis was reassessed in sorted memory (CD45RO + ) and naive (CD45RA + ) T cell subsets. In two patients from the high CTLp frequency group, Melan-A/Mart-1 27–35 –specific CTLp were mostly (patient 2) or only (patient 6) found in the CD45RO + memory T cell subset ( Table ). By contrast, in two patients from the low CTLp frequency group (patients 7 and 8), Melan-A/Mart-1 27–35 –specific CTLp were detected only in the CD45RA + naive T cell subset. Low frequency of CTLp to Melan-A/Mart-1 27–35 , detected in patients 3, 4, 5, 7, and 8, was not the result of nonspecific immune suppression preventing precursor growth or development of cytolytic function in LDA cultures. In fact, in these patients, the CTLp frequency of a different HLA-A*0201–restricted T cell epitope (the immunodominant Flu matrix 58–66 peptide) was in the same range, or even higher, than that found in a panel of HLA-A*0201 + healthy donors ( Table ). Taken together, these data indicate that expansion of the Melan-A/Mart-1 27–35 –specific T cell population occurs in a fraction of metastatic melanoma patients, as indicated by a high-frequency peripheral pool of antigen-specific T cells with a memory phenotype. In patients of the low CTLp frequency group, Melan-A/Mart-1–specific CTLs were activated in bulk culture with professional APCs (DCs and T2) versus nonprofessional APCs (monocytes), and then the frequency of CTL effectors was quantitated by LDA. As shown in Table , after 42 d of bulk culture, Melan-A/Mart-1 27–35 –specific effectors were expanded in patient 7 to a final frequency ranging from 1/63 to 1/142 after activation with peptide-loaded T2 or autologous DCs. No peptide-specific CTLs were found by LDA in cultures activated with peptide-loaded monocytes. Similar results were obtained with another patient in the low-frequency group (data not shown). In contrast, nonprofessional APCs (monocytes) could reactivate and expand Flu matrix 58–66 –specific T cells (a memory response that depends on previous viral exposure and does not require professional APCs for reactivation). In fact, a Flu matrix–specific CTL effector frequency of 1/1,190 was found in patient 7 after 21 d of bulk culture ( Table ), in comparison to a precursor frequency of 1/5,471 in fresh PBLs ( Table ). These data indicate that patients with low CTLp frequency of Melan-A/Mart-1 27–35 are endowed with only a naive immune repertoire, made up of rare precursors that require professional APCs for priming and proliferation. Kinetics of activation of Melan-A/Mart-1 27–35 –specific CTLs in bulk culture was compared in patients with low and high CTLp frequency. T2 cells (shown in Table to have the same APC efficiency as DCs) were used as stimulators after loading with Melan-A/Mart-1 27–35 or control tyrosinase 366–378 peptide. In T cell cultures from patients 1 and 2 (two patients with high CTLp frequency), Melan-A/Mart-1 27–35 specificity was already evident on day 21 (data not shown) and was confirmed on days 28 and 36 of culture ( Table ). Peptide specificity was documented by recognition of peptide-loaded 9742 LCL, HLA-A2–restricted lysis of HLA-A*0201 + Melan-A/Mart-1 + tumors (INT-MEL-8 and INT-MEL-9), and absence of HLA-restricted lysis on an HLA-A*0201 + Melan-A/Mart-1 − melanoma (INT-MEL-10) ( Table ). By contrast, T cell cultures from patients 3, 4, and 5 (all with low CTLp frequency) lacked peptide specificity against 9742 LCL targets, either on day 28 or 36. T cell cultures from patient 3 showed HLA-A2–restricted lysis of two Melan-A/Mart-1 + melanomas on days 28 and 36, but evidence of peptide specificity on peptide-loaded 9742 LCL was obtained only on day 49 of culture ( Table ). In other patients of the low CTLp frequency group, peptide specificity on 9742 LCL and HLA-A2–restricted recognition of tumors required up to 70 d of bulk culture to be obtained (data not shown). These data indicate that a high frequency of peptide-specific CTLp in blood of patients correlates with faster kinetics of generation of antigen specificity after in-vitro T cell activation with peptide-loaded APCs. CTL effector frequencies in bulk T cell cultures were compared in patients with low or high precursor frequency by LDA and T cell staining with Melan-A/Mart-1–HLA-A*0201 tetrameric complexes ( Table ). In day 28 bulk cultures of three patients of the high CTLp group, peptide-specific CTL effectors were between 1/10 and 1/24. By contrast, in day 28 bulk cultures from patients of the low CTLp group, the peptide-specific effectors were between 1/576 and 1/4,531. In two patients (patients 2 and 6), evaluation of CTL effector frequency by LDA was performed by two distinct readout systems: differential lysis of peptide-loaded or nonloaded 9742 LCL and differential recognition of HLA-A*0201 + Melan-A/Mart-1 + melanoma cells that were or were not preincubated with an anti–HLA-A2 mAb. Effector frequencies were similar by both readout systems ( Table ). Control LDA assays, performed on T cell cultures activated by empty T2 ( Table ), gave no detectable peptide-specific effector frequency (patients 1, 4, 5, 7, and 8). Furthermore, frequency evaluation by LDA and Melan-A/Mart-1–HLA-A*0201 tetrameric complexes gave similar results ( Table ). This indicated that essentially all T cells expressing a Melan-A/Mart-1–specific TCR, as identified by tetrameric complexes, could also be functionally identified by our LDA approach. In addition, T cell staining with tetrameric complexes allowed us to follow the evolution in culture of Melan-A/Mart-1–specific T cells during selection with peptide-loaded APCs. As shown in Fig. 1 , the number of peptide-specific T cells rose from 1/599 to 1/11 in a T cell culture from patient 6 and from 1/76 to 1/1.36 in the T cell culture of patient 2. This indicates that in the patients with high precursor frequency, T cell activation with peptide-loaded T2 cells leads to early and progressive expansion of peptide-specific T cells. Taken together, these data suggest that a high CTL effector frequency, after APC-mediated T cell selection, can be achieved only in patients with a high CTLp frequency in blood. Thus, presence of an expanded peripheral pool of T cells to a tumor antigen is an important requisite for efficient in vitro selection of antitumor T cells from peripheral blood of patients. T cells isolated from metastases of patients 1 and 2 (two patients of the high CTLp frequency group) and patient 3 (with low CTLp frequency) were tested for specificity after 3 wk of selection in bulk culture with Melan-A/Mart-1 27–35 –loaded T2 cells as APCs. The T cell lines from lesions of patients 1 and 2 specifically recognized 9742 LCL loaded with Melan-A/Mart-1 27–35 peptide and lysed the two Melan-A/Mart-1 + tumors INT-MEL-8 and INT-MEL-9 in an HLA-A2–restricted fashion ( Table ). By contrast, only nonspecific lysis on all targets by T cells isolated from a lymph node metastasis of patient 3 was observed. These findings were confirmed even after 36 d of culture (data not shown). Staining of TILs from the subcutaneous lesion of patient 1 with Melan-A/Mart-1–HLA-A*0201 tetrameric complexes revealed a frequency of 1/17.6 peptide-specific T cells , thus indicating a very high enrichment in comparison to frequency of CTLp to the same antigen detected in peripheral blood of the same patient (1/1,404; Table ). Thus, patients with an expanded peripheral pool of Melan-A/Mart-1 27–35 –specific T cells do have peptide-specific T cells in their metastatic lesions. Furthermore, the CTLp present in metastatic lesions could be readily activated by appropriate antigen presentation to acquire effector function with a fast kinetics of proliferation, suggesting absence of any irreversible functional block. To evaluate the relationship between the expanded peripheral pool of T cells to Melan-A/Mart-1 and in vivo response to tumor lesions, all available primary and metastatic lesions isolated during tumor progression from the nine patients were analyzed by immunohistochemistry. To this end, the brisk/nonbrisk/absent code for defining patterns of infiltrating T cells was adopted 1 2 . A common pattern emerged: in all patients, including those with high frequency of CTLp to Melan-A/Mart-1, evidence of tumor regression/necrosis was often completely lacking or, with few exceptions, appeared to involve only a minor portion of the area containing neoplastic cells in each lesion ( Table ). Moreover, tumor regression, when present, often appeared as areas of coagulative necrosis, sometimes admixed with hemorrhage, that were never infiltrated or immediately surrounded by CD3 + lymphocytes, even in the lesions containing brisk or nonbrisk CD3 + CD8 + T cells. Furthermore, with the exception of all lesions from patient 9, which lacked HLA class I antigens, including HLA-A2, all other lesions that could be analyzed expressed HLA-A2, suggesting that in these instances T cell epitope presentation was not impaired in tumor cells ( Table ). In patient 1, the primary lesion (lesion 1) and a satellitosis (lesion 2) were removed 2 mo before CTLp evaluation ( Table ). The first lesion had an absent pattern of CD3 + T cells and Melan-A/Mart-1 antigen expressed on 20% of the neoplastic cells. The satellitosis was nonbrisk for CD3 + T cells, but CD8 + T cells represented only 30% of them and Melan-A/Mart-1 was not expressed. A subcutaneous lesion (lesion 3) isolated 1 wk before CTLp evaluation was Melan-A/Mart-1 + and expressed HLA-A2 on the tumor and 30% of CD8 + cells among the nonbrisk CD3 + infiltrate. However, no evidence of tumor destruction was observed, even though this same lesion contained a high frequency of Melan-A/Mart-1–specific CTLp . A lymph node metastasis (lesion 4) was almost completely negative for Melan-A/Mart-1 and absent for CD3 + T cells. In the same patient, in spite of an expanded T cell population to Melan-A/Mart-1 in peripheral blood, three additional subcutaneous metastases developed within 6 mo of CTLp analysis. Two of these lesions expressed Melan-A/Mart-1, but no evidence of tumor regression or destruction was found, although all lesions contained a brisk CD3 + CD8 + infiltrate . In patient 2, a lymph node metastasis removed 6 d after CTLp analysis ( Table , lesion 9) showed tumor regression affecting 50% of the neoplastic tissue. Although this lesion contained Melan-A/Mart-1–specific CTLp ( Table ), the necrotic area appeared to be the result of an ischemic lesion and not of an immune response . In fact, the nonbrisk CD3 + CD8 + infiltrate did not surround nor infiltrate the necrotic area, which was instead surrounded by scattered granulocytes . The tumor cells were HLA-A2 + , and some areas showed a weak staining for Melan-A/Mart-1 , suggesting a possible tumor escape mechanism. In patient 6, an absent pattern of CD3 + T cells was found in an HLA-A2 + soft tissue metastasis lacking Melan-A/Mart-1 and removed 4 d after CTLp analysis ( Table , lesion 11), as well as almost no tumor regression but a 10% sclerosis. Again, the lack of Melan-A/Mart-1 suggests a possible tumor escape mechanism. In patient 9, three synchronous lesions were removed, including the primary tumor (lesion 12) and two metastases (lesions 13 and 14). All of these lesions were Melan-A/Mart-1 + and lacked HLA class I, including HLA-A2, suggesting another mechanism of tumor escape from immune surveillance. All of these lesions expressed an absent pattern of CD3 + T cells; no evidence of tumor regression was observed in the two metastatic lesions, and only 10% sclerosis was documented in the primary lesion. Furthermore, in patients 1, 2, 6, and 9, in spite of an expanded pool of Melan-A/Mart-1–specific T cells in peripheral blood, further disease progression occurred due to inoperable metastases at visceral organs or the brain. All of these patients died within 16 (patient 1), 1 (patient 2), 5 (patient 6), and 3 mo (patient 9) after CTLp evaluation, as summarized in Table . In addition, in 12/14 neoplastic lesions from the group of patients with low CTLp frequency, infiltrating T cells, Melan-A/Mart-1, or both were missing . Taken together, these data strongly suggest that an expanded pool of antigen-specific T cells in peripheral blood cannot overcome tumor escape mechanisms in neoplastic lesions, even when peptide-specific T cells are present in the neoplastic tissue, as shown for patients 1 and 2. By coupling a high efficiency LDA assay to dissection of memory versus naive T cell subsets, we obtained evidence that the Melan-A/Mart-1 27–35 peptide is immunogenic in vivo in a fraction of metastatic melanoma patients, as documented by the presence of an expanded peripheral pool of antigen-specific CD45RO + memory T cells. In the patients with an expanded T cell population to Melan-A/Mart-1 27–35 , the high CTLp frequency correlated with faster kinetics of CTL development and a higher number of effectors obtained in vitro after activation with peptide-loaded professional APCs in comparison to patients with low CTLp frequency. The first implication of our findings for immune intervention strategies is that activation of tumor-specific T cells by professional APCs will be much more efficient, in quantitative terms (total number of effectors that can be generated), in patients with an expanded peripheral pool of memory T cells than in patients with a low-frequency naive repertoire. The results obtained in the patients with high CTLp frequency are in agreement with data on memory phenotype of circulating CTLp to Melan-A/Mart-1 recently reported by D'Souza et al. 22 and with studies that have examined the response to viral antigens like those encoded by hepatitis C virus, herpes simplex virus, and Epstein-Barr virus 23 24 25 . In such studies, viral peptide–specific precursor frequency in infected individuals was 10–100-fold higher than in noninfected controls, and antigen-specific precursors were mostly in the CD45RO + subset. Furthermore, our results corroborate the findings indicating accelerated kinetics of Melan-A/Mart–specific CTL development in patients versus healthy donors 7 . In patients with low CTLp frequency, Melan-A/Mart-1 27–35 –specific precursors were found only in the CD45RA + naive T cell subset. No evidence of immunosuppression was found in these patients, as shown by analysis of frequency of Flu matrix 58–66 –specific CTLp in comparison to healthy donors. In addition, activation and expansion of Melan-A/Mart-1 27–35 CTLs could be obtained only by using professional APCs. These data indicate that these patients have a naive immune repertoire against Melan-A/Mart-1 27–35 , and expansion of Melan-A/Mart-1 27–35 –specific precursors did not occur during tumor growth or was transient and unable to generate memory T cells. Differences in the extent and mechanism of tumor antigen release 26 in tumor lesions may impact on antigen uptake and presentation by APCs, thus leading to priming of peptide-specific T cells only in some patients. In addition, in some but not in all patients, tumor cells may produce factors, such as vascular endothelial cell growth factor 27 , that inhibit APC differentiation and/or function. Furthermore, Melan-A/Mart-1 27–35 –specific precursors could be primed, rather than tolerized, by naturally occurring epitope mimics of Melan-A/Mart-1 27–35 in some but not all patients 28 29 . These mechanisms may hamper tumor immunogenicity, even in the presence of an antigenic tumor. Several reports have recently suggested that LDA may underestimate the frequency of antigen-specific T cells in comparison to techniques such as the ELISPOT (enzyme-linked immunospot assay) or staining antigen-specific T cells with MHC–peptide tetrameric complexes (for review see reference 30 ). In contrast with these concerns, in this study, evaluation of frequency of Melan-A/Mart-1–specific T cells in peripheral blood by LDA and tetramer staining provided similar values. In addition, we obtained a frequency range of Flu matrix–specific CTLp as high as that found by either ELISPOT or tetramer staining in previous studies 31 32 . The range of ∼1/5,000 for Flu matrix 58–66 –specific CTLp detected by our LDA assay in patients is at least 10-fold higher than that found by conventional LDA by other groups 33 34 . Those studies used an LDA technique based on 8–18 d culture time (instead of 28 d as in our study), PBMCs or B cells as APCs (instead of T2), and up to 4 × 10 3 targets in the split well assay (instead of 3 × 10 2 as in this study). Moreover, direct comparison of our LDA technique with tetramer staining on the same T cell cultures provided overlapping values in the frequency of Melan-A/Mart-1–specific effectors, both in high- and low-frequency cultures. This suggests that our modified LDA has improved sensitivity in detecting both high-frequency and low-frequency precursors. Furthermore, in agreement with a previous report 35 , comparison between LDA and tetramer staining provided direct evidence that all antigen-specific T cells (on the basis of tetramer staining) were indeed functional cytotoxic T cells able to recognize the relevant peptide (as determined by LDA), either when exogenously added to an LCL or when endogenously expressed in melanoma cells. In at least two patients of the high CTLp frequency subset, peptide-specific T cells were found in TILs from a subcutaneous and a lymph node metastasis. This indicated that in such patients, Melan-A/Mart-1 27–35 –specific T cells could home to neoplastic tissue. Activation of these TILs with peptide-loaded T2 cells in bulk culture resulted in Melan-A/Mart-1 specificity after only 3 wk of selection, a finding consistent with absence of any irreversible functional block of these cells and a high precursor frequency in these lesions. Tetramer staining of TILs from subcutaneous lesions showed that Melan-A/Mart-1 27–35 –specific T cells were 1/17.6 in comparison to 1/1,404 in peripheral blood of the same patient. This observation is in agreement with a recent report describing an expanded pool of Melan-A/Mart-1–specific T cells in metastatic tissue by tetramer staining 35 . Our findings also suggest that appropriate in vitro T cell activation can rescue antitumor function of peptide-specific T cells that infiltrate neoplastic lesions but that apparently do not exert antitumor activity in vivo. The observation that T cell activation with professional APCs could activate Melan-A/Mart-1–specific CTLs from both peripheral blood and tumor site suggests that antigen-specific vaccination approaches may reactivate and expand antitumor T cells in vivo. This is in agreement with the significant antitumor responses obtained by initial clinical studies of vaccination of melanoma patients with synthetic peptides plus adjuvants or with tumor antigen–loaded DCs 36 37 . Furthermore, our results indicate that high frequency of CTLp to a tumor antigen impacts on CTL generation. Thus, a possible relationship between an expanded pool of T cells to a tumor antigen (defined as high frequency of antigen-specific T cell precursors with a memory phenotype) before vaccination and clinical response to immune intervention should be evaluated in future studies. In spite of the presence of peptide-specific T cells in the tumor lesions and peripheral immunity to Melan-A/Mart-1 27–35 , the potential for immune response at the tumor site appeared impaired in most lesions of all patients tested. In fact, reduced/absent evidence of tumor regression was observed in the majority of the lesions available for investigation. In addition, even when areas of tumor regression were present, these areas were never associated with or surrounded by infiltrating CD3 + lymphocytes. In many instances, areas of regression were identified as coagulative necrosis characterized by nuclear loss and marked cytoplasmic eosinophilia in the absence of inflammatory infiltrate. This is a typical aspect of ischemic lesions suggesting vascular damage or inadequate blood supply as the initial mechanism leading to regression, rather than an immune-mediated mechanism. Several mechanisms may impair T cell response at the tumor site. Lack of epitope expression is a possibility supported by our findings and by a large set of reports (for review see reference 38 ), but several other mechanisms could be involved. For example, loss/defective function of TCR signaling molecules has been described in melanoma patients 39 . Activation of the defective T cells in the presence of IL-2 can rescue TCR signaling molecule expression and T cell function 40 . Similar mechanisms, based on defective TCR signal transduction, might explain why peptide-specific T cells infiltrating the tumor tissue in immunized patients may fail to destroy tumor cells in vivo while remaining responsive to in vitro activation. Taken together, these results suggest that in most metastatic lesions tumor escape mechanisms can hamper T cell–mediated immune response, even in lesions containing Melan-A/Mart-1 CTLp and in patients with an expanded peripheral T cell pool to the same antigen. The implication of these findings for immune intervention approaches is that means to overcome tumor escape mechanisms in neoplastic lesions may be as relevant as the attempts to induce/boost systemic and local T cell–mediated immunity to tumor antigens. | Study | biomedical | en | 0.999996 |
10477551 | The rearranged (rr)V2H + E + transgene was previously described as the γ transgene containing the EcoRI–SalI fragment of the G8 TCR-γ gene 22 25 . The rrV2H + E − transgene was identical except that the 2.8-kb KpnI–SalI fragment containing 3′E Cγ1 was removed from the 3′ end. The rrV2H − E + transgene lacked the 1.5-kb EcoRI–NcoI fragment containing HsA at the 5′ end. The rrV2H − E − transgene lacked both of these fragments. The γD(H + E + ) transgene was assembled from BALB/c DNA derived from phage clones. It included the 5-kb EcoRI fragment containing Vγ2 and HsA and a 15.5-kb MboI fragment containing Jγ1, Cγ1, and 3′E Cγ1 that extended from 4.8 kb upstream of Jγ1 to 4 kb downstream of Cγ1. Compared with germline DNA, the transgene lacked 18.5 kb of DNA between Vγ2 and Jγ1, including the Vγ3 and Vγ4 genes. The Vγ2 gene in the γD transgenics contained an XhoI linker at the ClaI site in the coding region that disrupted the reading frame and allowed discrimination between transgenes and endogenous genes. The γD(H + E − ) transgene was identical to γD(H + E + ) except it lacked the 2.6-kb KpnI–MboI 3′ fragment containing 3′E Cγ1 . The γD(H − E + ) transgene lacked the 3.5-kb EcoRI–XbaI 5′ fragment containing HsA. The γD(H − E − ) transgene lacked the 2.6-kb 3′ fragment containing 3′E Cγ1 and a 1.5-kb EcoRI–NcoI fragment containing HsA. The transgene constructs, free of vector DNA, were injected into fertilized (C57BL/6 × CBA/J)F 2 eggs. Transgenic founders were either analyzed directly or were backcrossed repeatedly to B6 mice (rrV2 lines) or CBA/J mice (γD lines; purchased from the National Cancer Institute, Bethesda, MD) to generate transgenic lines. Mice were bred and maintained in specific pathogen-free facilities at the University of California at Berkeley. The DNase I hypersensitive assays were performed on thymocytes and LPS blasts as described 26 except that the cells were lysed in a saponin solution 27 . The quantities of DNase I (Type IV; Sigma Chemical Co.) per tube were as follows: 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25, and 50 μg. The control tube contained water. The DNase I hypersensitive assays on liver were performed as previously described 28 . LPS blasts were made by incubating spleen cells, from which CD4 + and CD8 + cells had been depleted by complement lysis, with 40 μg/ml LPS ( Salmonella typhosa ; Difco Labs., Inc.) at a concentration of 2 × 10 6 cells/ml for 3 d. More than 90% of the resulting cells stained positive for the B cell marker B220. Peripheral T cells were prepared from a mixture of spleen and lymph node cells by passing the cells over nylon wool columns. To purify α/β and γ/δ T cells, the isolated peripheral T cells were combined with thymocytes, and the mixture was partially depleted of CD4 and CD8 cells by complement lysis followed by cell sorting with an Epics Elite flow cytometer (Coulter Immunology) using anti-γδ (GL3–FITC) and anti-αβ (H57.597–biotin) antibodies. Fetal thymus was timed by designating the day of the plug as day 0. The whole fetal thymus, including the capsule, was used to isolate RNA. CD4 − CD8 − double-negative (DN) thymocytes were prepared by complement lysis of whole thymocytes with anti-CD4 (RL172) and anti-CD8 (3.168.8 or AD415) antibodies and a mixture of guinea pig complement (GIBCO BRL) and rabbit complement followed by isolation of live cells on a Ficoll gradient. CD4 + CD8 + thymocytes (double-positives [DPs]) and CD4 + CD8 − and CD8 + CD4 − thymocytes (single-positives [SPs]) were sorted on an Epics Elite flow cytometer (Coulter Immunology) using anti-CD4 and anti-CD8 antibodies. The enriched DN populations of the γD lines employed for the analysis of transcription in different developmental stages were not sorted and hence were only ∼50% pure. For semiquantitative transcription analysis of γD lines, DN, DP, and SP thymocytes were sorted to >99% purity using anti-CD4 and anti-CD8 antibodies. Total RNA was prepared by the single step method using water-saturated phenol as described 29 . 20 μg of tRNA was added as a carrier. Genomic DNA was prepared from defined numbers of cells as described 29 . Lambda DNA (2.5 μg; New England Biolabs, Inc.) was added as carrier. RNase protection assays 30 were performed with a riboprobe generated using T7 RNA polymerase and a linearized pKS Bluescript™ vector (Stratagene Inc.) construct containing the KpnI–BsrI fragment (273 bp) spanning the V–J junction of the G8 γ gene. The control riboprobe specific for γ actin mRNA 31 was generated using SP6 RNA polymerase. Densitometric analysis was performed using a PhosphorImager ® (Molecular Dynamics). Serial threefold dilutions of DNA were prepared in the presence of 50 μg/ml bacteriophage lambda DNA (New England Biolabs, Inc.), and PCR reactions were performed as described 11 with the L2 and J1 primers 32 . The transgene contained an XhoI linker in the V2 coding sequence; thus, digestion of the products with XhoI distinguished the endogenous product from the transgene product. The sample dilutions were compared with a standard curve prepared with DNA from the DN2.3 hybridoma 33 , which contains two Vγ2 rearranged genes and four tubulin genes. A β tubulin PCR was used to normalize the samples. The bands were visualized by autoradiography, and their intensities were measured on a PhosphorImager ® . Reverse transcriptase (RT)-PCR was employed for a parallel analysis of transcript levels in the γD transgenic lines. The procedure was done as described using either oligo-dT or J1 primer for reverse transcription and the L2 and J1 primers (or tubulin primers) for PCR 32 , with or without RT, except that 1 μCi α-[ 32 P]dCTP was added during the PCR amplification step, and 28 cycles of amplification were performed. The PCR products were digested and analyzed as described for the genomic PCR. Anti–Vγ2 TCR (UC3-10A6) and anti–δ TCR (GL3) were purified and conjugated with biotin and FITC, respectively. Anti-CD8α–Tricolor was purchased from Caltag Labs., and anti-CD4–Red 613 was from GIBCO BRL. Unseparated thymocytes from adult mice were stained with all four antibodies in the first step and streptavidin–PE (Molecular Probes, Inc.) in the second step. Gated TCR-γ/δ + CD4 − CD8 − thymocytes were examined for Vγ2 expression on an Epics XL-MCL flow cytometer (Coulter Immunology). Initially, we compared the in vivo activity of two γ transgene constructs consisting of Vγ2, 4, and 3 gene segments upstream of the Jγ1-Cγ1 genes, all in their germline configurations. The two constructs were identical except that one lacked a 2.8-kb 3′ fragment that contains 3′E Cγ1 . We found that several independent transgenic lines of each type consistently underwent rearrangement of Vγ2 to Jγ1 in thymocytes and that both constructs were efficiently transcribed (data not shown; see below for a similar analysis). These data indicated that 3′E Cγ1 is not absolutely required for either transgene rearrangement or expression and raised the possibility that the constructs contained a second cis-acting enhancer-like element. A clue to the site of such an element came from a previous study of a transgene construct (19L5) containing a rearranged Vγ2–Jγ1Cγ1 gene lacking 3′E Cγ1 that was not expressed in vivo (reference 20 and Raulet, D., unpublished data). Compared with the constructs above, 19L5 lacked a 1.5-kb segment of DNA upstream of Vγ2 on its 5′ end. These data raised the possibility that a relevant enhancer element might lie on this 1.5-kb DNA segment 5′ of Vγ2. Examination of the region upstream of Vγ2 demonstrated a clear DNase I hypersensitive site, denoted HsA, in adult thymocytes . HsA mapped to the region 3 kb upstream of Vγ2, corresponding to the 5′ region that was absent from the 19L5 construct compared with the rearrangement constructs described above. The site was not DNase hypersensitive in B lymphocytes (LPS blasts, 90% B cells) or liver cells . In a parallel analysis, the 3′E Cγ1 region was weakly hypersensitive in normal adult thymocytes (data not shown). The DNase hypersensitivity of 3′E Cγ1 (designated HsE) was more clearly demonstrated in a transgenic line with 15 copies of an integrated TCR-γ transgene, called γB, that consists of 40 kb of contiguous germline DNA from the γ locus . HsE was also hypersensitive to DNase I in B cells of the transgenic mice but was not hypersensitive in liver cells. Several other hypersensitive sites, most of them weak, were also detected in the transgene, but these have not been corroborated in nontransgenic cells (data not shown). To systematically investigate the transcription-enhancing activities of HsA and 3′E Cγ1 in vivo, we compared four transgene constructs containing a prerearranged Vγ2–Jγ1Cγ1 gene . The rrV2H + E + construct containing both HSA and 3′E Cγ1 was previously described 25 . The rrV2H + E − construct contained HsA but lacked the 2.8-kb 3′ fragment containing 3′E Cγ1 , rrV2H − E + contained 3′E Cγ1 but lacked a 1.5-kb 5′ fragment containing HsA, and rrV2H − E − lacked both the 5′ and 3′ fragments. Founders were either killed and analyzed directly or bred to generate transgenic lines. Transgene copy numbers were determined by Southern blot analysis. In the cases where founder mice were analyzed directly, we determined transgene copy number in the cells being examined to minimize the effects of the transgene mosaicism that sometimes occurs in founder animals. A quantitative RNase protection assay was used to measure Vγ2 transcripts in RNA from peripheral T cells and thymocytes from the transgenic mice. The riboprobe spanned the unique V–J junctional region of the transgene, allowing the specific detection of transgene-encoded transcripts as full length protected products. γ Transcript levels were normalized by inclusion of a control γ actin probe in each reaction. The results demonstrated that the transgene was efficiently transcribed in peripheral T cells from all three rrV2H + E + lines and from all six rrV2H + E − lines . Similarly, transgene transcription was detected in thymocytes from all of these transgenic lines . Transcription was T cell–specific, as no transcripts could be detected in B cells or kidney cells from several representative transgenic lines (data not shown). In contrast to the transgenes containing HsA, transgene transcription could not be detected in either peripheral T cells or thymocytes of the five rrV2H − E − transgenic lines. Thus, the fragment containing HsA consistently enhances T cell–specific γ gene expression in chromatin templates in the absence of 3′E Cγ1 . In contrast to the consistent expression of transgenes containing HsA, sporadic expression was observed in the case of the rrV2H − E + transgene, which contained 3′E Cγ1 but not HsA. Of the 14 lines tested, expression was detected in 5 or 6 lines in peripheral T cells and in 7 or 8 lines in thymocytes . No expression of the transgene was detected in B cells or kidney cells from several representative transgenic lines (data not shown). Sporadic expression of the rrV2H − E + transgene was dependent on the 3′ fragment containing 3′E Cγ1 because, as already discussed, the rrV2H − E − transgene lacking this fragment was not expressed in five independent lines. Hence, 3′E Cγ1 can enhance transcription in chromatin templates but is subject to transgene position effects. To allow quantitative comparisons, transgene expression levels in peripheral T cells were plotted against transgene copy number. One unit of transcripts was defined as the level of transcripts directed by an endogenous rearranged Vγ2 gene. This value was determined by parallel analysis of the DN2.3 γ/δ cell line, which contains two rearranged Vγ2 alleles . In transgenic lines that contained HsA (rrV2H + E + and rrV2H + E − ), the graphs revealed a roughly proportional relationship between the number of integrated transgene copies and the levels of transgene expression . Furthermore, the slope of the graphs was ∼1, indicating that the level of transcripts per transgene copy was roughly the same as the level directed by an endogenous Vγ2 gene. Even an rrV2H + E + transgenic line with only two transgene copies exhibited a similar level of transgene expression per copy as the endogenous gene . In contrast, the rrV2H − E + transgene, which lacked HsA but contained 3′E Cγ1 , was transcribed at detectable levels in less than half of the lines . The lines where transcripts were detectable were all high-copy lines. No transcripts were detected in the lines harboring the rrV2H − E − construct. These results demonstrated that the transgenes that contained HsA exhibited position-independent, roughly copy number–dependent transcription of the transgene in peripheral T cells. In contrast, the transgene that contained 3′E Cγ1 but not HsA exhibited severe position effects. Transgene expression in γ/δ cells was investigated by determining transcript levels in sorted γ/δ cells in one transgenic line of each type and by assessing the effect of the transgene on the percentage of Vγ2 + cells among thymic γ/δ cells in several lines. Abundant transgene transcripts were present in γ/δ T cells from the three lines examined, representing an rrV2H + E + line, a high-expressing rrV2H − E + line, and an rrV2H + E − line . This result was also confirmed by RT-PCR assay using purified peripheral γ/δ T cells (data not shown). Similar levels of transcript were found in sorted α/β T cells from the three lines. The expression of the transgene in α/β T cells is probably due to the absence from the transgene of a transcriptional silencer that inhibits expression of endogenous γ genes in α/β T cells 24 34 (see Discussion). Flow cytometry was employed to determine the percentage of Vγ2 + thymic γ/δ cells in transgenic lines of each type . We chose lines that had the most similar transgene copy numbers to minimize the effect of gene dosage. In nontransgenic mice, ∼35–50% of thymic γ/δ cells expressed Vγ2. The percentage was unaffected in two rrV2H − E − lines (41–48%) but was elevated to 80–94% in the two rrV2H + E + transgenic lines. The percentage was also elevated in two rrV2H + E − lines (∼70%) and two rrV2H − E + lines that exhibited high levels of transgene transcripts in the thymus (∼80%). In contrast, two rrV2H − E + lines that were expressed poorly at the mRNA level also showed no enhancement in the percentage of Vγ2 + cells (30–37%). Thus, transgene expression at the mRNA level in bulk populations correlated with Vγ2 surface expression in γ/δ cells. Furthermore, the position effects exhibited by the rrV2H − E + transgene in bulk populations were recapitulated in the analysis of γ/δ cells. Normalization of the transcript levels determined by RNase protection to transgene copy number demonstrated that the rrV2H + E − transgene was expressed at higher levels in peripheral T cells than in thymocytes in all six transgenic lines, by an average of 4.5-fold . In contrast, the rrV2H − E + transgene was expressed at lower levels in peripheral T cells than in thymocytes in all the lines where expression could be detected, by an average of fivefold. In the rrV2H + E + lines, the transcript levels in thymocytes were similar to the levels in peripheral T cells, with one low-copy line exhibiting marginally higher (twofold) expression in thymocytes. These data suggested that HsA and 3′E Cγ1 are differentially regulated in peripheral T cells and thymocytes. To clarify the developmental activity of the two elements, we examined representative lines for transgene expression during thymocyte ontogeny and in subsets of adult thymocytes . The transgene with both elements, rrV2H + E + , was expressed well in fetal thymocyte populations from day 14–18 of gestation. Similarly, two rrV2H − E + lines that exhibited transgene expression in adult thymocytes also exhibited substantial transgene expression in fetal thymocytes. In contrast, the rrV2H + E − transgene was expressed very poorly in fetal thymocytes in both lines tested. We conclude that the HsA element displays poor enhancing activity in fetal thymocytes, whereas the 3′E Cγ1 element, when not subject to position effects, evinces relatively strong activity in fetal thymocytes. Transgene expression levels were also determined in adult immature CD4 − CD8 − (DN) thymocytes, immature CD4 + CD8 + (DP) thymocytes, and a mixture of the relatively mature SP CD4 + CD8 − and CD4 − CD8 + thymocytes . The rrV2H + E + transgene was expressed well in all of these cell populations. Consistent with the ontogeny data, all four rrV2H + E − transgenics tested exhibited poor expression in immature DN and DP thymocytes but strong expression in SP thymocytes and peripheral T cells. In contrast, in two lines where the rrV2H − E + transgene was expressed well in unseparated thymocytes, expression was relatively strong in the DN, DP, and SP populations but weak in peripheral T cells . These results suggest that 3′E Cγ1 , when not subject to negative position effects, functions well as an enhancer in immature thymocytes. In contrast, HsA by itself does not enhance transcription in immature thymocytes. As expected, little or no transgene expression was observed in DN thymocytes from two rrV2H − E + lines in which the transgene was expressed poorly in unseparated thymocytes and from the one rrV2H − E − line tested (data not shown). Four new transgenic recombination substrates were prepared to examine the role of HsA and 3′E Cγ1 in γ gene recombination . The γD construct consisted of a 5-kb genomic fragment containing HsA and Vγ2 attached to a 15.5-kb genomic fragment containing Jγ1, Cγ1, and 3′E Cγ1 ; γD(H + E − ) was identical to γD except it lacked 2.6 kb of DNA containing 3′E Cγ1 ; γD(H − E + ) contained the 3′E Cγ1 fragment but lacked 3.5 kb of DNA containing HsA; and γD(H − E − ) lacked the 3′E Cγ1 fragment as well as a 1.5-kb fragment of DNA encompassing HsA. In all of the transgenes, the Vγ2 gene contained a frameshift mutation to prevent expression of a functional protein. Rearrangement and expression of the transgenes was determined by semiquantitative PCR or RT-PCR, respectively, in thymocyte populations that had been enriched in DN cells (∼50% DN thymocytes). Vγ2–Jγ1 transgene rearrangements were easily detected in the single γD transgenic line that was examined . For comparative purposes, a separate analysis showed that the level of transgene rearrangement was one half to one third that of endogenous Vγ2 gene rearrangement levels after normalizing for gene copy. Approximately similar levels of rearrangement were detected in five of the six γD(H + E − ) transgenic lines, which lacked 3′E Cγ1 ; one line exhibited low levels of rearrangement. These findings corroborated the initial data in which 3′E Cγ1 was not necessary to support γ gene rearrangement in transgenic substrates. The role of HsA in stimulating recombination was suggested by the results with the γD(H − E − ) transgene, which was identical to γD(H + E − ) except that it lacked the HsA fragment. Rearrangement was undetectable in three of these transgenic lines and reduced by a factor of three to five in the remaining three lines. Hence, although low levels of rearrangement occurred in some transgenic lines in the absence of both HsA and 3′E Cγ1 , the fragment containing HsA stimulated high levels of rearrangement. Rearrangement of the γD(H − E + ) transgene was approximately normal in one transgenic line, undetectable in two lines, and reduced severalfold in a fourth line . This pattern of rearrangement, indicating clear position effects, cannot be clearly distinguished from the pattern observed in the γD(H − E − ) lines. Therefore, it is unclear from these data whether the 3′E Cγ1 element plays a discrete role in stimulating γ gene rearrangement (see Discussion). Transcripts of the rearranged genes were detected by RT-PCR in each of the γD, γD(H + E − ), and γD(H − E + ) lines where recombination was detected , and the relative levels were roughly correlated with the extent of rearrangement. In contrast, no such transcripts were detected in the γD(H − E − ) lines that exhibited low levels of rearrangement, supporting the earlier conclusion that transcription of the rearranged genes requires HsA and/or 3′E Cγ1 . To confirm that the developmental pattern of transgene transcription in the γD lines paralleled that of the rrV2 lines, sorted DN, DP, and SP thymocytes from γD(H + E + ) and γD(H + E − ) lines were assayed for transcripts of the rearranged transgene by semiquantitative RT-PCR . The results demonstrate that transgene expression was high in each population from the γD(H + E + ) line but was lower in DNs, undetectable in DPs, and high in SPs from the γD(H + E − ) line, consistent with the results from the rrV2 lines. The weak signal in the DNs of the γD(H + E − ) line is likely derived from the 5–10% of γ/δ T cells present in this population, as the transgene transcripts were hardly detectable in the CD3 − CD4 − CD8 − population of this line (data not shown). With the use of multiple restriction enzyme digests, we localized the DNase I hypersensitive site associated with HsA to a 462-bp PstI–NcoI fragment (data not shown). Although we have not proven that this small fragment contains the functional site defined by the transgenic studies, other studies have shown a colocalization of cis-acting functional sites and DNase I hypersensitive sites 35 . The sequence of this fragment revealed several consensus sites for known transcription factors, including sites for ebox proteins, myb, gata 3, lef/tcf, stat, and gaga factors . The data indicate that 3′E Cγ1 functions as an enhancer in vivo. In terms of enhancing transcription, 3′E Cγ1 seems to play a more important role than HsA in immature thymocytes. DNase I hypersensitivity of 3′E Cγ1 in thymocytes was easily detected in transgene templates. The hypersensitivity in B cells may be due to the absence from the transgene of “silencer elements” present in the endogenous locus, although we emphasize that the transgenes were not expressed in B cells. The endogenous 3′E Cγ1 site was clearly hypersensitive in a dendritic epidermal γ/δ T cell line (Goldman, J., and D.H. Raulet, unpublished data) but was difficult to detect in thymocytes, perhaps because endogenous γ gene expression is silenced in most thymocytes. Overall, the 3′E Cγ1 element has the properties of a typical non-LCR enhancer element in that it is active in transient transfection assays, exhibits DNase I hypersensitivity, and enhances transcription in vivo but is subject to transgene position effects. The HsA element is a T cell–specific enhancer-like element that promotes transcription of rearranged γ genes in mature T cells. In addition, HsA stimulates recombination of transgenic γ rearrangement substrates. HsA was hypersensitive to DNase I in thymocytes but not in B cells or liver cells. Although HsA exhibited clear enhancer activity in mature T cells when integrated as a multicopy transgene, it was devoid of enhancer activity in transient transfection assays in the PEER and Jurkat cell lines, in which 3′E Cγ1 was active (data not shown). Both of these cell lines are unlikely to represent immature cells where HsA is nonfunctional, because the Jurkat line, at least, appears to be relatively mature based on its capacity to produce cytokines after TCR cross-linking. It is possible that HsA only functions with a homologous (γ gene) promoter element or only in the context of chromatin. Other instances have been reported where an element enhanced transcription when integrated in chromatin but not in transient transfection assays 18 36 37 . HsA, when combined with 3′E Cγ1 as in the rrV2H + E + transgene, confers efficient transgene expression in cells that normally express γ genes, including DN thymocytes and purified γ/δ cells, but does not drive expression in non-T cells. The enhanced percentage of Vγ2 + thymic γ/δ cells with the various lines of transgenic mice provides further evidence that the transgenes are indeed expressed in γ/δ cells. Significantly, expression of the rrV2H + E + transgene was independent of transgene position effects, and the level of transgene expression was proportional to the number of transgene copies. Thus, the combination of HsA and 3′E Cγ1 exhibits several characteristics of LCRs. We have not demonstrated that these elements are effective in single transgene copies, as none of the relevant lines contained just a single copy. However, an rrV2H + E + line with two transgene copies exhibited high levels of transgene expression. Although the rrV2H + E + transgene was regulated appropriately in most respects, it was inappropriately expressed in α/β lineage T cells, unlike endogenous γ genes of this type. Previous studies provided evidence that the absence of expression of endogenous γ genes in α/β lineage cells is due to an associated “silencer” element 24 . Although the silencer has not been subsequently defined or localized in detail, we have recently shown that transgenes containing an additional 10 kb of flanking 3′ DNA compared with the rrV2H + E + transgene, when present at low copy number, are strongly downregulated in α/β but not γ/δ T cells (reference 34 and Kang, J., and D.H. Raulet, unpublished data). These data are consistent with the conclusion that transgene expression in α/β T cells observed here is due to a lack of cell type–specific repressive elements. Transgenes containing only HsA or 3′E Cγ1 were clearly expressed inappropriately. The transgene containing 3′E Cγ1 but not HsA exhibited severe position effects and was not expressed in a copy number–dependent fashion, suggesting that 3′E Cγ1 by itself is insufficient to open the chromatin. The transgene containing HsA but not 3′E Cγ1 was poorly expressed in some cells in which endogenous γ genes are expressed well, such as DN thymocytes and fetal thymocytes. Nevertheless, HsA by itself did stimulate transcription in peripheral T cells in every line tested and was expressed in a roughly copy number–dependent fashion in peripheral T cells. The effects of HsA suggest that it may isolate linked genes from the inhibitory effects of neighboring chromatin. The putative chromatin-opening activity of HsA is probably operative even in cells where HsA alone functioned poorly as an enhancer. In total or DN thymocytes, HsA without 3′E Cγ1 was expressed poorly, and 3′E Cγ1 without HsA was subject to position effects; together, the elements supported high-level position-independent expression in both populations . Thus, HsA may relieve position effects in immature thymocytes, cooperating with 3′E Cγ1 to yield maximal levels of expression. Consistent with the role of HsA as a chromatin-opening element, we found that the 3′E Cγ1 site was not DNase I hypersensitive in an rrV2H − E + transgenic line that expressed the transgene poorly but was hypersensitive in an rrV2H + E + transgenic line (data not shown). Other LCRs have been shown to involve cooperative elements that enhance transcription and exhibit chromatin-opening activity 12 13 16 17 37 38 39 40 . Hence, chromatin-opening elements may be at least partially separable from classical enhancers in several LCRs, including the TCR-γ LCR. In the absence of 3′E Cγ1 , HsA drove transcription in mature SP thymocytes and peripheral T cells but not immature thymocytes. This was true of all four lines tested. In contrast, 3′E Cγ1 by itself, when not subject to position effects, drove expression in DN, DP, and SP thymocytes but did so less well in peripheral T cells. Correspondingly, 3′E Cγ1 functioned relatively well in fetal thymocytes. It will be of considerable interest to address the developmental roles of these two elements in vivo, where different sets of Vγ genes are used in the fetal and adult stages. Other instances have been reported of lymphocyte receptor genes with multiple, developmentally regulated enhancer elements. For example, both the CD4 and CD8 loci contain elements that seem to function differently in mature versus immature T cells 41 42 43 44 45 46 47 . In addition to promoting transcription of γ genes, our results with the γD series of recombination substrates indicate a role for HsA in supporting rearrangement of γ genes, even in the absence of 3′E Cγ1 . These results are of interest given the fact that HsA is a poor enhancer in immature thymocytes, the population in which rearrangement presumably takes place. It will be of interest to assess in future studies whether rearrangement promoted by HsA in the absence of 3′E Cγ1 primarily involves the chromatin-opening activity of HsA or is associated with prior transcription of unrearranged Vγ genes. For technical reasons, we have been unable to address whether such germline transcription occurs from the transgenes. The γD(H − E − ) transgene underwent weak and sporadic rearrangement, as did the γD(H − E + ) transgene. These data alone were therefore insufficient to assess the role of 3′E Cγ1 in stimulating γ gene rearrangement. The finding that transgene rearrangement occurs to a limited extent in some lines lacking both elements is surprising, as enhancer elements are usually required for V(D)J recombination. It is possible that the transgene integrated into especially open chromatin in these lines. Alternatively, it remains possible that elements in the transgenes other than 3′E Cγ1 and HsA participate in stimulating γ gene rearrangement. However, it is notable that no transcription of the rearranged transgenes was detected, confirming the importance of 3′E Cγ1 and HsA in transcription. All of the enhancer elements identified to date in antigen receptor loci are located either downstream of the constant regions or within the J–C introns. The location of HsA between Vγ2 and Vγ5 is therefore a novel scenario for antigen receptor genes. A ramification of the inter-V region location of HsA is that rearrangements of Vγ5 to Jγ1 will delete the element. Therefore, HsA must be unnecessary for supporting transcription of rearranged Vγ5 genes. One possibility is that HsA is only necessary to initially open the chromatin surrounding the Vγ genes and that subsequent maintenance of an open configuration is controlled by other elements and/or factors. Alternatively, there may exist additional elements upstream of the Vγ5 gene that support chromatin opening in the relevant cells. As the Vγ5 gene is unusual in that it is believed to undergo rearrangement preferentially in intestinal epithelial lymphocytes rather than thymocytes 48 , it would not be surprising if the gene was regulated differently than the other Vγ genes. Finally, it is possible that the endogenous γ locus is sufficiently open in the absence of HsA for at least some cells to efficiently transcribe γ genes. It will be of interest to explore these possibilities by deleting HsA and/or 3′E Cγ1 at their endogenous locations. | Study | biomedical | en | 0.999997 |
