Figure 3Whole cell extracts of High Five insect cells individually expressing the wild - type ( WT ) or indicated alanine substitution FLAG - SHLD3C ( residues 126 - 250 ) variants and Strep - RIF1N ( residues 1 - 980 ) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation . Quantification of LacR / LacO assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to LacO arrays in U2OS 2 - 6 - 3 cells by mCherry - LacR - RIF1N . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 134 , 125 , 120 , 130 , 104 , 129 , 117 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . EV : empty vector . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . * * * : P = 0 . 0004 . * * : P = 0 . 002 . * : P = 0 . 02 . ns : P > 0 . 05 . Quantification of LacR - FokI assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to sites of DNA double - strand breaks induced by mCherry - LacR - FokI in U2OS 2 - 6 - 3 cells . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - FokI focus and the average nuclear intensity . Bars represent means ( n = 103 , 136 , 159 , 151 , 139 , 145 , 140 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Whole cell extracts of High Five insect cells individually expressing the wild-type (WT) or indicated alanine substitution FLAG-SHLD3C (residues 126-250) variants and Strep-RIF1N (residues 1-980) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.Quantification of LacR/LacO assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to LacO arrays in U2OS 2-6-3 cells by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 134, 125, 120, 130, 104, 129, 117 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). EV: empty vector. Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ***: P = 0.0004. **: P = 0.002. *: P = 0.02. ns: P > 0.05.Quantification of LacR-FokI assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to sites of DNA double-strand breaks induced by mCherry-LacR-FokI in U2OS 2-6-3 cells. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent means (n = 103, 136, 159, 151, 139, 145, 140 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ns: P > 0.05.


Figure 4Representative micrographs of immunofluorescence experiments ( from 3 biologically independent experiments ) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3 - KO cells . 3xFLAG - SHLD3 - complemented RPE SHLD3 - KO cells were treated with 200 nM aphidicolin ( Aphi ) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells . Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler . Pearson ' s correlation coefficients ( PCC ) were calculated for pixels within each cyclin A - negative , 53BP1 body - positive nucleus between the 53BP1 and FLAG channels . Each point represents the PCC value of an individual nucleus . Bars represent means . ( n = 120 , 130 for EV , SHLD3 from three biologically independent experiments ) . Analysis was performed using two - tailed Mann - Whitney test . * * * * : P < 0 . 0001 . Representative micrographs of immunofluorescence experiments analyzing localization of FLAG - SHLD3 alanine substitution variants stably expressed in RPE SHLD3 - KO cells by lentivirus transduction . Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells . Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3 - KO cells with or without 24 - hour 200 nM Aphi treatment . Bars represent means ± s . d . ( n = 3 biologically independent experiments with ≥ 30 nuclei imaged each ) .

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Figure 4Representative micrographs of immunofluorescence experiments (from 3 biologically independent experiments) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3-KO cells. 3xFLAG-SHLD3-complemented RPE SHLD3-KO cells were treated with 200 nM aphidicolin (Aphi) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells. Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler. Pearson's correlation coefficients (PCC) were calculated for pixels within each cyclin A-negative, 53BP1 body-positive nucleus between the 53BP1 and FLAG channels. Each point represents the PCC value of an individual nucleus. Bars represent means. (n = 120, 130 for EV, SHLD3 from three biologically independent experiments). Analysis was performed using two-tailed Mann-Whitney test. ****: P < 0.0001.Representative micrographs of immunofluorescence experiments analyzing localization of FLAG-SHLD3 alanine substitution variants stably expressed in RPE SHLD3-KO cells by lentivirus transduction. Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells.Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3-KO cells with or without 24-hour 200 nM Aphi treatment. Bars represent means ± s.d. (n = 3 biologically independent experiments with ≥ 30 nuclei imaged each).


Figure 5Left : Representation of the region of the RIF1 N - terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E - like domain . Residues 1 - 173 are highlighted . Right : Quantification of LacR / LacO assay measuring eGFP - SHLD3 recruitment to LacO arrays in U2OS 2 - 6 - 3 cells by the indicated truncated mCherry - LacR - RIF1N variants . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 148 , 123 , 122 for EV , 1 - 567 , 174 - 567 from 3 biologically independent experiments ) . Analysis was performed using Mann - Whitney two - tailed test . * * * * : P < 0 . 001 . Whole cell extracts of High Five insect cells individually expressing the FLAG - SHLD3C ( residues 126 - 250 ) and the indicated alanine substitution Strep - RIF1 ( residues 1 - 980 ) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation . Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . DNA damage was induced in U2OS 2 - 6 - 3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies . Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1 - labeled irradiation stripe and the average nuclear intensity . Only nuclei containing RIF1 stripes are quantified . Bars represent means ( n = 206 , 215 for RIF1WT , RIF1D28N from 2 biologically independent experiments ) . Analysis was performed using Welch ' s t - test . * * * * : P < 0 . 0001 .