10477552 | Homozygous viable motheaten (me v ) mice (C57BL/6J-me v strain) used in this study were from The Jackson Laboratory, propagated by heterozygote (+/me v ) sibling matings. Wild-type (+/+), +/me v , and homozygous (me v /me v ) mice were genotyped by PCR. The me v allele was detected (35 cycles; 1 min at 94°C, 1 min at 63°C, 1 min at 72°C) using two primers: 5′ GGT GGA GAA GAA AGG CCG GGA 3′ (me v –specific forward primer) and 5′ TAT CAG GCT TGG CAG CAA AC 3′ (common reverse primer). The wild-type allele was detected (35 cycles; 1 min at 94°C, 1 min at 64°C, 1 min at 72°C) using primers 5′ GGT GGA GAA AGG CCG GGT 3′ (wild-type SHP-1–specific forward primer) and 5′ TAT CAG GCT TGG CAG CAA AC 3′ (common reverse primer). Sex- and age-matched adult me v /me v mice and +/+ littermates (∼4 wk old) were used in this study. Total mouse bone marrow cells were aspirated from two femurs of each mouse. Total bone marrow cells were used for chemotaxis and suppression assays of HPCs. Total splenocytes were prepared by crushing spleens through iron meshes. Low density spleen mononuclear cells were recovered after density-cut on Lympholyte-M (Cedarlane Labs.). CD4 + T cells were isolated from the low density splenocytes by staining with biotin-labeled antibodies to CD4 (clone L3T4; PharMingen) followed by positively selecting the stained CD4 + T cells using streptavidin beads and magnets (Miltenyi Biotech). Isolated CD4 + T cells were ≥95% pure by CD4 expression. Total bone marrow cells were plated at 5 × 10 4 , and total splenocytes were plated at 5 × 10 5 /ml in 1.0% methylcellulose culture medium containing growth factors (30% vol/vol fetal bovine serum [Hyclone], 1 U/ml recombinant human erythropoietin [Amgen Biologicals], 50 ng/ml recombinant murine steel factor [Immunex Corp.], 5% vol/vol pokeweed mitogen mouse spleen cell–conditioned medium , and 0.1 mM hemin [Eastman-Kodak Co.]). Colonies derived from granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells were scored after 7 d of incubation in a humidified environment at 5% CO 2 and lowered (5%) O 2 as previously described 24 . The percentage of progenitors in S-phase was estimated by the high specific activity tritiated thymidine kill technique 24 . Absolute numbers of progenitors per organ were calculated based on the number of viable, total nucleated cells per femur or spleen, and on the number of colonies scored per number of cells plated. For assessment of suppression of CFU-GM, total bone marrow cells were plated in the presence of the 1.0% methylcellulose culture medium containing growth factors as noted above in the absence or presence of control medium, or purified recombinant preparations of murine TNF-α or IFN-γ, each at 10 ng/ml, or 100 ng/ml of various human chemokines (MIP-1α, MCP-1, exodus, SLC, TECK, MIP-4, CKβ-11, IL-8, IP-10, MIG, ENA78, GCP-2, lymphotactin, MCP-4 fractalkine, and MIP-1β). These cytokine/chemokine concentrations have previously been shown to be optimal for inhibition of colony formation by bone marrow progenitor cells from mice of different strains. IFN-γ, TNF-α, and most chemokines were purchased from R&D Systems, except for CKβ-11, which was purified from Chinese hamster ovary cells 25 . Quantitative chemotaxis assays using transwells (6.5-mm diameter, 5-μm pore size, polycarbonate membrane; Costar) have been described previously 11 12 . For chemotaxis of HPCs, 5 × 10 5 total bone marrow cells were used for each chemotaxis chamber. SDF-1 (obtained from Dr. Ian Clark-Lewis, University of British Columbia, Vancouver, Canada) was added to the lower chamber at various concentrations. Chemotaxis was allowed for 4 h. Input cells and cells migrating to the lower chamber were collected, and assayed for colony forming cells in methylcellulose culture containing the growth factors noted above. Cells from two transwells were combined to obtain enough numbers of HPCs for triplicated colony assays. Cells were plated at 5 × 10 4 per 1 ml methylcellulose culture medium. After 7 d, colonies deriving from CFU-GM were differentially counted using standard techniques under an inverted microscope 24 . Chemotaxis of mouse leukocytes has been described previously 12 . 5 × 10 5 low density mononuclear cells were used for input cells for chemotaxis of B and T lymphocytes and bone marrow macrophages. Chemotaxis was allowed for 3 h at 37°C. Input cells and cells migrating to the lower chambers were collected. For collection of bone marrow macrophages, cells attached to the bottom of culture plates were detached by incubating in 5 mM EDTA/PBS (pH 7.4) for 20 min followed by extensive pipetting. Input cells and cells migrating to the lower chamber were stained with antibodies (anti-CD11b/Mac-1–TRIcolor and anti-F4/80–FITC for bone marrow macrophages; anti-B220–PE or anti-IgM–PE, anti-CD4–Cychrome and anti-CD8–FITC for splenic lymphocytes) and analyzed by FACscan ® (Becton Dickinson) in a time-based manner for 20 s. Anti-CD4–Cychrome (clone L3T4), anti-CD8a–FITC (clone Ly-2), anti-B220 (clone RA3-6B2), and IgM-PE (clone Igh-6b) were purchased from PharMingen. Anti-F4/80–FITC was purchased from Serotec. Anti-Mac-1–TRIcolor was purchased from Caltag Labs. Migration of each subset was normalized for numbers of each subset of input cells. Actin polymerization assays were performed as previously described by others 26 with some modification. Cells were resuspended in RPMI 1640 medium supplemented with 0.1% BSA at 10 6 cells/ml. SDF-1 was added at various concentrations (2, 20, 200, and 2,000 ng/ml) to the cell solution, and at a peak time point (25 s after treatment with SDF-1), 0.1 ml of FITC-labeled phalloidin solution (4 × 10 −7 M FITC-labeled phalloidin, 0.5 mg/ml 1-α-lysophosphatidylcholine, and 18% formaldehyde in PBS, all from Sigma Chemical Co.) was added to 0.4 ml of cell solution to stain and fix cells. Cells were incubated for 10 min at 37°C, pelleted, and resuspended in 0.5 ml of 2% paraformaldehyde solution. Mean fluorescence was measured by FACscan ® . Spleen CD4 + T lymphocytes from wild-type and me v /me v mice were suspended in RPMI 1640 medium with 10% FCS and preincubated at 37°C for 10 min. The kinase assay was carried out as previously reported 27 . In brief, 5 × 10 6 cells were stimulated with SDF-1 at 2 μg/ml for 60 s and lysed in RIPA buffer. 250 μg of cell lysate was immunoprecipitated with anti–Erk 1 polyclonal antibody and protein A–Sepharose 4B beads. The immunocomplex was then washed twice with HNTG (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 1 mM Na 3 VO 4 ) and once with kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl 2 , 2 mM 2-ME, and 1 mM Na 3 VO 4 ). The reaction was carried out in a mixture containing 20 μl of kinase buffer, 1 mg/ml myelin basic protein, 80 μM ATP, and 0.075 μCi/μl [γ- 32 P]ATP at 30°C for 10 min. The beads were spun down, and 18 μl of supernatant was spotted onto P81 Whatman paper. The papers were then washed five times with 180 mM phosphoric acid and once with ethanol, then air dried and counted on a Beckman scintillation counter. For SHP-1 phosphorylation and association of SHP-1 with PI-3K, anti–SHP-1 polyclonal antibody (Santa Cruz Biotechnology) was used to immunoprecipitate SHP-1 from cell lysates of splenic CD4 + T cells or murine FDCP-1 cells activated as described above. Immunoprecipitates were resolved in SDS-PAGE and blotted with antiphosphotyrosine antibodies (Upstate Biotechnology, Inc.) and then with anti–SHP-1 antibody or anti–PI-3K P85 (Upstate Biotechnology, Inc.) as previously described 27 28 . Total RNA was isolated from various sources of cells, including bone marrow (total bone marrow cells) and spleen (low density mononuclear cells and isolated CD4 + T cells) using Trizol solution (GIBCO BRL). Multiprobe RNase protection template sets mCR5 (CCR1, CCR1b, CCR3, CCR4, CCR5, CCR2, L32, and GAPDH) and mCR6 (CXCR2, CXCR4, CXCR5, L32, and GAPDH) were purchased from PharMingen. High specific activity RNA probes were in vitro transcribed in the presence of [α- 32 P]UTP (3,000 Ci/mmol; 10 mCi/ml; Amersham Life Science) using T7 RNA polymerase included in the in vitro transcription kit (PharMingen) according to manufacturer's instructions. Probes and total RNA were allowed to hybridize and treated with RNAse A and T1, and followed by proteinase K treatment using RPA kit (PharMingen). The protected RNAs from the RNAse treatment were resolved on 8 M urea/5% acrylamide sequencing gels. Filters were dried, exposed on X-ray film (X-OMAT; Eastman-Kodak Co.), and analyzed for band radioactivity using a phospho-image analyzer (Molecular Image Analyzer; Bio-Rad). Each band's radio intensity was normalized for L32 internal control, and averaged expression levels from three to four independent experiments were plotted in Fig. 5 C. Student's t test was used to analyze data for significance. P < 0.05 was considered significant. SDF-1, expressed ubiquitously in many organs including bone marrow and lymphoid tissues, induces chemotaxis of HPCs. It has been suggested that SDF-1 is a chemoattractant that induces the homing and retaining of progenitor cells in bone marrow 11 29 . Thus far, SDF-1 is the only chemokine shown to induce chemotaxis of early stage myeloid and lymphoid progenitors. We examined the chemotactic responsiveness of CFU-GM (progenitor cells for granulocytes and macrophages) in bone marrow of me v /me v mice and +/+ littermates. The background migration of CFU-GM in +/+ mice and me v /me v mice was very low . In response to SDF-1, a typical bell-shaped chemotactic response was observed for +/+ progenitors . Bone marrow CFU-GM from me v /me v mice demonstrated a much higher chemotaxis rate (>50%) than their +/+ counterparts (slightly >10%). Also, me v /me v progenitors began to migrate at lower concentrations of SDF-1 than +/+ progenitors, demonstrating an increased sensitivity of me v /me v CFU-GM to the chemotactic effect of SDF-1. The suppressive activity of chemokines for proliferation of immature subsets of HPCs is a distinct biological activity from their chemotactic activity. Thus far, 21 chemokines that cross the CC, CXC, and C subfamilies have demonstrated such suppressive activity 30 . Among these chemokines, we tested 14 myelosuppressive chemokines (MIP-1α, MCP-1, exodus, SLC, TECK, MIP-4, CKβ-11, IL-8, IP-10, MIG, ENA78, GCP-2, lymphotactin, and MCP-4) for their effects on colony formation of bone marrow CFU-GM. These chemokines cross the CC, CXC, and C families of chemokines. As controls, we included two previously determined nonsuppressive chemokines (fractalkine, a CX 3 C member, and MIP-1β, a CC member). As shown in Fig. 2 , all 14 suppressive chemokines, but not the 2 nonsuppressive chemokines at an optimal concentration (100 ng/ml), significantly inhibited proliferation of +/+ littermate control CFU-GM. In contrast, none of the tested chemokines inhibited the proliferation of me v /me v myeloid progenitor cells . As additional controls for suppression, TNF-α and IFN-γ, previously shown to be suppressors of HPC proliferation 31 , were assessed. In contrast to the chemokines, TNF-α and IFN-γ demonstrated equally significant suppressive activities for bone marrow CFU-GM from both me v /me v and +/+ mice. The suppressive activities of chemokines are directly related to the percentage of HPCs in the S-phase of the cell cycle. The higher the percentage of HPCs in S-phase, the greater the inhibition by chemokines 2 . Therefore, we examined the cycling status (percentage in S-phase of the cell cycle) of CFU-GM, BFU-E, and CFU-GEMM in the bone marrow of me v /me v and +/+ control mice as estimated by high specific activity tritiated thymidine kill assay 24 . CFU-GM, BFU-E, and CFU-GEMM in the bone marrows of me v /me v mice were more actively proliferating (a greater percentage of HPCs in S-phase) than were these cells in +/+ mice ( Table ). Thus, the inability of CFU-GM, BFU-E, and CFU-GEMM in the marrow of me v /me v mice to respond to inhibition by chemokines could not be attributed to lack of cycling HPCs. The difference in cycling of CFU-GM, BFU-E, and CFU-GEMM in me v /me v and +/+ spleens was far greater than that of bone marrow progenitors demonstrating the especially enhanced proliferation states of me v /me v progenitors in spleen ( Table ). Since myeloid progenitor cells from me v /me v mice were more active in chemotaxis to SDF-1, and were not inhibited by a number of suppressive chemokines, it would be expected that the spleen and bone marrow of these mice would have more blood cells. In this regard, splenomegaly and abnormal hematopoiesis (excessive erythropoiesis and myelopoiesis) in the spleens of me v mice have been previously reported by others 32 . Thus we also examined the absolute numbers of myeloid progenitor cells in spleen and bone marrow ( Table ). Spleens from me v /me v mice had far greater numbers of myeloid progenitors (8.4 times more CFU-GM, 4.2 times more BFU-E, and 5.8 times more CFU-GEMM per organ) than did those from +/+ mice. Although the absolute numbers of progenitors in the marrows of me v /me v and +/+ mice were similar, the frequency of myeloid progenitors in marrow from me v /me v mice was higher than in +/+ mice, as the nucleated cellularity of me v /me v mice was significantly decreased (perhaps due to the smaller size of me v /me v mice). To determine whether mature leukocyte populations have any altered chemotactic responses, we tested leukocytes in spleen and bone marrow from me v /me v and +/+ mice. Spleen lymphocytes, including CD4 + T cells, CD8 + T cells, B220 + B cells, and bone marrow Mac-1 + F4/80 + macrophages were tested for their chemotactic response to SDF-1. We observed a twofold greater basal motility (an ability to transmigrate through a porous membrane independently of chemoattractants) in me v /me v CD4 + T cells than in their +/+ counterparts . In response to SDF-1, significantly more me v /me v CD4 + spleen T cells migrated than did +/+ CD4 + T cells. Splenic CD8 + T cells from me v /me v mice demonstrated a similar enhancement in chemotaxis to SDF-1 at all concentrations tested . B220 + B cells in spleen and bone marrow F4/80 + macrophages from me v /me v mice also showed significantly enhanced chemotaxis to SDF-1. However, in response to another chemokine, SLC, that is a more efficacious chemoattractant than SDF-1 for murine CD4 + T cells 12 , no enhancement of CD4 + T cell chemotaxis has been observed, demonstrating the differential effect of SHP-1 on chemotaxis to chemokines . Chemokines bind and activate G protein–coupled receptors followed by activation of various signaling molecules, leading to reorganization of actin cytoskeletal structures. Chemokine-dependent actin polymerization is an important event for cell motility, cell polarization, and formation of membrane structures such as uropods. The latter is implicated in adhesion to other cells and extracellular matrix proteins. To evaluate changes in intracellular events, we compared the basal and chemokine-induced levels of polymerized actin (F-actin) in cells from me v /me v and +/+ mice. The basal levels of F-actin in splenic lymphocytes and more specifically in splenic CD4 + T cells from me v /me v mice were greatly increased when compared with their +/+ counterparts . When treated with SDF-1 at various concentrations, +/+ splenic lymphocytes and CD4 + T cells increased cellular F-actin in a dose-dependent manner . Splenocytes and CD4 + T cells from the me v /me v mice demonstrated a similar trend of actin polymerization to +/+ counterparts in response to SDF-1. The overall F-actin content of me v /me v cells was greater than that of +/+ cells after activation with SDF-1, demonstrating greater cellular activity of actin polymerization. Mitogen-activated protein kinase (MAPK) is activated in response to SDF-1 in a CXCR4-overexpressed cell line 15 . We examined whether this downstream pathway of SDF-1 signaling was altered in me v /me v cells. MAPK activity in wild-type spleen CD4 + T cells was consistently induced in response to SDF-1 to 1.5-fold of basal level . In me v /me v CD4 + T cells, MAPK activity was induced to 2.5 fold of basal level, demonstrating an enhanced activation of MAPK. SHP-1 was constitutively tyrosine-phosphorylated at a low level, and did not get more phosphorylated in response to SDF-1 in splenic T cells or in a growth factor–dependent mouse cell line, FDCP-1, which underwent chemotaxis in response to SDF-1 (data not shown). We also observed that there was no induced association of SHP-1 with a known positive signaling factor, PI-3K, in response to SDF-1 in FDCP-1 cells (data not shown). Expression of chemokine receptors is tightly regulated depending on the types, and states of activation and differentiation of cells. We examined the possibility that the altered biological activities of chemokines might be attributed to changes in receptor expression in me v /me v versus +/+ cells. We performed RNase protection assays to measure transcripts of various mouse chemokine receptors, including the CXC chemokine receptors CXCR2, CXCR4, and CXCR5, and the CC chemokine receptors CCR1, CCR1b, CCR2, CCR3, CCR4 and CCR5 in cells from bone marrow and spleen. Expression of CXCR4, the receptor for SDF-1, did not change in me v /me v versus +/+ cells from bone marrow, spleen, and CD4 + T cells . Expression of CXCR2, a receptor for IL-8, GCP-2, GRO-α, -β, -γ, ENA-78, and NAP-2, was enhanced in leukocytes from bone marrow and spleen, and in spleen CD4 + T cells. The enhancement of expression of CXCR2 in me v /me v CD4 + T cells and splenocytes was especially notable . Expression of CXCR5, the receptor for BLC/BCA-1, was detected at high levels in +/+ splenocytes, and at low levels in me v /me v splenocytes. B cells were the major expression source of CXCR5 in +/+ splenocytes (data not shown), and the drastic decrease of CXCR5 expression appears to be due to deficiency of B cells in me v /me v spleen. In this regard, numbers of B220 + or IgM + B cells were heavily reduced in me v /me v spleen. B cells made up 40–50% of +/+ versus <15% of me v /me v total splenocytes. Expression of CCR1, a receptor for MIP-1α and RANTES, was greatly enhanced in me v /me v CD4 + T cells and splenocytes versus their +/+ counterparts. Expression of CCR2 (a receptor for MCP-1 to MCP-5) was enhanced in me v /me v CD4 + T cells versus +/+ cells. Expression of CCR3 (a receptor for eotaxin-1 and -2 and MCP-2 and -4), CCR4 (a receptor for TARC and MDC), and CCR5 (a receptor for MIP-1α and -1β and RANTES) was very low in +/+ and me v /me v CD4 + T cells and total bone marrow cells. In this paper we present evidence that SHP-1 is a novel regulator of chemokine-mediated biological effects. Two major biological activities, chemotaxis and suppression of proliferation, have been examined in this study. SHP-1–deficient immature and mature hematopoietic cells manifest enhanced chemotaxis to a CXC chemokine SDF-1. Actin polymerization and MAPK activation in SHP-1–deficient cells were also hyperresponsive to SDF-1. In contrast, the deficiency of SHP-1 abolished the sensitivity of immature progenitors to suppression by chemokines. Cellular responses to chemokines are presumably regulated by equilibrium of a number of positive and negative signaling factors. It has been reported that chemokine-mediated biological activities involve several positive factors including PI-3K 15 , mitogen-activated protein kinases (Erk 1 and Erk 2) 15 33 , adenylate cyclase 34 , and Janus kinase 2 (JAK2) 13 . As a negative signaling factor, serine/threonine kinases such as β-adrenergic receptor kinase 2 can act as a desensitization factor for chemokine receptor–mediated signaling 35 36 . SHP-1 negatively regulates a number of signaling pathways by dephosphorylating proteins on specific tyrosine residues. So far, the putative substrates for SHP-1 include receptors such as IL-3R 37 , B cell CD22 receptor 38 , B cell receptor Ig-α subunit 39 , killer cell inhibitory receptor 40 , platelet endothelial cell adhesion molecule-1 41 , IL-2Rβ 42 , and CD72 43 , as well as other intracellular proteins such as JAK1 and JAK3 42 , JAK2 44 , ZAP-70 45 , and p56 lck 46 . Chemokine-induced biological activities can be inhibited by specific tyrosine kinase inhibitors such as genistein (a general tyrosine kinase inhibitor) 14 47 , tyrphostin B42 (a specific JAK2 kinase inhibitor) 13 , and PD98059 (an inhibitor of the Erk pathways) 48 , suggesting the importance of tyrosine phosphorylation in chemokine-induced signaling. The effectiveness of these inhibitors depends on cell types, chemokines, and types of biological effects. For future study, it will be important to identify the target protein(s) of SHP-1 that can modify chemokine responses. The observations of a dichotomy in response of me v /me v cells to chemokines was of interest. Although me v /me v cells are enhanced in chemotaxis (hyperresponse), they are resistant to myelosuppressive chemokines in proliferation (hyporesponse). Chemotactic activity and myelosuppressive effect are very distinct biological activities in terms of kinetics, cell target specificity, and cellular signaling machinery. Specifically, 21 chemokines out of 34 that had been tested previously have myelosuppressive activity, inhibiting the proliferation of immature subsets of bone marrow cells that are responsive to stimulation by combinations of growth factors 30 . In contrast, only three chemokines, SDF-1, SLC, and CKβ-11, out of many that were analyzed, have been demonstrated to have chemotactic activities for human myeloid progenitors 10 11 49 50 . SDF-1 is a chemoattractant for CFU-GM, BFU-E, and CFU-GEMM 10 11 , whereas SLC and CKβ-11 are mainly chemotactic for the macrophage progenitors (CFU-M), a subset of CFU-GM 49 50 . Chemokine-induced chemotaxis occurs quickly and depends on chemokine gradients, whereas inhibition of myeloid progenitor cells by chemokines is dependent on concentrations rather than gradients of chemokines. Chemotaxis involves cell motility and cytoskeletal machinery to migrate, including reorganization of actin structures, adhesion to and detachment from substratum, and termination of movement by desensitization. On the other hand, inhibition of myeloid cell proliferation by chemokines presumably requires regulation of cell cycle and mitogenic signaling machinery. Although further studies on the differential signaling for these two biological processes are required, our results suggest that the mechanism of SHP-1 in regulation of suppression is probably different from that of chemotaxis. It is possible that SHP-1 dephosphorylates different target signaling proteins that are directly involved in or indirectly set optimal conditions for signaling for chemotaxis and suppression. The inability of me v /me v myeloid progenitor cells to respond to chemokines is reminiscent of the insensitivity of malignant myeloid progenitor cells from some patients with leukemia to suppression by chemokines 2 51 . The activity of chemokines can be regulated in two ways in the cells: modification of chemokine receptor expression and/or signaling pathways. In this study, we examined whether SHP-1 modulates chemokine receptor expression. Expression of CXCR4, the receptor for SDF-1, did not change in me v /me v versus +/+ cells. Thus it does not appear that the enhanced chemotactic response to SDF-1 can be attributed to enhanced CXCR4 expression. In bone marrow cells from me v /me v mice, we observed similar or slightly enhanced expression of many chemokine receptors, including those that are shown to bind many of the myelosuppressive chemokines. Thus lack of suppression of me v /me v progenitors in response to chemokines is probably not due to loss of chemokine receptor expression. Leukocytes in me v /me v mice show similar profiles of chemokine receptor expression to that of activated cells. In me v /me v CD4 + T cells, expression of a number of chemokine receptors (especially CXCR2, CCR2, CCR1, CCR2, and CCR3) is much higher than in the +/+ counterparts. It is difficult, at this time, to pinpoint the direct cause for the enhanced expression of some chemokine receptors in me v /me v T cells. However, there are several possibilities: (a) absence of SHP-1 directly induces expression of these chemokine receptors; (b) absence of SHP-1 activates cells and activated cells express more chemokine receptors; and/or (c) the pathological environment in me v /me v (e.g., autoimmunity) induces expression of cytokines that in turn drive the activation of T cells and induction of chemokine receptors. Taken together, our results suggest that SHP-1 deficiency in me mice alters myeloid progenitor and mature leukocyte responses to chemokines. | Study | biomedical | en | 0.999998 |
10477553 | The construction of site-directed transgenic (sd-tg) mice expressing the H and/or L chain genes coding for well-defined anti-DNA Abs has been described previously 22 23 . BALB/c 3H9Vκ8 sd-tg mice (3H9Vκ8/BALB/c) were crossed onto the MRL- lpr/lpr background and backcrossed three times to generate 3H9Vκ8 MRL- lpr/lpr mice (3H9Vκ8/ lpr ). Animals homozygous for the lpr gene were identified by two PCR assays using tail DNA. In brief, 1–2 mm of tail was snipped off and placed into 80 μl tail digestion buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 2.5 mM EDTA, 0.45% NP-40, and 0.45% Tween 20) containing 2 μl proteinase K (20 mg/ml; Boehringer Mannheim). After overnight incubation at 55°C, tail samples were boiled for 10 min and kept on ice. 1 μl of tail DNA was used for both PCR assays. The oligonucleotides used for these PCRs have been described previously 24 . They were Fas-I2F (forward: 5′ agcatagattccatttgct 3′), with Fas-Z8 (reverse: 5′-caaattttattgttgcgaca-3′) to identify the lpr allele, or Fas-I2B (reverse: 5′-agtaatgggctcagtgca-3′) to identify the wild-type fas allele. PCR amplifications were set up in 50-μl vol containing 1 U of AmpliTaq Gold™ (Perkin Elmer Corp.), 1× buffer II, 250 μM of each dNTP, 2.5 mM MgCl 2 , and 40 pmol of each primer. Amplifications were carried out in an OmniGene Hybaid thermocycler (Hybaid) under the following conditions: denaturation/enzyme activation for 9 min at 92°C; then 35 cycles of 30 s each at 94, 56, and 72°C; final elongation at 72°C for 7 min. B cell hybridomas were generated from unmanipulated spleen cells from a 2-mo-old 3H9Vκ8/ lpr mouse using SP2/0 myeloma cells 25 as the fusion partner. Spleen cells from 3H9Vκ8/BALB/c mice were stimulated in vitro for 3 d with 20 μg/ml LPS (Sigma Chemical Co.) and fused as previously described 13 . Hybridomas were plated at limiting dilution, and only wells bearing single colonies on 96-well plates containing <30 hybrids per plate were expanded for analysis. Isotypes were determined using an indirect solid-phase ELISA as previously described 26 . Plates were coated with anti-Ig Ab and developed with alkaline phosphatase–labeled anti-IgM, anti-IgG, anti-IgA, anti-κ, or anti-λ. The enzyme activity was revealed by the substrate p -nitrophenyl phosphate (Sigma Chemical Co.) and optical density was read at 405 nm. All commercial Abs were from Southern Biotechnology Associates, Inc. Samples were titrated, and concentrations were estimated by comparison to a standard curve. Binding to ssDNA was measured by solid phase ELISA. Immunolon ® 4 plates (Dynex Technologies, Inc.) were coated with 60 μl of histone-free protamine (Sigma Chemical Co.) at 500 μg/ml. Then, 60 μl of boiled and quickly chilled salmon sperm DNA (90 μg/ml) was allowed to bind to protamine-coated plates overnight at 4°C in a humid chamber. The next day, the plates were blocked with PBS containing 1% BSA. After washing with PBS-Tween, serum samples or hybridoma supernatants at various dilutions were added and incubated for 2 h at 37°C. To detect DNA binders, alkaline phosphatase–labeled mouse isotype-specific antibody was used (Southern Biotechnology Associates, Inc.), and the enzyme activity was revealed by the specific substrate p -nitrophenyl phosphate (Sigma Chemical Co.). Optical density was read at 405 nm. Binding to dsDNA was measured by a two-step solution phase ELISA as previously described 27 . In brief, antibody concentrations in the supernatants were first standardized to 2 μg/ml. Then, supernatants and protein-free calf thymus biotinylated DNA (1 μg/ml) were mixed, incubated at 37°C for 60 min, and transferred to microtiter plates (Immunolon ® 4; Dynex Technologies, Inc.) coated with pure avidin (10 μg/ml, 50 μl/well; Avidin D; Vector Labs., Inc.). After a 60-min incubation at room temperature, plates were washed, and DNA–Ab complexes were detected with alkaline phosphatase–conjugated goat anti–mouse isotype-specific Ab (Southern Biotechnology Associates, Inc.). Antinuclear specificities of mAbs and serum Igs were determined by indirect immunofluorescence staining of HEp-2 cells using an ANA test kit (The Binding Site, Inc.) according to the manufacturer's directions. Substrate cells were incubated with culture supernatants or serum at different dilutions for 20 min, washed in cold PBS/BSA 1%, and stained with Alexa™ 488 goat anti–mouse IgG (Molecular Probes, Inc.). Slides were viewed under a Zeiss LSM510 confocal microscope using a C-apochromat 40×/1.2 NA water immersion objective. Positive ANAs were further tested for their capacity to bind dsDNA of the trypanosoma Crithidia luciliae using an anti-nDNA antibody test kit (CrithiDNA; Antibodies Inc.) following the manufacturer's directions. The procedure is the same as that described for the ANA test. Genomic DNA was purified from individual hybrids as previously described 26 . Primers and conditions used for H and κ chain PCR assays have already been detailed . A LD/JHCH PCR was designed (see below) and used as a first approach to distinguish between 3H9V H replacement 22 and somatic mutation because somatic mutation in the CDR3 of the 3H9 sd-tg was shown to prevent amplification in the LD/CDR3 PCR. All hybrids negative in the LD/CDR3 PCR were tested with the LD/JHCH PCR. This PCR is not as specific as the LD/CDR3 PCR, because the JHCH primer is not sd-tg specific, and the 3H9 leader primer binds V genes with similar leader sequences. Hence, 3H9 sd-tgs with mutations in CDR3 as well as V H replacements where the invading V H gene uses a 3H9-like leader sequence will be detected in this assay. All PCR amplifications were carried out in a TouchDown thermocycler (Hybaid). All primers were made by the Princeton University Synthesizing/Sequencing Facility. Genomic DNAs from individual hybridomas were used as templates from which to amplify V H and Vκ genes. PCR amplifications were set up in a 30 μl vol containing 1 U of AmpliTaq Gold™ (Perkin Elmer) 1× buffer II, 200 μM of each dNTP (Boehringer Mannheim), 50 pmol of each primer, and 1.5 mM MgCl 2 (Perkin Elmer). The 5′ primers used to amplify V H regions were either LD3H9 (5′-ctgtcaggaactgcaggtaagg-3′) or VH5.3 (5′-(g/c)aggt(g/t)cagctgcag(g/c) agtctgg-3′) in combination with a primer located in the JH–CH intron (JHCH: 5′-cttctctcagccggctccctc-3′). For PCR detection of Vκ genes, the forward primers were either specific for Vκ8 (MW133: 5′-ggtacctgtgggacattgtg-3′) or degenerate Vs (5′-ggctgcag(c/g)ttcagtggcagtgg(a/g)tc(a/t)gg(a/g)ac-3′) 28 and the reverse primer was a primer specific for the Jκ–Cκ intron (MW176: 5′-tgccacgtcaactgataatgagccctctc-3′). Amplifications were carried out with a thermal reactor (subambient TouchDown; Hybaid) as follows: V H : 92°C for 9 min, followed by 38 cycles of denaturation for 45 s at 94°C, annealing for 50 s at 56°C, extension for 1 min and 40 s at 72°C, and a final extension of 7 min at 72°C. Vκ8: 92°C for 9 min, followed by 38 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 56°C, extension for 30 s at 72°C, and a final extension of 7 min at 72°C. Vs: 38 cycles of denaturation for 30 s at 94°C, annealing for 40 s at 65°C, extension for 1 min and 40 s at 72°C, and a final extension of 7 min at 72°C. Each fragment was size selected on a 1.5% agarose gel (Ultra Pure Agarose; GIBCO BRL) and purified by QIAquick gel extraction (Qiagen Inc.). Nucleotide sequencing was performed using the ABI Prism™ Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq ® DNA polymerase, FS, according to the manufacturer's directions (Applied Biotechnology, Inc.). Reactions were run on the Applied Biosystems 377 PRISM automated DNA sequencer (PE Applied Biosystems). Total RNA was isolated from hybridomas using the RNAeasy kit (Qiagen Inc.), and 10 μg RNA was converted into cDNA using avian reverse transcriptase (Promega Corp.) and an oligo-dT/random hexamer primer mixture. 1 μl of cDNA was then amplified by PCR using Vs 28 as a forward primer and Cκ-specific primer as a reverse primer (Cκ: 5′-tggatggtgggaagatg-3′). The PCR program was as follows: 35 cycles consisting of 20 s at 94°C, 40 s at 55°C, and 90 s at 72°C. A Taq enzyme activation step of 9 min at 92°C was performed before the first cycle, and a final extension step of 7 min at 72°C ended the program. PCR products were purified on a gel with the QIAquick gel extraction kit (Qiagen Inc.) and were sequenced with the ABI prism system. For each sequence identified as Vκ23, cDNA was also amplified using a Vκ23 primer specific for the framework (FW)1 (Vκ23: 5′-gatattgtgctaactcagtctccagccac-3′) and the same Cκ-specific primer. PCR products were sequenced as described above. Genomic DNA was extracted from the tails of two MRL- lpr/lpr mice as described above. 1 μl of DNA was used in a PCR containing 50 pmol of a Vκ23 forward primer specific for the FW1 region (Vκ23F: 5′-gatattgtgctaactcagtctccagccac-3′), 50 pmol of Vκ23 reverse primer complementary to the FW3 region (Vκ23R: 5′-gaggccagctgttactctgttg-3′), 1.5 mM MgCl 2 , 200 μM dNTPs, 3 μl 10× buffer II, and 1 U AmpliTaq Gold™ (Perkin Elmer). The PCR program was as follows: 92°C for 9 min, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and extension for 40 s at 72°C. A final extension step at 72°C was carried out for 7 min. PCR products were size selected on a 1.5% agarose gel (Ultra Pure Agarose; GIBCO BRL) and purified with the QIAquick gel extraction kit (Qiagen Inc.). Purified DNA fragments were cloned into the pGEM ® -T easy vector (Promega Corp.). JM109 competent cells were transformed by electroporation, and transformants were directly tested for the nature of the insert by PCR, using two different sets of Vκ23-specific primers. One set was made up of the Vκ23 primers used in the initial amplification (see above) and another set was made up of Vκ23 primers (Vκ23′F: 5′-gtgactccaggagatagc-3′; Vκ23′R: 5′-gttgataccagtgtaggtt-3′) specific for the new Vκ23 gene (Vκ23GL2) similar to our Vκ23 sequences (see text). Plasmid DNA from positive colonies of each group was sequenced as described above using a plasmid-specific T7 primer. Two Vκ23 germline sequences were identified. One was identical to the DP12 mAb sequence 4 and was not amplified by the second set of primers. The other one differed from the DP12 sequence by two nucleotides located in the FW1 and CDR2 and was amplified with the Vκ23′ primers. Sequences were analyzed for homology to the original 3H9 and Vκ8 transgenes as well as to published Ig gene segments using EMBL/GenBank databases and Kabat et al. 29 . The sequences of 3H9 V H gene and the Vκ8 gene can be found in EMBL/GenBank/DDBJ databases under the accession numbers M18234 (3H9) and M34742 (Vκ8). The Vκ23 sequences described in this paper are available from EMBL/GenBank/DDBJ under the following accession numbers: AF139842–AF139849. The accession numbers for the V H sequences presented in Fig. 3 B are AF145959–AF145963. We tested the hypothesis that mutations accumulated at the same rate and for equal duration at three loci. Under the assumption that all three loci mutate at the same rate, we reasoned that if the three genes had mutated for equal lengths of time, they should have equal numbers of mutations per base. We used the genealogical trees to determine the number of independent mutations (the total sum of the branch lengths in each tree) that occurred at each of the three loci. By this measure, 3H9, Vκ8, and Vκ23 loci accumulated M 3H9 = 34, M Vκ8 = 33, and M Vκ23 = 18 mutations , respectively, in sequences of length of L 3H9 = 363 bases, L Vκ8 = 339 bases, and L Vκ23 = 324 bases . We used only mutations in the coding sequences for this analysis. Assuming the hypothesis is true and an underlying Poisson distribution of mutations, we determined the mean number of mutations per base (λ 0 ) from the maximum likelihood estimate, in this case, the average: 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{{\hat {{\lambda}}}}}_{0}= \left \left(\frac{{\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{{\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}\right) \right =\frac{85}{1026}={\mathrm{0.08285.}}\end{equation*}\end{document} Under the null hypothesis (H 0 : mutation for equal duration at three loci), the number of mutations in each sequence ( M 3 H 9 , etc.) would be determined by the same value of λ 0 mutations per base estimated by λ̂ 0 above. Our hypothesis amounts to equating the three individual mutation frequencies λ 3H9 , λ Vκ8 , and λ Vκ23 to a common frequency λ 0 . Thus, the total number of mutations in the three sequence sets would be governed by Poisson distributions with means M 3 H 9 = L 3 H 9 λ 0 , M v κ8 = L v κ8 λ 0 , and M v κ23 = L v κ23 λ 0 . Put formally: H 0 : λ 3 H 9 =λ v κ8 =λ v κ23 =λ 0 , i.e., all have the same number of mutations per base, meaning the same duration of mutation. We tested the alternative: H A : λ v κ23 <λ 3 H 9 =λ v κ8 using the following statistic: 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{Z}}_{0}=\frac{ \left \left(\displaystyle\frac{{\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}{{\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}\right) \right - \left \left(\displaystyle\frac{{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}\right) \right }{\sqrt{\displaystyle\frac{ \left \left({\mathrm{{\hat {{\lambda}}}}}_{3{\mathit{H}}9}+{\mathrm{{\hat {{\lambda}}}}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right }{ \left \left({\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right ^{2}}+\displaystyle\frac{{\mathrm{{\hat {{\lambda}}}}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{ \left \left({\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right ^{2}}}}{\mathrm{.}}\end{equation*}\end{document} The next section describes how Z 0 was derived. The alternative hypothesis includes the statement that λ 3 H 9 =λ v κ8 , so we estimated their common value 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*} \left \left(\frac{{\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}{{\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}\right) \right ={\mathrm{{\hat {{\lambda}}}}}_{3{\mathit{H}}9}={\mathrm{{\hat {{\lambda}}}}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}{\mathrm{.}}\end{equation*}\end{document} λ̂ v κ23 is estimated by M v κ23 L v κ23 . Under H 0 , these two formulas estimate the same quantity. Thus under H 0 , the expectation of their difference: 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*} \left \left(\frac{{\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}{{\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}\right) \right - \left \left(\frac{{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}\right) \right \end{equation*}\end{document} is zero. To obtain a standardized (mean = zero, variance = one) statistic, namely Z 0 , under H 0 , we divided the difference by the square root of its estimated variance, which we calculated as follows. The variance of a difference is the sum of the terms' separate variances, which is: 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*}{\mathit{Var}} \left \left(\frac{{\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}{{\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}\right) \right +{\mathit{Var}} \left \left(\frac{{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}\right) \right = \left \left(\frac{{\mathit{Var}} \left \left({\mathit{M}}_{3{\mathit{H}}9}+{\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right }{ \left \left({\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right ^{2}} \right + \left \frac{{\mathit{Var}} \left \left({\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right }{ \left \left({\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right ^{2}}\right) \right = \left \left(\frac{{\mathit{Var}} \left \left({\mathit{M}}_{3{\mathit{H}}9}\right) \right +{\mathit{Var}} \left \left({\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right }{ \left \left({\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right ^{2}} \right + \left \frac{{\mathit{Var}} \left \left({\mathit{M}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right }{ \left \left({\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right ^{2}}\right) \right {\mathrm{.}}\end{equation*}\end{document} The variance of a Poisson deviate is equal to its mean, thus, 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*}{\mathit{Var}} \left \left({\mathit{M}}_{3{\mathit{H}}9}\right) \right =\overline{{\mathit{M}}}_{3{\mathit{H}}9}={\mathit{L}}_{3{\mathit{H}}9}{\mathrm{{\lambda}}}_{0}\end{equation*}\end{document} and similar formulas hold for Var ( M vκ8 ) and Var ( M vκ23 ). Thus, the variance of the difference is: 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*}\frac{{\mathrm{{\lambda}}}_{3{\mathit{H}}9}+{\mathrm{{\lambda}}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}}{ \left \left({\mathit{L}}_{3{\mathit{H}}9}+{\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}8}\right) \right ^{2}}+\frac{{\mathrm{{\lambda}}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}}{ \left \left({\mathit{L}}_{{\mathit{v}}{\mathrm{{\kappa}}}23}\right) \right ^{2}}{\mathrm{.}}\end{equation*}\end{document} This quantity is estimated by substituting the above estimates of the λ's for their true values. For these data, Z 0 = 2.063. We sampled the Poisson distribution with parameters M i , i = 3H9, Vκ8, Vκ23 to obtain deviated M 3 H 9 , M v κ8 , and M v κ23 , rigorously under H 0 . From these, a new λ̂ 0 was obtained as above, and finally, a new value of Z was derived. If the new Z was at least Z 0 , the event was tallied. The procedure was repeated 100,000 times. In these trials, Z 0 was equalled or exceeded only 1,780 times, requiring H 0 to be rejected at the 1.78% level. In nonautoimmune mice, 3H9Vκ8 B cells are anergized. Consequently, 3H9Vκ8 transgenic mice have no detectable DNA binding activity in their serum 12 . To assess whether such anergic B cells escape tolerance in autoimmune MRL/ lpr mice, sera from 3H9Vκ8/ lpr mice were tested for the presence of anti-DNA Abs. Anti-DNA Abs were readily detected by ELISA as well as by indirect immunofluorescence on Crithidia luciliae (data not shown). Furthermore, ANAs were detected in the sera of 3H9Vκ8/ lpr as early as 2 mo of age. None of these Ab specificities was detected in sera from 3H9Vκ8/BALB/c mice . Hybridomas from 3H9Vκ8/ lpr mice have many features that indicate 3H9Vκ8/ lpr B cells are activated rather than anergic, as they are in the BALB/c background. Unmanipulated 3H9Vκ8/ lpr B cells yield high frequencies of hybridomas, unlike 3H9Vκ8/BALB/c B cells, which require LPS stimulation or immunization for hybridoma formation. In this regard, 3H9Vκ8/ lpr B cells are similar to those of diseased, nontransgenic, MRL/ lpr mice that generally have high spontaneous fusion efficiencies 30 . A high percentage of spontaneous hybridomas from diseased mice produce autoantibodies such as anti-DNA and RF suggesting that self-antigens are driving fusable B cells 31 . Hybridoma panels from 3H9Vκ8/ lpr mice also have high frequencies of anti-DNA-secreting B cells. For example, in one hybridoma panel derived from a 2-mo-old 3H9Vκ8/ lpr mouse, 34 (71%) out of 48 hybridomas secreted anti-DNA Abs ( Table ). This is not surprising given that the tg Ab binds ssDNA, but of these 34 hybridomas, 9 bound dsDNA in ELISA, and of these, 5 displayed a homogeneous nuclear staining pattern on HEp-2 cells . Anti-dsDNA and ANAs are rarely found in hybridoma panels from LPS-activated B cells from 3H9Vκ8/BALB/c mice; instead, most of these hybridomas produce anti-ssDNA Abs (reference 13 and Table ). Finally, the presence of isotype-switched anti-DNA Abs in the serum of 3H9Vκ8/ lpr and the secretion of IgG by 40% of the hybridomas ( Table and Table ) are other indications of antigen activation. Strikingly, all the dsDNA/nuclear binders are IgG. Together, these results provide strong evidence that these mice are undergoing a (self-)antigen-driven immune response. Anti-dsDNA/ANAs in 3H9Vκ8/ lpr mice could be generated either by somatic mutation or by gene replacement. To distinguish mutation from replacement, we examined the status of the H and L chain sd-tgs. Those hybridomas that retain both the 3H9 H and Vκ8 genes but express unique specificities are likely to be mutated. Those that have lost 3H9 and/or Vκ8 gene(s) would most likely have specificities generated de novo. We tested for the presence of V H and Vκ transgenes in hybridomas from the 3H9Vκ8/ lpr mouse using a series of PCR assays . Out of 48 hybridomas studied, 38 (79%) were positive for both the 3H9 and Vκ8 genes. Of these double-positive hybridomas, some did not bind DNA, whereas others have acquired specificity for anti-dsDNA ( Table ), suggesting that the 3H9 and/or Vκ8 transgenes have undergone somatic mutation. Similar results were obtained from a hybridoma panel of a 4-mo-old 3H9Vκ8/ lpr mouse (Brard, F., and M. Weigert, unpublished data). The V H genes of 13 IgG-secreting hybridomas with changed specificity (anti-dsDNA/ANA + or non-anti-DNA) were amplified and sequenced. Of these, eight were 3H9 + Vκ8 + mutants and five were 3H9 − Vκ8 + . Their nucleotide and deduced amino-acid sequences are shown in Fig. 3a and Fig. b , and are summarized in Table . Mutations in the 3H9 sd-tg are located both in the coding and in the noncoding regions. The average number of mutations per V region is high, 12.5, with the most mutated V region having 22 mutations (hybridoma 28) and the least mutated having 6 (hybridoma 16) . In the JHCH intron, the average number of mutations within the 60 bp immediately 3′ of J H 4 is 2.4, with the most mutated sequence having 5 mutations. Of note, hybridoma 46 has a deletion of three codons in CDR2. In the same region, hybridoma 28 has a stretch of five nucleotides that are different from their germline counterparts. We think this most likely results from some form of insertion and deletion. Wilson et al. 32 described similar insertion/deletion mutants in Ig hypervariable loops. In their study, deletions occurred at tandem repeat sequences and inevitably destroyed one of the tandem repeats. Insertions usually involved duplications of the immediately adjacent sequence. In 3H9's CDR2, we have identified such a repeat sequence, A GA GAT G GA GAT , at the site of the insertion/deletion mutations. The deletion in hybridoma 46 removes one full repeat element plus one adjacent codon. The sequence of the insertion in hybridoma 28, GAGTC, is similar to the repeat motif GAGAT. Interestingly, the GAGAT motif contains the putative hot spot sequence RGYW 33 , and it is known that deletion/insertion events usually occur in the vicinity of mutational hot spots 32 . Mutations were also found in 10 of the Vκ8 sd-tgs . Here again, mutations were found throughout the sequence. The average number of mutations per V region was 9.2, ranging from 18 mutations (hybridoma 50) to 3 mutations (hybridoma 16). All together, many of the V H and V L genes sequenced from the hybridomas presented in Table have replacement mutations in CDRs. Thus, it is possible that the novel specificities of these antibodies are the result of mutation in the 3H9 and/or Vκ8 genes. Hybridomas 50, 79, 15, 83, 28, and 38 share eight mutations in their 3H9 sequences, therefore it is likely that they come from an expanded B cell clone. Ordinarily, B cells are defined as members of a clone based not only on the presence of shared mutations but also on sequence identity of V H CDR3. However, sequence identity of V H CDR3 is not an applicable criterion by which to judge clonality in transgenic models. Nevertheless, the following observations indicate that these six hybridomas are indeed derived from a single B cell clone: first, these hybridomas all express the same isotype, IgG2a. Second, these hybridomas also share 15 mutations in their Vκ8 and 3H9 sd-tgs, collectively. Third, each of these hybridomas maintains a germline H chain allele and has identical endogenous κ chain rearrangements (see below). Furthermore, it is unlikely that the shared mutations are entirely the result of positive selection and/or hotspot mutation, because only two of these shared mutations are found in the sequences of hybridomas that we know to be unrelated on the basis of rearrangement status ; moreover, 7 out of the 15 shared mutations in both H and L transgenes are nonselectable, either because they are silent or because they occur in introns. The most interesting feature of this clone is that all members share a mutation to the TAG nonsense codon in the Vκ8 sd-tg at the beginning of the CDR1 . Because these clones secrete an IgG2a/κ Ab, the untargeted κ allele must be rearranged and expressed. To determine the nature of the expressed L chain, Vκ mRNA from these six hybridomas was sequenced . All express a Vκ23 gene joined to Jκ2′, the MRL counterpart of Jκ2. Analysis of the V–J junction revealed that the first codon of Jκ2′ is deleted and a histidine is found in its place. This unusual junction has previously been observed in other Vκ23Jκ2′ sequences 18 34 . The histidine found at the junction may derive from either the end of the germline Vκ23 gene or the nucleotides immediately 3′ of the Vκ23 gene. To evaluate the number of mutations in the Vκ23 sequences, it was necessary to determine the germline sequence of the Vκ23 gene. Because GenBank did not contain an appropriate sequence, we cloned Vκ23 genes from MRL/ lpr tail DNA. Two potential candidates, Vκ23GL1 and Vκ23GL2 were identified. Our six hybridoma Vκ23 sequences are most similar to Vκ23GL2. Assuming that Vκ23GL2 is the bona fide germline counterpart, the Vκ23 sequences from our hybridomas had an average of 6.5 mutations per V region, with the most mutated V region having 10 mutations (hybridoma 28) and the least mutated having 5 (hybridomas 38 and 83). These six sequences have three mutations in common, and two other mutations are shared by at least four out of the six clone members. The shared V H and V κ mutations form a hierarchy characteristic of expanded clones. This hierarchy of mutations allowed us to construct a genealogy of the clone members . The 3H9 and Vκ8 trees have roughly the same height and shape. This similarity illustrates that the onset and rate of mutation are the same in H and L chain genes. Although the Vκ23 tree has a “branch morphology” similar to that of the 3H9 and Vκ8 trees, it appears to have a shorter “trunk.” Furthermore, the average number of mutations is less in Vκ23 than in either 3H9 or Vκ8 (6.5 compared with 14 and 13, respectively). These results indicate that the Vκ23 L chain started to mutate later than the Vκ8 or 3H9 transgenes, but that once initiated, Vκ23 mutation proceeded at a similar rate. Taken together, these mutation patterns and tree shapes imply that the Vκ23 gene was rearranged and expressed after the onset of mutation in the 3H9 and Vκ8 transgenes. To confirm this conclusion, a statistical analysis testing the hypothesis that mutations accumulated for equal durations at the three loci was performed. Implicit in this calculation is the assumption, based on the absence of clear evidence to the contrary, that the mutation rate was equal at each of these three loci. We also assumed that the number of mutations found in each tree would be Poisson distributed. We estimated mutation frequencies per base from the number of mutations divided by the number of bases in the corresponding sequence set. The frequency estimates for our 3H9 and Vκ8 trees did not differ significantly, and in fact were very close to each other. We combined the 3H9 and Vκ8 data and compared their combined frequency estimate to that obtained for the Vκ23 sequences. To test the significance of this difference, we combined all three data sets for an overall estimate of mutation frequency. We then simulated mutations in the three trees by generating Poisson deviates with means adjusted for each gene's particular sequence length. We tallied the number of times the difference in mutation frequencies observed this way, (i.e., rigorously, under the null hypothesis of identical mutation durations for each sequence), was as large or larger than their actual observed difference. Their observed difference was equalled or exceeded in only 1.78% of 100,000 trials. This allowed us to reject the hypothesis that all three Ig loci mutated for equal duration . Therefore, we conclude that the Vκ23 L chain gene was rearranged in the periphery and then started to undergo somatic mutation. All members of this clone have acquired dsDNA specificity, and five out of six are ANA + . The acquisition of dsDNA and antinuclear activity could be due to mutation in either 3H9 or Vκ23. In particular, the mutations to asparagine in FW3 and CDR3 in Vκ23 might create anti-DNA specificity 35 . Alternatively, the Vκ23 L chain itself may create the ANA specificity ( Table ), since ANAs and antihistone antibodies derived from autoimmune mice are often associated with Vκ23 L chains 18 19 34 36 37 38 . Earlier studies examined the regulation of the 3H9 H chain tg in lpr/lpr mice 18 19 . The 3H9 H chain transgenic without an accompanying L chain transgene is an excellent model for studying tolerance and loss thereof: the V H gene coding for 3H9 is the most popular V H among disease-associated anti-DNAs, constituting ∼20% of the >200 sequenced anti-DNAs from MRL/ lpr , (NZB × NZW)F 1 and (NZB × SWR)F 1 . An unusual but useful feature of 3H9 (and other anti-DNA H chains) is that association with different L chains modifies DNA binding. 3H9/L chain combinations fall into three broad classes: combinations that sustain ss/dsDNA like that of the original 3H9/Vκ4 combination, combinations that modify DNA binding as in the 3H9/Vκ8 combination that only binds ssDNA, and combinations that veto DNA binding as, for example, 3H9/Vκ12/13. A study of 3H9 H chain in combination with a broad variety of endogenous L chains shows that the proportion of the three classes is ∼30% ds/ss : 60% ss : 10% non-DNA binding 39 . Moreover, certain L chains in combination with 3H9 yield unique ANA patterns as illustrated in Fig. 2 for the 3H9/Vκ23 combination 21 . Thus, 3H9 sd-tg mice yield a spectrum of antibodies, making the 3H9 sd-tg MRL/ lpr a multipurpose system for studying the breakdown of tolerance. As might have been predicted, 3H9 tg lpr/lpr mice express the types of anti-DNAs that are ordinarily edited or inactivated. These include anti-ds/ssDNA and antibodies that are ANA + . But a limitation of this model is that the precursor to the pathogenic anti-DNAs expressed in this lpr/lpr tg cannot be established; hence, the site(s) at which tolerance is broken is unknown. The advantage of the 3H9Vκ8/ lpr mice described here is that the anti-DNA repertoire is limited to one specificity, anti-ssDNA, that in normal mice is known to be regulated by anergy 12 14 . That anti-dsDNAs are now expressed in the 3H9Vκ8/ lpr means either that anergic cells become activated or that anergy cannot be established in lpr/lpr mice. Based on the evidence for Fas-mediated regulation of peripheral B cells 40 41 , we favor the latter interpretation. The failure to establish or maintain anergy of anti-ssDNA leaves the lpr/lpr mouse with a population of B cells poised for the transition to autoimmune disease. Both in vivo and in vitro studies have shown that anti-DNAs such as 3H9Vκ8 can be substrates for mutation to dsDNA and nuclear antigen binding antibodies 39 42 . Here we show that this transition takes place during clonal expansion of 3H9Vκ8 B cells and results in the production of anti-dsDNA and ANAs typical of disease. In this regard, our results recapitulate the nature of MRL/ lpr and other autoimmune mice. But, during clonal expansion 3H9Vκ8/ lpr B cells are actually subjected to two forms of somatic diversification: first, expanded hybridomas have accumulated a high frequency of mutations. These include mutations to R and N that could account for the shift of 3H9Vκ8 specificity from ssDNA to ss/dsDNA and the acquisition of antinuclear specificity . The second form of diversification is L chain editing. This event is an indirect consequence of mutation: all members of the clone share a nonsense mutation in the Vκ8-Jκ5 gene, therefore IgGκ secretion requires L chain expression from the untargeted allele. This novel mechanism rescues a defunct B cell. Alternative explanations for this genotype, such as coexpression of both κ genes throughout the lifetime of the clone, are highly unlikely . V gene rearrangement during clonal expansion is not surprising in view of the evidence for RAG expression in germinal centers 43 44 45 . But the relevance of RAG-mediated recombination to the immune response at this stage of B cell development has not been established. RAG-induced DNA nicks 45 may just be another manifestation of programmed cell death. It is unlikely that secondary rearrangement would enhance an ongoing immune response, because changing either H or L usually will produce a new specificity 29 . Moreover, these new specificities will rarely, if ever, be propagated because of the lack of antigen and T cell help. Instead, RAG expression in germinal centers simply may be a gratuitous part of impending or actual cell death. This is suggested by the work of Hikida et al., who have shown that most of the B cells in germinal centers that express RAG are undergoing apoptosis 46 . In fact, apoptosis may have been the raison d'être for RAG to begin with. The earliest B and/or T cell receptor genes were probably intact V genes, not segmented V, D, and J genes. These genes may have included sequences homologous to recombination signals, and primitive vertebrates may have fixed genes coding for proteins that cut DNA at these sites. Such cuts may have served to inactivate V genes or even to kill cells, thereby enforcing allelic exclusion and/or helping to maintain steady state levels of lymphocytes. For a defunct B cell to be rescued by secondary rearrangement requires truly exceptional circumstances, but anti-DNAs such as 3H9/Vκ8 provide the conditions for rescue. First, L chain replacement does not always destroy DNA binding, because the H chain makes most of the antibody contacts to DNA 42 . Hence, specificity for DNA can be maintained or modified even though Vκ8 is replaced (i.e., a kind of impotent receptor editing). Second, the self-specificity of 3H9/Vκ8 (and the L chain alternatives that, with 3H9H chain, sustain DNA binding) ensures that these antibodies will always be exposed to a cognate antigen. That germinal centers are sites of apoptosis is relevant to this point because the germinal centers are considered possible sites where the target lupus autoantigens may be presented. Third, DNA can be thought of as a super-antigen, in the sense that it is associated with a wide variety of both self- and foreign proteins. Thereby, as long as a B cell's receptor binds DNA, that B cell can present a variety of T cell epitopes derived from these DNA-associated proteins. Take, for example, a receptor directed to the complex of DNA–histone. Even though L chain replacement converts the receptor to one that only binds DNA, the receptor will still bind chromatin and ultimately that B cell can present histone peptides. Thus, this kind of autoantibody will rarely be at a loss for T cell help. 3H9/Vκ8 descendents illustrate this strategy: the 3H9/Vκ23 receptor has acquired specificity for dsDNA yet still binds ssDNA, making it so that the revised B cell can still present the same T cell epitope(s) as the parental 3H9/Vκ8 B cell. These studies show that expression of anti-DNA in Fas-deficient mice is influenced in at least two ways: first, B cells that are anergic in normal mice become activated; second, anti-DNA antibodies can arise in defunct B cells by V gene replacement. Both processes lead to the development of a spectrum of autoantibodies. Activation of anti-ssDNA B cells leads to clonal expansion and mutation and some of these mutations lead to specificity for dsDNA. Thus, anergy is a major tolerance checkpoint in that it prevents low affinity anti-DNAs from mutating to pathogenic types. Secondary V gene rearrangement in B cells generates a new spectrum of specificities that will surely include autospecificities. These might be formed by novel VH/VL combinations as in antibodies associated with Vκ23. The fact that secondary rearrangements, or editing, create autoreactivity in the autoimmune MRL/lpr is paradoxical, because in normal B cell development each round of editing adds to the number of non-autoreactive, mature B cells an animal can generate from a given set of precursor cells. How can one account for this difference? The answer must lie in a cell's “motivation” for continuing to rearrange. An immature B cell from a normal individual wants to make a functional receptor that does not bind too strongly to surrounding (self-)antigens. A mature cell from an autoimmune individual, on the other hand, is attempting to secure positive selection signals by rearranging until it has a receptor that binds an antigen with high avidity. This notion stems from the work of Hertz et al., who have shown that mature B cells experiencing low affinity interactions with antigen tend to initiate recombination, whereas high affinity antigen interactions abolish recombinase activity in mature B cells 47 . They also point out that this type of peripheral regulation of receptor editing would tend to promote autoreactivity. What is not thoroughly considered by their work is the role of T cell help. And this is what may be special in the case of autoreactivity, especially of autoreactivity to DNA. Rescue of a cell that has become autoreactive through editing rearrangement in the periphery also requires T cell help (specific for autoantigens) that is extant in autoimmune mice. Genes that affect the death of autoreactive cells and the disposal of defunct B cells (such as the fas gene) have a broad influence on the regulation of autoimmunity. In addition to extending the survival (and subsequent activation) of anergic B cells, they may also permit the development of an inappropriate repertoire by secondary rearrangement. The ability to detect peripheral editing in MRL mice is probably greatly enhanced by the lpr mutation that extends the life of B cells which otherwise would die before having had enough time to make a new receptor. Moreover, genes involved in cell death may regulate the concentration or availability of self-antigen. Casciola Rosen et al. have shown that the self-antigens targeted in lupus are found on the surface of apoptotic cells 6 . This led them to suggest that failure to kill cells efficiently could influence self-tolerance by limiting the amount of self-antigen. This limitation may be particularly strict in the bone marrow microenvironment where the preimmune repertoire is developed. A shortage of tolerogen could explain why the spectrum of autoantibodies in systemic autoimmunity is biased toward molecules released during cell death and could also resolve the differences between regulation of antibodies directed to facultative and constitutive self-antigens. | Study | biomedical | en | 0.999998 |
10477554 | 13 patients (22–75 yr old) with advanced stage malignant melanoma and 10 healthy donors (22–73 yr old) were selected for this study on the basis of HLA-A2 Ag expression as assessed by flow cytometry of PBMCs stained with allele-specific mAb BB7.2 27 . Molecular HLA-A*02 subtyping performed by PCR–sequence-specific oligonucleotide probe (SSOP) 28 revealed that all HLA-A2 + individuals were A*0201. None of them had two different A*02 subtypes. PBMCs from healthy blood donors were obtained from buffy coats provided by the blood transfusion center in Lausanne, Switzerland. PBMCs were separated from heparinized blood diluted 1:2 with PBS by centrifugation over Ficoll-Paque (Pharmacia), washed three times, and cryopreserved in RPMI 1640/40% FCS/10% DMSO. Vials containing 10 7 PBMCs were stored in liquid nitrogen. Tetrameric complexes were synthesized as previously described 12 26 . In brief, purified HLA heavy chain and β2-microglobulin were synthesized using a prokaryotic expression system (pET; R&D Systems, Inc.). The heavy chain was modified by deletion of the transmembrane cytosolic tail and COOH addition of a sequence containing the BirA enzymatic biotinylation site. Heavy chain, β2-microglobulin, and peptide were refolded by dilution. The 45-kD refolded product was isolated by fast protein liquid chromatography, then biotinylated by BirA (Avidity) in the presence of biotin, adenosine 5′-triphosphate, and Mg 2+ (all from Sigma Chemical Co.). Streptavidin-PE conjugate (Sigma Chemical Co.) was added in a 1:4 molar ratio, and the tetrameric product was concentrated to 1 mg/ml. Tetramers were synthesized around two tumor antigenic peptides recognized by HLA-A*0201–restricted CTLs, both of which derive from melanocyte lineage-specific proteins. One peptide was the natural tyrosinase 368–376 epitope (the N370D variant, YMDGTMSQV, generated by Ag processing 29 ); the other was a modification of the Melan-A 26–35 epitope 30 . This modified epitope, ELAGIGILTV, carrying a substitution of Ala for Leu at position 2 from the NH 2 terminus (hereafter A27L), forms relatively stable complexes with HLA-A*0201 and is a more potent immunogen than is the natural Melan-A peptide 31 32 . Experiments have shown that tetramers synthesized around the A27L-modified epitope generally stained polyclonal and monoclonal Melan-A–specific CTL populations (data not shown and reference 12). However, we can not totally exclude the possibility that some T cell populations might not have been stained with the A27L peptide analogue containing tetramer. Finally, a third tetramer was synthesized around the HLA-A*0201–restricted influenza matrix Flu-MA 58-66 (GILGFVFTL) immunodominant peptide. A2/tyrosinase, A2/Melan-A, and A2/Flu-MA tetramers were used at 20 μg/ml. Anti-CD3 PE , CD8 FITC, PerCP , and CD45RA Cyc were obtained from Becton Dickinson; anti-CD45RO FITC was from DAKO Corp.; and anti-CD28 FITC was from Immunotech. Thawed PBMCs were cultured for 16–20 h in IMDM supplemented with 0.55 mM Arg, 0.24 mM Asn, 1.5 mM Gln, and 8% pooled human A + serum (complete medium). CD8 + lymphocytes were then purified from PBMCs in two rounds of positive selection by magnetic cell sorting using a MiniMACS device (Miltenyi Biotec Inc.) The resulting cells were >98% CD3 + CD8 + , and 10 6 were stained with A2/tetramers and FITC and Cychrome mAb conjugates in 50 μl of PBS, 2% BSA, and 0.2% azide for 40 min at 4°C. CD8 enrichment did not disturb the detection of tetramer + cells, either in terms of frequency or phenotype (data not shown). Cells were then washed once in the same buffer and analyzed immediately in a FACSCalibur ® (Becton Dickinson). CD45RA lo and CD45RA hi CD8 + T cells subsets from healthy donors HD 329 and HD 604 and from melanoma patients LAU 132 and LAU 203 were sorted using a FACStar ® (Becton Dickinson). These subpopulations were cultured at 10,000, 5,000, 2,500, and 1,250 cells/well (24 wells per condition) in complete medium plus 100 U/ml recombinant human IL-2, and stimulated at days 0 and 7 by autologous CD8 − PBMCs (10 5 /well) pulsed with 1 μM Melan-A 26–35 A27L peptide. At day 13, Ag recognition was assessed using T2 target cells (100 μl) labeled with 51 Cr and incubated in the presence or absence of 1 μg/ml of the antigenic Melan-A 26–35 A27L peptide for 1 h at 37°C and washed three times. Labeled target cells (10 3 cells in 50 μl) were then added to effector cells (50 μl) in V-bottomed microwells (50 μl). The effector cells were preincubated for 20 min at 37°C in the presence of unlabeled K562 cells (5 × 10 5 /well) to eliminate nonspecific lysis due to NK-like effectors. Cr release was measured in supernatant (40 μl) harvested after 4 h of incubation at 37°C. The percentage of specific lysis and the deduced frequency of CTL precursors (CTLp) present in each subset was calculated as previously described 33 . ELISPOT plates (Millipore) were coated overnight with antibody to human IFN-γ (Mabtech) and washed six times. 10 μg/ml peptide and 1.66 × 10 5 PBMCs per well in 200 μl Iscove's medium/8% human serum were added and incubated for 20 h at 37°C. Assays were performed in six replicates with either the Melan-A 26–35 A27L or the Flu-MA 58–66 peptide. The ILKEPVHGV Pol 476–484 peptide from the reverse transcriptase of HIV-1 was also included as a negative control. (All subjects in this study were HIV seronegative.) Cells were removed, and plates were developed with a second antibody to human IFN-γ (biotinylated) and streptavidin-alkaline phosphatase (Mabtech). The deduced frequency of peptide-specific CTLs in CD8 + T cells was calculated as: (mean no. of specific spots) / [(1.66 × 10 5 ) × (percentage of CD3 + CD8 + cells in PBMCs)], where the percent of CD3 + CD8 + cells in PBMCs was determined by flow cytometry on the same batch of cryopreserved cells. The baseline number, or cut off value, of nonspecific IFN-γ spots was calculated as the mean number of spots in the presence of the control HIV-1 peptide in 21 individuals + 3 SD. This value was 17 spots/10 6 PBMCs (mean = 5 spots/10 6 PBMCs, SD = 4), implying a lower specific detection limit of 1 in ∼60,000 PBMCs. Since the enumeration of Melan-A–specific lymphocytes with tetramers was directly performed on gated CD8 + lymphocytes, the cut off value and the frequencies determined by IFN-γ ELISPOT were all adjusted to reflect the percentage of CD8 + lymphocytes determined in the same PBMC batch by flow cytometry. This was performed to enable comparison with tetramer frequency values. The CD8-adjusted baseline spot value was 87 spots/10 6 CD8 + PBMCs (mean = 25 spots/10 6 CD8 + PBMCs, SD = 21). 13 HLA-A2 + patients with advanced stage malignant melanoma and 10 HLA-A2 + healthy donors were randomly selected for this study. Among melanoma patients, three presented concurrent vitiligo that developed either after a systemic treatment with intravenous IL-2 + Cis Platinum + IFN-α (patient LAU 155), during IFN-α therapy (LAU 156), or after isolated limb perfusion with high dose of TNF-α + melphalan (LAU 269). Highly enriched circulating CD8 + T lymphocytes (>98% CD3 + CD8 + ) from each individual were stained for flow cytometry with different A2/tetramers, two synthesized around melanoma-associated Ags, namely the Melan-A 26–35 A27L analogue and tyrosinase 368–376 , and one around the viral influenza matrix Flu-MA 58–66 peptide. As illustrated in Fig. 1 , circulating A2/Melan-A + and A2/Flu-MA + CD8 + cells were detected both in melanoma patients and healthy donors. In contrast, the frequency of A2/tyrosinase + cells was generally too low for direct ex vivo detection. However, we observed that a short in vitro Ag-driven expansion was sufficient to detect A2/tyrosinase + cells in the majority of A2/melanoma patients, confirming the presence of circulating tyrosinase specific CTLp 33a . To determine the levels of nonspecific A2/tetramer staining of circulating CD8 + T cells, a series of nine blood samples of randomly selected HLA-A2 − blood donors was analyzed ( Table ). Although this approach does not provide direct insight on the level of nonspecific epitope-based A2/tetramer staining in HLA-A2 + individuals, it allowed us to define a lower detection limit for tetramer staining in A2 + individuals. This lower detection limit was ∼0.04% of CD8 + T cells with A2/Melan-A tetramers (cut off = mean + 3 SD = 0.036), and <0.02% of CD8 + T cells with A2/Flu-MA tetramers (cut off = mean + 3 SD = 0.011). These detection limits for staining with A2/Melan-A tetramers are clearly lower for circulating cells than for tumor-infiltrated LNs (∼0.25%; calculated previously; reference 12). According to these limits, ex vivo circulating A2/Melan-A + CD8 + cells were found in significant numbers in 10 out of 13 melanoma patients, and in 6 out of 10 healthy donors. As we previously reported 34 , the frequency of A2/Melan-A + cells was generally very high in melanoma patients with concurrent vitiligo (mean = 0.23% of CD8 + cells). In contrast, the frequency of CD8 + cells stained with A2/Melan-A tetramers was comparable between melanoma patients without vitiligo and healthy donors (mean = 0.07%) ( Table ). Likewise, the frequency of A2/Flu-MA + cells was globally equivalent in all groups, with the exception of LAU 198, who had 1.65% of CD8 + cells stained with tetramers. In humans, cell surface expression of the CD45RA and CD45RO isoforms have been used to identify naive and memory T cells, respectively 13 14 15 . As circulating CD28 − CD8 + T cells present direct ex vivo cytolytic activity 19 20 and were recently proposed to correspond to effector-type CTLs 22 23 24 , individuals presenting significant amounts of circulating A2/Melan-A + and A2/Flu-MA + cells were phenotyped for CD28, CD45RA, and CD45RO surface expression by three-color flow cytometry analyses. Clearly, circulating A2/Melan-A + and A2/Flu-MA + CD8 + T cells from healthy donors displayed distinct phenotypes . Practically all of the A2/Melan-A + cells were CD28 + CD45RA hi /RO − (range: 84–95%), corresponding to a naive phenotype. In marked contrast, most of A2/Flu-MA + cells were CD45RA lo /RO + (range: 83–97%). Thus, the phenotype of A2/Flu-MA + cells corresponded to Ag-experienced memory T cells, compatible with the notion that the Flu-MA 58–66 peptide probably represents a recall Ag in these HLA-A2 + individuals. In addition, ∼20% of CD45RA lo /RO + A2/Flu-MA + cells from HD 099 and HD 604 presented a CD28 − phenotype (data not shown). In contrast to healthy individuals, the phenotype of A2/Melan-A + cells was heterogeneous in melanoma patients . In most of them (7 out of 10), A2/Melan-A + cells presented a uniformly naive CD28 + CD45RA hi /RO − phenotype (range: 81–95%), like those found in all healthy donors. However, 3 out of 10 patients either displayed >35% CD45RA lo /RO + (LAU 132 and 240), or >90% CD28 − CD45RA int (LAU 156) A2/Melan-A + cells . It is not possible with the current data to determine whether such phenotypic changes in Melan-A–specific lymphocytes may have occurred in response to peptide-based vaccination (LAU 132 received five rounds of vaccination with the Melan-A 26–35 peptide plus other melanoma-associated Ags with GM-CSF), or simply reflect non-Ag-specific changes after administration of cytokines such as GM-CSF or IFN-α (LAU 132 received GM-CSF concomitant with peptide administration, and LAU 156 was treated with IFN-α). Moreover, other melanoma patients included in this study who also received Melan-A 26–35 peptide vaccination with GM-CSF (LAU 269) or IFN-α therapy (LAU 267) did not present memory phenotype A2/Melan-A + cells in the circulating compartment. Altogether, phenotype and frequency of A2/Melan-A + cells were generally not correlated, since (a) memory phenotype cells detected in patients LAU 132 and 240 were not found at increased frequencies (0.07 and 0.04% of CD8 + T cells, respectively); and (b) high frequencies of A2/Melan-A + cells (>0.2% CD8 + T cells) in patients LAU 155, 233, and 269 as well as in healthy donor HD 604 were not of the memory phenotype. As an exception, LAU 156 presented high frequency of CD28 − A2/Melan-A + cells. The vast majority of A2/Flu-MA + cells displayed a memory-like CD28 + CD45RA lo /RO + phenotype in 11 out of 13 patients, as well as in all healthy donors . However, the A2/Flu-MA + cells presented both CD28 − CD45RA int (60%) and CD28 + CD45RA int (30%) phenotypes in LAU 156, and a CD28 + CD45RA int phenotype in LAU 269 (CD28 phenotype not shown). Initially, two healthy donors (HD 329 and 604) and two melanoma patients (LAU 132 and 203) were selected according to the phenotype of A2/Melan-A + cells: the vast majority of A2/Melan-A + cells from HD 329, HD 604, and LAU 203 were CD45RA hi (95, 95, and 94%, respectively); on the other hand, 46% of those from LAU 132 presented a CD45RA lo phenotype ( Table ). These phenotypes were independently examined by two functional assays, limiting dilution analysis (LDA) and IFN-γ ELISPOT. For LDA, CD45RA lo and CD45RA hi fractions of CD8 + T cells were sorted and stimulated twice with Melan-A 26–35 A27L peptide at limiting dilution conditions. After 13 d, the large majority of microcultures displaying Melan-A–specific CTL activity in HD 329, HD 604, and LAU 203 were detected in the progeny from the CD45RA hi subset (98, 99, and 90% of positive microcultures, respectively). In contrast, 73% of positive microcultures in LAU 132 were detected in the cells expanded from the CD45RA lo subset. Thus, the distribution of CTLp among the naive and memory subsets evaluated by functional LDA assays parallels that observed by flow cytometry phenotypic analysis, confirming the Ag specificity of the relatively low numbers of tetramer + lymphocytes. However, LDA underestimated the frequency of Melan-A–specific CTLp by a factor of ∼3 for CD45RA lo phenotype A2/Melan-A + cells (LAU 132), and to a higher extent by a factor of ∼13 in average for CD45RA hi phenotype A2/Melan-A + cells ( Table ). In parallel, the phenotype of Melan-A–specific cells was indirectly assessed by a 20-h IFN-γ ELISPOT assay. Given the limited number of cells available, this assay was not performed on CD45RA +/− sorted populations, but rather with unsorted PBMCs. As expected for truly naive CD8 + T cells, ex vivo Melan-A–specific IFN-γ producing cells were undetectable in PBMCs from HD 329, HD 604, and LAU 203 ( Table ). In contrast, for LAU 132, we found ∼100 IFN-γ–specific spots/10 6 CD8 + T cells, which represented a significant frequency above background levels (calculated as described below). To further investigate ex vivo IFN-γ production in response to challenge with Ag, both Melan-A– and Flu-MA–specific cells from all healthy donors and melanoma patients (except LAU 240 and 267) were analyzed . First, we determined a lower detection limit (cut off) for this assay. This cut off, based on the number of nonspecific spots obtained after stimulation with the irrelevant HIV-1 Pol 468–476 peptide of PBMCs from the 21 individuals analyzed, was ∼90 spots/10 6 CD8 + T cells (cut off = mean + 3 SD = 87, see Materials and Methods for details). With this detection limit, the frequencies of Flu-MA–specific IFN-γ–producing cells reached significant levels for 17 out of 21 individuals. Moreover, these frequencies correlated well with those calculated by tetramers , but were systematically underestimated (median, 3 times; min, 1.5 times; max, 15 times). In marked contrast, ex vivo Melan-A–specific cells generally did not produce IFN-γ, as expected for naive CD8 + T cells . Therefore, the apparent frequency of Melan-A–specific IFN-γ–producing cells was generally much lower than that obtained by tetramer staining (median, 30 times; min, 4 times; max, infinite). It is worth noting that, as some patients had a considerable fraction of A2/Melan-A + cells with an Ag-experienced phenotype , the frequencies of IFN-γ–producing cells upon stimulation with the Melan-A peptide analogue were less underestimated (seven and four times, respectively), when compared with direct counting with A2/Melan-A tetramers. To rule out the possibility that the relatively low numbers of Melan-A + lymphocytes detected in A2 + individuals was the result of some flow cytometry artifacts, circulating A2/Melan-A +/− CD8 + T cells from a healthy donor (HD 604) were directly sorted into tetramer + and tetramer − populations. After 15 d of mitogen-driven polyclonal expansion (1 μg/ml PHA-L, 100 U/ml IL-2, 10 ng/ml IL-7, and 5 × 10 5 /ml autologous CD8 − irradiated PBMCs), the tetramer + fraction exhibited 10% A2/Melan-A + cells, while the tetramer − fraction contained <0.02% A2/Melan-A + cells. As expected, both populations displayed a homogeneous CD45RA lo Ag-experienced phenotype (data not shown). Each cell fraction was subsequently tested for its lytic activity. The polyclonal A2/Melan-A + population specifically killed T2 target cells pulsed with the natural or the A27L analogue Melan-A 26–35 peptides, whereas the A2/Melan-A − population did not . This indicates the Ag specificity of cells stained with A2/Melan-A tetramers. Moreover, 9% of the whole A2/Melan-A + population specifically released IFN-γ in ELISPOT assays, whereas the number of IFN-γ spots was insignificant for the A2/Melan-A − population (data not shown). This confirms that release of IFN-γ may be restricted to Ag-experienced phenotype specific cells. To assess the fate of Melan-A–specific T cells in vivo, we followed Ag-specific lymphocytes by tetramer staining in a series of blood samples from patient LAU 132 taken over a period of 2 yr . In this patient, a primary skin melanoma of the lower limb was diagnosed in October 1994. Inguinal LN dissection revealed that 4 out of 6 nodes were infiltrated by melanoma cells. The patient was treated with isolated limb perfusion with melphalan, and subsequently received adjuvant IFN-α therapy until April 1996, at which time he underwent a second inguinal LN dissection (15 out of 16 positive LNs). The patient was tumor free from May 1996, then developed a brain metastasis diagnosed in December 1998. Immunization with melanoma-specific peptides was begun in June 1996; he received a first immunization cycle consisting of three or four weekly subcutaneous injections of 100 μg of each of the peptides Melan-A 26–35 , Tyrosinase 1–9 , Tyrosinase 368–376 , gp100 280–288 , gp100 457–466 , and influenza matrix Flu-MA 58–66 in PBS. Four additional immunization cycles were given in August and October 1996, then in March and June 1997. All cycles with the exception of the first included treatment with GM-CSF (daily subcutaneous injections of 75 μg, starting 4 d before peptide injection and covering the whole 3-wk immunization period). Before the first immunization cycle, A2/Melan-A + cells (0.04% of CD8 + T cells) presented a naive CD45RA hi phenotype. In marked contrast, 1 mo after the end of the first peptide injections and until the end of the second immunization cycle, half of the tetramer + cells presented an Ag-experienced CD45RA lo phenotype. This was accompanied by a small increase in the frequency of A2/Melan-A + cells (from 0.04 to 0.07% of CD8 + T cells). During the next year, the proportion of CD45RA lo A2/Melan-A + cells gradually decreased (from 51 to 23% of A2/Melan-A + cells), while the frequency of total A2/Melan-A + cells remained constant (∼0.07%). Moreover, the vast majority of A2/Melan-A + cells continuously displayed a CD28 + phenotype over time. Enumeration of tyrosinase-specific CD8 + lymphocytes using an available tetramer made with the Tyrosinase 368–376 peptide failed to reveal significant levels of positive cells in the samples tested . Using tetrameric complexes 35 , we have directly enumerated and phenotyped ex vivo melanoma-specific CD8 + T cells present in peripheral blood. This study reveals that circulating Melan-A–specific CD8 + T cells are generally present in high numbers both in melanoma patients and healthy individuals. These cells present a naive phenotype in healthy individuals, but may develop an Ag-experienced phenotype in some melanoma patients. Furthermore, Ag-specificity and phenotype of A2/Melan-A + cells were independently confirmed by functional assays. As recorded for one patient immunized with a Melan-A peptide, marked and reversible shifts in the proportion of memory-type circulating Melan-A–specific CTLs occurred in vivo. In contrast, circulating influenza virus–specific CTLs in most of the same individuals display a homogeneous memory phenotype. Our findings confirm and extend previous reports on the presence of circulating Melan-A–specific cells both in melanoma patients and healthy individuals 6 7 8 9 36 . However, the necessity of stimulating CTLp with Ag in order to detect Melan-A–specific cells had previously prevented a precise assessment of their frequency and phenotype ex vivo. We find here that circulating Melan-A–specific cells are indeed present in a large proportion (60%) of healthy individuals. Although this prevalence is in agreement with that measured in the previous studies (20–75% of healthy individuals), tetramer staining reveals for the first time that the frequency of circulating Melan-A–specific cells is much higher than had been anticipated (mean = ∼1/1,500 of CD8 + T cells) both in healthy individuals and melanoma patients. These frequencies are underestimated consistently by indirect assays, as demonstrated in this study by LDA and IFN-γ ELISPOT. Phenotypic analyses of A2/Melan-A + cells reveal that underestimation reflects the failure to detect primarily, although not exclusively, CD45RA hi -specific cells. Thus, naive phenotype cells are less efficiently stimulated during in vitro Ag-driven differentiation to effectors, as has been shown in experiments involving naive phenotype cells challenged by Ag-independent TCR cross-linking 37 38 . The frequencies of naive single epitope-specific CTLp have been estimated at ≤1/100,000 CD8 + T cells. Thus, it is striking that the mean frequency of circulating Melan-A–specific cells from HLA-A2 + healthy individuals is ≥60 times higher, in fact attaining the range of frequencies for single epitope-specific memory CTLs. In this regard, since Melan-A is a melanocyte lineage protein, it might be argued that enrichment of cells specific for the Melan-A 26–35 peptide could result from frequent priming events after common subclinical skin injuries in healthy individuals. Alternatively, the Melan-A homologous peptide gC 480–488 , derived from the unrelated glycoprotein C of the common pathogen HSV-1 39 , could also be responsible for the activation of Melan-A cross-reactive cells. However, the observation that ex vivo A2/Melan-A + cells from healthy individuals are of the naive CD28 + CD45RA hi /RO − phenotype does not support these hypotheses. It remains possible that CD28/CD45RA surface Ags do not reflect true human Melan-A–specific naive T lymphocytes. For instance, reversion of CD45RO + T cells to CD45RA + T cells after prolonged in vitro culture periods has been observed 40 41 . Although Flu-MA–specific CD8 + T cells from virtually all individuals studied displayed a memory CD45RO + phenotype, it remains to be ascertained whether such a conversion may have taken place in Melan-A–specific CD8 + T cells in vivo. Nevertheless, our results in no way rule out the possibility that Melan-A–specific cells in healthy individuals as well as in the majority of melanoma patients represent either truly naive T cells or cells that have been partially activated 42 43 . This raises the question as to how such relatively large repertoire of naive T cells is maintained in the periphery. For instance, what are the ligands and/or cytokines involved? Further phenotype and functional characterization of these cells are required to clarify these issues. Does tetramer staining of Melan-A–specific cells reveal differences between melanoma patients and healthy individuals? In terms of frequencies, Melan-A–specific cells were generally found in high numbers in patients, but these numbers were comparable with those measured in healthy individuals. In contrast, and as previously reported 34 , the frequency of Melan-A–specific cells was even higher in patients undergoing vitiligo. Previous reports have shown that development of vitiligo is more frequent in melanoma patients 44 , and that vitiligo may be associated with an ongoing immune response directed against melanoma cells 45 46 . This evidence suggests that melanocyte-specific CTLs can play a role in melanocytic destruction. From a clinical standpoint, the presence of high numbers of circulating Melan-A–specific cells found in the majority of HLA-A2 + melanoma patients represents a unique opportunity for vaccination protocols aimed at harnessing the potential power of these CTLs to migrate to and destroy tumors. In addition, polyclonal-specific T cell populations could easily be obtained by tetramer-guided sorting and used for adoptive transfer therapy 47 . The tetramer technology not only allows frequency analyses, but opens the possibility to characterize phenotypes corresponding to various differentiation stages of T cells. We observed that the majority of influenza virus–specific CTLs were CD45RA lo , whereas the melanoma-specific CTLs were largely CD45RA hi . Finally, CD45RA lo melanoma-specific CTLs were only observed in 3 out of 10 melanoma patients. The different expression of CD45 isoforms probably corresponds to different cellular activation status. Therefore, the fact that some patients, but no healthy individuals, had CD45RA lo /RO + Melan-A–specific cells may suggest that CTLs have been activated in vivo as part of immune activation against melanoma cells. When tracking a patient's immune response over the course of an immunotherapeutic treatment with Melan-A 26–35 , we recorded a marked but reversible shift in the proportion of memory-type Melan-A–specific cells. However, no changes in frequency or phenotype of Melan-A–specific cells were observed after a similar immunization schedule on a second melanoma patient (LAU 269). It is not possible at this time to establish whether peptide vaccination and/or GM-CSF administration were responsible for the phenotype shifts observed in the former patient. A larger group of vaccinated patients will be analyzed, as it is possible that this vaccination procedure is not efficient. Immunization with potent adjuvants and Melan-A peptide analogues with enhanced immunogenicity will be tested. Direct detection of tumor-specific lymphocytes in the periphery and in tumor-infiltrated LNs allowed us to obtain a better appraisal of a tumor-specific response in vivo. In tumor-infiltrated LNs, Melan-A–specific cells were enriched with frequencies ranging from 1/400 to 1/30 CD8 + LN cells and displayed an Ag-experienced (CD45RA lo /RO + ) phenotype in most cases, as compared with noninfiltrated adjacent LNs or LNs from patients with forms of cancer other than melanoma 12 . In contrast, circulating Melan-A–specific cells were generally not enriched in melanoma patients, as compared with healthy individuals and with normal LNs. Moreover, they infrequently presented Ag-experienced phenotypes. It is tempting to speculate that the differences observed in both frequencies and surface phenotype of Melan-A–specific CTLs are the consequence of selective in vivo activation of these cells at infiltrated LNs. It is also conceivable that primed cells are selectively accumulated at infiltrated LNs. Together, our findings emphasize the need to monitor both the tumor sites and the periphery to thoroughly evaluate the impact of natural or vaccine-induced tumor-specific CTL responses. | Study | biomedical | en | 0.999997 |
10477555 | We used the previously identified M. leprae sequence encoding an Nramp homologue to search the EMBL database using TBLASTN 20 . PCR on genomic DNA from M. tuberculosis (H37Rv) and BCG using Pfu polymerase (Stratagene, Inc.) was carried out with primers designed to introduce BglII restriction sites and a strong eukaryotic Kozak consensus (TTG GTG G to ATG ATG G, initiation codon underlined). PCR primers were as follows: 5′-GTA GCC AGA TCT ATG ATG GCG GGC GAA TTT CGG-3′ and 5′-GCG GTC AGA TCT TCA GCC GGT CAC CGT GAG ATA-3′ (BglII sites are underlined and the translational start codon is in bold). Cycle conditions were as follows: 1 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 66°C, 2 min at 72°C, with a final 4-min hold at 72°C. The reaction was supplemented with Mg 2+ (1 mM) and Betaine (1 M) (Sigma Chemical Co.). The PCR product from M. tuberculosis was subcloned into BglII sites in pSP, which contains 5′ and 3′ untranslated Xenopus β-globin sequences 21 and was verified by sequence analysis. Constructs containing both orientations of Mramp were obtained and designated pXmramp (sense) and pXpmarm (antisense). Xenopus laevis oocytes were prepared as described previously 22 . Capped cRNA encoding Mramp was transcribed (MEGAscript™ SP6; Ambion) from Xba1-linearized templates (pXmramp and pXpmarm), and oocytes were injected with cRNA (5 ng in 50 nl of water) or a corresponding volume of RNAse-free water. Fe 2+ and Zn 2+ uptake assays were performed after 48–96 h incubation in Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3 , 15 mM Hepes [pH 7.6], 0.3 mM Ca(NO 3 ) 2 .4H 2 O, 0.41 mM CaCl 2 .6H 2 O, 0.82 mM MgCl 2 .7H 2 O, 10 μg/ml penicillin, 10 μg/ml streptomycin) at 19°C 23 . 65 Zn 2+ uptake assays were performed on batches of 10–15 oocytes washed 4 times in freshly made up standard uptake medium (SUM: 90 mM KCl, 10 mM NaCl, 10 mM morpholinoethane sulfonic acid [MES], pH 5.0–pH 6.5; as before but with 10 mM Hepes at pH 7.0) and incubated in SUM for 1–4 h at room temperature with 100 μM 65 ZnCl 2 (Nycomed Amersham plc). Transport was terminated by washing oocytes in excess ice-cold SUM. 65 Zn 2+ uptake was quantitated in individual oocytes on a Wallac Wizard 1480 gamma counter and corrected for uptake into water or antisense cRNA–injected controls. 55 Fe 2+ uptakes were performed with 55 Fe 2+ (5 μM in 100 μM total Fe 2+ ; Nycomed Amersham plc) in SUM containing ascorbic acid (2 mM) to maintain iron in a ferrous state, and all studies used fresh solutions to minimize reoxidation of Fe 2+ . The amount of ferrous iron was verified by a ferrozine assay 24 . Quantitation of 55 Fe uptake was by scintillation counting (Wallac Microbeta Plus). Maximal expression of both 65 Zn 2+ and Fe 2+ uptake occurred between 2 and 4 d after microinjection. Competitions with divalent cations were performed using 65 Zn 2+ or Fe 2+ as permeants and 100-fold excess (10 mM) concentrations of CuCl 2 , MnCl 2 , FeCl 2 , ZnCl 2 , and MgCl 2 . All glassware was autoclaved after rinsing in 0.2 N HCl followed by deionized water to remove trace quantities of iron. Low Fe (∼5 μM) Sauton's medium for starter cultures consisted of asparagine (15 mM), MgSO 4 (1 mM), sodium citrate (4 mM), KH 2 PO 4 (7 mM), and glycerol (2% vol/vol, pH 7.1). Fe-depleted Sauton's medium (<1 μM) was prepared as above, omitting MgSO 4 , and stirred overnight at 4°C with Chelex 100 (10 g/l). After filter sterilization, the medium was supplemented (MgSO 4 [1 mg/ml equivalent to 8.3 mM final concentration], ZnSO 4 .4H 2 O [0.2 μg/l final concentration ∼ 1 nM], and MnCl 2 .4H 2 O [0.2 μg/l ∼ 1 nM]) to compensate for losses during chelation. Media containing “low Fe” (<1 μM), “medium Fe” (4 μM), or “high Fe” (48 μM) and “low Cu” (<0.5 μM), “medium Cu” (5 μM), or “high Cu” (69.8 μM) were prepared by additionally supplementing aliquots of this medium with ferric ammonium citrate (∼16% Fe content) or CuCl 2 . To ensure that concentrations of cations not being studied were above limiting concentrations, the Fe-modified media were supplemented with Cu (∼1 μM), and Cu-modified media with Fe (4 μM). Concentrations of Fe and Cu in these media were verified by ferrozine assay 24 and atomic absorption spectrophotometry, respectively. All chemicals were obtained from Sigma-Aldrich. Starter cultures in Dubos broth supplemented with 10% Dubos medium albumin (Difco) were initiated from glycerol stocks and grown at 37°C to mid-log phase. These were inoculated (1:10) into low iron Sauton's medium, grown for 1 wk (37°C, 5% CO 2 , without shaking), and subcultured to ensure complete depletion of iron and copper. 10 ml of these cultures was inoculated into Fe/Cu-depleted Sauton's medium (10 ml into 190 ml) supplemented to give low, medium, or high concentrations of iron or copper and grown for 5 wk. As BCG encodes an Mramp sequence identical to that of M. tuberculosis , we used BCG as a model to examine the expression of Mramp during intracellular infection. The human macrophage cell line, THP-1, was maintained as suspended cells and passaged at a density of 2–5 × 10 6 cells/ml. Before infection with BCG, the cells were passaged at least three times in antibiotic-free RPMI 1640 (ICN Biochemicals) supplemented with heat-inactivated FCS (10%), grown to a density of 2–5 × 10 6 cells/ml, and stimulated with PMA (20 mM; Sigma Chemical Co.) for 24 h to induce adherence. Nonadherent cells were removed by washing twice in PBS, and the resulting monolayers (∼3–5 × 10 7 cells/flask) were covered with supplemented RPMI 1640. Mid-log phase bacteria were pelleted from Dubos broth (500 g , 10 min), resuspended in medium, and sonicated (Rinco Ultrasonics) for 15 s (five 3-s bursts at 70% amplitude) to disaggregate bacterial clumps. The sonicate was added to macrophages (10 bacilli/macrophage) and left for 24 h (37°C, 5% CO 2 ). Extracellular mycobacteria were removed by decanting the supernatant and extensively washing the adherent cells twice in PBS. Efficiency of phagocytosis was estimated to be ∼30% by counting CFU in the collected washings and microscopic examination of Ziehl-Nielsen–stained macrophages. Macrophage viability (>90%) throughout these experiments was assessed by Trypan blue exclusion, with further details given in reference 25. After extensive washing of the macrophage monolayer, mycobacteria were recovered from differentially lysed THP-1 cells by the addition of 20 ml GTC solution (4 M guanidinium thiocyanate [GTC; Fluka] containing 0.5% sodium N -lauroylsarcosine, 25 mM sodium citrate, pH 7, and 0.1 M 2-ME) to each flask 25 . Total RNA was then extracted from the washed bacterial pellets as described previously 26 . First strand cDNA synthesis on total RNA used random hexamer primers (Promega) and Moloney murine leukemia virus reverse transcriptase (Superscript II™; GIBCO BRL) according to the manufacturer's instructions. Contamination of RNA by genomic DNA was excluded by PCR on RNA template made without reverse transcriptase and on template made from RNA pretreated with RNAse A (Qiagen). PCR was carried out using primers spanning the junction between the 3′ end of the Mramp gene and the open reading frame (ORF) immediately downstream under the following cycling conditions: 30 cycles of 1 min at 94°C, 2 min at 55°C, 3 min at 72°C. Primers were 5′-ACGATCACCCATAACAACAGG-3′ and 5′-CAGAGAACGACTTCACCAACC-3′. Templates for quantitative PCR assays were generated by oligonucleotide restriction site mutagenesis 27 , using M. tuberculosis genomic DNA as template, and corresponded to the following fragments: for Mramp , and for two “P”-type ATPases: yhho (503 bp, 79–582) and y39 (503 bp, 100–603). Spanning primer pairs were as follows: for Mramp , 5′-CTGGTTGCCGCGCTGAACATG-3′ and 5′-GAACTGAAACCCCATTCAGCCGGTCACCGT-3′; for y39 , 5′-GTTGGGGCCTCGAAGGAATTCGTTGACGCCGCA-3′ and 5′-CTTGCTGAACCACGCCAGCTT-3′; for yhho , 5′-ACCGGCTGAATGGGGTTTCAGTTCGACGCGGG-3′ and 5′-AACGAATTCCTTCGAGGCCCCAACCGCCAGCGC-3′. Primers were designed so that the Mramp antisense primer overlapped with the yhho sense primer, and the yhho antisense primer overlapped with the y39 sense primer, giving a tandemly organized construct after synthesis by PCR 28 . PCR cycle conditions, identical for each fragment, were as follows: 3 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 65°C, 2 min at 72°C, followed by a 4-min hold at 72°C. To discriminate competitor from target in tandem competitive (TC)-PCR reactions, a mutant asymmetrical Kpn1 restriction site was introduced ∼200 bp internal to the 5′ end of each fragment. These fragments were cloned in tandem into pGEM-T Easy (Promega). We chose “P”-type ATPase sequences ( y39 , putative Ca 2+ transporter; and yhho , atypical heavy metal transporter reviewed in Agranoff et al. ) in order to quantitate the relative amounts of Mramp expression in mRNA obtained from mycobacteria cultured in different ionic microenvironments. As an internal control for RNA preparations, we amplified 16S ribosomal RNA sequence using the primers 5′-CCC TTG TCT CAT GTT GCC AG and 5′-CTG GCA ACA TGA GAC AAG GG. First strand cDNA synthesis on total RNA was carried out as described above. Semiquantitative estimates of mRNA expression were derived by conventional reverse transcription (RT)-PCR carried out under identical conditions for each primer pair and identical starting cDNA concentrations for all three genes. Products were quantified using a GDS 7600 system (Ultraviolet Products) and normalized to the most abundant product for each gene. Precise quantitation of relative amounts of cDNA for Mramp and y39 under different growth conditions was carried out by TC-PCR. A series of PCR amplifications of each gene was carried out on a template mixture consisting of a fixed quantity of cDNA and serial dilutions (at 1/4 log intervals) of competitor molecule (linearized by Sac1 digestion). Products derived from competitor template could be distinguished from target product by KpnI digestion and were quantified as above. The plasmid competitor/cDNA ratio was calculated for individual reactions after heteroduplex correction and analyzed as published previously 29 . Comparison of radioisotope uptake rates between two conditions was by the Mann Whitney U test, and comparison of uptakes at different pH values and with competitors was after Box-Cox transformation (to normalize distributions) and used MANOVA (v5.2 SYSTAT). A single Nramp homologue (designated Mramp ) is located on cosmid MTY21C12 in a 5286-bp region containing 5 ORFs oriented in the same direction 30 . A potential ribosome binding site is immediately upstream of the first ORF with Mramp as the fourth gene in this series. To ascertain if Mramp is cotranscribed with neighboring ORFs, we carried out RT-PCR on total RNA extracted from cultured H37Rv using primers spanning the junction between the 3′ end of Mramp and the next ORF. A product of expected size (407 bp) was obtained (data not shown), indicating that Mramp is transcribed at least as a bicistronic operon. Database searches failed to identify functionally characterized homologues of this second ORF. Mramp encodes a predicted 428–amino acid protein with a molecular mass of 44.9 kD and 10 transmembrane segments consistent with proposed topologies for yeast homologues ( smf1 and 2 ) . Compared with eukaryotic homologues, the hydrophilic NH 2 -terminal region of Mramp, like those of other prokaryotic Nramp homologues, is shorter but exhibits a similar clustering of polar residues 31 . The COOH terminus is also shorter and probably lacks the two final transmembrane segments predicted in some eukaryotic homologues. The amphiphilic properties of transmembrane segments M3, M5, and M9, in which the polar and nonpolar residues are segregated to opposite faces of the predicted α-helices (possibly forming a transmembrane channel), are also found in Mramp. Sequence analysis of Mramp confirms that certain amino acid residues are highly conserved between all members of the Nramp family . Mramp sequences from M. tuberculosis and BCG are identical and are most closely related to other bacterial homologues (72.4, 40, and 40% sequence identities with M. leprae, Bacillus subtilis , and Escherichia coli , respectively), whereas comparison with eukaryotic homologues gives overall amino acid identities of 21–24%. There is an asymmetrical distribution of charged amino acid residues between the endo- and exofacial regions of Mramp. This is consistent with similar patterns of charge distribution observed in many integral membrane proteins 32 . Initially we cloned Mramp into pGEM-T Easy after mutating the mycobacterial GTG start codon to ATG without strengthening the Kozak consensus sequence. Microinjection of oocytes with RNA made from this construct induced up to twofold increases in 55 Fe 2+ and 65 Zn 2+ uptake compared with water-injected oocytes (data not shown). To optimize Mramp expression in oocytes, we retained the modified start codon, introduced a strong Kozak consensus, and cloned Mramp into a vector containing flanking X. laevis 5′ and 3′ untranslated regions (see Materials and Methods). In 10 independent experiments, RNA derived from this latter construct induced large increases (up to 22-fold) in the accumulation of 65 Zn 2+ by Mramp-expressing oocytes compared with water-injected or Mramp antisense–injected controls . To confirm that these induced 65 Zn 2+ uptakes were specific to Mramp , we also examined the uptake of 65 Zn 2+ in oocytes injected with RNA made from the Trypanosoma brucei hexose transporter . As expected, there was no increase in 65 Zn 2+ uptake associated with expression of THT1, confirming the requirement for Mramp to induce 65 Zn 2+ uptake. Conversely, we demonstrated 2′-deoxy- d [ 14 C]-glucose (2-DOG) uptake by THT1 but not by Mramp . To confirm that accumulation of 65 Zn 2+ continued beyond these experimental time points, we monitored 65 Zn 2+ uptake for up to 4 h. The increase in uptake of 65 Zn 2+ was linear during this period, which encompasses the uptake times of experiments shown (slope 3.8 ± 0.63, P < 0.001). In oocytes, translocation of divalent cations by DCT1 (the rat Nramp2 homologue) depends on cotransport of protons, with maximal activity of DCT1 at an extraoocytic pH of 5.5. To determine if cation transport by Mramp displays a similar pH dependence, we measured 65 Zn 2+ uptake by oocytes incubated in extracellular pH values between 5.0 and 7.0. In nine independent experiments, 65 Zn 2+ uptake by oocytes was confined to extracellular pH values between 5.5 and 6.5 and was completely abolished at pH 7 or 5. As a first step to determine the specificity of Mramp for divalent cations in the d -block series, we measured the uptake of 65 Zn 2+ in the presence of an excess of unlabeled Mn 2+ . Mn 2+ (10 mM) completely abolished uptake of 65 Zn 2+ , indicating that Mn 2+ competes with 65 Zn 2+ for binding to or uptake by Mramp. We next used 55 Fe 2+ as a permeant to investigate in detail the substrate specificity of Mramp for divalent cations. Mramp mediates large increases in the uptake of 55 Fe 2+ at pH 5.5 (up to 18-fold) above water-injected oocytes . This induced 55 Fe 2+ uptake is abolished by Mn 2+ and Cu 2+ . In contrast, Mg 2+ , a divalent cation that does not belong to the d -block series, enhanced 55 Fe 2+ accumulation by oocytes ninefold compared with 55 Fe 2+ uptake in its absence . Mycobacterial growth varied in media containing different concentrations of Fe 2+ and Cu 2+ . At relatively high concentrations (>45 μM), Fe 2+ and Cu 2+ inhibited bacterial growth by 29 and 38%, respectively . Mramp mRNA transcript was detectable in bacteria grown at all Fe 2+ and Cu 2+ concentrations tested , including a faint band in bacteria grown in Fe 2+ -depleted culture medium, which was quantifiable using the GDS 7600 system but is not visible on the photograph. mRNA for y39 was similarly detectable at all Fe 2+ and Cu 2+ concentrations. In contrast, mRNA for yhho gave much fainter bands in bacteria grown in high Fe 2+ concentrations and medium and high Cu 2+ concentrations, and was undetectable in other conditions. rRNA (16S) RT-PCR analysis of these templates confirmed that initial total RNA template quantities were comparable for all conditions . Semiquantitative analysis of PCR products from Mramp and y39 (a putative Ca 2+ -translocating P-type ATPase ) using identical template concentrations and PCR conditions showed large increases in mRNA for Mramp (∼50-fold) as Fe 2+ concentration increases from <1 to 48 μM . As Cu 2+ concentrations increase over a similar range, mRNA for Mramp increases ∼10-fold, and is maximal at 5 μM Cu 2+ . There is less increase in mRNA for y39 under these conditions (∼17- and 5-fold, respectively). To investigate the regulation of Mramp and y39 transcription more precisely, we used a ratiometric PCR technique called TC-PCR to quantitate mRNA for Mramp in relation to y39 in M. tuberculosis cultured in media containing these different concentrations of Fe 2+ and Cu 2+ . The mRNA ratios for Mramp / y39 fell fourfold (from 0.44 to 0.11) when Cu 2+ concentrations increased from 5 to 70 μM. The relatively low PCR yields of product from y39 except for bacteria grown in medium and high Cu 2+ concentrations precluded more accurate quantitation of Mramp / y39 mRNA ratios in these conditions. Fig. 5 C (top) shows that mRNA encoding Mramp is detectable in RNA extracted from intracellular BCG. For comparison, we show that Mramp mRNA is also detectable in axenically cultured BCG under identical PCR conditions. Comparability of template concentrations in this experiment was verified by RT-PCR analysis of rRNA (16S) . Although approximately 1.7 billion people (one third of the world's population) are infected with M. tuberculosis at any one time, it is striking that, in the absence of coinfection with HIV, fewer than 10% of these will develop active disease during their lifetimes 34 . Host genetic factors such as polymorphisms in Nramp1 clearly influence susceptibility to infection or disease caused by Mycobacteria species 35 . For example, tuberculosis in a tribally mixed Gambian population was associated with two pairs of Nramp1 polymorphisms 8 , and in another study involving members of Chinese and Vietnamese families with leprosy, haplotypes associated with Nramp1 -linked polymorphisms were observed to be distributed nonrandomly between affected and unaffected family members 9 . The Nramp family of proteins is highly conserved between bacteria and mammals, and two eukaryotic examples have been shown to transport divalent cations such as Fe 2+ and Mn 2+ 12 36 37 . Nramp1 is likely to perform similar transport functions to Nramp2, which mediates pH-dependent Fe 2+ uptake in heterologous expression studies and in vivo 12 36 . Defining the transport specificities for Nramp1 has proved difficult 38 , and all studies characterizing Nramp homologues have been carried out exclusively on eukaryotic sequences. Recent studies in RAW264.7 cells overexpressing Nramp1 suggest that Nramp1 does contribute to iron mobilization from vesicles 39 . Nramp1 also circumvents maturation arrest of phagosomes containing live BCG, permitting the increase in acidification normally seen in phagosomes containing killed BCG or latex beads 40 . These observations point to the possibility that competition for transition metal ions may be important in determining maturational dynamics of phagosomes as well as their lethality for certain intracellular pathogens. We studied a mycobacterial homologue of the Nramp family because of its potential relevance to intracellular survival. Nramp homologues are found in M. tuberculosis , M. leprae , M. smegmatis , and BCG 7 17 . These homologues (called Mramp) have been suggested to mediate the uptake of cations such as Fe 2+ , Mn 2+ , and Zn 2+ , which may be important in defence by microbial superoxide dismutase against the macrophage respiratory burst 17 18 . Our studies now provide direct evidence for a function of Mramp as a transporter of Zn 2+ and Fe 2+ . Furthermore, the enhanced uptake of 65 Zn 2+ and 55 Fe 2+ induced in oocytes expressing Mramp is abrogated by an excess of Mn 2+ and Cu 2+ , but not by unrelated divalent cations such as Mg 2+ , suggesting important interactions between Mramp and these transition elements. In spite of divergence in primary sequence between Nramp1, DCT1 (a rat intestinal homologue of Nramp2), and Mramp and their diverse phylogeny, all three sequences can mediate the uptake of Fe 2+ into Xenopus oocytes 12 . DCT1 transports other members of the transition metal series, and this broad specificity is also observed for Mramp. Therefore, Mramp represents a novel class of prokaryotic metal ion transporter with representatives in other bacteria such as E. coli and B. subtilis . We examined the pH dependence of cation transport by Mramp using 65 Zn 2+ as a permeant, because at pH > 6.0 it is difficult to manipulate the equilibrium between Fe 2+ and Fe 3+ even in the presence of reducing agents such as ascorbic acid 41 . There is a narrow range of extracellular acid pH values that allows Mramp-induced uptake of 65 Zn 2+ by oocytes. This pH range (5.5–6.5) coincides with estimates of ambient pH in the microenvironment of intraphagosomal mycobacteria 42 . This observation also provides evidence for the direction in which cation transport is likely to be taking place, namely from the relatively acidic phagosome into mycobacteria. By contrast, an uninfected phagolysosome (for example, one containing inert particles) has a significantly lower pH (<5.5 ), and would therefore be unlikely to allow efficient transport of divalent cations by Mramp. To assess the expression of Mramp in M. tuberculosis cultured axenically, we applied a precise assay to quantitate mRNA for Mramp obtained from organisms grown in media containing defined Cu 2+ and Fe 2+ concentrations. These studies permitted assessment of the growth characteristics of bacteria as well as the relative expression of mRNA for Mramp compared with mRNA encoding a putative Ca 2+ P-type ATPase (y39). Mramp is expressed poorly in bacteria grown in relatively Cu 2+ - and Fe 2+ -deficient media, and expression is enhanced at higher concentrations of these metal ions (≥5 μM). Similar patterns of mRNA expression are observed for the putative Ca 2+ -transporting P-type ATPase (y39), but in contrast, mRNA encoding an atypical heavy metal-translocating P-type ATPase (yhho) is barely detectable under any of the conditions tested . This stimulation of expression of mRNA for Mramp and y39 at higher ambient concentrations of Cu 2+ and Fe 2+ is associated with retardation of bacterial growth by ∼30%. mRNA for Mramp encoded by BCG is clearly expressed in the intracellular environment . We used BCG as a model for M. tuberculosis because M. tuberculosis is frequently cytopathic when cultured in THP1 cells, compromising yields of RNA. BCG is well recognized as a convenient model to study mycobacterial gene expression in these circumstances 25 . Mramp may act in concert with mechanisms inhibiting acidification of phagosomes to permit intracellular survival of mycobacteria. The deployment of Nramp1 in the host's phagosomal membrane is clearly important in defence against infection, as established by classical studies on the genetics of Nramp1. If Nramp1 also uses phagosomal protons to extrude cations, thereby competing with Mramp, the pH dependence of this phenomenon will be critical in establishing which of the two transporters (Mramp or Nramp1) functions most efficiently in the infected macrophage. Experiments to examine this hypothesis in greater detail can now be formulated on the basis of Mramp's function as defined by heterologous expression. Mramp is the first mycobacterial gene to be expressed in oocytes, exemplifying the utility of this system for the functional characterization of other prokaryotic transporters ( E. coli glycerol facilitator glpF, and E. coli water channel AqpZ 43 ). | Study | biomedical | en | 0.999996 |