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Figure 5Left: Representation of the region of the RIF1 N-terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E-like domain. Residues 1-173 are highlighted. Right: Quantification of LacR/LacO assay measuring eGFP-SHLD3 recruitment to LacO arrays in U2OS 2-6-3 cells by the indicated truncated mCherry-LacR-RIF1N variants. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 148, 123, 122 for EV, 1-567, 174-567 from 3 biologically independent experiments). Analysis was performed using Mann-Whitney two-tailed test. ****: P < 0.001.Whole cell extracts of High Five insect cells individually expressing the FLAG-SHLD3C (residues 126-250) and the indicated alanine substitution Strep-RIF1 (residues 1-980) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. DNA damage was induced in U2OS 2-6-3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies. Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1-labeled irradiation stripe and the average nuclear intensity. Only nuclei containing RIF1 stripes are quantified. Bars represent means (n = 206, 215 for RIF1WT, RIF1D28N from 2 biologically independent experiments). Analysis was performed using Welch's t-test. ****: P < 0.0001.


Figure 6Quantification of class switch recombination in stimulated CH12F3 Shld3 - / - cells stably expressing the indicated FLAG - SHLD3 variants through lentiviral transduction . Bars represent means ± s . d . , n = 3 biologically independent experiments . Analysis was performed using one - way ANOVA followed by Dunnett ' s multiple comparisons test against Shld3 - / - cells complemented with wild - type SHLD3 . * * : P < 0 . 01 , * : P < 0 . 05 , ns : P > 0 . 05 . Competitive growth assay of RPE BRCA1 - KO SHLD3 - KO cells complemented with 3xFLAG - SHLD3 variants . Growth of the indicated eGFP - expressing cells were compared to mCherry - expressing RPE BRCA1 - KO SHLD3 - KO cells transduced with empty vector after mixing 1 : 1 in 24 - well plates . Plates were imaged 1 , 5 , 9 , and 13 days after plating . Dotted black lines represent an eGFP : mCherry ratio of 1 . Points represent means ± s . d . ( n = 3 biologically independent experiments ) . Analysis was performed using a one sample t - test against a hypothetical eGFP : mCherry ratio value of 1 for the day 13 time point . * * : P < 0 . 01 , ns : P > 0 . 05 .

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Figure 6Quantification of class switch recombination in stimulated CH12F3 Shld3-/- cells stably expressing the indicated FLAG-SHLD3 variants through lentiviral transduction. Bars represent means ± s.d., n = 3 biologically independent experiments. Analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test against Shld3-/- cells complemented with wild-type SHLD3. **: P < 0.01, *: P < 0.05, ns: P > 0.05.Competitive growth assay of RPE BRCA1-KO SHLD3-KO cells complemented with 3xFLAG-SHLD3 variants. Growth of the indicated eGFP-expressing cells were compared to mCherry-expressing RPE BRCA1-KO SHLD3-KO cells transduced with empty vector after mixing 1:1 in 24-well plates. Plates were imaged 1, 5, 9, and 13 days after plating. Dotted black lines represent an eGFP:mCherry ratio of 1. Points represent means ± s.d. (n = 3 biologically independent experiments). Analysis was performed using a one sample t-test against a hypothetical eGFP:mCherry ratio value of 1 for the day 13 time point. **: P < 0.01, ns: P > 0.05.


Figure 1 ( B ) IHC for CDC50A and RIM1 on cultured neurons at DIV21 . The left panel shows CDC50A is expressed in both neuronal soma and processes . The right panel exhibits CDC50A colocalize to pre - synapses alongside neuronal processes . MAP2 , Microtubule - associated protein 2 , was used to identify neuronal cells and trace dendritic processes . RIM1 is an active zone protein , which marks pre - synapses . Left scale bar = 50 µm ; right scale bar = 2 µm . ( C ) Quantification of the percentage of RIM1 + pre - synapses colocalizing to CDC50A . N = 3 for biological replicates . ( E ) IHC for CDC50A and vGluT2 in the developmental dLGN at P14 , P21 and P30 . The left panels show the overview of immunostaining in the dLGN , scale bar = 200 µm . The right panels are the zoomed - in images , scale bar = 5 µm . Arrows indicate the colocalized CDC50A and vGluT2 . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . The result shows that a significantly higher percentage of CDC50A + / vGlut2 + synapses at P21 and P30 than P14 . N = 6 for biological replicates .