10477556 | Genomic DNA was extracted using TriReagent (Molecular Research Center, Inc.) according to the manufacturer's instructions. To detect α1 circles, a molecular beacon was used in combination with real-time PCR. The method of detection using molecular beacons 19 20 and a fluorescence detector system 21 22 23 24 has been previously described. Each 50-μl reaction contained 5 μl of DNA, and the final concentration of each component was as follows: 1.0× Taqman buffer A (Perkin-Elmer Corp.), 3.5 mM MgCl 2 , 0.4 pmol/μl of molecular beacon, 0.4 pmol/μl of each primer, and 1.25 U of AmpliTaq Gold DNA Polymerase (Perkin-Elmer Corp.). The primers (sense, 5′-GGATGGAAAACACAGTGTGACATGG-3′ and antisense, 5′-CTGTCAACAAAGGTGATGCCACATCC-3′) amplified a 208-bp product. The amplicon was sequenced and found to contain the appropriate motifs for a recombination signal sequence 12 13 . A molecular beacon was included in the reaction mixture throughout PCR to serve as a real-time detector for the amplified product. The molecular beacon was designed to recognize a region upstream from the signal joint. This beacon was specifically designed to have a hairpin structure, with a 6-bp stem and a 26-nucleotide target recognition loop, plus a fluorophore (FAM [6-carboxyfluorescein]) and quencher (DABCYL [4-dimethylaminophenlazo benzoic acid]) in close proximity to the two ends of the oligonucleotide. The target recognition sequence for the molecular beacon was 5′-GAGAACGGTGAATGAAGAGCAGACAG-3′. One cycle of denaturation (95°C for 10 min) was performed, followed by 45 cycles of amplification (94°C for 30 s, 60°C for 30 s, and 72°C for 30 s). PCR was carried out in a spectrofluorometric thermal cycler that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase while simultaneously carrying out programmed temperature cycles. The cycle number during PCR that yields a fluorescence intensity significantly above the background is designated as the threshold cycle (C T ). As shown here and reported elsewhere 21 22 23 24 25 , the C T is directly proportional to the log of the copy number of the target sequence in the input DNA. We have found that this α1 circle assay is efficient (<4 h), specific (see Results and Dicussion), sensitive (10 copies), dynamic (7-log range), and accurate (10% coefficient of variance). To normalize for cell equivalents in the input DNA, we used a separate real-time PCR/molecular beacon assay to quantify the CCR5 coding sequence, as it is known that this gene is present at only two copies per cell, i.e., there are no pseudogenes (Kostrikis, L. and D.D. Ho, personal communication). HIV-1–infected individuals were considered acutely infected if they were enrolled in treatment protocols within the first 90 d of symptoms of their acute HIV-1 infection. If enrolled after 90 d of symptoms, they were considered chronically infected. In the studies of the effect of treatment on α1 circle numbers, every patient was treated with a three- or four-drug combination. The regimens were standard dosages of zidovudine and lamivudine plus ritonavir, indinavir, nelfinavir, ritonavir/saquinavir, or abacavir/amprenavir. 13 of the chronically infected individuals were treated with the combination of stavudine, lamivudine, nelfinavir, saquinavir, and IL-2 (9 MU/day for 5 d in 6-week intervals for 8 cycles after day 28). Quantitation of CD3 + , CD4 + , CD8 + , and CD45RA + 62L + subpopulations in peripheral blood samples from HIV-1–infected individuals was done by four-color flow cytometry using the following mAbs: anti-CD3–PerCp (Becton Dickinson), anti-CD4–APC (Exalpha), anti-CD8–APC (PharMingen), anti-CD45RA–FITC (Becton Dickinson), and anti-62L–PE (Becton Dickinson). In the lymphocyte subsets study, a minimum of 10 5 naive or memory cells were isolated using a fluorescence-activated cell sorter, MoFLO (Cytomation, Inc.). Using four-color flow cytometry, naive cells were defined as CD45RA + and CD62L + , whereas memory cells were defined as CD45RO + for both CD4 + and CD8 + populations. The purity of the sorted cells was >99%, as analyzed by a FACSCalibur™ flow cytometer (Becton Dickinson). Quantitation of HIV-1 RNA in plasma was determined using the Roche Ultrasensitive Amplicor HIV-1 Monitor assay (lower limits of detection, 50 HIV-1 RNA copies/ml; Roche Molecular Systems). Comparisons between groups were done by nonparametric analysis using the Kolmogorov-Smirnov test (Statview, SAS Institute, Inc.). P values over 0.05 are regarded as not statistically significant. A real-time PCR assay with a molecular beacon detection system 19 26 was used to quantify α1 circles in DNA extracted from cells of interest. As shown in Fig. 1 B, α1 circles were detected in great abundance (∼10 5 copies/10 6 cells) in fetal thymic tissue but were not detected (<10 copies/10 6 cells) in nonlymphoid tissues or immortalized cell lines. In blood CD4 + and CD8 + lymphocytes, α1 circles were enriched in naive (defined as CD45RA + CD62L + cells) subpopulations by 10–50-fold compared with memory (CD45RO + ) subpopulations. The finding of low levels of α1 circles in memory cells was not unexpected, as these cells are derived from naive cells after antigen-induced proliferation during which TRECs are diluted. The specificity of the assay was further confirmed by the absence of α1 circles from sorted B cells and PBMCs of four individuals with severe DiGeorge syndrome or SCID . The numbers of α1 circles in PBMCs were assessed in a cohort of 532 normal, HIV-1–seronegative persons who ranged in age from infants to 95-yr-olds. Fig. 2 A shows that the mean number of α1 circles remained relatively stable at a high level of ∼10 5 /10 6 PBMCs for the first 10–15 yr of life. Stability of α1 circle numbers during early childhood was confirmed by studying sequential PBMC samples from 12 subjects followed from birth to age 3 or 4. Not a single child showed any significant decrease in α1 circle numbers over this period of observation (data not shown). Given that the total lymphocyte concentration decreases in the first 5 yr of life, if we expressed RTE concentration as the concentration of α1 circles per microliter of blood, then a decline in concentration of α1 circles per microliter of blood would be observed with time in this age range (data not shown). Between the teen years and age 20–25, a sharp drop of ∼1–1.5 logs was noted, followed by a gradual decline (downslope of ∼0.03/yr) thereafter. No differences were found between males and females. A total decrease of ∼2 logs over 10 decades was observed. It should be noted that even persons over the age of 70 have significant numbers of α1 circles in their PBMCs. In addition, when the results in Fig. 2 were converted to α1 circle numbers per microliter of blood, a drop in the concentration of RTEs was seen in the first 15 yr of life due to the known decrease in total lymphocyte numbers during this period. Overall, these results agree with the age-related decline in thymic lymphoid volume measured in an autopsy series 27 . The multiphasic decrease in α1 circles over time shown in Fig. 2 A, however, differs from the monotonic exponential decline reported recently 18 . The same trend in age-related decline is seen when the results are analyzed by box plots denoting the 90th, 75th, 50th, 25th, and 10th percentiles for each age category. The prominent transition observed for age 16–20 is consistent with prior findings that CD4 + lymphocyte regeneration after chemotherapy is severely impaired after the age of 20 28 29 . Fig. 2 B also demonstrates the considerable normal variation (1–1.5 logs) in α1 circle numbers within any age category. In a cross-sectional study, we compared 126 HIV-1–infected adults who had never received antiretroviral therapy with 88 age-matched seronegative controls. Of the HIV-1–infected individuals, 65 were studied during the acute stage of HIV-1 infection, i.e., within the first 90 d of infection. As shown in Fig. 3 A, both acutely and chronically infected individuals have significantly lower α1 circle numbers than controls ( P < 0.05). Many HIV-1–infected individuals, however, have α1 circle numbers in the normal range, in contrast to the previous suggestion that every HIV-1–infected adult has a low concentration of RTEs in blood 18 . We performed our studies on PBMC samples, whereas the study by Douek et al. 18 measured the number of TRECs in separated CD4 + and CD8 + T cell populations. Note as well the absence of supranormal α1 circle numbers from infected individuals to support the suggestion of a “thymic rebound” that had been raised by imaging studies on HIV-1–infected humans 10 or histopathologic examinations of SIV-infected macaques 6 . Among HIV-1–infected individuals, no significant correlation was observed between α1 circle numbers and concurrent CD4 + lymphocyte count or plasma viral load ( P > 0.05; data not shown). A more prominent impact of HIV-1 infection on the number of α1 circles was seen in a similar cross-sectional comparison of 42 HIV-1–infected children (ages 0–10 yr) with 124 age-matched seronegative controls . The HIV-1–infected children were either on no therapy or monotherapy with a nucleoside analogue, typically zidovudine. The median α1 circle value for HIV-1–infected children was 3.7-fold lower than that in normal subjects, with ∼50% having values below the normal 10th percentile. A low α1 circle number in PBMCs of HIV-1–infected persons could be due to either decreased thymic output or to an increased rate of cell death or proliferation. No mechanistic discrimination can be made from these cross-sectional analyses. The consequence of HIV-1 infection on the number of α1 circles was then assessed longitudinally in 16 homosexual men who were enrolled in an HIV-1 natural history cohort. They were all initially HIV-1 seronegative but became infected during the course of observation. Cryopreserved PBMC samples pre- and postseroconversion (up to ∼7 yr of follow-up, and without the use of antiretroviral drugs except for zidovudine for four individuals in their last few time points) were assayed for α1 circles. A considerable person-to-person variation was again noted. As exemplified by the five cases shown in Fig. 3 B, some individuals demonstrated a progressive decline in α1 circle numbers, whereas others had sequential values that were essentially no different from their preinfection baselines. The data from each case were then analyzed by linear regression, and the slope of the change in α1 circle numbers over time was individually calculated . Although the slope in half of these individuals did not show an appreciable decline, in the other half there was a downslope (>0.30/yr) that was >10 times steeper than the normal rate of decline. What might account for these dichotomous results was not immediately evident. However, the slope of the decline in α1 circle numbers correlated significantly with each patient's rate of decrease of CD4 lymphocytes . The precise mechanistic implication of this correlation remains unclear, as it could be interpreted to mean that the rate of loss of CD4 + T cells is linked either to a faster rate of drop in thymic output or to a faster rate of death or proliferation for RTEs. We studied the impact of HAART on the number of α1 circles in PBMCs of 74 previously drug-naive individuals (mean age, 36; ages ranged from 24 to 59). 27 patients began treatment within the first 90 d of their HIV-1 infection, whereas 47 were chronically infected for varying lengths of time. DNA extracted from sequential PBMC samples (generally ≥5) from each case were assayed for α1 circles. Variable patterns of response to HAART were observed, in contrast to the nearly complete response rate previously reported 18 . As displayed in Fig. 4 A, those individuals with baseline values of >2,200 α1 circles/10 6 PBMCs (the median value for age-matched normal controls) in general did not have a rise in α1 circle numbers while receiving HAART. In contrast, individuals with baseline values ≤2,200 typically showed increases during treatment. These differences were quantified by calculating the slope of the regression line for all α1 circle data points from each individual. For example, in the cohort of chronically infected individuals, the mean slope of the change in α1 circles during HAART was +1.13/yr for those with a low baseline value and −0.51/year for those with a normal baseline value . That the overall slope difference between individuals with low baselines and those with normal values was statistically significant ( P < 0.05, t test) suggests that an increase in the number of RTEs during HAART is seen primarily in individuals with an existing impairment. The thymus is known to express high levels of CXCR4 30 31 32 , the major entry coreceptor for syncytium-inducing (SI) strains of HIV-1 33 . Both X4 and R5 viral isolates can replicate in SCID-hu mice, although X4 viruses replicate with greater cytopathicity 30 . Perhaps individuals with SI viruses have a lower concentration of RTEs due to increased thymic destruction by SI viruses, especially in light of the observation that such individuals have a faster rate of CD4 + T lymphocyte loss 34 35 36 . However, in preliminary studies, we have noted that many individuals with low α1 circle numbers have no detectable SI viruses in their blood (data not shown). Additional work will be necessary to define the mechanistic pathways by which the concentration of RTEs in blood is affected in some and spared in others. Important clues might come from studies to understand why HIV-1–infected children are more likely than adults to have a lower number of RTEs. Some have suggested that certain infected infants have an immunophenotype resembling that seen in congenital athymic defects 37 38 . The application of new assays to measure TRECs to this clinical setting could be particularly revealing. The sequential α1 circle numbers of eight individuals on an identical HAART regimen (standard dosages of zidovudine, lamivudine, abacavir, and amprenavir; median duration follow-up, 450 d) were converted to α1 circle copies per volume of blood based on concurrent total lymphocyte counts in blood. These results were then compared with the CD4 + and CD8 + naive (CD45RA + CD62L + ) T cell numbers . As has been reported 39 40 41 42 , mean CD4 + T lymphocyte counts increased more significantly in the initial months of therapy, followed by a slower rise thereafter (mean total increase of 218 cells/μl). Mean CD4 + naive cell numbers climbed steadily throughout the treatment period (mean total increase of 93 cells/μl), as did CD8 + naive cells (mean total increase of 65 cells/μl), despite a clear drop in total CD8 + T lymphocyte counts (mean total decrease of 250 cells/μl). Six of the eight individuals had low levels of α1 circles before therapy (i.e., <2,200 α1 circles/10 6 PBMCs). The kinetics of the changes in α1 circles per microliter of blood did not, however, temporally correspond to those of CD4 + and CD8 + naive cells. There was an initial rise in the concentration of α1 circles within the first 90 d of therapy followed by a subsequent decrease. More importantly, the total number of RTEs, as reflected by α1 circle measurements (<10 copies per microliter of blood), was numerically insufficient to account for the rise seen in CD4 + and CD8 + naive lymphocytes. This could be partly explained by proliferation of RTEs before or soon after emigration from the thymus, a phenomenon reported to occur in the mouse 43 . Alternatively, this discrepancy could suggest that much of the observed rise in CD45RA + CD62L + T cells after HAART is not due to direct outpouring from the thymus. Using a simple and accurate real-time PCR/molecular beacon assay, we have shown that the number of RTEs in blood remains high for the first 10–15 yr of life, followed by a sharp drop in the late teenage years and a gradual decline thereafter. HIV-1 infection was found to lower the concentration of RTEs in a subset of but not all adult individuals. The rate of loss of RTEs correlated directly with the rate of CD4 + T lymphocyte decline. However, it remains unclear whether the abnormality seen in some HIV-1–infected individuals is due to decreased thymic output or to an increased death or proliferation rate of thymic emigrants. Because many HIV-1–infected persons had normal numbers of α1 circles, including some with low CD4 + T cell counts, it is difficult to invoke thymic regenerative failure as a generalized mechanism for the depletion of CD4 + lymphocytes. HAART had no appreciable effect on the number of α1 circles in those individuals whose baseline levels were already within the normal range. On the other hand, significant increases were observed in those with a preexisting impairment. These increases, however, were numerically insufficient to account for the rise in naive (CD45RA + CD62L + ) CD4 + and CD8 + T lymphocytes. This finding indicates that the rise in naive T lymphocytes during HAART may not be a direct measure of thymic output; instead, it may largely reflect either peripheral expansion of naive lymphocytes without a phenotypic switch 7 or the reversion of phenotype from memory cells 7 44 45 . This conclusion is consistent with the observed increase in CD45RA + CD62L + T lymphocytes after HAART in individuals who had been thymectomized 11 . We therefore caution against the use of such phenotypic markers as a direct indicator of thymic output. | Study | biomedical | en | 0.999996 |
10477557 | B7-1 −/− , B7-2 −/− , and B7-1/B7-2 −/− mice were generated by gene targeting as described previously 5 6 . B7-deficient mice were backcrossed from the 129/S4SvJae background to the C57BL/6 background for at least six generations before use in experiments, and were genotyped as described previously 5 6 . CD28 −/− mice were purchased from The Jackson Laboratory. Mice used for controls were either wild-type or heterozygous littermates or C57BL/6 mice purchased from The Jackson Laboratory. Brigham and Women's Hospital and Harvard Medical School are accredited by the American Association of Laboratory Animal Care (AALAC). Mice were cared for in accordance with institutional guidelines in a pathogen-free animal facility. MOG 35-55 (MEVGWYRSPFSRVVHLYRNGK) and MOG 92-106 (DEGGYTCFFRDHSYQ) were synthesized by Dr. David Teplow at the Biopolymer Facility (Center for Neurological Diseases, Brigham and Women's Hospital) on an Applied Biosystems 430A peptide synthesizer using fluorenylmethoxycarbonyl (F-MOC) chemistry. The peptides were >90% pure, as determined by HPLC. Groups of 6–8-wk-old female mice (three to six per group) were immunized with 100 μg of MOG 35-55 emulsified 1:1 in CFA supplemented with 400 μg of Mycobacterium tuberculosis H37RA (Difco Laboratories) in the two flanks subcutaneously. 200 ng of pertussis toxin (List Biological Laboratories) was injected intravenously on the day of immunization and 2 d later. Wild-type mice were immunized with 100 μg of MOG 35-55 in CFA (Difco Laboratories). 10 d later, draining lymph nodes were harvested, and lymphocytes were cultured with 30 μg of MOG 35-55 and 20 ng of murine rIL-12 (Genetics Institute). After 4 d of culture, cells were washed and resuspended in PBS for transfer. Wild-type or B7-1/B7-2 −/− recipients received 10 7 cells intravenously, as well as 200 ng of pertussis toxin immediately after cell transfer and 2 d later. Mice were observed daily for clinical signs of EAE up to 35 d after immunization or cell transfer, and scored on a scale of 0–5: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and fore limb paralysis; and 5, moribund state. Mean clinical score was calculated by averaging the scores of all mice in each group, including animals that did not develop EAE. Brains and spinal cords were removed and fixed in 10% formalin. Paraffin-embedded sections were stained with Luxol fast blue–hematoxylin and eosin for light microscopy. Inflammatory foci were counted in the meninges and parenchyma by an unbiased observer as described previously 7 . Mice were immunized subcutaneously with 100 μg of MOG 35-55 emulsified 1:1 in CFA (Difco Laboratories). Draining lymph nodes were harvested 10 d later, and single cell suspensions were prepared. Whole lymph node cells were cultured in 96-well plates at 5 × 10 5 cells/well with a range of concentrations of MOG 35-55 or MOG 92-106 peptide in HL-1 media (BioWhittaker). Plates were pulsed with [ 3 H]thymidine (New England Nuclear) at 1 μCi/well on day 3 of culture for the final 18 h. Mean incorporation of thymidine in DNA was measured in triplicate wells by liquid scintillation counting . Lymph node cells were prepared and cultured as above with media alone or 50 μg of MOG 35-55 peptide. Supernatants were harvested at 24, 48, and/or 72 h of culture. Cytokines (IL-2, IFN-γ, TNF-α, IL-4, and IL-10) were determined by sandwich ELISA, as described 8 . mAb pairs and recombinant cytokine standards were purchased from PharMingen. The following mAb pairs were used: IL-2 (JES6-1A12, capture; JES6-5H4 detection); IFN-γ (R4-6A2 capture; XMG1.2 detection); IL-4 (BVD4-1D11 capture; BVD6-24G2 detection); IL-10 (JES5-2A5 capture; SXC-1 detection); and TNF-α . To examine the role of the B7–CD28 costimulatory pathway in the initiation of MOG-induced EAE, we immunized wild-type C57BL/6 mice and C57BL/6 mice lacking CD28 or either B7-1 or B7-2 or both B7 costimulators with MOG 35-55 and observed the mice for clinical signs of EAE. Mice deficient in either B7-1 or B7-2 developed EAE with comparable severity and time course to wild-type mice . EAE incidence, time of onset, and maximal clinical score were not significantly different among wild-type, B7-1 −/− , and B7-2 −/− mice ( Table ). All three groups had >90% clinical disease incidence. EAE onset occurred 13–16 d after immunization and reached maximal severity at a clinical score of 2.5–3.0 (severe hind limb weakness or paralysis). These results indicate that the presence of either B7-1 or B7-2 alone is sufficient to provide the costimulation necessary to support the development of MOG-induced EAE in C57BL/6 mice. In contrast, mice lacking both B7-1 and B7-2 were strikingly resistant to EAE induction . In 3 separate experiments involving 18 B7-1/B7-2 −/− mice, none of the B7-1/B7-2 −/− mice developed clinical signs of EAE during the 35-d observation period, whereas 90% of wild-type littermates developed EAE. Similar to the B7-1/B7-2 −/− mice, mice lacking CD28 were also resistant to EAE induction . Only 2 of 13 (15%) CD28 −/− mice developed clinical signs of EAE. In these two CD28 −/− mice, the time of onset of EAE was delayed, and the maximal disease score was also less than in wild-type mice ( Table ). The brain and spinal cords of wild-type, B7 −/− , and CD28 −/− mice were examined histologically at the end of the clinical observation period (day 35 after immunization). Wild-type, B7-1 −/− , and B7-2 −/− mice had similar numbers of inflammatory lesions in the meninges and CNS parenchyma . More than 80% of mice in each of these groups demonstrated inflammatory lesions. In contrast, EAE inflammatory foci were observed in approximately half of B7-1/B7-2 −/− and CD28 −/− mice, and the numbers of foci were significantly reduced in both groups ( Table ). Thus, the lack of clinical disease in B7-1/B7-2 −/− mice was accompanied by a marked reduction in CNS inflammation. Strikingly, in the seven B7-1/B7-2 −/− mice that exhibited inflammatory foci ( Table ), these foci were limited almost exclusively to the leptomeninges . Thus, the absence of both B7-1 and B7-2 resulted in a disproportionate reduction of parenchymal compared with meningeal infiltrates. To determine whether the phenotype of responding T cells to MOG 35-55 was altered in mice lacking either or both B7 costimulators, we restimulated draining lymph node cells with MOG 35-55 in vitro and measured proliferative responses and cytokine secretion. For these studies, draining lymph nodes were obtained 10 d after immunization with MOG 35-55. Proliferation and cytokine production were evaluated on days 1, 2, and 3 of in vitro culture using a range of peptide concentrations. As shown in Fig. 3 , the proliferative responses of draining lymph node cells obtained from immunized wild-type and B7-1 −/− mice were comparable. No consistent or significant differences in IFN-γ and TNF-α production were observed in B7-1 −/− lymph node cultures compared with wild-type. Although the proliferative responses of B7-2 −/− lymph node cells were modestly reduced at high peptide concentrations, they produced more IFN-γ than wild-type cells . IL-2, IL-4, and IL-10 were below detection limit for all cultures. The modest differences in T cell proliferation and cytokine production in B7-1 −/− and B7-2 −/− lymph node cultures are similar to the minimal variations in clinical and histological manifestations of EAE among wild-type, B7-1 −/− , and B7-2 −/− mice. In contrast, EAE-resistant B7-1/B7-2 −/− mice exhibited markedly impaired proliferative responses to MOG 35-55 compared with wild-type mice . Even at the highest peptide concentration examined, the proliferation of B7-1/B7-2 −/− lymph node cells was only 25% of wild-type levels. Despite the reduced proliferation, B7-1/B7-2 −/− lymphocytes produced significantly increased levels of IFN-γ (>30-fold increase) with faster kinetics. Although IFN-γ was detectable in wild-type cultures only after 48 h of stimulation, IFN-γ was present at high levels in B7-1/B7-2 −/− cultures by 24 h. IFN-γ production by B7-1/B7-2 −/− cells remained much higher than wild-type throughout the 3-d culture period. TNF-α production was also increased in B7-1/B7-2 −/− cultures. However, in contrast to IFN-γ, B7-1/B7-2 −/− lymphocytes secreted higher levels of TNF-α only in the first 24 h of culture. By 48 h, TNF-α was detectable in wild-type cultures while TNF-α production in B7-1/B7-2 −/− cultures had declined. IL-2, IL-4, and IL-10 were below detection limits. These results suggest that B7-1/B7-2 −/− T cells are primed to MOG 35-55 in vivo, but that the expansion of these T cells is impaired. Furthermore, the increased levels of IFN-γ and TNF-α in B7-1/B7-2 −/− lymphocyte cultures indicate that EAE resistance in these mice is not likely to be due to a defect in Th1 cytokine production. To evaluate whether B7 costimulation has a role in the effector phase of EAE, wild-type MOG 35-55–specific T cells were adoptively transferred into wild-type and B7-1/B7-2 −/− mice. B7-1/B7-2 −/− mice developed less severe EAE upon adoptive transfer compared with wild-type . EAE onset was delayed in B7-1/B7-2 −/− mice. Severity and duration of clinical disease in B7-1/B7-2 −/− mice was also significantly reduced. Histologic evaluation demonstrated that the B7-1/B7-2 −/− recipient mice had fewer CNS inflammatory infiltrates compared with wild-type mice, with a greater reduction of parenchymal compared with meningeal foci ( Table ). Thus, B7 costimulation is required both for the initiation and/or expansion of T cells in the peripheral immune compartment and for the effector phase of EAE to mediate tissue injury and enable disease progression. In this study, we have examined the role of B7–CD28 costimulation and its relative importance for the induction and effector phases of MOG-induced EAE using mice deficient in B7 costimulators or CD28. We have demonstrated that: (a) mice lacking either B7-1 or B7-2 develop EAE comparably to wild-type mice; (b) mice lacking both B7-1 and B7-2 or CD28 are highly resistant to EAE induction; (c) resistance to EAE is not due to a lack of Th1 cytokine production; and (d) B7 costimulatory molecules are critical for the effector phase of EAE, since EAE is markedly reduced in B7-1/B7-2 −/− recipients after adoptive transfer of MOG-specific wild-type T cells. Taken together, these results indicate that the B7–CD28 costimulatory pathway is required for the development of MOG 35-55–induced EAE in C57BL/6 mice. B7 costimulators have critical roles both in the initial activation and expansion of MOG-reactive T cells and in their activation within the target organ. Our studies with mice lacking either B7-1 or B7-2 alone demonstrate that B7-1 and B7-2 have overlapping roles in MOG-induced EAE. This contrasts with EAE studies in SJL mice in which in vivo administration of anti–B7-1 antibodies reduced disease whereas anti–B7-2 antibodies either had no effect or exacerbated disease 2 3 4 . Since the timing and level of B7-1 expression is unchanged in B7-2 −/− mice and, likewise, B7-2 expression is unaffected in B7-1 −/− mice (our unpublished data), the difference in conclusions drawn from the two systems is not likely to be attributable to alterations in B7 expression in B7-1– or B7-2–deficient mice. Several factors may have confounded the results of antibody blocking studies. These include Fc receptor–mediated effects, inadequate penetration of antibodies in vivo, and varying affinity of the antibodies to their ligand. Genetic backgrounds may also influence the respective roles of B7-1 and B7-2 in the induction of EAE. The relative importance of B7-1 and B7-2 may differ between MOG-induced EAE in C57BL/6 mice and proteolipid protein (PLP) or myelin basic protein (MBP)-induced EAE in SJL mice. Induction and level of B7-1 and B7-2 expression may differ on various genetic backgrounds. Genetic analyses have identified chromosomal regions containing the CD28/CTLA-4 and B7-1/B7-2 loci as important for the development of EAE 9 . Further studies are needed to determine whether polymorphisms within the CD28/CTLA-4 or B7-1/B7-2 loci lead to altered expression of these genes or to altered protein function. Such differences in the B7-CD28/CTLA-4 costimulatory pathway on different genetic backgrounds could contribute to the relative role of B7-1 versus B7-2 or CD28 in the pathogenesis of EAE and other autoimmune diseases. Cytokines play a key role in initiating, propagating, and regulating tissue-specific autoimmune injury. The role of cytokines in regulating EAE is complex. Although there is evidence that Th1 cells that produce proinflammatory cytokines (IFN-γ and TNF-α) are critical for the induction of EAE, direct administration of anti–IFN-γ in vivo enhanced EAE 10 11 . And although intrathecal injection of IFN-γ induced local inflammation 12 , systemic administration of IFN-γ suppressed EAE development 13 . Recent studies using cytokine- or cytokine receptor–deficient mice suggest that rather than promoting CNS inflammation, IFN-γ and TNF-α may be important in limiting the extent and duration of EAE. Mice lacking IFN-γ or IFN-γ receptor develop more severe MOG-induced EAE than wild-type littermates 14 15 16 . TNF-α −/− mice develop severe MOG-induced EAE with extensive inflammation, demyelination, and high mortality 17 18 19 . Our results show that although B7-1/B7-2 −/− mice are resistant to MOG-induced EAE, peripheral T cells from these mice produce increased levels of IFN-γ and TNF-α. Whether resistance to EAE in B7-1/B7-2 −/− mice is due to lack of expansion of encephalitogenic precursors or to excessive production of IFN-γ and TNF-α is not clear. However, our observations of increased IFN-γ or TNF-α by B7-1/B7-2 −/− T cells and EAE resistance in B7-1/B7-2 −/− mice are consistent with studies that report exacerbated EAE in mice lacking IFN-γ or TNF-α. 14 15 16 17 18 19 . Our data are also consistent with studies demonstrating a major defect in Th2 but not Th1 cytokine production in the absence of both B7-1 and B7-2 or CD28 20 21 . Our results suggest that impaired T cell expansion could, in part, be responsible for EAE resistance in B7-1/B7-2 −/− mice, since it appears that MOG-specific T cells are primed in these mice but that their expansion is markedly reduced. However, in addition to controlling T cell expansion, B7 molecules may also be important in regulating migration of encephalitogenic T cells into the CNS and/or their reactivation within the CNS. The greater proportionate reduction of parenchymal, as opposed to meningeal, infiltrates in B7- and CD28-deficient mice compared with those in wild-type mice also suggests that B7 interactions may affect inflammatory cell homing to different CNS compartments. Furthermore, the adoptive transfer experiments demonstrate for the first time that, in addition to playing a key role in the initial expansion and differentiation of T cells, B7 costimulation is required for mediating inflammation during the effector phase of EAE. The demonstration of a key role of B7 costimulation in the effector phase of EAE has important therapeutic implications, since most autoimmune diseases are diagnosed after initial responses to the autoantigens. Our results provide impetus for testing B7 blockade as a therapeutic approach in the effector phase of human autoimmune diseases. | Study | biomedical | en | 0.999998 |
10477558 | Development of FPR knockout mice has been described previously 21 . Mice used in this study were from FPR +/− × FPR +/− matings of an F1 backcross of FPR +/− 129/Sv with wild-type C57Bl/6 mice. Leukocytes were harvested from the peritoneal cavity of FPR +/+ and FPR −/− mice after thioglycollate (TG) irritation, as described previously 21 . Cells obtained after 3 h were >90% neutrophils, whereas cells obtained after 72 h were >90% macrophages, as determined by the morphologic appearance of Diff-Quick-stained preparations. Cells were lysed, and total RNA was prepared using the RNA STAT-60 protocol (Tel-Test, Inc.). RNA (10 μg) was separated on a denaturing agarose gel, and Northern blots were prepared by standard methods 24 . Blots were hybridized with a full-length Fpr-rs2 ORF probe, which was labeled with 32 P-dCTP using a random-primer DNA labeling kit (Boehringer Mannheim). To control for RNA loading, the blots were also probed separately with a 49-bp oligonucleotide from the 5′ terminal end of the actin gene, which was labeled with a DNA 5′ end labeling kit (Boehringer Mannheim). The Fpr-rs2 ORF was amplified from genomic clone 7 22 using the upper strand primer 5′-ATATAAGCTTGCCACC ATG GATTATAAAGA TGATGATGATA AA GAATTC GAATCCAACTACTCCATCCATCTGAATG-3′, which contains a Flag epitope tag (underlined), an EcoRI site (italicized), and an ATG initiating codon (bold); and lower strand primer 5′-CG CTCGAG TCA TGGGGCCTTTAACTCAATGTCTG-3′, which contains an XhoI site (italicized sequence) and the termination codon (bold). The 1.1-kb PCR fragment was then ligated into pCR2.1. Sequence fidelity was verified, and the correct insert was then subcloned between the NotI and XhoI sites of pcDNA3 (Invitrogen). Human embryonic kidney (HEK) 293 cells were maintained in DMEM with 10% FBS. 10 7 cells in log phase were electroporated with 20 μg of plasmid DNA using a GenePulser (Bio-Rad Laboratories). Cell colonies resistant to 2 g/liter G-418 (GIBCO BRL) were isolated and expanded in DMEM with 10% FBS and 2 g/liter G-418. Cells (∼10 7 /ml) were incubated in HBSS and 2.5 μM Fura-2 AM (Molecular Probes, Inc.) for 30–60 min at 37°C in the dark. The cells were then washed with HBSS and resuspended at 10 7 cells/ml. 4 × 10 6 cells were stimulated in a total volume of 2 ml in a continuously stirred cuvette at 37°C in a fluorimeter (Photon Technology, Inc.). fMLF, MLF, and recombinant C5a were obtained from Sigma Chemical Co. The chemokines IL-8, macrophage-inflammatory protein (MIP)-1β, and monocyte-chemotactic protein (MCP)-3 were obtained from Peprotech. The data were recorded every 200 ms as the relative ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm. For some experiments, cells were incubated with 250 ng/ml pertussis toxin for 4 h before functional assay. HEK 293 cells were detached from flasks by replacing media with 0.05% trypsin (Quality Biological) and incubating at room temperature for ∼1 min. DMEM containing 20% FBS was added, and the cells were harvested, pelleted, and resuspended at 10 6 cells/ml in RPMI 1640 supplemented with 1% BSA and 20 mM Hepes. fMLF was loaded at varying concentrations in the lower compartment of a 48-well microchemotaxis chamber (NeuroProbe). To distinguish chemotaxis from chemokinesis, additional experiments were carried out in which an equal concentration of fMLF was tested simultaneously in the upper and lower compartments. The chamber was soaked in 1% SDS overnight and washed before each experiment. A polyvinylpyrrolidone-free polycarbonate filter (10- and 3-μm pores for HEK 293 cells and neutrophils, respectively) was used. For experiments with HEK 293 cells, the filters were coated with 0.05 mg/ml rat tail collagen in RPMI 1640 and 70 mM Hepes for 2 h and dried before each experiment. The filter, coated side down, was placed between the upper and lower compartments of the chamber, and 50 μl of 10 6 HEK 293 cells/ml was loaded in the upper compartment. The chemotaxis chamber was incubated at 37°C, 100% humidity, and 5% CO 2 for 5 h for HEK 293 cells and 45 min for neutrophils. The filter was then removed, washed, fixed, and stained. Cells that migrated through the filter were counted microscopically under high power. All conditions were tested in triplicate. Previously, we reported that Fpr-rs2 is expressed in unfractionated peripheral blood leukocytes 22 . To determine the expression pattern in finer detail, we used Northern blot hybridization to probe total RNA from peritoneal cells elicited 3 and 72 h after instillation of TG. The elicited peritoneal cell populations were markedly enriched in neutrophils and macrophages at 3 and 72 h, respectively ( > 90% pure). The residual 10% of cells were mainly mononuclear cells and neutrophils in the 3- and 72-h cell populations, respectively. Specific mRNA bands were detected in both the 3- and 72-h TG-elicited peritoneal cells. In both cases, two classes of transcripts ( ∼ 1.5 and 1.9 kb) were observed, and the signal strength was similar for each class within each sample. However, signals from the 3-h cells were much stronger than from the 72-h cells . Note that 72-h RNA was deliberately overloaded relative to 3-h RNA, as revealed by hybridization with an actin probe. A reasonable interpretation of these results, consistent with the functional data that follows, is that Fpr-rs2 is primarily expressed in neutrophils, and the weak signal in the 72-h cells is due to neutrophils, which make up a small minority of the total cell population. An alternative explanation is that monocytes express Fpr-rs2 in the circulation, but not after extravasation. To assess whether Fpr-rs2 encodes a functional FPR, we performed a gain-of-function genetic test by measuring fMLF-induced intracellular Ca 2+ flux in HEK 293 cell lines stably transfected with expression plasmids containing the Fpr-rs1 or Fpr-rs2 ORFs. In the initial screen, we tested 16 G-418–resistant HEK 293 cell colonies derived from the same transfection, all of which responded to 10 μM fMLF. In contrast, nonformylated MLF at 1 μM, C5a at 10 nM, and the chemokines IL-8, MIP-1β, and MCP-3 at 100 nM or greater did not induce a response. Also, Fpr-rs1 –transfected cells, maintained in the same selective conditions, consistently failed to respond to fMLF at concentrations as high as 100 μM . Higher concentrations could not be tested for technical reasons due to the hydrophobicity of fMLF. Based on this result, we infer that Fpr-rs2 encodes an FPR, which we provisionally name FPR2. Four cell lines that gave particularly strong responses, designated L4-7, L4-14, L4-15, and L4-16, were selected for further study. In each case, the calcium flux responses were concentration dependent and saturable, with an EC 50 of 5.3 ± 0.3 μM . HEK 293 cells transfected with mouse FPR also exhibited a calcium flux response to fMLF that was concentration dependent and saturable; however, the EC 50 , ∼50 nM, was much lower than for FPR2 . This value is consistent with that previously reported for expression of mouse FPR in frog oocytes in a calcium-release assay 20 . When FPR2-transfected cells were sequentially stimulated with 100 μM fMLF, no response was observed after the second stimulation, suggesting complete receptor desensitization by the first stimulus . The fMLF response could also be abolished by pretreatment of FPR2-expressing cells with pertussis toxin, suggesting that the receptor is coupled to a G i -type G protein . As a control of cell integrity, we stimulated toxin-treated cells with 10 μM ATP, which induced a calcium flux at levels equivalent to the untreated cells. To test the mechanism of fMLF induction of calcium flux in FPR2-transfected cells, we carried out radioligand binding assays using a 3 H-fMLF probe. Although human and mouse FPR-expressing cells exhibited specific binding, cells expressing FPR2 did not (data not shown). This is not surprising, since the highest concentration of 3 H-fMLF that could be meaningfully tested was 640 nM, which is below the threshold of detection of calcium flux induced by fMLF in FPR2-transfected cells. In contrast, the threshold for induction of calcium flux by fMLF in human and mouse FPR-transfected cells is ∼0.5 and 10 nM, respectively. We conclude that FPR2 is most likely a low-affinity FPR. Given the reactivity of FPR2 to fMLF observed in the calcium flux assay, we next tested its ability to mediate chemotaxis. FPR2-expressing cells migrated in a concentration-dependent manner in response to fMLF with a threshold of ∼1 μM. The EC 50 was ∼5 μM. In all experiments, the upward phase of the concentration–response curve was consistently superimposable with that of the calcium flux assay for the same FPR2-expressing cell line. Mouse FPR-expressing HEK 293 cell migration followed a clear-cut bell-shaped concentration–response curve whose EC 50 was shifted ∼10–100-fold to the left relative to the FPR2 curve . The activity was specific for both receptors, since HEK 293 cells transfected with the related gene Fpr-rs1 and selected with G-418 did not exhibit concentration-dependent migration in response to fMLF when tested with concentrations ranging from 0.1 nM to 100 μM . To distinguish chemotaxis from chemokinesis, equal concentrations of fMLF were added to the upper and lower chambers of the chemotaxis apparatus. In this configuration, no dose-dependent cell migration was observed . Therefore, FPR2 can not only cause intracellular signaling, but can also use those signals to elicit a chemotactic action by the cell. To determine whether FPR2 may operate as a second mouse neutrophil fMLF receptor in primary cells, we examined fMLF responses by cells from FPR knockout mice. Previously, we reported that neutrophils from these mice failed to respond to fMLF either in calcium flux or chemotaxis assays at concentrations as high as 1 μM, and noted that this correlated well with the concentration–response relationship for HEK 293 cells expressing mouse FPR in the calcium flux assay 21 . However, having now discovered that the threshold for calcium flux in FPR2-expressing cells was ∼1 μM, we retested neutrophils from these animals at higher concentrations and observed a concentration–response relationship that was virtually identical for FPR −/− neutrophils versus HEK 293 cells expressing FPR2 for both calcium flux and chemotaxis . The EC 50 value in FPR −/− neutrophils was ∼6 μM for calcium flux, in close agreement with the value of 5.3 μM cited above for FPR2-expressing HEK 293 cells . The chemotaxis concentration–response curves were also very similar . Calcium flux and chemotaxis experiments were also performed on neutrophils from wild-type mice using an expanded concentration range relative to what was used in our previous report, 1 nM to 100 μM . In the calcium flux assay, the response saturated at 1 μM fMLF, which corresponds to the saturation concentration for mouse FPR and the threshold concentration for FPR2 . For chemotaxis, we observed an unusual, multiphasic concentration–response relationship, which was equivalent to the sum of the individual relationships for mouse FPR and FPR2 tested separately in HEK 293 cells . A first peak was clearly resolved and consistently observed between 0.5 and 50 μM, and had an optimum of 5 μM. The curve then passed through a minimum at 50 μM, and then consistently increased at 100 μM. The second peak could not be fully resolved due to artifacts induced by DMSO required to solubilize fMLF at higher concentrations. However, its existence is strongly corroborated by a dose-dependent increase in migration of neutrophils from the FPR −/− mice over the same concentration range. In this study, we have identified dual concentration–response optima for normal mouse neutrophils in chemotaxis induced by the proinflammatory chemoattractant fMLF. For leukocytes, chemoattractant concentration–response relationships are classically described by a bell-shaped curve with a single optimum; the presence of a second optimum as we have described is highly unusual. The functional characteristics of FPR versus FPR2, the second mouse neutrophil low-affinity fMLF receptor subtype that we have identified, in conjunction with analysis of neutrophils from mice lacking the high-affinity fMLF receptor FPR, strongly suggest a molecular explanation for this anomaly in which FPR and FPR2 account for the low and high concentration–response optima, respectively. The significance of this result relates to the dilemma of how leukocytes navigate through the highest portions of chemoattractant concentration gradients in which their high-affinity chemoattractant receptors are likely to become deactivated through receptor phosphorylation and/or sequestration mechanisms 25 26 . Our data suggest the hypothesis that distinct high- and low-affinity receptors for the same chemoattractant may work as a relay to sensitize the cell throughout the gradient, allowing it to arrive at the focus of inflammation. Most work on leukocyte chemotactic receptors has focused on the identification of receptors that bind ligand in the low nanomolar range, so-called “high-affinity receptors,” because binding is easy to measure with available radioligand probes. However, potentially important low-affinity ligand–receptor interactions have also been identified, such as multiple CXC chemokines for CXCR1 27 and MIP-1β and MCP-1 for CCR1 28 . In this regard, the CXC chemokine neutrophil-activating peptide 2 (NAP-2) is particularly interesting since, like fMLF in our study, it appears to activate neutrophil chemotaxis via two concentration–response optima by high-affinity binding to CXCR2 and low-affinity binding to CXCR1 29 . Alternative mechanisms of navigation may also exist, such as usage of high-affinity receptors at different points during the migration path through differential spatial expression of cognate chemoattractants, or by receptor recycling, or by a combination of these mechanisms. In this regard, chemokines may be particularly important for fine-tuning leukocyte migration to inflammatory sites. In humans, the two functional FPRs, FPR and FPRL1R, also have high and low affinity, respectively, for fMLF 7 10 12 . Consistent with this, in calcium flux assays, fMLF is ∼100-fold more potent at FPR versus FPRL1R in vitro. This hierarchy is analogous to that for mouse FPR and FPR2. However, one significant difference is that, to date, FPRL1R has not been reported to be a chemotactic receptor, and its biological function remains unknown. Therefore, the discovery of FPR2 represents the first time that two chemotactic FPRs have been observed in a single species. FPRL1R also binds lipoxin A 4 with high affinity 15 , to our knowledge the only example of a receptor with both peptide and lipid ligands. In mice, the related gene Fpr-rs1 encodes a receptor, LXA 4 R, that binds lipoxin A 4 23 . Therefore, it would appear that differential expansion of an ancestral FPR gene occurred during evolution between humans and mice, resulting in two mouse receptors that split the functions of human FPRL1R: FPR2 mediates responses to N -formylpeptides, whereas LXA 4 R mediates responses to lipoxin A 4 . This theory will need to be tested by additional experiments addressing the specificity of FPR2 for lipoxin A 4 and LXA 4 R for fMLF. In addition, these receptors will need to be tested for their specificity for SAA and HIV-1 T21, which act at human FPRL1R, and HIV-1 T20, which acts at FPR 16 17 18 . Moreover, alternative N -formylpeptides besides fMLF must be considered as potential physiological ligands for both FPR and FPR2. In conclusion, since FPR and FPR2 are both expressed in neutrophils and are differentially sensitive to fMLF, we propose that they may act sequentially during the inflammatory response: FPR recruits neutrophils in the low concentration portion of an N -formylpeptide gradient, whereas FPR2 operates closer to the inflammatory focus where concentrations are expected to be highest and FPR is more likely to be desensitized. We are currently developing an FPR2 knockout mouse to test this hypothesis further, as well as to test the physiological role of FPR2 and its relationship to human chemoattractant signaling. | Study | biomedical | en | 0.999999 |
10477747 | Cell lines were obtained from the American Type Culture Collection unless otherwise noted. IMR90 normal human fibroblast cells were grown in MEM Alpha containing 10% FBS. MCF7 human breast cancer and SF268 human astrocytoma cells (National Cancer Institute, Division of Cancer Treatment and Diagnosis, Developmental Therapeutics Program, National Institutes of Health) were grown in RPMI 1640 containing 10% FBS. Indian muntjac, Muntiacus muntjak , normal skin fibroblasts were grown in F-10 Ham's nutrient mixture containing 20% FBS. Xenopus laevis A6 normal kidney cells were grown in medium NCTC-109 containing 15% deionized water and 10% FBS. Cultures were maintained at room temperature, ∼24°C, in an atmosphere of 5% CO 2 . Drosophila melanogaster epithelial cells, a gift of C. Wu (National Cancer Institute, Division of Basic Sciences, Laboratory of Molecular Cell Biology, National Institutes of Health, Bethesda, MD), were grown in Schneider's Drosophila medium containing 10% heat-inactivated FBS at room temperature. Anti-γ was prepared by Genosys Biotechnologies Inc. The peptide CKATQAS(PO 4 )QEY was synthesized, conjugated to keyhole limpet hemocyanin, and injected into rabbits. The immune serum from the third bleed was passed through a column containing immobilized CKATQASQEY to absorb antibodies to unphosphorylated H2AX. Cells growing in 10-cm dishes, on Labtek II slides (Nalge Nunc International), or on coverslips were exposed to the indicated amount of ionizing radiation from a 137 Cs source in a Mark I irradiator (J.L. Shepherd and Associates). Doses >20 Gy were given at a rate of 15.7 Gy/min. Doses of 2 and 0.6 Gy were given in 1 min. Except for the source of UVA irradiation, the method of Limoli and Ward 1993 was followed. MCF7 cells, grown in the presence or absence of 0.4 μM bromo deoxyuridine (BrdU) 1 and 2.4 μM thymidine for 3 d, were subcultured onto No. 1 1/2 coverslips that had been gently scribed with lines by a diamond pencil. After growth for 24 h, the cells were incubated with Hoechst dye 33258 for 5 min. The coverslips were mounted on a glass slide with a 0.5-mm-thick silicone gasket (Electron Microscopy Sciences) to form a chamber which was filled with PBS. The slides were kept on ice until placed on the stage of the microscope fitted with a LaserScissors™ Module 390/20 (Cell Robotics, Inc.). This laser emits at 390 nm, a wavelength at which the Hoescht dye has substantial absorption. An image of the chosen field of cells containing an inscribed line was recorded and printed. A proposed path of the laser was traced on the print. The laser was operated at various power outputs (100% = 20 μj/pulse) and focused through a 100× objective to a 0.5-μm-diameter circle in the focal plane of the cells with the pulse rate set at 10 pulses/s. The laser was guided by means of a joystick along the traced path at a maximum rate of 8 μm/s. After irradiation, the coverslips were transferred to a culture dish with growth media for 30 min at 37°C before fixation. After processing for laser scanning confocal microscopy, the coverslips were mounted on slides and the irradiated cell groupings were found with the aid of the inscribed lines. Cells were grown on Labtek II slides or coverslips. After irradiation and recovery at 37°C, the cell preparations were fixed in 2% paraformaldehyde in PBS for 5 min, washed in PBS, permeabilized in 100% methanol at −20°C for 5 min, washed, blocked with 8% BSA for 1 h, incubated with the γ-H2AX first antibody at 800-fold dilution for 2 h, washed, incubated with a Cy2-conjugated goat anti–rabbit second antibody (Jackson Immunolabs) at 200-fold dilution for 1 h, washed, mounted with or without propidium iodide, and viewed with a PCM2000 laser scanning confocal microscope (Nikon Inc.) using a 100× objective. Optical sections (0.5 μm) through the thickness of the sample were imaged and combined in a maximum projection with Simple32 software (Compix Inc.) so that all of the visible foci and bands in a nucleus or mitotic figure were recorded. The projection was saved as a BMP file and brought into Paint Shop Pro 5 (Jasc Software, Inc.) and Powerpoint (Microsoft Corp.) for presentation. Polyvinylidene difluoride (PVDF) membranes containing transferred proteins were blocked with 1% dried nonfat milk for 1 h, incubated with the γ-H2AX first antibody at 12,000-fold dilution for 2 h, washed, incubated with peroxidase goat anti–rabbit second IgG (Calbiochem-Novabiochem Corp.) at 3,000-fold dilution for 1.5 h and washed. Anti-γ binding was visualized by chemiluminescence . To examine the spatial distribution of γ-H2AX in the chromatin of irradiated cells, a polyclonal antibody (anti-γ) was raised in rabbits against a synthetic phosphorylated peptide containing the mammalian γ-H2AX COOH-terminal sequence. On immunoblots of total protein extracts from irradiated MCF7 cells, anti-γ detected one band at the position expected for γ-H2AX . No binding was detected in irradiated samples when the immunizing peptide was present as competitor or with preimmune serum . Although anti-γ binding was not apparent in unirradiated MCF7 samples at film exposures optimal for γ-H2AX detection , small amounts of binding were detectable on highly exposed immunoblots. Results from our laboratory show that γ-H2AX is present in apoptotic cells with fragmented DNA (Rogakou, E.P., W. Nieves-Neira, C. Boon, Y. Pommier, and W.M. Bonner, manuscript submitted for publication), indicating a possible source of γ-H2AX in unirradiated cultures. Another possibility is that anti-γ cross-reacts slightly with unmodified H2AX. However, two-dimension gels which separate these two protein species showed that anti-γ bound only γ-H2AX with no detectable cross-reaction to unmodified H2AX. This result indicates that the binding of anti-γ in extracts of unirradiated cells is due to the presence of γ-H2AX in some of the cells in those cultures. H2AX as well as H2A1, the most plentiful of the H2A species in mammals, can be phosphorylated on serine residue 1 and acetylated on lysine residue 5 . H2AX molecules modified in the NH 2 -terminal region bound anti-γ only when they were γ-modified . Also noticeable was the lack of anti-γ binding to H2A1, whose sequence, except for the COOH-terminal motif, is almost identical to the H2AX. Since the H2AX COOH terminus is highly conserved, immunoblots were prepared from irradiated cell cultures of various species to examine whether anti-γ could detect γ-H2AX homologues. Anti-γ detected one band that migrated as expected for the appropriate γ-H2AX homologue not only in other examined mammalian species such as the mouse, rat, hamster, (data not shown), and M . muntjak , but also in X . laevis , D . melanogaster , and S . cerevisiae . These experiments show that not only H2AX itself, but also its phosphorylation in response to ionizing radiation, have been highly conserved during evolution. With the demonstration that anti-γ is specific for γ-H2AX, the distribution of γ-H2AX in irradiated cells was examined. Cells of the normal human fibroblast line IMR90 and the human breast cancer line MCF7 both responded to ionizing radiation with the formation of discrete foci containing γ-H2AX throughout the nuclei . Some cells of both lines contained foci in the absence of irradiation; most of the unirradiated MCF7 cells contained one to two foci , whereas fewer of the IMR90 cells did . The amount of γ-H2AX present in these foci in unirradiated cells is evidently below the level of detection of the immunoblots shown in Fig. 1 A, but may account for the signal seen on highly exposed immunoblots. No foci were apparent in unirradiated or irradiated cells when 1 μM immunizing peptide was included in the first antibody solution (data not presented). The relationship between the presence of γ-H2AX in unirradiated and irradiated cells is being examined. With IMR90 cells, foci were apparent 3 min after irradiation with 0.6 Gy , persisted at 15–60 min , then decreased in number at 180 min . With MCF7 cells, the time course of foci appearance and disappearance was similar . A more detailed analysis of IMR90 cells is presented in Fig. 3 , in which individual nuclei in fields of cells were scored for the number of foci. Compared with the unirradiated control cells, which contained an average of 1.5 ± 2.4 (14 nuclei) foci per nucleus, all the IMR90 cells 3 min after exposure to 0.6 Gy contained numerous small foci, with an average of 16.3 ± 3.6 (11 nuclei) foci per nucleus. The foci became fewer in number but better defined after 15 min, 10.1 ± 3.9 (17 nuclei) foci per nucleus; 30 min, 11.6 ± 5.3 (18 nuclei) foci per nucleus; and 60 min, 11.4 ± 6.1 (15 nuclei) foci per nucleus. After 180 min recovery, the number of foci again decreased to 4.8 ± 3.3 (17 nuclei) foci per nucleus, and at 270 min there were 4.5 ± 5.3 (26 nuclei) foci per nucleus. In addition, after 270 min recovery, 2 of the 26 scored nuclei appeared to be free of foci, possibly suggesting that in these two cells all of the introduced DNA double-strand breaks had been rejoined. This time course obtained by counting foci is very similar to that obtained by Rogakou et al. 1998 , in which mass formation of γ-H2AX was measured after CHO cells had been irradiated with 200 Gy. This similarity suggests that the processes of γ-H2AX appearance and disappearance are the same at these two very different amounts of ionizing radiation. Larger amounts of radiation resulted in larger numbers of foci in both IMR90 and MCF7 cultures. Compared with IMR90 cultures 15 min after 0.6 Gy, with 10.1 to ± 3.9 (17 nuclei) foci per nucleus, IMR90 cultures 15 min after 2 Gy contained 24 ± 5.7 (10 nuclei) foci per nucleus. For MCF7 cultures, the comparable numbers are 12.2 ± 5.7 (26 nuclei) foci per nucleus 15 min after 0.6 Gy, and 27.1 ± 10.8 (24 nuclei) foci per nucleus 15 min after 2 Gy. For both IMR90 and MCF7 cultures, the values for 2 Gy are somewhat less than expected for linear proportionality with respect to the amount of radiation. This discrepancy may be at least partly explained if two foci at different levels in the nuclei overlap in the maximum projection and are scored as a single focus. This overlapping is more likely as the number of foci increases. In addition, it is relevant to mention that the SD values are presented as a means of displaying the range of the values in different nuclei, not as a measure of reproducibility. DNA breakage by ionizing radiation is a stochastic process, and thus the values of foci per nucleus are expected to follow a Poisson distribution. The distribution of values shown in Fig. 3 for each time point as well as the calculated SDs are consistent with those of Poisson distributions. Each Gy introduces one double-strand break per 0.2 × 10 9 bp DNA when irradiated cells are analyzed before any repair can take place . In typical mammalian cells with a diploid DNA content between 6 × 10 9 bp in G1 and 12 × 10 9 bp in G2, this number corresponds to 30–60 DNA initial double-strand breaks per Gy depending on the cell cycle position of the cell. About 60% of these initial breaks are transient with rejoining half-lives on the order of minutes, whereas the other 40% are much more persistent with rejoining half-lives on the order of hours . The transient fraction may be composed of multiply nicked DNA molecules that maintain a continuous double helix in vivo but which are pulled apart under the in vitro assay conditions . Thus, depending on their position in the cell cycle, IMR90 cells are predicted to contain 18–36 initial and 7–14 persistent DNA double-strand breaks after exposure to 0.6 Gy. At 3 min after irradiation, most of the IMR90 cells displayed between 10 and 20 foci , with an average of 16.3 ± 3.6 foci per nucleus, values near the low number of expected initial breaks (18–36) and the high number of expected persistent breaks (7–14). At 15 and 30 min after irradiation, most of the IMR90 cells displayed between 5 and 15 foci with averages of 10.1 ± 3.9 and 11.6 ± 5.3 foci per nucleus, respectively. These values agree well with the number of expected persistent breaks (7–14). As there is likely to be a wide range in the severity of the DNA damage at locally multiply damaged sites , perhaps some of the small γ-H2AX foci seen at 3 min after irradiation cease growing or disappear because those DNA double-strand breaks were rejoined very quickly. These observations show that there is a close correlation between the numbers of γ-H2AX foci and the numbers of expected DNA double-strand breaks, leading to the conclusion that each γ-H2AX focus may represent a DNA double-strand break in vivo. If each γ-H2AX focus identifies a DNA double-strand break, then the two should coincide. To determine this, advantage was taken of the finding that γ-H2AX was formed when DNA double-strand breaks were introduced into cells by the BrdU dye–UVA light procedure of Limoli and Ward 1994 . A UVA pulsed laser (390 nm) commonly used in LaserScissors™ devices can be substituted for the UVA fluorescent light source (365 nm), and has the advantage of permitting the illumination of specific partial nuclear volumes. When MCF7 cells with BrdU-containing DNA were exposed to the laser in the presence of the dye, those nuclear regions traversed by the laser at 1, 10, and 30% relative power contained γ-H2AX foci . γ-H2AX formation was dependent on the presence of BrdU; when BrdU was absent but dye still present, γ-H2AX foci were consistently found only in the cells traversed with the laser at 30% relative power . This experiment demonstrates that γ-H2AX foci form at the sites of DNA double-strand breaks. Notably, there appears to be no difference in the efficacy of 1, 10, and 30% relative power in the formation of γ-H2AX foci , possibly because even at 1% the exposure was more than sufficient to lead to the complete conversion of H2AX in the path of the laser to γ-H2AX. Limoli and Ward 1994 used exposures of up to 3 kJ/m 2 , whereas Rogakou et al. 1998 used 10 kJ/m 2 . The LaserScissors™ at 1% relative power produces 0.2 μj/pulse in a focused region ∼0.5 μm in diameter, or 1,000 kJ/m 2 per pulse, values at least 100 times more than those used in the earlier studies with UVA fluorescent bulbs. Thus, it is quite possible that extensive formation of γ-H2AX occurs in the beam path at 1% relative power and that more is not formed at the higher power settings. It is relevant to mention that cells are generally quite transparent to these UVA wavelengths, thus most of the laser radiation probably passes completely through the cell, inflicting little if any damage except for that absorbed by the dye. It is also notable that there are more γ-H2AX foci in the regions adjacent to the path of the microbeam laser in the cells grown with BrdU than in those without , probably because the BrdU and dye present in the former make the whole nucleus very sensitive to any UVA light that might be scattered from the laser beam. Thus, to limit DNA damage to more defined regions, it may be more appropriate to omit BrdU and use higher laser power settings. Mitotic MCF7 cells were present in some of the cultures analyzed in Fig. 2 . The mitotic cell noted in Fig. 2 J (m) is of particular interest because this culture was fixed only 3 min after irradiation, a result indicating that γ-H2AX foci form on mitotic chromosomes as well as on interphase chromatin. When fields of cells from MCF7 cultures containing both mitotic and interphase cells were analyzed for γ-H2AX foci 15 and 225 min after exposure to 0.6 Gy, the number of foci was decreased at the latter time in both interphase and mitotic cells . These results show that the kinetics of γ-H2AX foci appearance and disappearance are similar whether cells are interphase or mitotic. Because of the large number of small chromosomes in human mitotic cells, it is not possible to consistently visualize γ-H2AX foci on individual chromosomal arms. However, individual chromosome arms can be easily visualized in fibroblasts of the Indian muntjac, M . muntjak , a small deer with the typical mammalian DNA complement divided into 6 chromosomes instead of the 46 in humans . Interphase nuclei and condensed chromosomes from muntjac cells exposed to 0.6 Gy were found to show distinct γ-H2AX foci similar in number and intensity to those seen in the MCF7 cells . In addition, it is possible to see the relationship of γ-H2AX foci to the chromosome arms. A study was performed with muntjac cell cultures exposed to 0.6 Gy and permitted to recover for various times . The fields were searched for mitotic cells, which were imaged. A mitotic figure with discretely visible arms taken from each recovery time period is presented in Fig. 6 . γ-H2AX foci, although not detectable after 0.3 min , were detectable as small punctate foci after 1 min and continued to grow in number and size until 30 min . At 90 min, the γ-H2AX foci were fewer in number but similar in size to those seen at 30 min. Since muntjac cells contain ∼90% of the DNA per cell as human cells , similar numbers of γ-H2AX foci per Gy would be expected in the two. If each half mitotic figure contains the G1 complement of DNA, one would expect ∼6 initial and 6 persistent DNA double-strand breaks in each. The number of γ-H2AX foci visible in each half of the 9- and 30-min mitotic figures is higher than that found in the IMR90 cells , and nearer the expected value for initial rather than persistent DNA double-strand breaks. This difference might reflect a greater sensitivity of detection of small γ-H2AX foci in mitotic cells due to the greater compaction of the chromatin; mitotic cells often display more distinct foci than do interphase cells . The difference could also be due to differences in DNA double-strand break detection and rejoining between interphase and mitotic cells, or to differences between human and muntjac cell metabolism. The IMR90 cells contained more foci at 3 min after 0.6 Gy than later , whereas the opposite was the case for the muntjac mitotic cells . These findings indicate that these types of differences do exist, but whether they are due to differences in detection sensitivity, DNA compaction, species metabolism, or other factors requires further study. These findings show that DNA double-strand breaks are rapidly detected and marked in condensed chromosomes as well as in interphase chromatin. The mitotic cells imaged in Fig. 6 appear to have been dividing normally when fixed. However, in cultures fixed at 90 min after irradiation, defective mitotic cells could be found . Three mitotic cells containing six isolated chromosomal arm fragments are shown, each with a large γ-H2AX focus at one end (indicated by green arrows). In addition, no isolated chromosomal arms lacking a terminal γ-H2AX focus were found in any mitotic figure. These results provide direct visual confirmation that γ-H2AX forms en masse at the sites of DNA double-strand breaks. Ionizing radiation introduces a delay in cell cycle progression in early G2. Kimler et al. 1981 determined that in CHO cells, the duration of the G2 delay could be calculated as 1 h per Gy for cells that were irradiated before prophase. The arrest point was calculated to be 17 min before metaphase and 49 min before completion of cytokinesis. Similar values had been found for mouse leukemic cells , indicating that these values may apply to mammalian cells in general. Thus, the M . muntjak cells exposed to 0.6 Gy could have arrested in early G2 for ∼30 min, then entered mitosis and progressed toward cytokinesis, but were unable to complete cytokinesis because chromosome arms abandoned during anaphase blocked the constriction of the cleavage furrow. Collectively, these data reveal the presence of an immediate, substantial, and evolutionarily conserved response of cells to the introduction of DNA double-strand breaks. This response involves the formation of γ-H2AX on chromosomal regions encompassing megabase lengths of DNA adjacent to break sites. No mammalian cell line, normal or repair-defective, has been found that lacks the ability to form γ-H2AX when exposed to ionizing radiation. Cell lines from patients with ataxia telangiectasia, Werner's syndrome, Bloom's syndrome, and Nijmegen breakage syndrome all form γ-H2AX after exposure to ionizing radiation . M059J human, scid mouse, and V3 hamster cells that lack the DNA-activated protein kinase catalytic subunit (DNA-PK cs ) form γ-H2AX by gel analysis, and show γ-H2AX foci after exposure to ionizing radiation (Rogakou, E.P., C. Boon, and W.M. Bonner, unpublished results). A KU70 knockout cell line also forms γ-H2AX. The lack of mutant cell lines unable to form γ-H2AX indicates that it may fulfill essential functions in organisms throughout the evolutionary scale. It has been reported that when DNA double-strand breaks were introduced into regions of fibroblast nuclei by partial volume irradiation, MRE11, one of the proteins involved in DNA double-strand break rejoining, was found in these regions 30 min after irradiation . Maximal amounts of γ-H2AX are formed in irradiated human fibroblasts by 10 min, amounts corresponding to regions of chromatin containing about 2 × 10 6 bp of DNA and 2,000 γ-H2AX molecules . One possible role of these γ-H2AX foci at the sites of DNA double-strand breaks could be to serve in recruiting proteins that are involved in rejoining DNA ends such as MRE11 or RAD50 to those sites, either directly, through binding to the γ-H2AX COOH terminus, or indirectly, through an altered regional chromatin structure. With the γ-H2AX antibody, regions of chromatin containing DNA double-strand breaks may be isolated and components that interact with those regions may be characterized. The γ-H2AX domains seen on chromosome arms are similar in appearance to chromosome bands and also appear to stop enlarging after 30 min, even though some DNA double-strand breaks are still present. Although other explanations are possible, these findings may suggest the existence of units of higher-order chromatin structures that are involved in monitoring DNA integrity. | Study | biomedical | en | 0.999997 |
10477748 | An NH 2 -terminal truncated MVP was subcloned in two steps into the EcoR1 and XbaI restriction sites of pEG202 (kindly provided by Dr. Roger Brent, Molecular Science Institute, Berkeley, CA). The resultant plasmid, plex-MVP, and the reporter plasmid pSH-18 were then transformed into yeast cells of the EGY48 strain (Trp − Leu − His − Ura − , LacZ − ). These cells were then transformed with a HeLa cell acid fusion cDNA library in the pJG7-4 expression vector (constructed by J. Gyuris, Mitotix, Cambridge, MA, and kindly provided by Dr. Roger Brent), and about 1 million transformants were plated onto dropout media lacking Trp, His, and Ura containing glucose. Positive clones were selected by replica plating onto dropout media lacking Trp, His, Ura, and Leu containing galactose and X-gal. About 256 clones were selected in the initial screen, but upon rescreening only 6 clones were able to coactivate the lexA-responsive LEU2 and lacZ reporter genes of EGY48 on galactose containing selection media. Putative interactor plasmids were rescued by transformation into KC8 cells. The six putative clones were tested for the specificity of interaction by retransformation into EGY48 along with the reporter plasmid pSH-18 and either the plex-MVP or plex-bicoid as bait plasmids. Three clones specifically interacted with plex-MVP only (the other three interacted with both baits suggesting their interaction was nonspecific). The three interactor clones (8, 15, and 21) were sequenced and determined to be independent overlapping clones of p193. All of the interactor clones contain the 3′ terminus of p193 beginning at bases 4515 (clone 15), 4633 (clone 21), and 4791 (clone 8). In vitro binding assays using glutathione S -transferase (GST) fusion proteins and in vitro translated MVP were carried out as described . Vaults were purified from monkey liver as described previously . Purified vaults were fractionated onto four 6% SDS polyacrylamide gels, stained with copper, and the appropriate bands were excised. An estimated 26 pmol of the 193-kD vault protein was sent to Dr. William S. Lane (Harvard Microchemistry Facility, Cambridge, MA). Peptide sequences were determined on a Finnigan TSQ-7000 Triple Quadrapole Mass Spectrometer. Previously, NH 2 -terminal sequence analysis on p193 protein purified from bovine spleen vaults and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories) was carried out by Dr. Audree Fowler (UCLA Protein Microsequencing Facility, University of California, Los Angeles School of Medicine, Los Angeles, CA). Although the degenerate peptide sequence was not useful for cloning, the sequence verified the NH 2 terminus determined by 5′ rapid amplification of cDNA ends (RACE). To isolate the cDNA encoding p193, a human cDNA library (kindly provided by Dr. Owen Witte) was screened as described previously . A total of 500,000 recombinants were screened with a randomly primed probe to the interactor clone 15 . 71 positive clones were identified. Restriction analysis determined the longest clone to be a 3-kb EcoR1 fragment . Reverse transcription followed by PCR was used to isolate bases 663–2492. The first strand cDNA was synthesized using SuperScript II (Life Technologies, Inc.) and random primers (16-mers, kindly provided by Dr. Dohn Glitz, University of California, Los Angeles) from total HeLa cell RNA. The following primers were used for reverse transcription PCR: p193RB5 (5′-CCCCCGAATTCGTGGATGTCTTGCAGATATTTAGAGTT-3′), p193+2225 (5′-TTGGGAGATAGGCAGCAGACAAACCGATGT-3′), p193RT5 (5′-CCTTATAAGCCCCTGGACATCAC-ACCACCTCC-3′), and p193-RBD3 (5′-CCCGGATCCGGCCTTGGTGCTGCTGGAA-3′). The PCR products were digested with either EcoRI or Eco RV and BamHI , and cloned into the corresponding sites in pBluescript SK+ (Stratagene). The 5′ end of the cDNA clone (bases 1–663) was obtained by 5′ RACE according to the manufacturer's instructions (Life Technologies, Inc.) except poly(A) + RNA from 293 cells was used in place of total RNA. The gene-specific primers were GSP1 (5′-TCTGCCCAAATCATCTCTACTAAA-3′) and GSP2 (5′-GAGTGCTTGAATTCATGACTTCCTCC-3′). The amplified RACE product was digested with EcoRV (base 663) and SalI (a site from the abridged universal anchor primer) and was cloned into the corresponding sites in pBluescript SK+. A complete p193 cDNA clone was assembled from various restriction fragments. The NH 2 terminus was tagged with VSVG (a 14 aa sequence, YTDIEMNRLGK, from vesicular stomatitis virus glycoprotein) and subcloned into the expression vector, pSVL (Amersham Pharmacia Biotech). COS cells were transiently transfected with the lipid reagent DMRIE (Life Technologies, Inc.) following the manufacturer's guidelines. A multiple tissue Northern blot containing 2 μg of poly(A) + human RNA was purchased from Origene and hybridized following their protocol with a randomly primed p193 probe (bases 385–880). The blot was stripped twice and hybridized first with a randomly primed MVP probe (bases 1–330) and then with a human β-actin cDNA probe supplied by Origene. Preparation of HeLa cell extracts (S100 and P100) and discontinuous sucrose gradient fractionation of the P100 extracts were carried out as described . The 100,000 g pellet was resuspended by dounce homogenization with a Teflon pestle. Both S100 and P100 extracts and sucrose gradient fractions were resolved by SDS-PAGE and transferred to Hybond membrane (Amersham Pharmacia Biotech). The equivalent of 1.2 × 10 6 cells were represented in each lane of the S100 and P100 extracts. Equivalent aliquots of each of the sucrose gradient fractions were represented. The membrane was incubated with affinity-purified anti-p193 antibody (1:500), followed by an HRP-conjugated secondary antibody and visualized by ECL (Amersham). Two fragments of the p193-containing aa 408–611 (p193rbd) or 1471–1724 (p193int) were expressed in the pET expression system (Novagen) or as GST fusion proteins (Amersham Pharmacia Biotech). The p193int (pET) protein was purified on a His-bind column (Novagen) and injected into a rabbit. Conversely, the p193rbd (pET) protein was present in the insoluble fraction and was purified on an SDS-polyacrylamide gel; the appropriate fragment was excised, minced, and injected into the same rabbit. A p193 containing GST fusion protein was coupled to Affi-Gel 15 resin (Bio-Rad Laboratories) to make an affinity column. Antiserum was initially purified on protein A as described . After affinity purification on Affi-Gel 15 columns, the antibody protein concentrations were too low to be measured by protein assay and appropriate dilutions were determined by immunoblot analysis. Affinity-purified Ig was used at 1:500 for immunoblot analysis. The catalytic domain (aa 255–611) of p193 was amplified by PCR and inserted into the EcoR1 and XhoI sites of the pET28b expression vector. His tagged p193cat (aa 255–611) protein was purified on a His-bind affinity column. Poly(ADP-ribose) activity assays were carried out as described previously . Reactions contained 1 μg of purified p193 cat or 3 μg of purified rat liver vaults. Vaults were purified from rat liver as described . Reactions were incubated at 25°C for 30 min in assay buffer (0.1 ml) containing 50 mM Tris-HCl, pH 8.0, 4 mM MgCl 2 , 0.2 mM DTT, 1.3 μM [ 32 P]NAD+ (4 μCi; New England Nuclear), and 1 mM of unlabeled NAD+. Some assays contained the PARP inhibitor 3-aminobenzamide (3ABA) at 1 mM final concentration. Reactions were stopped by the addition of TCA containing deoxycholate (as a carrier) to a final concentration of 20% and 0.8 mg/ml, respectively. Precipitated proteins were suspended in SDS loading buffer, and fractionated by SDS-PAGE. Proteins were visualized by Coomassie blue stain and exposed to PhosphorImager screens (Molecular Dynamics). HeLa cells or human foreskin fibroblasts were grown on coverslips until ∼50–70% confluent, then cells were fixed in 4% paraformaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl 2 , pH 6.9), permeabilized and postfixed in methanol . The DNA damage experiments were carried out on human foreskin fibroblasts that were exposed to a UV Stratalinker 1800 (Stratagene) using energy settings 50, 100, 150, and 200 μJ. UV-treated cells were allowed to recover for 24 h before immunostaining. Cells were stained with DAPI (Sigma Chemical Co.) at 0.5 μg/ml. The following antibodies were used for our immunofluorescence studies: affinity-purified anti-p193 polyclonal IgG was used at 1:100; affinity-purified anti–rat vault polyclonal IgG was used at 1:100; a monoclonal anti-MVP (LRP56; kindly provided by Dr. Rik Scheper, Academic Hospital, Vrije Universiteit, Amsterdam, The Netherlands) IgG was used at 10 μg/ml; a monoclonal anti-p53 (BP53; Sigma Chemical Co.) was used at 1:200; a polyclonal anti-PARP (Boehringer Mannheim) antibody was used at 1:2,000; and a monoclonal anti-tubulin was used at 1:200. Cells were incubated for 1 h in primary antibody followed by a 30-min incubation with goat anti–rabbit Cy3 (1:250 dilution; Jackson ImmunoResearch Laboratories), goat anti–mouse Cy3 (1:250 dilution; Jackson ImmunoResearch Laboratories), or goat anti–mouse FITC (1:200 dilution; Jackson ImmunoResearch Laboratories). COS cells were transfected with the VSVG-tagged p193 cDNA using the lipid reagent DMRIE (Life Technologies, Inc.). Transiently transfected cells were fixed as described above and immunostained with a monoclonal antiserum to the 14 aa VSVG epitope tag, anti-VSVG (P5D4; Sigma Chemical Co.) antiserum at 1:500, followed by incubation with a goat anti–mouse Cy3 (1:250 dilution; Jackson ImmunoResearch Laboratories). Fixed cells were mounted in polyvinyl alcohol–based mounting medium and viewed with a Nikon FXA epifluorescence microscope. To identify cellular proteins which interact with the MVP, we pursued a yeast two-hybrid strategy . A cDNA sequence encoding the rat MVP , missing the NH 2 -terminal 67 aa, was inserted into the expression vector, pEG202. The resultant plasmid plex-MVP encodes a hybrid protein containing the DNA-binding domain of lexA fused to MVP residues 68–885. We then transformed the yeast strain EGY48 containing the lacZ reporter (pSH18-34) and lex-MVP along with a galactose-inducible HeLa acid fusion cDNA library. About 1 million library transformants were screened, and 6 clones were isolated that coactivated the lexA-responsive LEU2 and lacZ reporter genes of EGY48. Three of the isolates interacted specifically with lex-MVP in a yeast two-hybrid retransformation assay where an irrelevant protein (lexA-bicoid) was used as a negative control ( Table ). Nucleotide sequence analysis of the three isolates identified a previously determined nucleotide sequence of unknown function . The three overlapping clones encoded the COOH terminus, beginning at aa 1471, 1510, and 1562, respectively . The region encoding aa 1562–1724 was designated the MVP interaction domain , since it is the smallest domain that we have tested that interacts with MVP. The results of the two-hybrid assay were consistent with the results from an in vitro binding assay using a GST fusion protein. In this experiment, the p193 MVP interaction domain (p193int) was expressed in Escherichia coli as a GST fusion protein and was then bound to glutathione beads. The beads were incubated with reticulocyte lysate containing in vitro–translated 35 S-labeled MVP, and washed. Binding was assessed by fractionation on SDS-PAGE, followed by PhosphorImager analysis . Concurrently, highly purified vaults from monkey liver were fractionated by SDS-PAGE, stained, and the appropriate gel fragments (p193) were excised and sent to William S. Lane (Harvard Microchemistry Facility) for peptide sequence analysis. One peptide sequence was obtained (AALKNGETAEQLQK) and was determined to correspond to nucleotides 639–680 of the KIAA0177 by a TBLASTN search of the nonredundant nucleotide sequence database . These results confirmed the identity of the KIAA0177 sequence to be a truncated form of the 193-kD vault protein. The 5′ end of the p193 cDNA clone was obtained using 5′ RACE. The predicted NH 2 terminus was confirmed by earlier NH 2 -terminal aa sequence analysis of p193 protein purified from bovine spleen vaults carried out by Audree Fowler (UCLA Protein Microsequencing Facility). The bovine NH 2 -terminal sequence MTV(L/G)IFAN(S/L)(T/P)F(Q/V)L verifies the NH 2 terminus of the p193 protein . The sequence differences between the human and bovine p193 proteins probably represent species-specific variation. Although the degenerate sequence was not useful for cDNA cloning, it allows us to conclude that we have identified the authentic NH 2 terminus. The composite p193 cDNA is 5490 bases, with a short untranslated 5′ end; the coding region encompasses bases 107–5281 and encodes a protein of 1724 aa. Fig. 1 shows the 1724 aa sequence encoded by the p193 cDNA. The size of the predicted protein was calculated to be 192.7 kD. A PROSITE protein sequence analysis of the aa sequence revealed several interesting features, thus allowing the sequence to be separated into four domains . First, aa 1–94 were identified as a BRCT domain . BRCT domains were first identified in the BRCA1 gene and later were determined to define a superfamily of cell cycle checkpoint DNA damage response proteins . The BRCT domain is thought to be important for protein–protein interactions. Domain II is discussed below. Domain III is formed by aa 616–706 and 877–919. This domain shares 30 and 27% identity, respectively, with the inter–α-trypsin inhibitor heavy chain–related protein, a novel human plasma glycoprotein . Domain IV is the MVP interacting domain . The second domain, aa 209–563, shares 29% identity with the catalytic subunit of PARP . PARP is a nuclear protein that can be divided into three domains: the NH 2 -terminal DNA binding domain (containing two zinc fingers), a central automodification domain, and a COOH-terminal catalytic domain . The catalytic subunit binds to NAD+, hydrolyzes the nicotine moiety, and polymerizes the ADP-ribose group in response to DNA damage. Poly(ADP-ribose) is attached mainly to PARP, but also to other substrates including histones H1 and H2B . A number of drugs have been shown to bind to the active site of the catalytic subunit, thus blocking NAD binding . The minimum region necessary for PARP to retain catalytic activity is a 40-kD fragment . The crystal structure of the catalytic fragment of PARP has been determined . Based on the crystal structure, the residues that form the NAD binding pocket are conserved between PARP and p193 . These data suggest that this region of the p193 will form a similar binding pocket, which could have catalytic activity. To determine whether p193 has PARP activity, the catalytic domain of p193, aa 255–611, were expressed in E . coli as a His-tagged fusion protein and purified. An in vitro PARP activity assay, which measures the addition of radiolabeled ADP-ribose to protein acceptors with [ 32 P]NAD+ used as a substrate, was carried out. A Coomassie stain of the gel before exposure to a PhosphorImager screen shows that equal amounts of proteins were used in all of the assays . Like PARP, the catalytic domain of p193 contains ADP-ribosylation activity, and it ADP-ribosylates itself . This activity is heat inactivatable . The addition of unlabeled NAD+ (1 mM) decreased the level of labeled ADP-ribose polymers added to p193 (255–611) about threefold . To confirm that the labeling reaction with p193 was analogous to PARP-catalyzed poly(ADP-ribosyl)ation, the PARP-specific inhibitor 3ABA was included in a reaction. Modification of p193 (255–611) was decreased about twofold in the presence of the inhibitor . Furthermore, modified p193 (255–611) reacted with a monoclonal anti–poly (ADP-ribose) antibody (data not shown), consistent with it carrying ADP-ribose polymers. These data indicate that p193 (255–611) is a PARP. Next, we wanted to investigate whether full-length endogenous p193 within the vault particle would possess enzymatic activity. Highly purified vault particles were incubated with [ 32 P]NAD+ in the presence and absence of inhibitor or unlabeled NAD+. The most prominently modified protein in purified vaults was the MVP. However, there was some labeling in the vicinity of the p193 and a high molecular weight smear was also detected . Modification of all of these products was competed for by the addition of unlabeled NAD+ and partially competed by the addition of the inhibitor 3ABA. These data indicate that full-length p193 is a PARP that is active in the vault particle with at least one specific substrate, MVP. We determined the expression of p193 by Northern blot analysis of human tissues, including brain, heart, kidney, spleen, liver, and leukocytes . In all tissues, except brain, a 5.4-kb mRNA was readily detectable in 2 μg of poly(A) + RNA. The highest level of expression was seen in kidney, with about equal levels detectable in spleen and liver. The p193 mRNA tissue expression pattern is similar to that of MVP; however, the level of expression in individual tissues is variable, as there is a higher level of MVP mRNA in spleen compared with liver . A polyclonal anti-p193 antibody was generated from bacterially expressed fragments of p193 . The anti-p193 antibody recognizes a single protein species of 193 kD by immunoblot analysis . To compare the subcellular distribution of p193 with MVP, extracts from tissue culture cells were isolated and fractionated on a discontinuous sucrose gradient followed by immunoblot analysis . Vaults are cytoplasmic particles that typically pellet with the microsomes at 100,000 g . Detergent-lysed HeLa cells were centrifuged at 20,000 g , resulting in a nuclear (N) pellet. The supernatant was further fractionated by centrifugation at 100,000 g and the supernatant (S100) and pellet (P100) fractions were analyzed by immunoblotting with anti-p193 antibody. Interestingly, unlike MVP, which primarily fractionates with the P100, all of the fractions contained the p193 protein . We should note that the N fraction does not represent purified nuclei, and a certain portion of the cells are in mitosis at any given time, so the amount of p193 detected by immunoblotting may not be comparable to that seen by immunofluorescence (see below). Further fractionation of the P100 extract on a discontinuous sucrose gradient revealed that the majority of p193 sedimented to the 45/50% sucrose layer, coinciding with the pattern observed for the MVP . These results suggest that all of the p193 protein in the P100 fraction is associated with the vault particle. Immunoblot analysis of vaults purified from rat liver revealed that the p193 vault protein is recognized by the anti-p193 antibody, further confirming its association with vaults . The shifted mobility of p193 in vaults purified from rat liver is probably due to species-specific differences. To determine the intracellular distribution of p193 in tissue culture cells, we carried out immunostaining using affinity-purified anti-p193 antibody. Vaults have a punctate cytoplasmic distribution . They have also been localized to lamellipodia and at the tips of actin filaments at or near adhesion plaques . Immunofluorescence patterns observed for p193 have a similar punctate cytoplasmic distribution. However, a variable number of nuclear speckles are also detectable with anti-p193 antibody that is not seen with anti-vault antibody . Double-immunofluorescence images revealed coincident staining of p193 and MVP in the cytoplasm, but no coincident staining is detected in the nucleus. The lack of completely coincident staining of p193 and MVP in the cytoplasm is not surprising, as our fractionation studies showed that not all of the p193 is associated with vaults. Transfection of COS cells with a cDNA encoding p193 containing a VSVG epitope tag (VSVG-193) revealed that the recombinant protein is distributed similarly to endogenous p193 . The p193 localization pattern is very different from that seen for anti-PARP antiserum, which showed a mostly nuclear staining pattern . Interestingly, in mitotic cells a portion of the p193 immunoreactivity localizes to the mitotic spindle , like β-tubulin , with merged images depicting their colocalization . Vault staining of mitotic cells is diffuse and punctate throughout the cytoplasm (data not shown). Only a portion of the p193 is localized to the mitotic spindle, as another exposure also shows the diffuse punctate p193 staining in the cytoplasm , presumably representing the p193 in vaults. Next, we determined whether the distribution of p193 or vaults varied in response to DNA damage. Human foreskin fibroblasts were UV irradiated (100 μJ), and the distribution of p53, p193, and vaults was monitored by immunofluorescence . p53 is known to be activated in response to DNA damage and is upregulated and localizes to the nucleus . No change in either the distribution of p193 or vaults was detectable in the UV-treated cells . Identification of the 193-kD vault protein completes the molecular characterization of the repertoire of proteins that form the basic vault particle. The identities of both the 193- and 240-kD vault proteins have led to unexpected but tantalizing findings. The determination that the 240-kD protein is identical to the telomerase-associated protein, TEP1, suggests that this protein may have a more general role in RNP structure, function, or assembly . The data presented here demonstrates that the p193 provides vaults with an enzymatic activity. We show that p193 is a PARP that ADP-ribosylates itself and the major vault protein in purified vaults. Based on this data we propose that p193 be named VPARP for v ault p oly( A DP-ribose) p olymerase. To date there are only two other proteins that contain poly(ADP-ribosyl)ation activity: PARP and tankyrase. PARP is a nuclear protein that has been studied for nearly 20 yr . It is activated in response to DNA damage, where it binds to DNA at single or double-strand breaks and covalently attaches ADP-ribose moieties derived from NAD+ to itself and other nuclear proteins . PARP has also been shown to be a target of caspases during apoptosis, where it is cleaved near the DNA binding domain . Although PARP knockout mice are viable, they are more sensitive to gamma irradiation and treatment with the alkylating agent N -methyl- N -nitrosourea . Mice lacking PARP have recently been shown to be resistant to pancreatic β-cell destruction and development of type I diabetes induced by streptozocin . Recently, another PARP, tankyrase, was identified through its ability to interact with TRF1 . TRF1 is a mammalian telomeric protein that binds to double-stranded telomere repeat containing DNA at chromosome ends . Overexpression of TRF1 induces telomere shortening, whereas expression of a dominant negative TRF1 results in longer telomeres . These data indicate that TRF1 is a negative regulator of telomere length. TRF1 is not a component of telomerase; therefore, it must interact with other proteins that mediate the interaction of telomerase with telomeres. One such protein may be tankyrase. Tankyrase was shown to ADP-ribosylate itself and TRF1, and this modification abolishes the ability of TRF1 to bind to telomeric DNA sequences in an in vitro gel shift assay. These results suggest that poly(ADP-ribosyl)ation may negatively regulate telomere length. Since the precise function of vaults is unknown, it is difficult to assess the impact of poly(ADP-ribosyl)ation on its function. However, it may enhance or negate vault interaction with other proteins in the cell, or it may allow for changes in vault conformation, i.e., opening and closing of vaults. A BRCT domain has been identified at the NH 2 terminus of VPARP. More than 50 distinct proteins have been identified that contain a BRCT domain, and many of these proteins have defined roles in the cellular response to DNA damage . BRCT domains are thought to mediate protein–protein interactions and are usually found at either the NH 2 or COOH termini of proteins. Some proteins contain multiple copies of the BRCT domain. Interestingly, PARP also contains a BRCT domain in the central automodification domain upstream of the catalytic domain . The BRCT domain in PARP is separated from the catalytic domain by ∼145 aa, similar to the distance that separates these two domains in VPARP (115 aa). In some respects, multidrug resistance could be considered a response to DNA damage. Many chemotherapeutic drugs are DNA-damaging agents (e.g., doxorubicin and mitoxanthrone). The upregulation of vaults in some types of multidrug-resistant cancers , along with p193's homology to PARP, suggested that vaults may have a role in DNA damage response. However, we have shown that when cellular DNA is damaged by exposure to UV light sufficient to activate p53, the distribution of vaults and VPARP remains unchanged. In addition, we have determined that VPARP activity is not activated by damaged DNA in extracts using an in vitro ADP-ribosylation assay (data not shown). It seems reasonable to propose that like PARP, VPARP activity will be activated by some as yet undetermined signal. Other functional PARPs must exist, as PARP-deficient mouse cells have recently been shown to synthesize ADP-ribose polymers in response to the DNA-damaging agent, N -methyl-nitro-nitrosoguanidine . Northern blot analysis revealed a single VPARP mRNA that is heterogenously expressed in human tissues, with the highest amounts detectable in kidney. MVP showed a similar pattern of expression, although levels varied depending on the tissue being examined. Subcellular fractionation of tissue culture cells revealed that ∼90% of the MVP is present in the 100,000 g pellet (P100, crude vault fraction) . However, only a portion of VPARP, TEP1, and vRNA are associated with the 100,000 g vault particle fraction . Factors governing vault particle formation, function, or assembly have not been determined. It is possible that like TEP1, VPARP may be a shared protein interacting with other cellular proteins. TEP1 is associated with telomerase activity , and may have a more general role in RNP structure, function, or assembly . The role of vRNA in vault particle function has not yet been defined. However, previous studies have demonstrated that the vRNA is not a structural component of the vault particle . Here we show that VPARP provides vaults with an enzymatic activity, and that this activity will likely be important in VPARP's vault-independent function. There are three potential nuclear localization signals in VPARP and a portion of the VPARP protein is localized to the nucleus by subcellular fractionation and in a variable number of nuclear speckles by immunofluorescence. VPARP is probably associated with other cellular proteins and substrates that have not yet been identified. The localization of VPARP, but not of vaults, to the mitotic spindle is particularly intriguing, inasmuch as neither vaults nor VPARP appears to associate with interphase microtubules in HeLa cells. Vaults have been reported to associate with microtubules in neurite extensions of differentiated PC-12 cells , and sea urchin vaults were originally discovered because of their ability to copurify in vitro with egg microtubules through several cycles of polymerization and depolymerization , although in adult sea urchin coelomocytes, sea urchin vaults do not appear to associate with microtubules by double immunofluorescence. It is possible that a putative sea urchin VPARP could mediate the vault association with egg microtubules in vitro, and that the assembly and/or disassembly of egg microtubules copurify selected microtubule-associated proteins specifically associated with the mitotic spindle. Examples of other proteins that selectively associate with mitotic spindle microtubules, but not interphase microtubules, include human Eg5 and protein phosphatase γ1 . The association of human Eg5 with the mitotic spindle requires phosphorylation of a specific threonine residue by p34 cdc2 . Protein phosphatase γ1 is an isoform of protein phosphatase 1 (PP1), a family of serine/threonine phosphatases that has many important regulatory functions in mammalian cells . Recently it was recognized that the three isoforms (α1, δ1, and γ1) are each localized to distinct sites in both mitotic and interphase cells . These findings suggest that these distinct localizations may allow the various isoforms to control multiple cellular processes. Precisely how VPARP is recruited to the mitotic spindle is unknown. Posttranslational modifications to VPARP such as phosphorylation, self-modification by conjugation of poly(ADP-ribose) moieties, or interactions with other protein(s) during mitosis are all candidate mechanisms that could mediate interaction with the mitotic spindle. The key question that remains to be addressed in future studies is whether the spindle-associated VPARP is enzymatically active, and if so, what is the function of such localized PARP activity. It is clear that vaults, like Pandora's box, contain surprises in addition to their enigmatic contents. | Study | biomedical | en | 0.999996 |
10477749 | Simian kidney COS-7 cells and human mammary MCF-7 cells were cultured in DME supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in an atmosphere of 5% CO 2 . For experiments involving the use of the human mineralocorticoid receptor (MR), the cells were kept in DME containing 3% charcoal treated FCS to remove residual steroids. The expression vector containing the wild-type human GR and MR have been described by Giguère et al. 1986 and Arriza et al. 1987 . The recombinants GMM, GGM, and GGΔ have been described by Arriza 1991 . The plasmid pcDNA3RAP46 was constructed by insertion of the coding sequence of human RAP46 into the plasmid pcDNA3. The expression plasmid pT2 contains sequences encoding two copies of the hemagglutinin influenza (HA)-tag 5′-AA GCT TCC ACC ATG ATC TTT TAC CCA TAC GAT GTT CCT GAC TAT GCG GGC TAT CCC TAT GAC GTC CCG GAC TAT GCA GGA TCT ACT CGA GAG GAT CCG GTA CCT ATC TAG A-3′ cloned between the HindIII and XbaI sites of the plasmid pcDNA3 (Invitrogen Corp.). The constructs pT2RAP46 and pT2RAP46ΔC47 were constructed by inserting blunt-ended BamHI/XbaI fragments from pcDNA3RAP46 and pcDNA3RAP46ΔC47 (lacking the last 47 COOH-terminal amino acids) into XhoI and XbaI sites of pT2 after a fill-in reaction with DNA polymerase I (Klenow fragment). The constructs pT2RAP46Δ40 and pT2RAP46Δ70, containing deletion mutants of the first 40 and 70 NH 2 -terminal amino acids of RAP46, were constructed by PCR amplification with XhoI and BamHI ends and insertion into the corresponding sites of pT2. The construct p46N69GFP, encoding the first 69 NH 2 -terminal amino acids of RAP46 fused to the NH 2 terminus of the green fluorescence protein (GFP) sequence, was obtained by subcloning a HaeIII/PvuII fragment from pT2RAP46 into the blunt-ended HindIII site of pcDNA3.1hGFP. The pcDNA3.1hGFP construct contains sequences encoding the humanized GFP (CLONTECH Laboratories, Inc.) between the HindIII and XbaI sites of pcDNA3.1 (Invitrogen Corp.). The reporter plasmid pGL3MMTV encodes the firefly luciferase gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat region (−241 to +137), cloned as a BamHI/BglII fragment from the plasmid pHCwt CAT . The reporter plasmid −517/+63 Coll-Luc already has been described by Heck et al. 1997 and Schneikert et al. 1996 . The control plasmid for transfection pTKRenilla Luc was obtained from Promega and the expression vectors GR–GFP and GFP–MR have been described by Carey et al. 1996 and Fejes-Tóth et al. 1998 . The GFP plasmids GRΔ491-515GFP and GR1-515GFP were constructed by exchanging the GR sequence in the construct GR–GFP with the GRΔ491-515 and GR1-515 sequences . 100,000 MCF-7 cells were transiently transfected by the calcium phosphate coprecipitation procedure in 3.4-cm-diam plates with 2 μg reporter plasmid, 0.1 μg pTKRenilla Luc, and 2 μg expression vector. After 5 h of incubation with DNA and 2 min shock with 10% glycerol in PBS, the cells were further incubated for 36 h. Thereafter, they were harvested and cellular extracts were prepared for luciferase assay as described by Schneikert et al. 1996 . Renilla luciferase assay was performed with the dual luciferase reporter assay system from Promega, according to the manufacturer's instructions. COS-7 cells were transfected by electroporation as previously described by Klocker et al. 1992 with 16.5 μg DNA containing 2 μg reporter plasmid, 0.5 μg pTKRenilla Luc, 7 μg wild-type or mutant RAP46, and 7 μg receptor expression vector. COS-7 cells were transiently transfected with either empty expression vectors or expression vectors encoding GR, RAP46, and RAP46Δ70. After 36 h incubation in DME containing 3% charcoal treated FCS, the cells were trypsinized and resuspended in the same medium. 200,000 cells in 100 μl medium were incubated for 90 min at room temperature with different concentrations (0–100 nM) of [ 3 H]dexamethasone (81.0 Ci/mmol; Nycomed Amersham Inc.) in the absence and presence of a 1,000-fold M excess of unlabeled dexamethasone. After incubation, the cells were washed three times with 1 ml PBS and recovered by centrifugation (3,800 g for 3 min). The cell pellet was then lysed in 5 ml scintillation fluid and the radioactivity determined by liquid scintillation counting. Immunofluorescence experiments were performed as previously described by Herscovics et al. 1994 and the photographs were taken with an LSM 410 invert Zeiss confocal microscope. Electrophoretic mobility shift and immunoblots assays were performed as described previously by Gast et al. 1995 . RAP46 is a protein initially isolated by virtue of its association with the GR in an interaction-screening assay . Since then, association of RAP46 and the GR has been shown in vitro in a glutathione S–transferase pull-down assay . To determine whether these two proteins associate in vivo, we performed immunofluorescence experiments with RAP46 and the GR in the absence and presence of hormone. To demonstrate the specificity of these interactions, control experiments were carried out with the MR, a structural and functional homologue of the GR . When expressed in COS-7 cells, most of the RAP46 protein resided in the cytoplasm as determined by laser confocal microscopy. Antibodies that recognize COOH-terminal epitopes or the HA-tag on this protein clearly showed cytoplasmic localization of RAP46 . In certain regions of the cytoplasm, RAP46 colocalized with the unliganded GR or MR . In the absence of hormone, the MR was identified in the cytoplasm, as well as in the nucleus , in agreement with the results of Fejes-Tóth et al. 1998 . In the presence of ligand when the GR and MR are translocated from cytoplasm into the nucleus, RAP46 was transported with the GR , but not with the MR , showing a specific in vivo association of GR and RAP46. Note that cells containing only RAP46, but not the GR, do not translocate into the nucleus in the presence of dexamethasone . Through its association with the GR and MR in the cytoplasm, RAP46 may alter the ligand binding properties of these receptors in line with its cochaperone activity . We therefore analyzed the hormone binding properties of these receptors in the presence of RAP46 in whole cells and in cytosol preparations. These studies revealed only a slight change in the ligand binding activity of the receptors in the presence of RAP46. For example, Scatchard plot analysis showed an insignificant change in the dissociation constant (K d ) of the receptor for dexamethasone from 20 to 17 nM in the presence of RAP46 . A slight (20%) reduction in the maximum hormone binding capacity (B max ) of the receptor was also detected . This downregulation of the B max is minimal compared with our previous report of a strong RAP46-mediated inhibition of transactivation by the GR . Thus, RAP46 must exert its negative regulatory function at other stages in the action of the GR. In the liganded state, when both the GR and MR are in the nucleus, RAP46 downregulated the transactivation function of only the GR, but not that of the MR . This repression was independent of whether dexamethasone, cortisol, or aldosterone was the activating ligand . The lack of RAP46 effect on transactivation by the MR is possibly due to the different cellular localizations of these two proteins in the presence of ligand . We therefore repeated the transfection experiments with the MR and the RAP46 isoform BAG-1L, which is constitutively localized in the nucleus . In this study, transactivation by the MR was not repressed by BAG-1L, although the response of the GR was inhibited . This indicates a fundamental difference in the action of RAP46 towards the MR and the GR. GR–MR chimeric constructs were therefore used to identify the regions of the GR necessary for repression by RAP46 . Two chimeric GR–MR constructs (GMM and GGM), as well as a truncated GR construct (GGΔ), were used in these analyses. Transactivation by the chimeric MR receptor (GMM) containing the NH 2 -terminal sequence of the GR in the presence of either aldosterone, dexamethasone, or cortisol was not repressed by RAP46 . This may be due to the absence of the hinge region (amino acids 491–515) of the GR that we previously identified as an interaction domain for RAP46 and demonstrated its necessity for RAP46-mediated inhibition of transactivation by the GR . The presence of this domain in the chimeric construct GGM still did not allow RAP46 to repress the transactivation function of this mutant receptor , demonstrating the need for the presence of the hormone binding domain (HBD) of the GR. This finding was further confirmed by the use of the truncated receptor (GGΔ), which contains the interaction domain, but lacks the HBD of the GR. In cotransfection experiments, this mutant GR constitutively transactivated a glucocorticoid responsive gene construct, but its activity was not repressed by RAP46 . Further control experiments with chimeric receptors MGG and MMG were performed. Transactivation by MGG containing both the hinge region and HBD of the GR was repressed by RAP46, but not transactivation by MMG containing only the HBD of the GR, but not the hinge region of the GR (results not shown). These results, together with our previous findings on the hinge region , demonstrate that this region, together with the HBD of the GR, are both required for RAP46-mediated inhibition of transactivation by the GR. To further determine the role of the HBD in the repression of the transactivating function of the GR by RAP46, we asked whether this region is involved in the GR-mediated recruitment of RAP46 into the nucleus. In laser confocal immunofluorescence experiments, we showed that the GGΔ mutant receptor lacking the HBD is permanently localized in the nucleus in the absence and presence of hormone, as already reported . However, in >75% of the cells, RAP46 was colocalized with the receptor in the nucleus . Thus, the inability of RAP46 to repress transactivation by GGΔ is not due to the preclusion of this protein from the nucleus. Rather, it must be due to the absence of an important function in the HBD required for the negative action of RAP46. To find out how RAP46 is recruited into the nucleus by the GR, we used the mutant GR with a deletion of the hinge region (amino acids 491–515) in the confocal immunofluorescence experiments. In this study, both RAP46 and the mutant receptor were localized in the cytoplasm in the absence of hormone. But, in the presence of dexamethasone, the ability of the GR to recruit RAP46 was severely compromised . Only ∼50% of the cells containing the mutant receptor and RAP46 showed colocalization of the two proteins in the nucleus . In the remaining cases, RAP46 was localized in the cytoplasm while the liganded receptor was translocated into the nucleus . This demonstrates that the hinge region of the GR is indeed involved in the transport of RAP46 into the nucleus, although this process could be aided by other regions of the receptor. These regions are most likely contained in the HBD since its deletion, as shown above, slightly decreased nuclear transport of RAP46. As RAP46 does not drastically alter the hormone binding properties of the GR in the cytoplasm, and it is translocated into the nucleus by the GR, its major site of action must be in the nucleus. Previously, we have shown that it inhibits the DNA binding activity of the receptor , but the mechanism involved is not known. We therefore deleted the NH 2 -terminal sequences of this protein encompassing the sequence motif [EEX 4 ] 8 to determine the effect of the truncated proteins on DNA binding by the GR and GR-mediated transactivation at the MMTV promoter. While RAP46 inhibited DNA binding by the GR, mutants with truncation of the first 40 or 70 NH 2 -terminal amino acids, eliminating five or all eight of the repeat-motif, partially or completely abolished this negative effect of RAP46 on DNA binding, as well as transactivation by the GR . Control immunoblot experiments with extracts of the transfected cells showed that the GR and RAP46 constructs were adequately expressed in this experiment . Note that the apparent complete inhibition of DNA binding by the GR in Fig. 5 B is due to the very short autoradiographic exposure time of the gel. Longer exposures showed that DNA binding was drastically repressed, but not completely abolished (results not shown). The two RAP46 NH 2 -terminal mutants behaved as the wild-type RAP46 in several experiments. This was demonstrated by RAP46Δ70, which slightly decreased the maximum hormone binding capacity of the GR without significantly affecting the K d for dexamethasone, as we have reported already . In the presence of ligand, this mutant, as in the case of the wild-type RAP46, was also recruited into the nucleus by the GR . However, unlike the wild-type RAP46, it did not downregulate DNA binding by the GR, nor did it repress GR-mediated transactivation at the MMTV promoter . Thus, COOH-terminal sequences of RAP46 are required by the GR for the recruitment of this protein into the nucleus. To determine the sequences at the COOH terminus necessary for the recruitment, we used a mutant RAP46 lacking the last 47 amino acids known to bind hsp70/hsc70 in immunofluorescence experiments . This mutant, unlike the NH 2 -terminal deletion construct, was not transported into the nucleus , indicating that the extreme COOH-terminal sequence of RAP46 is required to get this protein into the nucleus. Interestingly, deletion of this COOH-terminal sequence also abolished the repressive action of RAP46 on DNA binding by the GR (results not shown). This demonstrates the necessity for nuclear transport of RAP46 for its negative regulatory activity. These results, together with our findings from the experiments with the NH 2 -terminal deletion mutant RAP46Δ70 , demonstrate that although nuclear transport of RAP46 is necessary, it is not sufficient for inhibition of DNA binding by the GR. This function requires the NH 2 terminus of RAP46. To determine the contribution of the NH 2 terminus of RAP46 to downregulation of GR activity, we fused the first 69 amino acids to GFP and asked whether such a construct was sufficient in repressing GR activity. In immunofluorescence experiments, this fusion protein was found both in the cytoplasm and the nucleus (results not shown). In transactivation studies carried out in GR- and RAP46-positive human mammary tumor MCF-7 cells , overexpression of RAP46 inhibited transactivation by the GR, as we had previously reported . However, overexpression of GFP alone had no effect on GR response, whereas the N69GFP construct increased transactivation by the endogenous GR . This shows a dominant–negative effect of the N69GFP protein on the repressive action of endogenous RAP46 in GR-mediated transactivation. This dominant–negative effect was further demonstrated in DNA binding studies involving the GR. In this assay, the N69GFP fusion protein, but not GFP alone, abolished the repressive action of RAP46 on DNA binding by the GR . These studies confirm that RAP46 uses its NH 2 -terminal sequence for the repression of GR activity, possibly through interaction with other factors since its negative effect could be competed by an excess of the N69 sequence fused to GFP. To further determine the significance of the [EEX 4 ] 8 motif in the negative action of RAP46, we examined the effect of the RAP46 isoform BAG-1L (which also carries this sequence motif) on DNA binding by the GR and transactivation by this receptor at the MMTV promoter. In these experiments, both RAP46 and BAG-1L negatively regulated the DNA binding and transactivation functions of the GR to the same extent . BAG-1, the isoform of RAP46 that lacks a considerable portion of the repeat motif, had no effect, as we have already shown for its closely related homologue RAP46Δ40 . Since the nuclear action of the GR involves both positive and negative regulation of gene expression , we examined the effect of RAP46 and BAG-1L on the negative regulatory activity of the GR. This was achieved by measuring the ability of the GR to repress the expression of a human collagenase I gene construct induced by the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA). This study showed that transrepression was unaffected by RAP46, whereas BAG-1L only slightly (30%) abrogated this function of the GR . These effects remained unchanged, even when different amounts of the two constructs were transfected (results not shown). These findings show that the transactivating and transrepressing functions of the GR are regulated differently by RAP46. They further demonstrate that the major nuclear action of RAP46, and its isoform BAG-1L, on GR activity is the repression of the transactivation function of this receptor. hsp70 and hsp90 provide the GR with the proper conformation for ligand binding and, therefore, play an important role in the ligand-mediated activation of this receptor . In this study we have shown that RAP46, a protein that binds hsp70 to inhibit its ability to refold denatured proteins , downregulates transactivation by the GR. This negative regulation does not involve inhibition of the ability of the receptor to bind hormone. In hormone binding studies, RAP46 did not alter the K d of the receptor for its ligand. We therefore concluded that the negative regulation of GR activity by RAP46 must occur through other mechanisms. We demonstrated by immunofluorescence experiments that in the presence of ligand, RAP46 is recruited from the cytoplasm into the nucleus by the GR, but not by the MR. As RAP46 binds hsp70/hsc70, the nuclear transfer could occur via this heat shock protein, especially since it is reported to be transported into the nucleus by both the GR and MR . However, if RAP46 were to be transported via its interaction with hsp70/hsc70, it would be expected to be recruited into the nucleus by both receptors. Since only the GR, but not the MR, recruited RAP46 into the nucleus, it is very likely that the nuclear transport is mediated by a specific association of RAP46 with the GR. Previously, we have shown in glutathione S–transferase pull-down experiments that the hinge region of the GR (amino acids 491–515) is necessary for the association of RAP46 with the GR. In those experiments, we showed that deletion of this region abolished RAP46-mediated downregulation of transactivation by the GR . In the present study, we have shown that deletion of the hinge region partially decreased the ability of RAP46 to be transported into the nucleus by the GR. This means that the hinge region has at least two functions. First, it contributes to nuclear recruitment of RAP46 and second, it also plays a role in inhibition of GR-mediated transactivation. As the hinge region of the GR differs considerably in sequence from that of the MR, it could explain why RAP46 is not translocated into the nucleus by the MR and why transactivation by this receptor is not repressed by RAP46. In our study to find out which region of RAP46 is required for recruitment into the nucleus, we used a mutant that lacks the last 47 COOH-terminal amino acids known to bind hsp70/hsc70 . This protein was not translocated into the nucleus by the GR, implicating the hsp70/hsc70 binding site in the nuclear transport of RAP46. Thus, binding of hsp70/hsc70 to RAP46 may be involved in the nuclear transport and the heat shock protein must interact differently with the GR and MR to account for the differences observed in the recruitment of RAP46 into the nucleus by these two receptors. Alternatively, hsp70/hsc70 may not be necessary at all, but the same sequence used for binding the heat shock protein may be involved in a direct interaction with the GR. This would mean that RAP46 must compete with hsp70/hsc70 for binding to the GR. Our experiments so far do not distinguish between these possibilities. We have shown that, although nuclear recruitment of RAP46 is necessary, it is not sufficient for negative regulation of GR activity. A RAP46 protein lacking the first 70 amino acid residues was transported into the nucleus by the GR, but nevertheless failed to inhibit transactivation by the receptor. This indicates that the inhibitory function of RAP46 requires its NH 2 -terminal sequences. This region of RAP46 is possibly involved in interaction with other proteins, since a fusion protein containing this segment exerted a strong dominant–negative function and relieved the downregulatory action of RAP46 on transactivation by the GR. The first NH 2 -terminal 70 amino acids of RAP46 contain a sequence motif [EEX 4 ] 8 that may contribute to the downregulation of transactivation by the GR since BAG-1L which contains this motif, but not BAG-1, also repressed GR-mediated transactivation. BAG-1, on the other hand contains two copies, rather than the eight copies of the [EEX 4 ] motif. Thus, whatever the function of this sequence, several copies, rather than a duplication, are necessary for the manifestation of its repressive function. The [EEX 4 ] 8 sequence is highly enriched in threonine and serine residues (27% of the total sequence). Our preliminary in vivo labeling studies showed that RAP46 is a phosphoprotein, with almost all its phosphorylated residues localized at the [EEX 4 ] 8 sequence in the first NH 2 -terminal 70 amino acid residues (Schneikert, J., and A.C.B. Cato, unpublished). This finding is rather striking and highly suggestive of a role of phosphorylation in the action of the NH 2 terminus of RAP46. Inhibition of GR-mediated transactivation by the corepressor calreticulin has been shown to be dependent on inhibition of DNA binding brought about by the interaction of this protein with sequences in the DNA binding domain of the receptor . Distinct sequences in the DNA binding domain of the GR are not recognized by RAP46 for the inhibition of transactivation by the GR. The site of interaction of RAP46 on the GR is the hinge region . However, the HBD of the GR also contributes to the negative action of RAP46. From our studies, we conclude that the negative action of RAP46 is effected through a prior nuclear transport of this protein via a process that requires the COOH-terminal region (the hinge region and, to some extent, the HBD) of the GR, as well as the last 47 COOH-terminal amino acids of RAP46. In the nucleus, RAP46 inhibits DNA binding by the receptor through a mechanism that is not quite clear at the moment. The fact that the HBD of the GR binds hsp70/hsc70, which is also recognized by RAP46, and all three proteins are found in the nucleus, argues in favor of at least a tertiary protein complex in the negative regulatory action of RAP46. Other proteins may be part of this complex as well, especially protein binding to the NH 2 -terminal region of RAP46. We have demonstrated that the inhibitory action of RAP46 is specific for those actions of the GR requiring DNA binding. Thus, transactivation, which is dependent on DNA binding by the GR is repressed, but not GR-mediated repression of the activity of the transcription factor AP-1, which does not require DNA binding by the receptor . We cannot, however, rule out the possibility that the use of TPA for the activation of AP-1 in our experiments disrupts the RAP46 complex necessary for inhibiting GR action. The biological significance of the negative regulatory action of RAP46 are manifold. We previously have shown that RAP46-mediated inhibition of transactivation by the GR correlates with inhibition of glucocorticoid induced apoptosis . We also observed that T cells that are resistant to glucocorticoid-induced apoptosis express relatively high levels of RAP46 . Conversely, conditions that downregulate the endogenous levels of RAP46, such as exposure of thymoma cells to the immunosuppressant rapamycin, enhanced the transactivation potential of the GR and reduced GR-mediated apoptosis . In the present study we have shown, by the use of a dominant–negative mutant construct, that the transactivation function of the GR in the human mammary carcinoma MCF-7 cells is negatively regulated by endogenous RAP46. As neoplastic cells generally express relatively high levels of RAP46 and BAG-1L , our results on the inhibition of GR-mediated transactivation by these proteins may provide mechanistic explanations for the reported cases of glucocorticoid resistance in neoplastic transformation. This point would have to be taken into consideration in studies on glucocorticoid resistance in disorders such as acute lymphocytic and nonlymphocytic leukemia . | Study | biomedical | en | 0.999996 |
10477750 | Hela cells were grown in DME supplemented with 10% FBS. Hela cells adapted for growth in suspension were cultured in SME supplemented with 10% FBS and 1% pluronic acid. K562 suspension cultures were grown in RPMI 1640 supplemented with 10% FBS. All cells were grown in a humidified 37°C chamber at 5% CO 2 . Centrifugal elutriation was performed using a Beckman centrifuge (J6-MC) with the JE5.0 rotor as described . Samples were taken from each fraction and analyzed for cell cycle distribution by FACS. Hela cells arrested in mitosis were obtained by growing them in the presence of nocodazole (0.06 μg/ml) for 12–16 h. Both normal and nocodazole-blocked mitotic cells were harvested from the culture dishes by mechanical shakeoff. Full-length gfp-hBUBR1 expression construct was described previously . The mutant gfp:hBUBR1Δkinase construct was made by deleting nine amino acids (amino acids 795–803, KVSSQPVPW) from the most conserved kinase subdomain II . Deletion was accomplished by PCR-based mutagenesis. Transient transfections were performed by lipofection using LT2 (PanVera) as described . Hela cells were plated onto gridded coverslips and were synchronized at the G1/S boundary by a double thymidine block. Before injection, the media was replaced with fresh Hepes buffered DME plus 10% FBS. Cells were injected 1–2 h after they were released from the G1/S boundary. Microinjection was performed with a semi-automatic injector and the Eppendorf automated micromanipulator . For each coverslip, 200–300 cells were injected with each antibody mix over a course of ∼30 min. Multiple coverslips were injected to accommodate time course experiments where samples had to be taken at different times. The affinity-purified hBUBR1 and CENP-E antibodies used for the injection experiments have been previously described . Nonimmune IgG was isolated from sera, which was originally used to affinity purify hBUBR1 antibodies, and verified by immunofluorescence and Western blots to no longer contain detectable levels of hBUBR1 antibodies. Antibodies were kept in Ca ++ - and Mg ++ -free PBS and were filtered through a 0.22-μm Millipore microfiltration cup before use. To visualize the cells that had been injected with antibodies, cells were fixed for 7 min in 3.5% paraformaldehyde in PBS and permeabilized in 0.2% Triton X-100 for 3–5 min before proceeding with antibody incubations. To minimize loss of loosely attached mitotic or apoptotic cells, the coverslips were centrifuged at 200 g for 3 min in a clinical centrifuge (GPKR; Beckman). For time-lapse microscopy, Hela cells were plated onto 35-mm glass bottom microwell dishes (MatTek Corp.), and cells were monitored with a 60× PlanApo objective mounted on a Nikon Eclipse TE300 inverted microscope. Cells were kept at 37°C with a 35-mm tissue culture dish stage warmer regulated by a temperature controller (TC-102; Medical Systems Corp.). Time-lapse images were captured using a Sensys CCD camera (Photometric) that was controlled by Image-Pro Plus software (Phase 3 Imaging Systems). Cells on coverslips were fixed, permeabilized, and stained as described . Injected rabbit antibodies were detected with Cy5-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories). Endogenous hBUBR1 was visualized with a rat anti–hBUBR1 antibody that was generated and affinity-purified the same way as the previously described rabbit hBUBR1 antibodies . Anticentromere autoimmune serum was a gift of K. Sullivan (Scripps Research Institute, La Jolla, CA). Microtubules were stained with anti–α-tubulin monoclonal antibody (Sigma Chemical Co.). The primary antibodies were visualized using an appropriate secondary antibody that was coupled to Cy2 or Texas red (Jackson ImmunoResearch Laboratories). DNA was stained with 0.1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI). Stained cells were examined with either a 40 or 100× PlanNeofluor objective mounted on a Nikon Microphot SA that was equipped with epifluorescence optics. Images were captured with a TEC-1 CCD camera (Dage-MTI) that was controlled with IP LabSpectrum v2.0.1 (Scanalytics Inc.) and contrast enhanced with Adobe Photoshop 5.0 (Adobe Systems Inc.). Cells were harvested, washed, and lysed in NP-40 lysis buffer (1% NP-40, 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM DTT) supplemented with protease and phosphatase inhibitors as described previously . Cell lysates were centrifuged at 10,000 g at 4°C for 15 min. The protein concentrations of the lysates were determined (BCA protein assay; Pierce Chemical Co.) and adjusted to the same protein concentration. Immunoprecipitation was performed with 300 or 500 μg of lysate and the final concentration of hBUBR1 antibody was ∼2 μg/ml. For FPLC separation, S100 was loaded onto a Superose 6 column (Amersham Pharmacia Biotech Inc.), and proteins were eluted in NP-40 lysis buffer with protease and phosphatase inhibitors. Fractions containing cyclosome/APC were combined, concentrated with a Centricon 100 (Amicon Inc.), and incubated with hBUBR1 antibodies. To solubilize hBUBR1 that is associated with the kinetochore, chromosomes were boiled in 2% SDS for 2 min, diluted 10-fold with NP-40 lysis buffer, clarified, and processed for immunoprecipitation. Antibody was incubated with lysates for 3 h at 4°C, and then 30 μl of 50% slurry of protein A–Sepharose (Repligen) was added and incubation proceeded for another 30 min. The beads were recovered by low speed centrifugation, washed five times in 0.5 ml of ice-cold lysis buffer before SDS gel sample buffer was added. In some cases, hBUBR1 immunoprecipitates were incubated with 400 units of λ protein phosphatase (New England Biolabs Inc.) for 30 min at 30°C in buffer supplied by the manufacturer. Samples from the immunoprecipitates or whole cell lysates were separated through 4–12% gradient SDS-PAGE, transferred onto Immobilon P (Millipore), and then processed for Western blots. Antibodies to cyclosome/APC subunits hscdc16 and hscdc27 were gifts of Dr. P. Hieter (University of British Columbia, Vancouver, British Columbia, Canada). APC7 antibodies were a gift from Dr. J.M. Peters (IMP, Vienna, Austria). Rabbit hBUB3 antibodies were generated against GST:hBUB3 fusion protein and affinity-purified as described . Primary antibodies were detected with alkaline phosphatase–conjugated anti-rabbit secondary antibodies used at 1:30,000 (Sigma Chemical Co.), and then processed for CDP-Star chemiluminescence detection (Tropix). hBUBR1 was immunoprecipitated from lysates containing identical amounts of protein as described above. After washing the pelleted beads five times in NP-40 lysis buffer, the beads were washed three times in kinase buffer (50 mM Tris-Cl, pH 7.4, 10 mM MgCl 2 , 10 mM β glycerophosphate). Beads were incubated in kinase buffer that contained 50 μM ATP and 20 μCi γ-[ 32 P]ATP at 25°C for 15 min. Cold ATP was added to a final concentration of 0.5 mM and the reaction was allowed to proceed for another 15 min before SDS sample buffer was added. The immunoprecipitation–kinase samples were boiled and separated through a 4–15% gradient gel, fixed, dried, and then subjected to autoradiography. Quantitation was done using a Bio-Imaging analyzer . Two complementary approaches were used to examine if hBUBR1 function is required for the mitotic checkpoint in Hela cells. Our first strategy to inhibit hBUBR1 activity was to microinject hBUBR1 antibodies into cells and test the response of the injected cells to the microtubule inhibitor nocodazole. The hBUBR1 antibodies used for the injection experiments have been shown not to cross-react with hBUB1 as determined by Western blots and immunoprecipitations . Hela cells synchronized at the G1/S boundary were microinjected with hBUBR1 or nonimmune antibodies shortly after they were released from the G1/S boundary. Approximately 2 h before the synchronized cells were expected to enter mitosis, nocodazole was added to the medium and the injected cells were sampled at various times during the ensuing 8 h . Mitotic cells were scored by the presence of condensed chromosomes which aggregated near the center of the cell when the spindle is abolished by nocodazole . Approximately 12 h after release from the G1/S boundary, most cells have entered mitosis. At this time, there was no significant difference between the mitotic indices of the cells that were injected with hBUBR1 or the nonimmune antibodies. Thus, injection of antibodies did not interfere with mitotic entry. Nonimmune antibodies were diffusely distributed throughout the mitotic cell and did not interfere with the localization of hBUBR1 at kinetochores of the unaligned chromosomes . In contrast, the injected hBUBR1 antibodies were concentrated at the kinetochores of the unaligned chromosomes . The presence of hBUBR1 at kinetochores of these cells was independently confirmed by staining with hBUBR1 antibodies . When cells were examined at later times after nocodazole treatment, the mitotic index of cells injected with nonimmune IgG remained high, whereas the mitotic index of cells injected with hBUBR1 antibodies was about threefold lower . The decrease in the mitotic index of cells that were injected with the hBUBR1 antibodies was accompanied by a fivefold rise in the number of interphase cells with aberrantly shaped nuclei relative to the negative control . The flattened morphology as well as the presence of nuclei indicated that these cells exited mitosis . The presence of paired centromeres, as revealed by staining with an anticentromere autoimmune (ACA) serum, suggested that either the chromosomes did not separate or had separated but re-replicated . To directly confirm that inhibition of hBUBR1 abrogated the mitotic checkpoint and caused cells to exit mitosis, time-lapse videomicroscopy was used to follow the fate of nocodazole-treated cells that were microinjected with hBUBR1 antibodies. Fig. 1 C shows that within 30 min after the cell entered mitosis, the cell began to flatten out. 2 h later, the presence of a nucleus demonstrated that it had clearly exited mitosis . At this level of resolution, we could not determine how rapidly cells injected with hBUBR1 antibodies exited mitosis. Uninjected cells on the same coverslip as well as cells injected with nonimmune antibodies remained blocked in mitosis for up to 16 h (data not shown). To obtain independent verification of the microinjection results, we attempted to disrupt endogenous hBUBR1 function by targeting an hBUBR1 mutant to kinetochores. We created a mutant by deleting nine amino acids (residues 795–803) from subdomain II of the conserved catalytic core of the hBUBR1 kinase. Western blots confirmed that both mutant and wild-type hBUBR1 were expressed at equivalent levels (data not shown). Both mutant and wild-type gfp:hBUBR1 were transfected into synchronized Hela cells shortly after they were released from the G1/S boundary . Nocodazole was added ∼2 h before the cells were expected to enter mitosis. By 12 h after release from the G1/S boundary, mitotic cells expressing both mutant and wild-type gfp-hBUBR1 at their kinetochores were identified . After overnight incubation in the presence of nocodazole, the mitotic index of cells expressing the mutant gfp:hBUBR1 was ∼2 times lower than for wild-type gfp:hBUBR1–transfected cells . In concordance with the microinjection results, many of the cells expressing the mutant hBUBR1 exited mitosis and formed aberrant shaped nuclei . The accumulation of the gfp:hBUBR1Δkinase in the cytoplasm of these aberrant interphase cells is consistent with the distribution of endogenous hBUBR1 in interphase cells as previously reported . The microinjection and transfection experiments demonstrate that hBUBR1 kinase is likely to be an essential component of the mitotic checkpoint. We have previously shown that hBUBR1 can form a complex with the kinetochore motor CENP-E . If hBUBR1 can monitor kinetochore microtubule interactions mediated by CENP-E as part of its normal checkpoint function, disruption of hBUBR1 might cause cells to exit mitosis prematurely. Alternatively, hBUBR1 might regulate CENP-E functions that are important for chromosome alignment. In this case, loss of hBUBR1 should interfere with CENP-E functions at the kinetochore and block cells in mitosis with unaligned chromosomes . To test the importance of hBUBR1 during normal mitosis, the fates of synchronized cells that were either injected with hBUBR1 antibodies or transfected with the gfp:hBUBR1Δkinase were determined. Approximately 12 h after release from the G1/S boundary, cells that were injected with nonimmune IgG were found in prometaphase and metaphase with the majority being at metaphase . Immunofluorescence staining revealed that the metaphase cells exhibited detectable levels of hBUBR1 at their kinetochores despite the presence of nonimmune antibodies throughout the cell . For cells injected with the hBUBR1 antibody, most of the mitotic cells were in prometaphase. The injected hBUBR1 antibodies were found throughout the cell including kinetochores . The presence of hBUBR1 antibodies at the kinetochores did not interfere with CENP-E localization . Although a few metaphase cells injected with hBUBR1 were seen, we consistently identified anaphase cells that contained lagging chromosomes . These cells appeared to be in late anaphase as they contained a mature cleavage furrow that was concentrated with CENP-E . The presence of lagging chromosomes suggested that these cells exited mitosis in the presence of unaligned chromosomes and gave rise to the high frequency of aberrantly divided cells that accumulated at later time points . The nuclei of the cells injected with hBUBR1 antibodies were abnormally shaped and DNA was invariably found in between the two divided cells . The DNA in between the cells was derived from chromosomes because they contained centromeres as determined by ACA staining . However, we cannot distinguish whether the foci of ACA staining represent separated centromeres or fragmented centromeres. This cut phenotype was specific for hBUBR1 antibodies as this was not observed in uninjected cells or cells injected with nonimmune antibodies. In fact, hBUBR1 antibody injection did not produce any normal telophase cells . Similar results were obtained when synchronized Hela cells were transfected with the mutant hBUBR1Δkinase. In transfected cells that had progressed into mitosis, the mutant hBUBR1 was concentrated at kinetochores . At later times, there was a large increase in aberrantly divided cells with the cut phenotype . Both mock and wild-type gfp:hBUBR1–transfected cells divided normally and did not exhibit the cut phenotype (data not shown). The combined data suggest that disruption of hBUBR1 function caused the cells to exit mitosis in the presence of unaligned chromosomes. We have previously shown that disruption of CENP-E functions at kinetochores prevented chromosome alignment and arrested cells in mitosis for >12 h . To test whether hBUBR1 is required for this checkpoint, we coinjected hBUBR1 and CENP-E antibodies into synchronized Hela cells, and then compared the fates of these cells to those that were injected with CENP-E antibodies alone. Injection of CENP-E antibodies blocked the assembly of CENP-E onto kinetochores but did not interfere with kinetochore localization of hBUBR1 or the distribution of other centromere components that were recognized by ACA . As shown previously, the mitotic block induced by injection of CENP-E antibodies is the direct result of disruption of kinetochore function and not bipolar spindle formation . Cells coinjected with CENP-E and hBUBR1 antibodies entered mitosis. Immunofluorescence staining confirmed that hBUBR1 remained at kinetochores of the injected cells. 16 h after release from the G1/S boundary, we counted cells that were either in mitosis, telophase, aberrantly divided cut, or apoptosis. Cells injected with just CENP-E antibodies were arrested in mitosis with unaligned chromosomes . No cells were found to be apoptotic, in telophase, or cut. Examination of the cells that were coinjected with hBUBR1 and CENP-E antibodies showed an ∼5-fold reduction in the number of mitotic cells relative to cells injected with CENP-E alone . The reduction of mitotic cells was accompanied by a large increase in the number of aberrantly divided cells that exhibited the cut phenotype along with a slight increase in apoptotic cells. Normal telophase cells that were coinjected with the hBUBR1 and CENP-E antibodies were not found. These data showed that hBUBR1 is an essential component of the mitotic checkpoint that is sensitive to kinetochore functions that were specified by CENP-E. Having established that hBUBR1 is an essential component of the mitotic checkpoint, we wanted to investigate its mechanism of action by characterizing its expression and kinase activity as a function of the cell cycle. hBUBR1 was immunoprecipitated from K562 erythroleukemic cells and Hela cells that were separated into different phases of the cell cycles by centrifugal elutriation. Portions of the immunocomplexes were used to determine steady-state levels of protein and kinase activity. We assayed the immunoprecipitated hBUBR1 for autokinase activity as a recent study showed that a transfected hBUBR1 exhibited autokinase activity . However, hBUBR1 isolated from K562 and Hela (data not shown) exhibited no detectable autokinase activity or kinase activities towards exogenous substrates (data not shown) even though it was expressed throughout the cell cycle of both cell types . hBUBR1 levels remained fairly constant throughout the cell cycle of K562 cells, whereas hBUBR1 levels in Hela cells fluctuated with the cell cycle. hBUBR1 level was lowest in G1 and steadily increased as cells progressed towards mitosis. If hBUBR1 is a mitosis-specific kinase, its activity would have escaped detection because the vast majority of the cells that were elutriated into the late fractions were in G2 and only a very minor fraction was in mitosis. Therefore, we compared the hBUBR1 kinase activity between interphase and mitotically arrested Hela cells. hBUBR1 autokinase activity remained undetectable in interphase cells, but was highly active in mitotically arrested cells . The same profile of hBUBR1 kinase activity was obtained with exogenous substrates such as myelin basic protein and a carboxyl-terminal fragment of CENP-E (data not shown). The increase in hBUBR1 kinase activity in mitotic cells was not due to increased expression of the protein or its association with the hBUB3 subunit relative to interphase cells . However, hBUBR1 mobility was reduced in mitotically blocked cells relative to the interphase cells . The upshift was due to phosphorylation as the electrophoretic mobility of hBUBR1 was no longer retarded after phosphatase treatment . In vivo labeling experiments showed that hBUBR1 was also phosphorylated during interphase but became hyperphosphorylated in mitosis (data not shown). To further test if the hyperphosphorylation of hBUBR1 was related to the mitotic checkpoint, we compared hBUBR1 in mitotically blocked cells to cells that were released from the block and had exited mitosis . The majority of hBUBR1 was hyperphosphorylated in mitotically blocked cells relative to normal mitotic cells. 1 h after their release from the mitotic block, hBUBR1 was no longer hyperphosphorylated . In the previous experiments, hBUBR1 kinase activity was determined in cells that had already been blocked in mitosis for many hours. As a checkpoint kinase, hBUBR1 should respond to spindle defects even after cells have reached metaphase. To test this, we isolated mitotic Hela cells by mechanical shakeoff, and then exposed them to nocodazole for various times. Microscopic analysis revealed that >90% of the mechanically detached cells were in metaphase with some prometaphase and very few (<1%) anaphase cells. hBUBR1 was immunoprecipitated from cell lysates that were harvested at different times of incubation with nocodazole, and then assayed for kinase activities and protein expression . Comparison of hBUBR1 kinase activity between metaphase cells and those that were exposed to nocodazole showed that kinase activity was rapidly stimulated within 15 min of nocodazole treatment . At this time, the spindle was absent and chromosomes were unaligned (data not shown). hBUBR1 kinase activity reached a peak at 30 min after nocodazole treatment and gradually declined. By 4 h, hBUBR1 kinase activity had decayed to a level that was slightly above that of normal metaphase cells. During the course of the experiment, cells were confirmed to be blocked in mitosis by phase-contrast microscopy and expression levels of cyclin B . Western blots of the hBUBR1 immunoprecipitates revealed that the levels of hBUBR1 and hBUB3 remained fairly constant over the course of the experiment . The increase of kinase activity was not due to changes in levels of hBUBR1 or its subunit, hBUB3. However, we detected an increase in the level of hyperphosphorylated hBUBR1 (as determined by retarded migration) between metaphase cells and those that were incubated in nocodazole . The increase in the level of hyperphosphorylated hBUBR1 did not correlate with its peak kinase activities . The hBUBR1 kinase activities described above were derived from the soluble pool of proteins and excluded those that were associated with kinetochores. As the kinetochore is a critical component of the checkpoint, we probed the insoluble fractions that were enriched in chromosomes for hBUBR1 to see how this population responded to spindle disruption . Western blots revealed that hBUBR1 levels increased slightly after 30 and 60 min in nocodazole, but returned to the initial level after 2 and 4 h of nocodazole treatment. The most dramatic change was that hBUBR1 was quantitatively hyperphosphorylated within 15 min of spindle disruption and this high level of phosphorylation was maintained throughout the block. Next, we examined the native size of hBUBR1 between interphase and mitotically blocked cells. Gel filtration chromatography revealed that hBUBR1 exists primarily in an ∼500-kD complex in interphase cells . In mitotically arrested cells, hBUBR1 was associated with the ∼500-kD complex, but was also found in a larger complex that overlapped with the cyclosome/APC . The possibility that hBUBR1 was associated with the cyclosome/APC was tested by immunoprecipitating hBUBR1 from the fractions containing the cyclosome/APC and probing for the presence of the APC subunits hscdc27, hscdc16 , and APC7 . The results showed that all three of the APC subunits, coimmunoprecipitated with hBUBR1 in the mitotically blocked lysates . Although some hBUBR1 was detected in the cyclosome/APC containing fractions in interphase cells , this was not associated with the cyclosome/APC , and most likely resulted from smearing of the 500-kD complex in the gel filtration column. This finding along with the absence of cyclosome/APC subunits in immunoprecipitates from nonimmune IgG demonstrated that the cyclosome/APC specifically associates with hBUBR1 only in mitosis. We estimated the proportion of the cellular pool of the cyclosome/APC that was associated with hBUBR1 in mitotically arrested cells by comparing the relative levels of various APC subunits in hBUBR1 immunoprecipitates and the remaining supernatant. When ∼30% of the total pool of hBUBR1 was immunoprecipitated from mitotic lysates, roughly 16–20% of the total pool of hscdc16, hscdc27, and APC7 was also found in the precipitate. Assuming that the remaining pool of hBUBR1 that was not immunoprecipitated can also associate with the APC, we can extrapolate that ∼50% of the total pool of APC can be associated with hBUBR1. Since the gel filtration data show that ∼50% of the total pool of hBUBR1 comigrates with the APC, we estimate that about 25% of the total pool of APC is associated with hBUBR1 in mitosis. The results from this study provide details about the function and biochemical properties of hBUBR1 kinase. We demonstrate that hBUBR1 is essential for the mitotic checkpoint as disruption of hBUBR1 function prevented cells from arresting in mitosis in the presence of nocodazole. Instead, the cells exited mitosis without dividing and formed highly aberrant nuclei that contained unsegregated chromosomes. The behavior of hBUBR1-defective cells is very similar to that exhibited by cells with defective MAD1, MAD2, or BUB1 functions. Depletion of MAD2 and MAD1 from Xenopus egg extracts resulted in the inability to block mitosis in the presence of nocodazole . Introduction of anti-MAD2 antibodies into cells abrogated their ability to arrest in mitosis in the presence of nocodazole or taxol . Overexpression of a mutant form of mBUB1 and hBUB1 prevented cells from arresting in mitosis in the presence of nocodazole. Our studies also showed that hBUBR1 provided an essential function during normal mitosis by preventing cells from prematurely exiting mitosis. It was unlikely that disrupting hBUBR1 functions directly interfered with chromosome alignment since we were able to identify metaphase cells that were injected with hBUBR1 antibody or expressed the hBUBR1 kinase–defective mutant. As the frequency of such metaphase cells was rare, the most likely fate of many of the prometaphase cells that we saw was premature exit from mitosis. This interpretation is supported by the identification of aberrant anaphase cells with lagging chromosomes. The lagging chromosomes most likely were chromosomes that had not achieved alignment at the metaphase plate when cells decided to exit mitosis. This defect would explain the high frequency of abnormally divided cells that contained chromatin trapped by the cleavage furrow. At present, we have not directly determined precisely how rapidly cells progress through mitosis when hBUBR1 function is inhibited. However, studies have shown that disruption of mBUB1 appeared to accelerate cells through mitosis by ∼25 min . Likewise, microinjection of MAD2 antibodies into prophase human keratinocyte cells caused them to precociously exit mitosis before chromosomes were properly aligned . High resolution videomicroscopy showed that disruption of MAD2 in human keratinocyte cells accelerated mitotic progression by 6.5 min. Despite the similarity in the outcomes of all these studies, it remains to be seen how MAD2, BUB1, and BUBR1 relate to each other at a mechanistic level. As all of these proteins are localized at the kinetochore, they may act in parallel by monitoring different activities of the kinetochore or work as an integrated complex to generate the wait anaphase signal from the kinetochore. These two possibilities are supported by the fact that the two BUB1-related kinases do not share overlapping functions as neither one is able to maintain the mitotic checkpoint alone. Our data show that one checkpoint function of hBUBR1 is to monitor kinetochore functions that are specified by CENP-E. When kinetochores are depleted of CENP-E, cells arrest in mitosis with unaligned chromosomes but a normal spindle. Our data suggest that hBUBR1 activity at the kinetochore is important for generating the wait anaphase signal. This possibility is supported by the finding that hBUBR1 was still present on kinetochores that lacked CENP-E. Furthermore, microinjection of hBUBR1 antibodies abrogated the mitotic arrest and caused cells to exit mitosis. If hBUBR1 was contributing to the wait anaphase signal from the CENP-E–depleted kinetochores, it must be able to do so when it is not in direct contact with CENP-E. Under normal circumstances, we speculate that interactions between CENP-E and microtubules can directly regulate hBUBR1 activity by altering its interactions with CENP-E. Although such a feedback mechanism can link kinetochore attachments to the checkpoint, the threshold level of CENP-E activity that is required to activate or inactivate hBUBR1 activity remains to be determined. We have shown that when kinetochore–microtubule attachments of metaphase cells are dissolved by nocodazole, the most dramatic change was the quantitative hyperphosphorylation of the kinetochore-bound hBUBR1. Perhaps the phosphorylation state of hBUBR1 at the kinetochore is related to the number of kinetochore microtubules that are attached to CENP-E and the level of tension that results from these interactions. At metaphase, the levels of both CENP-E and hBUBR1 have been shown to be reduced along with other checkpoint proteins. Under these circumstances, there may be insufficient hBUBR1 kinase activity that is generated from the kinetochore to sustain the checkpoint components that lie downstream of the kinetochore. Studies have shown that it takes ∼30 min from the time the last chromosome becomes aligned at the metaphase plate until the onset of anaphase . This may be the time that is required for the checkpoint signal to fall and to allow cyclosome/APC activity to reach a critical level that is necessary to drive cells into anaphase. Before this critical level of cyclosome/APC activity is reached, the checkpoint can be reactivated by unattached kinetochores to once again inhibit cyclosome/APC. Although we proposed that hBUBR1 kinase activity is sensitive to the mechanical activities of the kinetochore, activation of its kinase may require additional biochemical changes such as mitosis-specific phosphorylations. As kinetochores are now known to also contain cdc2 , MAP kinase , and hBUB1 (mBUB1) kinases , any one of these may be important for regulating hBUBR1 kinase at the kinetochore. We infer from the interaction between hBUBR1 and cyclosome/APC that hBUBR1 might provide global checkpoint functions by directly inhibiting cyclosome/APC. This is supported by the finding that partially purified hBUBR1 from Hela cells inhibited ubiquitination activity of the APC in vitro (Sudakin, V., and T. Yen, unpublished results). By comparing the amount of APC that coimmunoprecipitated with hBUBR1 to that in the remaining supernatant, we estimated that 25% of the total pool of APC was bound to hBUBR1. As MAD2–p55cdc was also found to be associated with only a fraction of the total pool of APC, it is possible that the hBUBR1–APC complex represents a different subpopulation of the APC. As hBUBR1 kinase activity is stimulated when the spindle is disrupted, hBUBR1 may directly phosphorylate and inhibit cyclosome/APC activity or phosphorylate specific substrates so that they are no longer recognized by the cyclosome/APC. As proposed for MAD2–p55cdc , the interactions between hBUBR1 and the cyclosome/APC should be inherently unstable so that cells can proceed into anaphase when the checkpoint is extinguished. As hBUBR1 appears to form a fairly stable complex with the cyclosome/APC in cells arrested in mitosis, its kinase activity may be labile. This is supported by the observation that the in vitro hBUBR1 kinase activity had decayed when it was isolated from cells whose spindle had been disrupted by nocodazole for an hour as compared with the mitotic cells that were examined 30 min earlier. As the phosphorylation status of the soluble pool of hBUBR1 did not strongly correlate with its kinase activity, additional factors may influence hBUBR1 kinase activity. Despite the decay in the soluble pool of hBUBR1 kinase activity, sufficient kinase activity was probably maintained in the mitotically arrested cells by the continued presence of unattached kinetochores. The cumulative results obtained for hBUBR1 suggest that it can provide checkpoint functions at the local and global levels. In the simplest model, unattached kinetochores contain active hBUBR1 kinase that can rapidly activate the soluble pool of hBUBR1 through autophosphorylation. This then leads to inhibition of the cyclosome/APC and exit from mitosis. This model of hBUBR1 function shares features proposed for MAD2–p55cdc and MAD2/MAD1 . The overall view is that a soluble pool of checkpoint proteins is used to block cyclosome/APC activity, but the inhibitory status of these checkpoint proteins must be maintained through interactions with the unattached kinetochore. We propose that hBUBR1 can accomplish this through an autokinase loop that may also include MAD2–p55cdc complex. As MAD2 is not known to be hyperphosphorylated in mitotically blocked cells, hBUBR1 might phosphorylate one of its associated subunits such as p55CDC or MAD1 . Biochemical analysis of yeast MAD1 showed that it is hyperphosphorylated in mitotically arrested cells and that this is dependent on the MPS1 and BUB1 kinases . Although vertebrate MAD1 does not undergo phosphorylation-dependent shifts in its migration in SDS-PAGE , it does not eliminate the possibility that MAD1 is hyperphosphorylated in mitosis. It is possible that in vertebrate systems, MAD1, MAD2, p55CDC, and hBUB1 act in parallel with hBUBR1 or through more complex interactions. Clearly, it will be important to clarify the biochemical relationship amongst these proteins to resolve whether they act together or along separate pathways in vertebrate cells. | Study | biomedical | en | 0.999997 |
10477751 | Mass cultures of sympathetic neurons were derived from the superior cervical ganglia of postnatal day 1 Sprague-Dawley rats, as previously described , except that neurons were dissected into plating Ultraculture medium (serum free) containing 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Biowhittaker). Neurons were plated in the medium supplemented with 3% rat serum (Harlan Sprague Dawley Inc.), 50 ng/ml mouse 2.5 S NGF prepared from mouse salivary gland (Cedarlane Labs, Ltd.), and 0.5% cytosine arabinoside (Sigma Chemical Co.). For 3[4,5-dimethylthio-zol-2-yl]2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) assays and biochemistry, neurons were plated on tissue culture dishes (Falcon Plastics) coated with rat-tail collagen. For microscopy, cells were plated on 16-well glass chamber slides (Nunc Inc.) coated first with collagen/poly- d -lysine, followed by a second coating of collagen. Neurons were plated at a density of 2,500–3,000/well of a 96-well plate for the MTT assays and 40,000 cells/well of a 6-well plate for biochemistry. In all cases, neurons were cultured for 5 d in the above-mentioned medium. After 5 d of culture, cells were washed three times for 1 h in Ultraculture supplemented with 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. After the washes, cells were induced in the same media used for the washes, supplemented with NGF or KCl in the presence or absence of kinase inhibitors. MTT and TdT-mediated dUTP nick end labeling (TUNEL) assays were performed 48 h after induction, and cell extracts for Western blotting were taken 15 min after induction. The kinase inhibitors KN-62, K-252a, LY294002, and PD98059 were obtained from Calbiochem-Novabiochem Corp. Nifedipine was obtained from BIOMOL. For adenoviral infection, cells were grown for 3 d in plating medium as described earlier and then switched into similar media containing the desired MOI (multiplicity of infection = pfu/cell) of adenovirus and no cytosine arabinoside. Infection was allowed to proceed for 24 h, after which the cells were switched back to plating media containing cytosine arabinoside without virus for an additional 24 h before the washout and induction with NGF or KCl. Recombinant adenoviruses expressing dominant-negative Ras (N 17 -Ras), dominant-negative Akt (dnAkt; Boudreau, M., C. Tudan, and D.R. Kaplan, manuscript submitted for publication), and dnTrkA were amplified, purified, and titered as previously described . Survival assays were performed 48 h after washout and induction of neurons as previously described . In brief, 20 μl of MTT reagent was added to the medium in each well of a 96-well plate containing the cultured neurons. After a 2.5 h incubation at 37°C, the medium/MTT mixture was removed and the cells were lysed with 100 μl of isopropanol containing 2 μl/ml of concentrated HCl. The absorbance of the lysate at 570 and 630 nm was determined using a Biotek model EL X -800 UV plate reader (Mandel Scientific Inc.). For the TUNEL experiments, cells were briefly rinsed in PBS, pH 7.2, and fixed for 15 min in 4% paraformaldehyde (Sigma Chemical Co.), 0.25% glutaraldehyde (Fluka AG), and 0.2% Triton X-100 (Sigma Chemical Co.) in PBS, pH 7.2. Cells were then permeabilized with 0.5% Triton X-100 for 5 min and washed three times with PBS. TUNEL reaction was performed for 1 h at 37°C. Each 100 μl of TUNEL reaction mixture contained 20 μl of TdT buffer, 1.5 μl of TdT enzyme (both from Promega Corp.), and 1 μl of biotin-16-dUTP (Boehringer Mannheim Corp.). After the TUNEL reaction, cells were rinsed three times in PBS and incubated for 45 min at room temperature with Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) diluted 1:500 in PBS. Cells were then counterstained for 1 min with Hoechst 33258 (Sigma Chemical Co.) and diluted 1:1,000 in PBS. Cells were washed three times with PBS after each of these incubations and then mounted. For each treatment, random images were captured and processed. Digital image acquisition and analysis was performed with the Northern Eclipse software (Empix Inc.) using a Sony XC-75CE CCD video camera. Sympathetic neurons were rinsed briefly in cold TBS and then lysed in TBS lysis buffer supplemented with Mini Complete protease inhibitor cocktail (Boehringer Mannheim Corp.) and 1.5 mM sodium vanadate. Lysates were scraped into Eppendorf tubes and rocked for 10 min at 4°C. Samples were then cleared by centrifugation. Protein concentration was determined by the BCA assay (Pierce Chemical Co.) using BSA as a standard. For immunoprecipitation, samples were diluted into immunoprecipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% vol/vol NP-40, 0.5% wt/wt sodium deoxycholate, 0.1% wt/vol SDS, 5% vol/vol glycerol, 10 mM sodium fluoride, 5 mM EGTA, 1 mM EDTA, and 30 mM glycerolphosphate) containing the primary antibody, and incubated with gentle agitation for 4 h at 4°C. 30 μl of protein A–Sepharose beads (Pharmacia Biotech, Inc.), which were preincubated 1 h in cold immunoprecipitation buffer, were added and the samples were further incubated overnight at 4°C. After immunoprecipitation, beads were washed four times in 3% NETF buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 3% vol/vol NP-40). The NETF buffer was then replaced with 2× sample buffer and placed in a boiling water bath for 10 min. Immunoprecipitated samples were then separated by SDS-PAGE. For SDS-PAGE, samples were diluted in sample buffer and placed in a boiling water bath for 5 min. Equal amounts of protein were separated on 7.5–15% gradient gels and transferred onto nitrocellulose overnight at 100 mA. For all antibodies, except antiphosphotyrosine (which was blocked with 3% BSA in TBS), blots were blocked in 5% skim milk (Carnation) in TBS overnight at 4°C. Primary antibodies used included antiphosphotyrosine (mAb 4G10; Upstate Biotechnology Inc.), anti-panTrk 203 , antiextracellular signal-regulated kinase (anti-ERK) 1 and 2 (pAb C-18; Santa Cruz), anti-tyr/thr phosphorylated ERK1 and 2 (Promega Corp.), anti-Akt and antiphosphoserine 473 Akt (New England Biolabs). For Western blots, secondary antibodies used were HRP-conjugated anti-mouse (1:10,000) and anti-rabbit (1:10,000) pAbs (Boehringer Mannhiem Corp.). All incubations were performed in 2.5% skim milk in TBS + 0.1% Tween-20 (Sigma Chemical Co.). For detection, blots were washed with TBS and antibody localization visualized using the ECL chemiluminescence kit (Nycomed Amersham Inc.). 100 μg of total protein was immunoprecipitated, except that after immunoprecipitation, beads were washed twice in 3% NETF buffer, followed by two washes in NETF buffer without NP-40, and a single wash in reaction buffer (20 mM MOPS, pH 7.2, 25 mM β-glycerolphosphate, 5 mM EGTA, 2 mM EDTA, 20 mM MgCl 2 , 2 mM sodium orthovanadate, 1 mM dithiothreitol, PKA inhibitor peptide [Upstate Biotechnology, Inc.], and 5% glycerol). The beads were resuspended in 20 μl of reaction buffer and 10 μl of myelin basic protein cocktail (2 mg/ml MBP in reaction buffer) for ERK assays, or histone H2B cocktail (1.6 mg/ml histone H2B in reaction buffer) for Akt assays. The reactions were initiated with 10 μl of 50 μM γ[ 32 P]ATP (∼3,000 cpm/pmol) in a final volume of 40 μl and incubated for 20 min at 30°C. The reactions were terminated with the addition of 5× SDS sample buffer for 5 min and loaded onto an SDS-PAGE gel. After transfer of protein, the membrane was immunoblotted for the appropriate protein, exposed to film, and the bands excised and subjected to Cerenkov counting. Akt peptide assays were conducted similarly, except an Akt specific substrate peptide was used in place of histone H2B. In brief, the beads were suspended in 20 μl reaction buffer with 10 μl Akt-1 substrate peptide (RPRAATF; Santa Cruz) dissolved as 1 mg/ml in assay buffer, and the reaction was started with the addition of 10 μl of 250 μM γ[ 32 P]ATP (3,000 Ci/mmol prepared in assay dilution buffer). The reaction proceeded for 20 min at 30°C and was stopped by spotting 25 μl on 2.5-cm P81 filter paper (Whatman Inc.) and the remaining protein was charged with loading buffer for Western analysis. The P81 filter papers were washed 10 times in 0.75% phosphoric acid and counted in the scintillation counter to monitor incorporation of γ[ 32 P]ATP. To test the hypothesis that neurotrophins and neuronal activity together regulate neuronal survival in a fashion distinct from either alone, we focussed on postnatal day 1 sympathetic neurons of the neonatal rat superior cervical ganglion (SCG). Survival of these neurons, which are cultured at the time when they undergo naturally occurring cell death in vivo, can be maintained with either NGF or depolarization as induced by chronic KCl treatment . To perform these experiments, sympathetic neurons were selected for five days in 50 ng/ml NGF, were switched to varying concentrations of NGF and/or KCl for two days, and survival was then measured using MTT assays, which measure mitochondrial function. As previously reported, maximal sympathetic neuron survival was obtained with either 10 ng/ml NGF or 50 mM KCl . Interestingly, when suboptimal concentrations of NGF and KCl were added together, these two stimuli acted synergistically to promote neuronal survival . Specifically, while 2.5 ng/ml NGF supported only 29% survival and 5 or 10 mM KCl supported no survival, the combination of 2.5 ng/ml NGF plus 5 or 10 mM KCl supported 46 and 59% neuronal survival, respectively. Similarly, 5 ng/ml NGF caused 53% survival, but the addition of 10 mM KCl, which on its own has no survival effect, supported ∼100% neuronal survival . One possible explanation for this synergy is that KCl causes enhanced synthesis and/or secretion of a nonneurotrophin growth factor, thereby conditioning the media and causing enhanced survival. If this hypothesis were true, the synergistic survival should be density-dependent. To test this possibility, sympathetic neurons were plated in 50 ng/ml NGF at densities of 350 and 1,500 cells/well for five days . Neurons were then switched to varying concentrations of NGF and/or KCl for an additional two days. The survival of these cultures was measured by MTT assay. This analysis demonstrated that the cell density had no effect on synergistic survival mediated by NGF and KCl. To confirm that the MTT assays were accurately measuring increases in neuronal survival, we performed similar experiments, but measured the extent of neuronal apoptosis rather than mitochondrial function. 48 h after being switched into various concentrations of NGF and/or KCl, neurons were stained for apoptotic nuclei using TUNEL-labeling and stained for all nuclei using Hoechst 33258 . We then determined the percentage of TUNEL-negative cells in representative fields from each survival condition. This analysis confirmed the results obtained with the MTT assays. Specifically, 57% of cells treated with 5 ng/ml NGF were TUNEL-negative and 17% of cells treated with 10 mM KCl were TUNEL-negative . In contrast, when neurons were cultured in 5 ng/ml NGF plus 10 mM KCl, the number of TUNEL-negative neurons was increased to the same level as observed with 10 ng/ml NGF or 50 mM KCl . Thus, suboptimal amounts of NGF and KCl synergistically support sympathetic neuron survival in a density-independent fashion. Previous work with cortical neurons indicates that depolarization-induced survival requires an autocrine neurotrophin loop . To determine if a similar mechanism could explain the functional synergy observed here, we asked whether KCl-mediated survival requires Trk receptor activation. Lysates of neurons maintained in varying concentrations of NGF or KCl were immunoprecipitated with anti-panTrk, and the level of Trk receptor activation monitored by Western blot analysis with antiphosphotyrosine . Both 5 and 10 ng/ml NGF led to a robust increase in Trk autophosphorylation, but Trk autophosphorylation was undetectable in the presence of KCl, as previously reported by ourselves and others . We then determined whether TrkA activation was necessary for KCl-mediated survival using a recombinant adenovirus that expresses a kinase-inactive form of TrkA that dimerizes with, and inhibits the activity of, endogenous TrkA . NGF-selected neurons were infected with this dnTrkA adenovirus at various MOIs, and were then switched to 10 ng/ml NGF or 50 mM KCl. MTT assays revealed that dnTrkA caused a concentration-dependent decrease in NGF-mediated survival, but had no effect on KCl-mediated survival . In contrast, infection with a control adenovirus had no effect on survival in either condition . We confirmed that Trk activity is not necessary for KCl-induced survival using the pharmacological Trk antagonist, K-252a ; neurons were exposed to varying concentrations of NGF or KCl plus or minus 200 nm K-252a, and were then analyzed biochemically for TrkA activation, using MTT assays and TUNEL to monitor survival. Western blot analysis of anti-panTrk immunoprecipitates with antiphosphotyrosine revealed that, as predicted, K-252a completely eliminated NGF-mediated TrkA autophosphorylation. MTT assays revealed that 200 nM K-252a decreased NGF-dependent sympathetic neuron survival by 60%, but had no effect on survival mediated by KCl . In addition, 200 mM K252a did not support survival in the absence of NGF or KCl . TUNEL confirmed these results; in 10 ng/ml NGF, K-252a treatment led to the same percentage of TUNEL-negative neurons as did NGF withdrawal (10%), while having no effect on the number of TUNEL-negative nuclei in 50 mM KCl . Finally, we confirmed that KCl-mediated, but not NGF-mediated, sympathetic neuron survival requires activation of L-type calcium channels, using the L-type calcium channel blocker, nifedipine . TUNEL-labeling of sympathetic neurons maintained in NGF or KCl plus or minus 1 μM nifedipine revealed that this drug completely inhibited KCl-, but not NGF-mediated survival . Moreover, biochemical analysis confirmed that nifedipine had no effect on Trk receptor activation in the presence of NGF or KCl . Thus, KCl-dependent survival requires activation of L-type calcium channels, and NGF-dependent survival requires TrkA. To determine potential intracellular convergence points for KCl and NGF-mediated survival, we examined two downstream substrates; the ERKs (MAP kinases), which are activated in sympathetic neurons by depolarization and by NGF , and the serine–threonine kinase Akt, which is activated by TrkA , and is required for NGF-mediated sympathetic neuron survival . To perform these experiments, NGF-selected sympathetic neurons were acutely stimulated with varying concentrations of NGF or KCl for 15 min, and then analyzed biochemically. Western blot analysis of neuronal lysates with phospho-specific ERK antibodies revealed that both NGF and KCl caused a dose-dependent increase in ERK phosphorylation, as monitored by phospho-specific antibodies for tyrosine and threonine (tyr/thr) or for tyrosine alone . We also performed in vitro kinase assays using myelin basic protein as a phospho-acceptor substrate to monitor ERK activity. This analysis revealed that while both NGF and KCl induced increased phosphotransferase activity of ERK in a concentration-dependent fashion, higher levels of activation were observed with NGF than with KCl . Similar results were obtained when Akt activity was assessed. Western blot analysis with a phospho-specific (serine 473) Akt antibody revealed that NGF and KCl both caused a concentration-dependent increase in Akt phosphorylation, with NGF being more effective than KCl . To more accurately measure Akt activity, we also performed in vitro kinase assays using two substrates, histone H2B and the Akt specific substrate PRPAATF. With histone H2B, results were similar to those obtained using the phospho-Akt antibody . Results differed slightly, however, with the Akt-specific substrate; in this case, both NGF and KCl increased Akt phosphotransferase activity in a dose-dependent fashion to approximately similar levels . Thus, somewhat surprisingly, depolarization was able to increase Akt activity to levels similar to NGF-induced TrkA activation at doses where both NGF and KCl mediate maximal survival. Together, these results indicate that depolarization leads to robust activation of both the ERKs and Akt in sympathetic neurons. Since both of these substrates are known downstream targets of Ras , and because Ras previously has been shown to be activated by calcium influx , we hypothesized that depolarization-induced neuronal survival required Ras-dependent stimulation of either the PI3-kinase–Akt pathway or the MEK–ERK pathway. To test this hypothesis, we selectively blocked Ras and/or each of these two signaling pathways. We first determined whether KCl-mediated survival required Ras activation, using an adenovirus expressing a dominant-inhibitory form of Ras . NGF-selected neurons were infected with 200 MOI of adenovirus, expressing either dnRas or green fluorescent protein (GFP), and then were switched to KCl for two days. MTT assays revealed that dnRas expression decreased survival 60% in the presence of 50 mM KCl, a decrease similar to that observed in 10 ng/ml NGF . The GFP adenovirus had no effect on neuronal survival. We next determined if either of the two downstream Ras pathways, PI3-kinase–Akt or MEK–ERK, were also essential for KCl-induced survival using two pharmacological agents; LY294002, which selectively inhibits PI3-kinase , and PD98059, which selectively inhibits MEK . NGF-selected neurons were maintained for 48 h in 50 mM KCl or 10 ng/ml NGF plus or minus 100 μM LY294002 or 75 μM PD98059, and were then analyzed by MTT assays and TUNEL. MTT assays revealed that inhibition of PI3-kinase with LY294002 reduced KCl-induced sympathetic neuron survival by ∼40%, while treatment with PD98059 had no effect . The effect of cotreatment with both drugs was similar to LY294002 alone . In contrast, LY294002 completely inhibited survival mediated by NGF , as previously reported . TUNEL confirmed these results; PD98059 had no effect on numbers of TUNEL-negative neurons in either KCl or NGF, whereas LY294002 decreased the proportion of TUNEL-negative neurons to 42 and 10% respectively, in the presence of 50 mM KCl and 10 ng/ml NGF . To confirm that this concentration of PD98059 inhibited MEK, in spite of having no effect on neuronal survival, we examined the downstream MEK substrates, the ERKs. Western blot analysis of sympathetic neurons treated with 10 ng/ml NGF or 50 mM KCl plus or minus 75 μM PD revealed that, as predicted, this drug inhibited both tyrosine/threonine and tyrosine phosphorylation of the ERKs, but had no effect on Akt phosphorylation . Thus, the MEK–ERK pathway is apparently not required for depolarization-induced sympathetic neuron survival. Since PI3-kinase is necessary for 40–50% of KCl-mediated neuronal survival, we next determined whether KCl-induced survival also required the PI3-kinase substrate Akt, using an adenovirus expressing dnAkt from a TTA-inducible promoter (Boudreau, M., C. Tudan, and D.R. Kaplan, unpublished data). NGF-selected sympathetic neurons were infected with 10 to 100 MOI dnAkt adenovirus and 50 MOI TTA-expressing adenovirus (which transactivates the promoter for dnAkt), and were then maintained in 50 mM KCl for two days. As a control, neurons were infected with 50 or 200 MOI of the TTA adenovirus alone. MTT analysis revealed that dnAkt reduced KCl-mediated survival ∼40%, whereas the TTA adenovirus on its own had no effect . In contrast, similar experiments with 10 ng/ml NGF revealed that, as previously reported , dnAkt completed inhibited NGF-induced survival . Together, these data indicate that blocking either Ras, PI3-kinase, or Akt is sufficient to reduce KCl-mediated survival by 40–50%. To test whether these results reflect the presence of a linear Ras–PI3-kinase–Akt survival pathway, we determined whether inhibiting Ras or PI3-kinase blocked the KCl-induced phosphorylation of Akt. Initially, we examined Ras: neurons were infected with the dnRas adenovirus and were then acutely activated with 50 mM KCl or 10 ng/ml NGF for 15 min. Western blot analysis with antiphospho-Akt revealed that dnRas blocked Akt activation in response to 50 mM KCl , suggesting that Akt activity is dependent upon Ras activity. Similarly, inhibition of PI3-kinase with 100 μM LY294002 revealed that treatment with LY294002 completely blocked the KCl-induced phosphorylation of Akt, as assayed either by Western blot analysis with antiphospho-Akt or Akt kinase assays using the Akt-specific substrate . Thus, calcium influx through L-type calcium channels causes activation of a Ras–PI3-kinase–Akt pathway that is essential for ∼50% of KCl-induced neuronal survival. On the basis of these data, we hypothesized that the synergistic survival seen with NGF and KCl might be mediated by a convergent stimulation of the Ras–PI3-kinase–Akt pathway. To test this hypothesis, NGF-selected neurons were induced for 15 min with 5 ng/ml NGF plus 10 mM KCl, a combination that mediated survival synergistically , and were then analyzed biochemically. Western blot analysis with antiphospho-Akt, followed by quantitative densitometry, revealed that while 10 mM KCl had no detectable effect on Akt phosphorylation , the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to levels of Akt phosphorylation that were higher than either of these concentrations of NGF alone . Similar results were obtained when ERK phosphorylation was examined. 10 mM KCl did not detectably increase ERK tyrosine/threonine phosphorylation above controls, but the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to significantly higher levels of ERK phosphorylation than did treatment with either concentration of NGF alone . We then determined the functional necessity of this convergent activation for sympathetic neuron survival. We first examined Ras: NGF-dependent neurons were infected with the dnRas adenovirus, were switched to 5 ng/ml NGF plus 10 mM KCl, and survival was determined two days later by MTT assays. This analysis revealed that inhibition of Ras decreased the synergistic survival ∼50%, a result similar to that seen with NGF or KCl alone . Coincident with the decrease in neuronal survival observed with dnRas, we also observed a partial decrease in the downstream activation of Akt and the ERKs, as assessed by Western blots with phosphorylation-specific antibodies . We then examined the necessity for PI3-kinase or MEK in synergistically-mediated survival using the pharmacological inhibitors LY294002 and PD98059. MTT assays of neurons maintained in 50 ng/ml NGF with 10 mM KCl plus or minus one of these inhibitors revealed that inhibition of PI3-kinase with LY294002 completely blocked neuronal survival, whereas inhibition of MEK with PD98059 had no effect . Cotreatment with both LY294002 and PD98059 gave results similar to those observed with LY294002 alone . Thus, PI3-kinase activity is essential for all of the synergistic survival effects seen with NGF plus KCl. Finally, we examined the necessity for Akt using the dnAkt adenovirus: neurons were infected with 50 MOI of the TTA-expressing adenovirus and 10 to 100 MOI of the dnAkt adenovirus, and then switched to 5 ng/ml NGF plus 10 mM KCl for two days. MTT assays revealed that inhibition of Akt completely blocked the ability of NGF plus KCl to maintain sympathetic neuron survival . Together with the LY294002 experiments, these data indicate that the synergistic survival observed with depolarization and NGF is mediated via convergence onto the PI3-kinase–Akt pathway. Together, these data indicate that Ras–PI3-kinase–Akt is one of the survival pathways induced by activation of neuronal L-type channels, and that this pathway is essential for the synergy between NGF and depolarization. A second, calcium-activated pathway that might also be involved in neuronal survival involves calcium/calmodulin-dependent protein kinase II . To determine the potential importance of this pathway for sympathetic neuron survival, neurons were maintained in various concentrations of NGF and/or KCl plus or minus 10 μM KN-62, a specific pharmacological blocker of CaMKII . MTT assays two days later revealed that 10 μM KN-62 dramatically reduced neuronal survival in 50 mM KCl, but had no effect on survival mediated by 10 ng/ml NGF or by 5 ng/ml NGF plus 10 mM KCl . TUNEL assays confirmed this result: the number of TUNEL-negative cells was similar in NGF plus or minus KN-62, but a large decrease in TUNEL-negative cells was observed when KN-62 was added to 50 mM KCl . Thus, in addition to a Ras–PI3-kinase–Akt pathway, calcium influx through L-type channels also mediates neuronal survival through a CaMKII-dependent pathway. To determine whether Akt activation was also downstream of CaMKII, we assessed KCl-mediated Akt phosphorylation in the presence of 10 μM KN-62. Western blot analysis revealed that inhibition of CaMKII with KN-62 had no effect on Akt phosphorylation as induced by NGF, KCl, or combinations of NGF plus KCl . In contrast, KN-62 dramatically reduced KCl-induced ERK tyrosine/threonine phosphorylation, but had no effect on NGF-induced ERK phosphorylation . Interestingly, KN-62 partially blocked ERK phosphorylation induced by 5 ng/ml NGF plus 10 mM KCl , indicating that the synergistic activation of ERKs seen in NGF plus KCl is partially due to CaMKII. Together, these data indicate that: CaMKII is a component of an important depolarization-induced survival pathway in sympathetic neurons; Akt is not downstream of CaMKII in this survival pathway; and this CaMKII pathway is not an important player in the synergistic survival observed with NGF and KCl. Instead, NGF and depolarization apparently converge onto a distinct Ras–PI3-kinase–Akt pathway to synergistically regulate survival. Data presented here demonstrate that combinations of trophic support (NGF) and neuronal activity (KCl-induced depolarization) have a positive, synergistic effect on the survival of peripheral sympathetic neurons at concentrations of NGF and KCl that are themselves suboptimal for neuronal survival. Moreover, these experiments demonstrate that the synergistic survival is due to convergence onto the PI3-kinase–Akt survival pathway, and that in the case of KCl, activation of this pathway is a consequence of calcium-mediated Ras activation . In contrast, although KCl-mediated survival also requires concomitant activation of CaMKII, this protein is dispensable when both KCl and NGF are both present , and it is not involved in activating Akt under any of the conditions studied here. This convergent activation of Akt, a signaling protein known to be essential for the survival of several neuronal populations , may provide a general mechanism for coordinating the survival effects of growth factors and neural activity throughout the developing nervous system. These findings have important implications for neuronal survival in the developing and mature injured nervous system. During development, ∼50% of central and peripheral neurons undergo apoptosis. Although neurotrophin signaling has traditionally been considered to be the major mechanism for the matching of neurons to their targets , a large body of work indicates that establishment of appropriate neural activity is also important . Evidence presented here demonstrates that, when NGF is limiting, levels of depolarization that themselves have no survival effect, synergize with NGF to support sympathetic neuron survival. Since NGF concentrations in vivo during naturally occuring sympathetic neuron death are limiting , our findings imply that developing neurons that are active will have a competitive survival advantage over those that are not, even when both are exposed to similar limiting amounts of target-derived NGF. This mechanism, therefore, ensures selection of neurons that pathfind their way to an appropriate target early enough to sequester target-derived NGF and participate in a functional circuit. The validity of such a model derives from the finding that inhibition of either pre- or postganglionic activity is sufficient to enhance developmental sympathetic neuron apoptosis . Does this mechanism generalize to neurons other than sympathetic neurons? Results presented here demonstrating synergistic survival effects of neurotrophins and depolarization are strikingly similar to those previously reported for central neurons, such as retinal ganglion cells , which require both growth factors and either depolarization or neurotransmitters for their survival in culture. Thus, central and peripheral neurons may not be as different in this regard as previously thought. It may simply be that cooperative interactions between growth factors and activity are always essential for survival of central neurons, whereas they are only essential for peripheral neurons when trophic support is suboptimal. However, since NGF concentrations are suboptimal during development and neural activity is essential for survival during naturally occurring sympathetic neuron death , then these apparent differences may be a function of culture conditions, rather than a reflection of the in vivo situation. In this regard, convergent activation of Akt by growth factors and activity may be just as important to central neurons as to peripheral neurons, since Akt is a necessary survival protein for CNS neurons, such as cerebellar granule cells . It is also clear, however, that in the case of central neurons, growth factors and depolarization collaborate to regulate neuronal survival by more than one mechanism . How do NGF and KCl converge to activate Akt? Neurotrophin binding to TrkA leads to activation of the PI3-kinase–Akt pathway . The activation of PI3-kinase is thought to occur both via Ras and Gab-1 . Moreover, Ras, PI3-kinase, and Akt activity have all been shown to be essential for NGF-mediated survival of sympathetic neurons . With regard to KCl, calcium influx has been shown to activate Ras via Ras–GRF and, depending on the neuronal context, may or may not activate PI3-kinase . In this paper, we demonstrate that calcium influx also activates Akt, and that this activation involves both Ras and PI3-kinase . Moreover, our data demonstrate that synergistic survival is dependent on Ras, PI3-kinase, and Akt activity, indicating that NGF and KCl converge directly on these proteins to coordinately regulate neuronal survival. The synergistic activation of signaling proteins by two growth factors previously has been observed in a number of systems, such as Erk 2 by stem cell factor and IL-3 , and p38MAPK by IL-2 and IL-12 . Such biochemical synergy can occur on several levels. We favor a hypothesis in which KCl causes a nonlinear activation of Ras or PI-3 kinase beyond the activation or localization observed by NGF alone, stimulating the production of PI3-kinase–derived second messenger molecules that function as activators of PDK1, PDK2, and Akt itself, and which recruit Akt to the plasma membrane, an event necessary for increasing Akt activity . The synergistic activation of Akt would therefore be due to the combined actions of multiple Akt activators, some of which are activated linearly and others nonlinearly. In a recent study examining depolarization-induced survival of neuroblastoma cells, Yano et al. 1998 showed that in neuroblastoma cells, calcium influx mediates cell survival via a PI3-kinase–independent CaMK kinase–Akt pathway. In contrast, our findings indicate that depolarization-induced Akt activation in primary sympathetic neurons is totally dependent upon PI3-kinase. Moreover, although KCl-mediated sympathetic neuron survival is highly dependent on CaMKII, this protein is dispensable for the Akt-dependent synergistic survival effect seen with NGF and KCl. These findings do not imply that CaMKII is unimportant for sympathetic neurons exposed to both NGF and depolarization. In fact, data presented here indicate that CaMKII activity is essential for the synergistic ERK activation seen in response to these two extrinsic cues. Although this ERK activation is not essential for sympathetic neuron survival, it may well be essential for other neuronal responses. In particular, the MEK–ERK pathway is required for neurite extension in PC12 cells , and our recent data indicate that MEK is equally important for growth of sympathetic neurons in vivo and in vitro (Zirrgeibel, U., D. Lederfein, J. Atwal, J. Toma, F. Miller, and D. Kaplan, unpublished data). Thus, the biochemical convergence of NGF and KCl on the ERKs may well be important for neuronal growth and plasticity . The involvement of CaMKII in this convergence fits well with its proposed role in neuronal plasticity . In summary, these findings demonstrate that, like central neurons, NGF and depolarization synergistically regulate the survival of peripheral sympathetic neurons under conditions where trophic support is suboptimal, such as during naturally occurring neuronal death. This synergistic survival effect is mediated by intracellular convergence on a Ras–PI3-kinase–Akt pathway. Since Akt is an essential survival molecule for several neuronal populations, this intracellular convergence may represent a general mechanism for coordinating the survival effects of growth factors and neural activity throughout the nervous system. | Study | biomedical | en | 0.999995 |
10477752 | Mouse Pam212 keratinocytes, an immortalized cell line spontaneously derived from a primary keratinocyte culture and GP+E 86 cells producing retroviruses that express either V12H-Ras or the catalytic subunit of PI3K targeted to the plasma membrane (p110CAAX) were grown in DME supplemented with 10% FCS (GIBCO BRL), 100 μg/ml ampicillin, 32 μg/ml gentamicin, and 100 μg/ml amphotericin B (Sigma Chemical Co.). Cells were grown at 37°C in a humidified 5% CO 2 atmosphere. Retroviruses encoding V12H-Ras with a neomycin resistance marker or controls with the neomycin resistance marker alone have been previously described . Retrovirus encoding p110CAAX was generated by subcloning the BamHI insert from pSG5-p110CAAX into pLXSP3. pLXSP3 was a gift from A. Sewing (Imperial Cancer Research Fund, London, UK) and was generated by ligating the HindIII-XbaI fragment of pBabe puro, containing the puromycin resistance gene, into pLXSN. Infective retroviruses were obtained by growing GP+E 86 cells to confluence and collecting the culture medium. Supernatants were filtered through 0.45-mm pores and frozen as aliquots in the presence of polybrene. Retrovirus-containing supernatants were added to subconfluent cultures of Pam212 cells and replaced 16 h later with fresh medium. Pools of infected cells were obtained after selection with 400 μg/ml G418 (Calbiochem-Novabiochem Co.) or 1.5 μg/ml puromycin (Boehringer Mannheim) for 7 d, during which time all control noninfected cells died. More than 200 colonies were pooled from each infection. The following antibodies were used: rat monoclonal anti-mouse E-cadherin (ECCD-2; a gift from Dr. M. Takeichi, Kyoto University, Kyoto, Japan); mouse monoclonal anti–β-catenin, mouse monoclonal anti–α-catenin, mouse monoclonal anti-GSK3β, and mouse antiphosphotyrosine (PY20) (Transduction Laboratories); mouse monoclonal anti–c-H-Ras (Ab-1) and mouse monoclonal anti-pan Ras recognizing the normal and activated forms of H-Ras (Oncogene Science, Inc.); mouse monoclonal anti–6-His (CLONTECH Laboratories); rabbit anti–GSK3β-P-Ser 9 antiserum (Chemicon International Inc.; provided by Dr. F. Wandosell, Centro de Biología Molecular, Madrid, Spain); rabbit polyclonal anti-GST (Sigma Chemical Co.); rabbit antipeptide antiserum anti–α-catenin (VB1) and rabbit antipeptide antiserum anti–β-catenin (VB2) ; rabbit polyclonal anti-APC (N-15) (Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti–PI3 kinase p85 and anti–PI3 kinase p110α (Upstate Biotechnology); and mouse monoclonal anti–β-tubulin (Amersham). Secondary antibodies included: BODIPY-conjugated goat anti–rat IgG, anti–mouse IgG and anti–rabbit IgG (Molecular Probes Inc.); AMCA-conjugated rabbit anti–rat IgG, Cy5-conjugated donkey anti–rabbit IgG (Jackson ImmunoResearch Laboratories) and peroxidase-conjugated sheep anti–rat IgG, anti–mouse IgG and anti–rabbit IgG (Amersham). Recombinant constitutively active Ras (V12Ras), dominant negative Ras (N17Ras), C3 transferase and dominant negative Rac (N17Rac) were prepared as glutathione-S-transferase (GST) fusion proteins in Escherichia coli , purified using glutathione-Sepharose beads, thrombin cleaved, dialyzed, and concentrated as described . The activity of each batch of recombinant proteins was tested in fibroblasts and keratinocytes as described . Recombinant GST–β-catenin fusion protein was generated by isolation of full-length human β-catenin cDNA from plasmid pQE32 (a gift of Dr. J. Behrens) by SmaI-SalI digestion and ligation into pGEX-5X-2 plasmid (Pharmacia Biotech). Recombinant His-p85α wt fusion protein was produced in COS cells as described and purified with His-Trap columns (Pharmacia Biotech) following the instructions of the manufacturer. Confluent patches of keratinocytes were injected essentially as described and visualized by mixing the recombinant proteins with dextran conjugated to Texas red (mol wt 10,000; Sigma Chem-ical Co.). After microinjection, cells were fixed in either cold methanol or 3.7% buffered formaldehyde for 5 min at 4°C and rinsed in PBS. Staining with the different antibodies was performed as described . In extraction experiments, cells grown on coverslips were treated with NT buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl 2 , 5 mM Ca 2 Cl, 1% NP-40, 1% Triton X-100) for 15 min on ice before fixation and staining. Preparations were viewed either by confocal microscopy or in an Axiophot photomicroscope (Carl Zeiss). Confocal images were obtained using a laser scanner attached to a Nikon microscope (Optiphot 2). Pictures were processed using the Adobe Photoshop 4.0 software (Adobe Systems, Inc.). Whole cell extracts were obtained from F-25 flasks of 80–90% confluent cells. For total protein extracts, cells were washed twice in cold HMF-Ca buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM Ca 2 Cl) and extracted in 1 ml of S buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl 2 , 5 mM Ca 2 Cl, 2% SDS) containing protease and phosphatase inhibitors (2 mM PMSF, 20 μg/ml aprotinin, 1 mM N -ethylmaleimide, 1 mM sodium orthovanadate, 2 mM hydrogen peroxide) for 5 min at room temperature under continuous agitation. Soluble and insoluble fractions were obtained as previously described using the NT buffer containing the inhibitors described above. Immunoprecipitations of soluble fractions were carried out with different antibodies as previously described and analyzed in 7.5% or 12% SDS-PAGE gels. In P-Tyr immunoprecipitations, controls were performed by adding 5 mM P-Tyr or P-Ser (Sigma Chemical Co.) to soluble fractions. Immunoblotting of the different fractions and immunoprecipitates was performed by transferring the gels to Immobilon P membranes (Millipore Corp.), incubation with the appropriate antibodies, and development with an enhanced chemiluminescent kit (Amersham). Cells were grown in F-25 flasks to 80% confluence in normal growth medium, washed three times in HMF-Ca buffer, and pulse-labeled for 1 h in 1 ml of methionine- and glutamine-free minimal essential medium (GIBCO BRL) supplemented with 4 mM glutamine, 10% FCS and 100 μCi [ 35 S]methionine-cysteine (trans-label; Amersham; 1,000 Ci/mmol). Labeled cells were rinsed three times in normal growth medium containing an excess of cold methionine (0.15 mg/ml), chased in this medium for the indicated times, and the soluble extracts subjected to β-catenin immunoprecipitation. The labeled immunoprecipitates were resolved in 7.5% SDS-PAGE gels, transferred to Immobilon P membranes, and exposed to autoradiography. Bands corresponding to β-catenin were identified by immunoblotting. 35 S-labeled β-catenin detected at the different experimental points was quantified by scanning and digitalization of the autoradiograms with Adobe Photoshop 4.0 and integration with NIH Image 1.62f software. The integrated density obtained at the different times was normalized to that obtained at time 0 h in each experiment. Metabolic labeling of keratinocytes with [ 32 P]orthophosphate was performed as previously described with slight modifications. 20 h after plating the cells, monolayers were labeled in media without phosphate supplemented with 500 μCi/ml of [ 32 P]orthophosphate (acid-free, 5 μCi/ml; Du Pont Company; NEN Life Science Products) for 4 h at 37°C in a CO 2 incubator. After labeling, cells were washed twice with PBS and lysed with NT buffer, containing a cocktail of phosphatase (1 mM sodium orthovanadate, 50 mM sodium fluoride, and 2 mM hydrogen peroxide) and protease inhibitors for 30 min at 4°C with gentle rotation. Extracts were prepared as described, immunoprecipitated with anti–β-catenin antibodies (Transduction Laboratories), and resolved on 7.5% SDS-PAGE gels. Protease and phosphatase inhibitors were maintained during all the immunoprecipitation procedures. Total [ 32 P]phosphate labeling was detected by autoradiography, and the total β-catenin protein was determined in the same gel by immunoblotting. 32 P-labeled and total β-catenin were quantified by scanning and digitalization of the autoradiograms and blots as described. Phosphoamino acids were analyzed based on the method of Boyle et al. 1991 with minor modifications. 32 P-labeled phosphorylated β-catenin bands were excised from membranes and hydrolyzed with 6 N HCl at 110°C for 2 h. The hydrolysates were lyophilized using a speed-vac concentrator and resuspended in 10 μl of pH 1.9 buffer (2.2% formic acid, 7.8% acetic acid, pH 1.9) containing 2 μg each of cold phosphoamino acid standards. The samples were spotted onto thin-layer cellulose plates and amino acid separation was performed on an apparatus running at 1,500 V for 45 min in the first dimension with the pH 1.9 buffer. The second dimension thin layer chromatography was carried out in a mixture of n-buthanol/acetic acid/H 2 O (100:22:50). The individual amino acids were identified by comparison with the internal standards detected by ninhydrin staining. The position of 32 P-labeled phosphoamino acids were determined by autoradiography. Activity of GSK3β was tested in the absence and presence of 50 mM lithium chloride in crude cell extracts prepared following the procedure recently described and using the specific phosphopeptide GSM (RRRPASVPPSPSLSRHS S HQRR, where S indicates a phosphoserine introduced during synthesis) (a gift of Dr. A.J. Harwood, MRC Laboratory for Molecular Cell Biology, London, UK). The difference between the activity obtained in the absence and presence of lithium chloride was represented as the specific GSK3β activity. Pull-down assays were performed using soluble extracts obtained in NT buffer after preclearing with glutathione-Sepharose 4B (Pharmacia Biotech). 200 μl of the precleared extracts were incubated with 100 μl of purified GST–β-catenin fusion protein (0.2 μg/μl) for 2 h at 4°C. For in vitro protein binding assays, 200 ng of both GST–β-catenin and His-p85α wt were mixed in 500 μl of immunoprecipitation buffer and incubated 1 h at 4°C. In all cases, interacting β-catenin–GST complexes were collected by addition of glutathione-Sepharose 4B, washed twice with immunoprecipitation buffer, and finally resuspended in Laemmli sample buffer. Complexes were analyzed in 7.5% SDS-PAGE gels and immunoblotted with the indicated antibodies. Nearly subconfluent cells were transfected in duplicate in P-60 dishes with 4 μg of the pRSV-LacZ containing a β-galactosidase reporter gene and 4 μg of either pTOPFLASH or pFOPFLASH containing multimerized wild-type or mutated Lef-1/Tcf binding sites, respectively, and a luciferase reporter gene (a gift of Dr. H. Clevers, University Hospital, Utrecht, The Netherlands). Transfection was performed with lipofectamine plus (GIBCO BRL). Luciferase and β-galactosidase activities were measured 24 h after transfection. To analyze short-term effects of activated H-Ras on the E-cadherin/catenin complexes, we chose the murine epidermal keratinocyte cell line Pam212, obtained after spontaneous immortalization of a primary keratinocyte cell culture . This cell line maintains all the epidermal characteristics of keratinocytes, expresses a normal H- Ras protooncogene, and is nontumorigenic when injected into athymic nude mice . The dominant active form of H-Ras (V12Ras; 0.5 μg/μl) protein was microinjected in confluent Pam212 keratinocytes showing stable cell–cell contacts, and the cells were fixed and stained for different proteins after 1–24 h of incubation. As shown in Fig. 1 , Fig. 2 Fig. 3 Fig. 4 h after microinjection E-cadherin and α-catenin were completely absent from the cell–cell contacts established between microinjected cells. At the same time, V12Ras induced a strong cytoplasmic staining of endogenous β-catenin . In addition, a diffuse nuclear labeling for β-catenin could be detected in some of the microinjected cells . Microinjection of Pam212 cells with a dominant negative H-Ras protein, N17Ras induced no alterations in the adhesion complexes or β-catenin localization . Our data indicate the specificity of V12Ras effects and are in agreement with previous data using anti–Ras antibodies in human keratinocytes (Braga, V.M.M., M. Betson, and N. Lamarche-Vane, manuscript submitted for publication). 16 h after V12Ras microinjection in Pam212 cells, E-cadherin and α-catenin remained absent from the cell–cell contacts and β-catenin was still faintly detected in the cytoplasm (data not shown), indicating that dominant active H-Ras induces dismantling of E-cadherin/catenin complexes and cytoplasmic accumulation of β-catenin in mouse epidermal keratinocytes. The possible participation of Rho and Rac in the dissociation of E-cadherin complexes induced by V12Ras in confluent mouse keratinocytes was also investigated. However, inhibition of endogenous Rho or Rac affected primarily the adhesion to substratum, resulting in the detachment of the cells from the dish within 2 h without affecting cell–cell contacts (data not shown), in contrast to our previous studies in human keratinocytes . These results might reflect a modulation on the response of cadherin receptors to inhibition of small GTPases because of the cellular context and/or to specific effects of the extracellular matrix . Previous studies have implicated PI3K and MAPK activities in dismantling E-cadherin/catenin complexes induced by activated V12Ras in other systems . In murine keratinocytes, the involvement of PI3K in the destabilization of E-cadherin/catenin complexes induced by activated H-Ras was confirmed by microinjection analysis in the presence of specific inhibitors. Preincubation of Pam212 cells during 30 min with the PI3K inhibitor wortmannin at 200 nM, blocked the effect of V12Ras on the adherens junctions . To further investigate the effect of activated Ras and the involvement of PI3K in the destabilization of the E-cadherin/catenin complexes, we generated stable transfectants of Pam212 cells expressing H-V12Ras by retroviral transduction. Cells were pooled from three independent infection assays, which showed similar results. The solubility of E-cadherin/catenin complexes in control PamNeo (Neo)- and V12Ras-overexpressing (Ras) cells was first analyzed by immunostaining after detergent extraction of the cells. As showed in Fig. 2 b, E-cadherin and β-catenin were preserved at cell–cell contacts of control Neo cells after treatment with NT buffer . Both proteins were removed from cell–cell contacts by the detergent treatment in Ras cells , indicating that H-Ras activation promotes solubilization of the cadherin complexes. This solubilization was prevented by preincubation of Ras cells with wortmannin during 1 h , further supporting the involvement of PI3K activity in H-Ras–induced destabilization of the adhesion complexes. Immunoblotting analysis of the distribution of the different components of the E-cadherin/catenin complexes into the detergent-soluble (S) and insoluble (I) fractions of control neomycin (Neo)- and V12Ras (Ras)-transduced cells is shown in Fig. 3 a. E-cadherin and β-catenin were found in both the soluble and insoluble fractions in Neo cells, whereas in Ras-expressing cells, most of the E-cadherin and β-catenin and an increased proportion of α-catenin were detected in the soluble fraction. Quantification of the data obtained in three independent experiments indicated that 8% and 11% of E-cadherin and β-catenin, respectively, were detected in the insoluble fractions of Ras cells. In contrast, in control Neo cells, 39% of E-cadherin and 29% of β-catenin were associated to the insoluble fraction. In addition, the distribution in soluble and insoluble fractions of a control protein, β-tubulin, was not disturbed upon V12Ras overexpression . These results confirm that the components of the E-cadherin complexes were weakly associated to the actin cytoskeleton in the Ras-expressing cells. Immunoblot analysis of total protein extracts of Neo and Ras cells showed that the level of p21 protein increased about 10-fold in PamV12Ras cells . Total protein levels of E-cadherin and α-catenin decreased in V12Ras-overexpressing cells (Ras) as compared with control cells (Neo), but no changes in β-catenin levels were observed . On the other hand, soluble E-cadherin of either Neo and Ras keratinocytes was able to associate in a similar manner to the α- and β-catenin components . Immunoprecipitation of tyrosine-phosphorylated proteins in the soluble fraction of Pam212 keratinocytes showed a similar degree of β-catenin tyrosine phosphorylation in both Neo and Ras cells and the absence of tyrosine phosphorylation in E-cadherin and α-catenin in both cell lines. These results indicated that stable expression of dominant active H-Ras decreases the total levels of E-cadherin and α-catenin without affecting the level of β-catenin and, more significantly, induces the redistribution of the different components to a more soluble cytoplasmic pool. To get further insights into the H-Ras–induced cytoplasmic relocalization of β-catenin, we analyzed the participation of PI3K. Cell extracts obtained from control (Neo) and PamV12Ras (Ras)-transduced cells were immunoprecipitated with antibodies against the regulatory subunit of PI3K (p85α), and the presence of β-catenin was analyzed by immunoblotting. As shown in Fig. 4 a, β-catenin was detected in the p85α immunocomplexes from control Neo cells, but a significant increase of associated β-catenin was detected in the immunocomplexes of Ras cells. However, the level of total (data not shown) and immunoprecipitated p85α was similar in both cell types. The reverse experiment, immunoprecipitation with anti–β-catenin antibodies and immunoblotting with anti-p85α , also showed an increase in β-catenin/p85α association in Ras cells. In addition, the strong association of β-catenin with the PI3K complex in Ras cells was also detected when the p110α catalytic subunit was immunoprecipitated . Interestingly, the interaction of p110α with its regulatory subunit, p85α, was also significantly increased in Ras cells. The interaction of β-catenin with PI3K was confirmed in pull-down experiments with recombinant GST–β-catenin. As shown in Fig. 4 b, an excess of recombinant GST–β-catenin interacts similarly with endogenous p85α derived from protein extracts of Neo and Ras cells. These results suggest that endogenous p85α, from either control or V12Ras cells has the same ability to associate with recombinant β-catenin. The quantitative differences found in the in vivo β-catenin–p85α interaction might reflect different properties of endogenous β-catenin in control and Ras-expressing cells. GST–β-catenin recombinant protein also showed a strong interaction with E-cadherin and α-catenin in the pull-down assays , indicating the functionality of the protein in in vitro interactions. A direct interaction of β-catenin with the regulatory subunit of PI3K, p85α, was further demonstrated in in vitro binding assays using recombinant GST–β-catenin and His-p85α wt fusion proteins . Taken together, these results indicate the ability of β-catenin to associate with PI3K in Pam212 keratinocytes. This interaction is significantly increased in V12Ras-overexpressing cells, where both the regulatory and catalytic subunits of PI3K form part of this novel β-catenin complex. The above results prompted us to examine the interaction of β-catenin with other known partners in Neo versus Ras cells. Because of the relevance of the β-catenin–APC interaction in the regulation of cytoplasmic β-catenin levels, we investigated whether activated H-Ras could influence such interaction. High levels of soluble β-catenin coprecipitated with APC in control Neo cells , whereas very low levels of β-catenin were detected in the APC immunocomplexes of PamV12Ras cells , even though the level of APC was similar in both cell types . On the other hand, similar levels of GSK3β protein were detected in whole cell extracts from control Neo and Ras cells , but no stable association of β-catenin with GSK3β was found in either cell line . To investigate whether the disturbed interaction of β-catenin with APC induced by activated H-Ras was due to inhibition of GSK3β, we analyzed its activity in crude cell extracts from control PamNeo and PamV12Ras cells. Ras-transformed cells from three independent infection assays (Ras1, 2, and 3) showed a level of GSK3β activity sensitive to lithium chloride similar or slightly higher to that of control cells . In addition, endogenous phosphorylation of GSK3β at Ser9 in Ras-expressing cells was slightly higher than in control Neo cells . These results indicate that the overall activity of GSK3β in Pam212 cells was not significantly decreased by overexpression of activated H-Ras. Nevertheless, a slight increase of about twofold in basal Lef-1/Tcf–dependent transcriptional activity was observed in Ras-transformed cells , suggesting a weak activation of β-catenin signaling in these cells. To further analyze the status of β-catenin in control and Ras-transformed keratinocytes, we performed in vivo phosphorylation analysis on both cell types after 4 h of metabolic labeling. The level of β-catenin phosphorylation in PamV12Ras cells resulting from two independent infections, Ras1 and Ras2, was significantly lower than that of parental Pam212 cells . Quantitive analysis of the phosphorylated and total immunoprecipitated β-catenin indicated that the ratio of [ 32 P]β-catenin in Ras1 and Ras2 keratinocytes was 6% and 17% relative to that of the parental cells. In addition, the phosphoamino acid analysis of immunoprecipitated β-catenin showed that P-Ser was the major phosphorylated amino acid, and, as expected, the relative P-Ser content of β-catenin in Ras-transduced cells was much lower than that of parental cells . The P-Tyr content of β-catenin could not be detected in the phosphoamino acid analysis, probably because of the lower stability of P-Tyr residues to acid hydrolysis or to a low level of P-Tyr labeling in the 4-h pulse, in contrast to the steady state levels detected in the immunoprecipitation analysis . These results indicate that activated H-Ras induces cytoplasmic accumulation of hypophosphorylated β-catenin and inhibits its interaction with APC in keratinocytes, through a mechanism apparently independent of stable interactions with or reduced activity of GSK3β. To get additional information on the involvement of PI3K in the stabilization of cytoplasmic β-catenin, Pam212 cells overexpressing the membrane-bound form of p110α subunit of PI3K (p110CAAX) and puromycin-resistant control cells were generated by retroviral transduction. Pam-p110 cells from two independent infections (p110S1 and p110S2) behaved similarly to PamV12Ras cells with respect to β-catenin and APC content. Thus, no changes in the total level of both proteins and a strong reduction in the level of β-catenin–APC interaction were observed in p110 cells, when compared with control puromycin-resistant (Puro) cells. The effect of p110α expression on β-catenin localization was further investigated by microinjection of recombinant GST–β-catenin fusion protein. 4 h after microinjection, a strong cytoplasmic and nuclear accumulation of β-catenin was observed in PamV12Ras (Ras) and Pamp110α (p110) cells . In both cell types, β-catenin staining was also detected at the cell–cell contacts of the microinjected cells. In contrast, β-catenin staining was exclusively detected at the cell–cell contacts after microinjection of the GST–β-catenin fusion protein in the parental Pam212 (Pam) cells , possibly because of its quick degradation or incorporation into junctions. In Ras and p110 microinjected cells staining with anti–GST antibodies showed localization of the exogenous protein at the membrane, cytoplasm, and nucleus but only at the membrane junctions in Pam cells (data not shown). On the other hand, staining of E-cadherin at the cell–cell contacts was not modified in p110 cells , indicating that PI3K activity alone is not sufficient to disrupt cell–cell adhesion in mouse keratinocytes. The stabilization of endogenous β-catenin in cells overexpressing either V12Ras or p110-CAAX was confirmed by pulse–chase experiments. As can be observed in Fig. 8 a, β-catenin was quickly degraded in both control puromycin (Puro)- and neomycin (Neo)-transduced cells. In contrast, the metabolic stability of endogenous β-catenin was significantly increased in V12Ras (Ras)- and p110CAAX (p110)-overexpressing cells . Quantification of the autoradiograms shown in Fig. 8 a indicated a half-life for β-catenin of ∼1 h in control Pam cells, and of >4 h in V12Ras- and p110CAAX-overexpressing cells . These results demonstrate that the stability of cytoplasmic β-catenin is increased by the expression of V12Ras and constitutively active PI3K in Pam212 keratinocytes. Alterations in the expression or function of the E-cadherin/catenin adhesion system occur frequently in a wide variety of human carcinomas . Indeed, a causal role for the loss of E-cadherin has been recently demonstrated during the transition from adenoma to invasive carcinoma . The molecular mechanisms underlying the loss of expression or functionality of individual components of the cadherin/catenin complexes in tumorigenesis are still poorly understood. The implication of β-catenin in the Wnt signaling pathway has opened new avenues in the study of the modulation of the cadherin–adhesion complexes during tumor progression . However, it is still largely unknown if other signaling pathways frequently activated in tumor cells and, more specifically, H-Ras activation can influence the signaling activity of β-catenin. Our results indicate that activation of H-Ras induces the dismantling of E-cadherin/catenin complexes and the stabilization of hypophosphorylated cytoplasmic β-catenin through signaling pathway(s) involving PI3K that lead to the inhibition of β-catenin–APC interaction and to a stable interaction of β-catenin with PI3K complex. In addition, activation of PI3K is sufficient to promote the stabilization of cytoplasmic β-catenin and its nuclear translocation. H-Ras expression in mouse epidermal keratinocytes induced a decrease in the total levels of E-cadherin and α-catenin without significantly affecting the levels of β-catenin, as previously described in intestinal and mammary cells . In addition, in V12Ras-expressing keratinocytes, most of the E-cadherin and associated β-catenin are found in the detergent soluble fraction, indicating a weak interaction of the E-cadherin/catenin complexes with the cytoskeleton in those cells. We suggest that activation of H-Ras can affect the E-cadherin/catenin complexes in epidermal keratinocytes via signaling mechanisms different from those leading to increased P-Tyr, in contrast to previous reports using other cell types . Despite the biochemical effects on cadherin complexes induced by activated H-Ras in mouse keratinocytes, no significant differences could be observed in the morphological phenotype of the parental Pam212, control PamNeo, and PamV12Ras cells when growing at medium high density. These results differ from those previously reported after Ras transformation of MDCK cells or mammary MCF10A cells , where a more epitheloid or fibroblastic phenotype was sometimes observed. These differences may be related to the level of activated H-Ras expression obtained in the different studies or may be specific of the cell system analyzed. In this sense, it is worth mentioning that tyrosine phosphorylation of catenins plays a positive role in the stratification process in differentiated keratinocytes . In spite of those differences, our results indicate that even in situations where a full phenotypic transformation is not observed, activated H-Ras is able to significantly modify the E-cadherin/catenin complexes as in the murine Pam212 epidermal keratinocytes. Thus, the modification of the adhesion complexes might be an early event driven by H-Ras transformation. Our microinjection studies are in agreement with those showing that V12Ras leads to the loss of E-cadherin and β-catenin from cell–cell contacts in MDCK cells, dependent on both PI3K and MAPK activities . However, no cytoplasmic localization of β-catenin was observed and the α-catenin status was not analyzed in this study. These differences can be due to different fixation and permeabilization procedures or to the cell systems analyzed. In addition to the involvement of PI3K in the Ras-dependent destabilization of cadherin complexes and relocalization of β-catenin, our results provide evidence for a strong association of cytoplasmic β-catenin with PI3K both in vivo and in vitro. In vitro binding studies clearly show a direct interaction between β-catenin and p85α, , suggesting that the in vivo interactions might be mediated by the regulatory subunit of PI3K. In fact, p85α is able to interact with endogenous β-catenin in control Pam212 cells, and this interaction is significantly increased in V12Ras-overexpressing cells. Interestingly, concomitant with the increased association with the regulatory subunit, association of the catalytic subunit of PI3K with β-catenin is strongly induced in PamV12Ras . These observations suggest that the PI3K heterodimer is involved in the stabilization of cytoplasmic β-catenin induced by activated H-Ras in keratinocytes. The involvement of PI3K in the Ras-induced β-catenin stabilization is further supported by analysis of Pam212 keratinocytes overexpressing the membrane-bound catalytic subunit of PI3K (Pamp110 cells). In these cells as well as in PamV12Ras cells, microinjected recombinant β-catenin is stabilized in the cytoplasm and translocated to the nucleus after 4 h of microinjection. In contrast, the exogenous catenin is apparently degraded or incorporated into junctions in control cells . Furthermore, the pulse–chase analysis demonstrates that expression of V12Ras or p110CAAX significantly reduced the turnover of endogenous β-catenin. These results indicate that constitutive activation of PI3K is sufficient for stabilization of β-catenin and its translocation to the nucleus, although not for disruption of cell–cell junctions. One interesting possibility is that other pathways activated by V12Ras, like MAPK, are required for initial dismantling of adhesion complexes, and that cytoplasmic β-catenin is then stabilized because of its interaction with activated PI3K. This is now being investigated in further detail. Here, we provide evidence to support that H-Ras-induced stabilization of β-catenin occurs through interference with its APC interaction and decrease in the levels of serine phosphorylation. The ability of β-catenin to associate with APC is significantly reduced in keratinocytes stably expressing V12Ras. However, in contrast to previous reports, our present results indicate that the stabilization of hypophosphorylated cytoplasmic β-catenin induced by activated H-Ras might occur by mechanism(s) independent of stable interaction with GSK3β or significant alterations in the total GSK3β activity . Inhibition of additional kinase(s) or activation of phosphatase(s) may be involved in the dephosphorylation of β-catenin upon Ras activation. In this sense, it has been reported that β-catenin can also be phosphorylated by diacylglycerol-independent protein kinase C isoforms , and that its stability is modulated by PP2A phosphatase . In Ras-expressing mouse keratinocytes β-catenin might transiently interact with distinct cytoplasmic partners for its translocation to the nucleus. Interestingly, in our Ras-expressing keratinocytes only a slight increment in Lef-1/Tcf–dependent transcriptional activity is observed, suggesting that additional factors are needed to fully induce β-catenin transcriptional activation . Regarding the mechanism(s) leading to β-catenin stabilization, it is not known whether interaction with PI3K can modulate its association to APC or directly contribute to its stabilization in the cytoplasm. Interestingly, β-catenin–APC interaction is also blocked in p110CAAX-overexpressing keratinocytes , but similar levels of total GSK3β activity have been observed in controls, PI3K and V12Ras transformants (Espada, J., and A. Cano, unpublished results). We speculate that p85α (and/or p110α) might compete with other β-catenin partners, such as APC or E-cadherin, rendering the molecule inaccessible for the ubiquitin-proteasome degradation. On the other hand, PI3K activation by H-Ras can further increase the β-catenin–PI3K interaction. The possibility of a recruitment of β-catenin into vesicles through PI3K interaction cannot be discarded, although it is not supported from the immunofluorescent staining . In summary, our results show a direct effect of H-Ras activation on the stabilization of β-catenin cytoplasmic pools in epidermal keratinocytes. This effect is mediated by the PI3K effector and involves a novel β-catenin–PI3K complex and the inhibition of β-catenin–APC interaction. Together with recent data from other groups , our results highlight the role of PI3K as a main regulator of different signaling pathways impinging on the modulation of the E-cadherin–mediated adhesion and β-catenin signaling. They also indicate that H-Ras activation can induce β-catenin signaling and, thus, can contribute to the present knowledge on the molecular mechanisms of cancer development. | Study | biomedical | en | 0.999997 |
10477753 | The sea urchin Lytechinus pictus was obtained from Marinus, Inc. and the sand dollar Echinarachnius parma was used on site at the Mount Desert Island Biological Laboratory. Gametes were obtained by intracoelemic injection of 0.5 M KCl, and after fertilization the fertilization envelopes were removed either by passage through Nitex membranes or by treatment with 1 M glycine. Zygotes were then cultured in filtered sea water at 16–17°C. Cortical cytoskeletons and whole cell extracts were prepared essentially as described in Walker et al. 1997 . In brief, at time points before or following fertilization, 100 μl of eggs was washed once in isolation buffer (20 mM Pipes, pH 7.3, 5 mM MgCl 2 , 5 mM EGTA, 1 M glycerol, 5 mM sodium vanadate, 25 mM NaF) supplemented with 10 μg/ml soybean trypsin inhibitor, benzamidine, leupeptin, α2-macroglobulin, aprotinin, and 1 mM PMSF. Half of the washed cells were resuspended, vortexed, and snap frozen in 20 vol of EB (80 mM β-glycerophosphate, 10 mM MgCl 2 , 10 mM EGTA plus protease inhibitors), and the other half were lysed for 10 min on ice in 20 vol of isolation buffer containing 0.5% NP-40 and protease inhibitors. The detergent-extracted embryos were then homogenized in a loose-fitting dounce homogenizer, and washed three times by pelleting and resuspension in isolation buffer minus detergent. The washed cortices were then resuspended in 50 μl of isolation buffer and snap frozen in liquid nitrogen. To assay for p34 cdc2 activity, whole cell and cortical fractions were thawed on ice, diluted fourfold in EB, and a fraction was set aside to determine the protein concentration. 10 μl of the diluted fraction was then mixed with 10 μl of a reaction mix containing 1 mg/ml histone H1, 200 μM [γ- 32 P]ATP (2 Ci/mmol), 20 μM H-7, and the mixture was incubated at 20°C for 20 min. The reactions were stopped by addition of boiling 2× SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE, and following Coomassie blue staining and drying, phosphorylation was analyzed by autoradiography and scintillation counting. Histone phosphorylation was normalized to the extract protein concentrations as measured by BCA assay (Biorad). To test cortical H1 kinase for sensitivity to kinase inhibitors, cortices prepared from dividing zygotes were diluted into EB and assayed for H1 kinase activity in the presence or absence of 5 μM roscovitine, 10 μM olomoucine, 10 μM isoolomoucine, 20 μM H-7, 10 μM ML-9, or 10 μM genistein. 1 ml packed eggs was incubated in the presence of 5 mCi [ 32 P]orthophosphoric acid in 4 ml phosphate-free sea water for 60 min at 15°C. Eggs were then washed free of label, and fertilized. In some experiments, eggs were fertilized and cultured in the presence of 32 P up until nuclear envelope breakdown, at which time eggs were washed free of label. Incubating eggs before or after fertilization had no effect on the patterns of phosphate incorporation into myosin regulatory light chain. At time points through the first mitosis, cortical cytoskeletons were prepared as described above. In some experiments, the detergent soluble fraction was clarified at 14,000 g and with 10 μl of either normal rabbit serum or rabbit anti–egg myosin antibodies for 2 h at 4°C. The immune complexes were harvested with protein G agarose (Amersham Pharmacia Biotech) and washed three times in TBS containing 1% Triton X-100, 25 mM NaF, and 5 mM sodium vanadate. The resultant cortices or immunoprecipitates were resolved by SDS-PAGE, transferred to Immobilon P membranes, stained with Coomassie blue R-250, and phosphorylated polypeptides were visualized by autoradiography. Regulatory light chains were identified as bands comigrating with purified light chains or light chains immunoprecipitated from sea urchin whole cell extracts with myosin heavy chain antibodies. Light chains were excised from the immobilon membranes, and nonspecific sites were blocked in 0.5% polyvinylpyrridone in 100 mM acetic acid for 1 h at 37°C. Light chains were then digested overnight with 160 μg/ml TPCK-trypsin in 50 mM ammonium bicarbonate, pH 8.0, at 37°C. The peptide digests were then subjected to four rounds of lyophilization and resuspension in water to remove the bicarbonate. Samples were then resuspended in pH 1.9 buffer (2.2% formic acid, 7.8% acetic acid) and spotted onto a cellulose TLC plate. Samples were subjected to electrophoresis for 60 min at 1,000 V using a Hunter high voltage electrophoresis system (C.B.S. Scientific Co.), and then subjected to liquid chromatography in a second dimension in phosphochromatography buffer (37.5% n -butanol, 25% pyridine, 7.5% acetic acid) for 5 h. The plates were then dried and the peptide digests visualized by autoradiography. Tryptic peptide assignments were based on Satterwhite et al. 1992 . Purified brush border (provided by Dr. Karl Fath, University of Pittsburgh, Pittsburgh, PA) or sea urchin egg myosin II were phosphorylated in vitro with either gizzard myosin light chain kinase (MLCK) (provided by Dr. R. Adelstein, National Institutes of Health, Bethesda, MD) or the catalytic fragment of PKC (Calbiochem) according to Satterwhite et al. 1992 , and subjected to peptide digestion and phosphopeptide analysis. When PKC- or MLCK-phosphorylated brush border and sea urchin egg myosin light chain were mixed and subjected to phosphopeptide analysis, the resultant maps were superimposable. These digests served as standards to identify phosphopeptides from in vivo–phosphorylated samples. To determine whether p34 cdc2 phosphorylation of myosin light chain affects the association of myosin II with the cortical cytoskeleton, cortical cytoskeletons were prepared from interphase zygotes (∼30 min after fertilization), when cortical kinase activity was low . 30 μl suspensions of cortices were incubated either alone or in the presence of 10 −8 M PKC or p34 cdc2 –cyclin B complex and 200 μM [γ- 32 P]ATP (2 Ci/mmol) for 25 min at 20°C. The reactions were clarified for 5 min at 14,000 g . The supernatants were removed and the cortices resuspended in a matching volume of isolation buffer. SDS-PAGE sample buffer was added to both fractions, and after boiling, equal volumes were loaded onto 12% SDS-PAGE gels, transferred to Immobilon membranes, and light chains were detected by autoradiography and immunoblotting with rabbit anti–egg myosin antiserum that recognizes both heavy and light chains. To arrest cells in mitosis, a truncated form of Arbacia punctulata cyclin B (Δ90 cyclin) was expressed in bacteria and purified as described previously . As an additional purification step, the enriched Δ90 cyclin fraction (∼80% pure) was clarified and applied to a Superose 12 FPLC column, and peak fractions were >90% homogenous. The purified cyclin was dialyzed against injection buffer (10 mM Hepes, 100 mM potassium aspartate, pH 7.2), and concentrated to ∼2 mg/ml with Microcon™ concentrators (Amicon). The activity of the recombinant protein was assessed in vitro before microinjection by testing the ability of Δ90 cyclin to arrest Xenopus cycling extracts in mitosis. For microinjection of Δ90 cyclin, E. parma embryos were fertilized, stripped of their fertilization envelopes and hyaline layers, and cultured through the first division at 16°C. Two cell embryos were then held in place with a holding pipette, and one blastomere was injected with injection buffer alone or injection buffer containing Δ90 cyclin; injected blastomeres were then marked with a small volume of Wesson oil. The uninjected blastomere served as a time control. Embryos were then scored for cleavage. Injection volumes varied between 0.5 and 4%, resulting in an intracellular concentration of ∼1 μM Δ90 cyclin. To confirm that Δ90-injected blastomeres were arrested in mitosis, and because sand dollar embryo chromosomes are difficult to discern by standard light microscopy, injected blastomeres were cultured in injection chambers in the presence of 1 μg/ml Hoescht No. 33342, and chromatin condensation was observed by fluorescence microscopy. To assess the contractile state of the cortex in Δ90-arrested cells, blastomeres were injected and incubated until prophase spindles were visible in the uninjected blastomeres. A needle was then lowered onto the injected cell parallel to the plane of the coverslip, bisecting the cell and pressing the spindle poles towards the cell surface. Another needle was placed adjacent to the injected blastomere to hold the embryo in place. An alternative method for altering the geometrical relationship between the spindle poles and the surface was to draw control or Δ90-injected blastomeres into a fire-polished pipette with an internal diameter between 40 and 65 μm. Bright field images were recorded on a Leitz diavert microscope with Tech pan film ASA 200, and figures were prepared using Adobe Photoshop ® software. Previous work in the laboratory has characterized several protein kinases associated and active within the actin-based cortical cytoskeleton of dividing sea urchin blastomeres. Among these are the tyrosine kinases abl and fyn, a mitogen-activated protein (MAP) kinase, and as yet uncharacterized kinases of 42, 45, and 84 kD. A histone H1 kinase activity was also detected whose peak activity was detected in cortices prepared from zygotes undergoing cytokinesis. In addition, p34 cdc2 was detected in cortices by Western blotting. To further examine the kinetics of cortical H1 kinase activity, zygotes of the sand dollar E. parma were harvested at various times after fertilization, and whole cell and detergent-extracted cytoskeletons were prepared for analysis. As shown in Fig. 1 A, a consistent delay in peak activity was observed in the cytoskeletal fractions when compared with whole cell H1 kinase activities through the first two cell cycles. Although the actual times at which cleavage occurred varied slightly from one experiment to another due to differences in the temperature at which the zygotes were cultured, cortical H1 kinase activity peaked on average 10–15 min after whole cell levels had reached maximum levels. Observation of embryos collected at this time by interference contrast microscopy indicated that cells had entered anaphase and many had begun dividing. These differential kinetics between whole cell and cytoskeletal-associated H1 kinase activity was not species-specific, but were also observed in all species of sea urchins tested ( Strongylocentrotus purpuratus, L. pictus, Lytechinus variegatus ). To confirm that this activity was attributable to p34 cdc2 and not other kinases present in the cortex, we tested the cortical H1 kinase activity in the presence of a battery of kinase inhibitors, including several derivatives of 6-dimethylamino purine (6-DMAP), whose effects are specific to cyclin-dependent kinases, including p34 cdc2 . As shown in Fig. 1 B, cortical H1 kinase activity was sensitive to the CDK inhibitors roscovitine, olomoucine, and butyrolactone (data not shown), but not the inactive isomer isoolomoucine or the PKC/PKA inhibitor H-7. Cortical H1 kinase activity is also insensitive to the MLCK inhibitor ML-9, and genistein (data not shown). In addition, measurements of specific activities indicate that cortical H1 kinase activity was enriched on average 2.5–3-fold over whole cell levels. Results of H1 kinase assays indicated that cytoskeletal-associated p34 cdc2 activity cycled with kinetics delayed with respect to whole cell levels, and that this delayed activity extended into anaphase and cleavage. In light of data suggesting that p34 cdc2 may regulate the timing of cytokinesis through the modulation of myosin II activity , we asked whether the extended p34 cdc2 activity is reflected in vivo by myosin regulatory light chain (LC20) phosphorylation in the cortical cytoskeleton of sea urchin embryos. Smooth muscle and cytoplasmic myosin light chains may be phosphorylated on five residues: serines 1 and 2, threonine 9, threonine 18, and serine 19 . Whereas PKC phosphorylates LC20 on serines 1 and 2 and threonine 9 , p34 cdc2 phosphorylates serines 1 and 2 only . In contrast, MLCK phosphorylates both threonine 18 and serine 19 . These differential phosphorylation sites may be resolved by phosphopeptide mapping as illustrated in Fig. 2 , where purified myosin II was phosphorylated in vitro by PKC or MLCK. Two phosphopeptides were detected in PKC phosphorylated light chains, corresponding to serines 1 and 2 and threonine 9 , whereas a single phosphopeptide corresponding to serine 19 was visible in light chains phosphorylated by MLCK . Peptide digests from in vitro–phosphorylated brush border or sea urchin egg myosin II were superimposable when phosphorylated by the same kinases (data not shown), indicating that the sea urchin homologue of regulatory light chain also contained these positive and negative regulatory sites. Using metabolic labeling and phosphopeptide mapping, we followed the phosphorylation of regulatory light chain in the cortical cytoskeleton of sea urchin zygotes through mitosis. Synchronous, metabolically labeled cultures were monitored by interference contrast optics, and samples were collected at the time of nuclear envelope breakdown, metaphase, anaphase, and cleavage. Cortical cytoskeletons were prepared from each sample, and after SDS-PAGE and transfer to polyvinylidene difluoride (PVDF) membranes, light chains were subjected to tryptic digestion and peptide mapping. As shown in Fig. 2 , there was little detectable light chain phosphorylation up until anaphase, at which time there was a dramatic increase in a single phosphopeptide . Mixing tryptic digests from MLCK-phosphorylated light chains with in vivo–labeled LC20 digests prepared from telophase zygotes identified the phosphopeptide in anaphase and telophase as the activating MLCK site . Serine 19 phosphorylation decreased after cleavage (110–125 min after fertilization), and the fluctuations in cortical LC20 phosphorylation could not be attributed to differences in myosin II recruitment to the cortex, since Western blotting of in vivo–labeled cortices revealed that levels of myosin II remained constant throughout the cell cycle (data not shown). Although the increase in LC20 phosphorylation on activating residues is consistent with data from cultured cells where there is an increase in serine 19 phosphorylation upon anaphase onset , we were surprised to find no evidence of serine 1,2 phosphorylation on light chains associated with cytoskeletal myosin heavy chain at any time during mitosis , especially in light of our data regarding p34 cdc2 activity associated with cytoskeleton . Light chain phosphorylation on serine 1,2 has been detected in vivo in tissue culture cells arrested in mitosis using microtubule-destabilizing drugs , as well as Xenopus and sea urchin extracts. This apparent discrepency might be explained by either: (a) the soluble and cytoskeletal myosin populations were subject to differential regulation; (b) the presence of a phosphatase activity that prevented our detection of cdc2 phosphorylation of light chain; or (c) a selective destabilization or solubilization of myosin filaments from the cortical cytoskeleton following serine 1,2 phosphorylation . To control for the first two possibilities, sea urchin eggs were labeled in vivo, samples collected from cultures during metaphase, and LC20 was immunoprecipitated from detergent-soluble supernatants using anti–myosin heavy chain antibodies and subjected to tryptic digestion and phosphopeptide analysis. As shown in Fig. 3 A, a major phosphopeptide could be detected in samples prepared from metaphase zygotes that comigrated with phosphopeptides derived from in vitro–phosphorylated LC20 . A second, minor phosphopeptide could be detected comigrating with threonine 9–containing phosphopeptides. These results suggested that our experimental conditions did allow for the detection of serine 1,2 phosphorylation in vivo, and suggested that serine 1,2 modulation of myosin II activity may be dependent on the cytoplasmic compartment in which the myosin molecules reside. Finally, experiments were performed in vitro to control for the possibility that p34 cdc2 phosphorylation of LC20 might selectively destabilize myosin II association with the cortical cytoskeleton, and thus preclude our detection of serine 1,2 phosphorylation in the cortex. Cortices were prepared from zygotes at times when cortical kinase activity is low in comparison to dividing cells (20–50 min after fertilization) , and treated with purified p34 cdc2 –cyclin B or PKC in the presence of [γ- 32 P]ATP. The reactions were then clarified by low speed centrifugation, and the supernatant and pellet (cortical) fractions examined by autoradiography and Western blotting with an anti–egg myosin antibody that recognizes both heavy and light chains. As shown in Fig. 3 B, there was no detectable increase in soluble myosin as the result of either p34 cdc2 or PKC treatment. Autoradiography confirms that LC20 phosphorylation was detectable in both p34 cdc2 - and PKC-treated cortices, confirming that the cytoskeletal-associated myosin light chains were accessible to the soluble kinases. Thus, LC20 phosphorylation on inhibitory sites did not appear to correlate with an increase in myosin solubility. Results of in vivo labeling and phosphopeptide mapping suggest that despite the enriched and extended levels of p34 cdc2 activity associated with the cortex, cytoskeletal myosin II was not subject to a light chain-based negative regulation during mitosis. In an effort to directly assess the light chain–based model for the timing of cytokinesis in vivo, we asked whether the cortex could be induced to form a cleavage furrow in the presence of chronically extended p34 cdc2 activity. Towards these ends, a truncated, nondegradable form of cyclin B was produced in Escherichia coli (Δ90 cyclin) . Nondegradable forms of cyclin B have been introduced in cultured cells as well as Xenopus and sea urchin eggs . In each case, chromatin remains condensed, and cleavage is arrested. However, because there are dramatic effects on microtubule dynamics and spindle behavior in Δ90 cyclin–arrested cells, it is difficult to attribute the inhibition of cytokinesis to either a suppression of myosin II–based contractility or a failure to deliver the cleavage stimulus to the cortex. To differentiate between these possibilities, recombinant Δ90 cyclin was produced in bacteria, purified to homogeneity, and tested in Xenopus cell–free extracts for its ability to arrest cycling extracts in a mitotic state (data not shown). Δ90 cyclin was then concentrated and injected into blastomeres of two cell E. parma embryos. Injection of Δ90 into blastomeres shortly before nuclear envelope breakdown resulted in mitotic arrest in 81% of the cells injected ( n = 42). In six cases where Δ90 cyclin was injected during metaphase, cells divided but arrested in the following cell cycle. In contrast, 93% of cells injected with buffer alone ( n = 31) went on to develop past the mesenchymal blastula stage. Examination of injected cells revealed that although injected blastomeres failed to divide, the mitotic apparatus was still visible and spindle poles underwent an anaphase B–like separation as reported previously , and in some cases, the spindle poles split to form three or four individual aster centers . Arrested blastomeres remained viable for up to 3 h, at which time the cells underwent membrane blebbing, and died soon after . Vital staining with Hoescht No. 33342 revealed that throughout this period, chromatin remained condensed. To ask whether cells arrested in mitosis were capable of forming cleavage furrows, Δ90-injected blastomeres were manipulated such that the spindle poles were placed in close proximity to the cell surface. To perform this manipulation, two opposing needles were brought down upon the surface of the injected blastomere, and pressed down so that two aster centers were isolated within a confined space and pushed against the surface. As shown in Fig. 4 , a unilateral furrow formed between the spindle poles. If one or both needles are removed once furrowing commences, the furrow progressed to near completion. Arrested blastomeres induced to furrow in this fashion remain arrested, and underwent no further divisions. Furrowing occurred ∼5 min after application of the needles, resembling the normal kinetics of cleavage furrow induction in E . parma embryos . Of the 14 Δ90-arrested blastomeres manipulated in this fashion, 8 were induced to furrow. Of the blastomeres that failed, two had asters that were normal to plane of the pipettes (and the coverslip) such that the furrow would have to ingress along the long axis of the bisected cell. The remaining four cells had spindle poles that had separated >45 μm, a distance determined previously in normal E . parma blastomeres to be too great to induce furrowing . The physical manipulation of the asters in Δ90-arrested cells suggested not only that the cortical cytoskeleton retains the capacity to assemble a contractile ring in the presence of chronically elevated MPF levels, but also suggested that the timing of cytokinesis may be a function of the spindle's capacity to deliver the signal to the surface. To further explore this notion, a second method was employed to alter the geometry of normal and Δ90-arrested cells. Blastomeres were carefully drawn into a fire-polished capillary pipette, resulting in a cylindrical cell and a reduced distance between the spindle pole and the cell surface . As shown in Fig. 5 , when an uninjected blastomere was drawn into a pipette, the mitotic apparatus is usually drawn into the distal portion of the cell. Just after the appearance of anaphase asters , a cleavage furrow was induced and furrowing was complete before the spherical control had commenced cleavage, even though anaphase onset and astral microtubule elongation occurred simultaneously in the two cells. When Δ90-arrested cells were drawn into a pipette, cleavage furrows could also be observed. In the embryo shown in Fig. 6 , the aster centers could not be clearly delineated, but a localized contraction was induced adjacent to a cleared zone (arrow), and because the spindle was aligned slightly oblique to the axis of the cylinder, the furrow attempted to progress along the long axis of the cell. Similar results have been obtained with normal cylindrical cells when the axis of the spindle is normal to the long axis of the cell . Similar results were obtained using injected mRNA in L. pictus and Dendraster excentricus . Furrows induced in cylindrical, Δ90-injected embryos were irregular, and while none (5/5 blastomeres) progressed to completion, contractility activity was observed in all cells whose capillary diameter was not >60 μm. In an effort to understand the role of protein phosphorylation in the temporal and spatial regulation of cleavage furrow formation, we sought to address how p34 cdc2 kinase affects the timing of contractile ring assembly in embryonic cells. Results of this study indicate that despite enriched and prolonged levels of p34 cdc2 activity associated with the cortical actin cytoskeleton, there is no appreciable phosphorylation of myosin regulatory light chain on residues shown to be inhibitory for myosin II motor activity . Additionally, micromanipulation and microinjection studies with nondegradable forms of cyclin B indicate that cells arrested in mitosis are capable of forming cleavage furrows, but do not do so unless mitotic apparatus is in direct contact with the cell surface. Together, these results represent a critical assessment of the respective roles of p34 cdc2 and myosin II regulation in the timing of cytokinesis, and suggest that while the programmed destruction of p34 cdc2 activity may indeed act as the timer for cytokinesis, the timing of cytokinesis is not accomplished by a suppression of myosin II–based contractility. Mapping of cortical and whole cell H1 kinase activity indicates that p34 cdc2 activity associated with the actin cytoskeleton cycles with kinetics delayed with respect to global MPF levels . Whether this activity is sequestered within a specific subdomain of the cortex (i.e., polar versus equatorial), or what the functional significance of this delayed activity is in regards to the spatio-temporal regulation of contractile ring formation remains unknown at this time. The notion that cyclin destruction does not proceed uniformly throughout the cytoplasm has been recently demonstrated in Drosophila embryos where cyclin destruction begins at the spindle poles and spreads to the spindle midzone, after which cyclin disappears from the cytoplasm . Actin-associated MPF activity may represent a sequestered fraction of activity that is last to undergo ubiquitin-mediated destruction. In amphibian eggs, MPF activation is not only spatially regulated, but is also associated with a reorganization of the cortical cytoskeleton. A series of surface contraction waves (SCWs) originate from the animal pole in a cell cycle–dependent manner during the early cleavage cycles of frog and salamander embryos . Subsequent mapping of surface contractile behavior and p34 cdc2 activity indicates that the wave of MPF activation originating at the animal pole runs concomitantly with a relaxation of the cortex (SCWa) . Conversely, cyclin B destruction is accompanied by a cytochalasin-insensitive contraction wave (SCWb) . While these waves run concomitantly with the division cycle, the biochemical nature of these cycles of cortical relaxation and contraction, as well as their relatedness to contractile ring formation, is still unclear . It is yet to be determined whether myosin light chain phosphorylation also accompanies either the relaxation or contraction waves. Additionally, the actin-binding proteins caldesmon and spectrin have both been shown to be substrates of p34 cdc2 , and this modulation negatively regulates the interactions of these proteins with the actin cytoskeleton . It is conceivable then that the cortical relaxation observed with MPF activation is attributable to the modulation of filament binding and cross-linking proteins. With regard to echinoderm eggs, the inverse trend towards increased cortical stiffness during mitosis does not seem to correlate either to myosin light chain phosphorylation in the cortex , or to the ability of the cortex to respond to signals from the mitotic apparatus . Further characterization of cortical H1 kinase activity in echinoderm eggs, as well as identification of other cortical substrates for p34 cdc2 , will reveal whether the differential activation and inactivation of cortical p34 cdc2 is related to the spatial regulation of MPF activity seen in Xenopus eggs. In vivo analysis of myosin light chain phosphorylation reveals that whereas there was evidence of p34 cdc2 phosphorylation on light chains associated with soluble myosin II, cortical-associated myosin was under no such regulation despite the presence of p34 cdc2 activity associated with the actin cytoskeleton . Control experiments suggest that the absence of serine 1,2 phosphorylation in cortical LC20 is not due to altered solubilities of phosphorylated myosin, accessibility of serines 1 and 2 to phosphorylation, or artifacts of preparation that would preclude our detection of serine 1,2 phosphorylation . In vitro, regulatory light chain is a poor substrate for p34 cdc2 when associated with myosin heavy chain , yet robust serine 1,2 phosphorylation can be detected in sea urchin and Xenopus extracts, as well as in whole cell extracts from metabolically labeled tissue culture cells . However, the induction of cleavage furrows in Δ90-injected cells argues that the model proposing p34 cdc2 -mediated suppression of myosin II activity and thus cytokinesis may no longer represent a viable one for the timing of cytokinesis, regardless of the cytoplasmic compartment in which the regulation occurs. Indeed, a recent study carried out in fission yeast indicates that mutations in the light chain phosphorylation sites have no effects on cytokinesis . It is possible that differential regulation of cytoskeletal and soluble myosin II may contribute to the tight spatial regulation of myosin activation and contractile ring formation in embryonic cells. Embryos generally contain large stores of contractile proteins required for the rapid series of cell divisions that accompany early development, and ∼90% of myosin II is soluble in the sea urchin egg (Shuster, C., unpublished observations). Inactivation of this large soluble pool may contribute to spatial regulation of cleavage furrow formation by limiting the myosin filaments that may be activated or recruited to the cleavage furrow. If this is indeed the case, serine 1,2 phosphorylation would likely accomplish this by lowering the affinity of myosin for actin, and not by affecting the ability of MLCK to phosphorylate light chain . Another issue regarding the regulation of myosin II during cell division centers on the role of serine 19 phosphorylation during cytokinesis. Studies using phosphoepitope-specific antibodies for serine 19 as well as phosphorylation-sensitive biosensors detect an increase in serine 19 phosphorylation upon anaphase onset that concentrates in the equatorial zone as well as in the margins as the cells respread following mitosis . The kinetics of serine 19 phosphorylation in the cortical cytoskeleton of sea urchin embryos resemble those seen in cultured cells . And although there is a correlative relationship between serine 19 phosphorylation and cytokinesis, studies of regulatory light chain function in Dictyostelium argue that light chain phosphorylation may be altogether dispensable for contractile ring function . While basal levels of actin-activated ATPase activity may be sufficient for contractile ring formation in Dictyostelium grown either on substrate or in suspension , the role of serine 19 phosphorylation in mammalian cells as well as in echinoderm eggs has not been thoroughly evaluated. Mutation of both MLCK sites in Drosophila results in defects in cytokinesis and ring canal formation resembling the light chain–null ( spaghetti-squash) phenotype , suggesting that activating phosphorylation is required for cytokinesis. Testing the functional significance of serine 19 phosphorylation during cell division in animal cells, and the unequivocal identification of the modifying kinase are both areas of intense interest and investigation . The induction of cleavage furrows in Δ90-arrested cells supports the notion that the cortex is capable of responding to contractile stimuli even under conditions of chronically elevated MPF levels, and corroborate biochemical data indicating that cortical myosin II is not under a light chain–based suppression during mitosis . Introduction of nondegradable cyclin B (Δ90 cyclin) into dividing echinoderm eggs or tissue culture cells results in a stereotypic series of spindle movements where sister chromatid separation proceeds normally as does anaphase B spindle movements, but the nuclear envelope does not reform and cytokinesis does not occur . Spindle pole separation becomes quite exaggerated, and in the case of echinoderm embryos, spindle poles split to form up to four asters . As a means of assessing whether the cortical cytoskeleton can respond to signals from the spindle in the presence of high MPF levels, the geometrical relationship between the spindle and the cell surface was altered in Δ90-arrested blastomeres . Under conditions where aster centers were placed adjacent to the cortex, furrows could be induced at the equatorial zone between the spindle poles . The induction of cleavage furrows was not only dependent upon reducing the distance between the spindle pole and the surface, but also on the interastral distance, where furrowing could not be induced in cells where extreme anaphase B spindle pole separation could not be compensated for by pushing the asters against surface (data not shown). In this sense, our data support the argument made by Wheatley et al. 1997 that the cytokinesis defect in Δ90-injected cells is due to exaggerated anaphase B movements that reduce the capacity of the microtubules (astral microtubules in sea urchin eggs, midzone microtubules in tissue culture cells) to stimulate cleavage furrows. However, we observed many cases ( n = 15) where spindle poles split to form multiple asters , all of which had interastral distances which would normally support furrow formation (29–35 μm), but the cells did not divide unless the asters were physically displaced toward the cortex. Thus, it appears that spindle integrity alone does not explain the reversible inhibition of cleavage furrows in Δ90-arrested blastomeres. The necessity of bringing the asters of Δ90-injected cells into close proximity with the cortex suggests that at least one determinant of the timing of cytokinesis is the geometrical relationship between the spindle poles and the surface. The spatial relationship between the spindle poles and the surface in normal and geometrically or chemically altered cells, and the respective effects on the timing of cleavage furrow formation is illustrated in Fig. 7 . Under normal conditions, the cleavage plane is specified shortly after the metaphase–anaphase transition . With the onset of anaphase and the decline of p34 cdc2 activity, there is an extensive elaboration and elongation of astral microtubules along with an accompanying loss of spindle birefringence . Contact between astral microtubules and the surface requires as little as 1 min to specify the position of the furrow, and after a brief latent period the contractile ring is induced . Under conditions where the distance between the spindle poles and the surface is reduced, as in the case of a cylindrical cell , contractile rings are induced and progress to completion before spherical controls . Thus, while both spherical and cylindrical cells enter anaphase at the same time, the timing of furrow formation is a function of the distance that the putative cleavage stimulus has to travel to reach the surface, and not an abrupt cell cycle transition. Chemical modulations of astral microtubule elongation extend this notion. If astral microtubule elongation is inhibited with reagents such as urethane , cytokinesis does not occur unless the distance between the spindle pole and the surface is reduced by micromanipulation . One explanation for the reversible inhibition of cytokinesis observed in Δ90-arrested blastomeres is that the physical displacement of the spindle poles towards the surface compensates for the normal elaboration of the astral microtubules during anaphase . Collectively, the induction of cleavage furrows under these conditions implicates the delivery of the cleavage stimulus via astral microtubule elongation as an important determining factor in the timing of cytokinesis. Therefore, by implication, the regulation of microtubule dynamics represents an indirect mechanism by which the timing of cytokinesis may be specified by p34 cdc2 . The rates of microtubule turnover shift dramatically during mitosis , and the 10-fold increase in microtubule catastrophe rates seen in cell-free extracts is dependent on p34 cdc2 . Indeed, p34 cdc2 –cyclin B has been shown to bind and phosphorylate both microtubule-associated protein 4 and p77 echinoderm microtubule-associated protein , and MAP kinase family members have also been implicated in the regulation of microtubule dynamic instability . Phosphorylation lowers the affinity of MAPs for the microtubule, resulting in increasing catastrophe rates . However, additional factors such as Op18 , the kinesin-like protein XKCM1 , and the microtubule-severing protein katanin may also play crucial roles in regulating microtubule stability during mitosis. The notion that microtubule turnover remains at a mitotic state in Δ90-arrested cells is supported by observations of injected sea urchin zygotes with polarization optics , where the spindle undergoes normal anaphase chromosome separation, yet the spindle poles remain birefringent. Understanding how the activities of these factors change in relation to the fall of MPF activity during anaphase may identify critical regulatory events that lead to the stabilization of astral microtubule arrays and induction of contractile ring assembly. | Study | biomedical | en | 0.999997 |
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