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Figure 1(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates. (E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.


Figure 2 ( C ) IHC for CDC50A . CDC50A protein intensities were reduced after neuronal inactivation ( day 7 ) , and was recovered after removing neuronal inhibitors ( day 13 ) . Scale bar , 10 µm . ( D ) Relative CDC50A intensities of neurons . N = 5 for biological replicates , mean ± SD , * p < 0 . 5 , * * * * p < 0 . 0001 by one - way ANOVA with Holm - Sidak ' s multiple comparisons test . ( E ) CDC50A expression at RGC inputs after 5 - day consecutive TTX treatment . Scale bar , 10 µm . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s test .

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Figure 2(C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test. (E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.


Figure 3 ( A ) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls . Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days . CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment . Scale bar , 10 µm . ( D ) IHC of Cdc50a - RNAi neurons exhibited pSIVA colocalized with synapsin I , a general synapse marker . The dotted square was zoomed - in shown on the right side . Arrows indicated where pSIVA colocalized with synapsin I . Scale bar , 5 µm .

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Figure 3(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.


Figure 4 ( D ) Representative IHC images of CDC50A expression at synapses . Scale bar , 5 µm . Arrows indicate VGluT2 + RGC inputs . ( E ) Relative CDC50A expression at mCherry + synapses in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SD , * p < 0 . 05 by Student ' s t - test . ( F ) PSIVA staining in the dLGN . Exposed PS was detected by pSIVA on RGC inputs in Cdc50a - RNAi mice . Scale bar , 5 µm . Arrows indicate pSIVA + / mCherry + RGC inputs . ( G ) Quantification of the percentage of pSIVA + / mCherry + RGC inputs in total mCherry + RGC inputs . N = 4 for biological replicates , mean ± SEM , * * p < 0 . 01 by Student ' s t - test .

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Figure 4(D) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs. (E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test. (F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.


Figure 5 ( F ) Maximum intensity z - projection images of vGluT2 + synapses in the core region of Cdc50a RNAi mice and controls . Right panel showed enlarged synapses in the boxed region . Scale bar , 20 µm . ( H ) Maximum intensity z - projection images of vGluT2 + synapses in the shell region . Enlarged images ( right panel ) shows large " complex encapsulated " boutons . Scale bar , 20 µm . ( I ) Quantification of vGluT2 + synapse density . N = 3 for biological replicates , mean ± SD , Student ' s t - test .

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Figure 5(F) Maximum intensity z-projection images of vGluT2+ synapses in the core region of Cdc50a RNAi mice and controls. Right panel showed enlarged synapses in the boxed region. Scale bar, 20 µm.(H) Maximum intensity z-projection images of vGluT2+ synapses in the shell region. Enlarged images (right panel) shows large "complex encapsulated" boutons. Scale bar, 20 µm. (I) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, Student's t-test.


Figure 6 ( D ) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN . Scale bar , 20 µm . ( E ) Quantification of CTB488 + inputs ( non - infected areas ) versus CTB647 + inputs ( infected areas ) in Cdc50a RNAi mice and controls . N = 3 for biological replicates , mean ± SEM , * * * * p < 0 . 0001 , two - way ANOVA with Sidak ' s multiple comparisons test .

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Figure 6(D) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN. Scale bar, 20 µm. (E) Quantification of CTB488+ inputs (non-infected areas) versus CTB647+ inputs (infected areas) in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SEM, **** p < 0.0001, two-way ANOVA with Sidak's multiple comparisons test.


Figure 7 ( A ) The confocal images , 3D - rendered images and orthogonal views of microglia engulfing vGluT2 + synapses in controls and Cdc50a - RNAi mice . Scale bar , 10 µm .

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Figure 7(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm.


Figure 8 ( A ) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice ( Gpr56fl / fl ; Cx3cr1cre / + ) and controls ( Gpr56 + / + ; Cx3cr1cre / + ) . Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA . The yellow squares indicated the AAV2 non - infected areas and the dotted outlines indicated the AAV2 infected areas . Scale bar , 200 µm . ( B ) Confocal images of vGluT2 + synapses in the AAV2 non - infected areas and infected areas of the dLGN . Scale bar , 20 µm . ( C ) Quantification of vGluT2 + synapses in the non - infected areas and infected areas of microglial GPR56 conditional knockout mice and controls . N = 4 for biological replicates , mean ± SD , * p < 0 . 05 , * * * * p < 0 . 0001 by two - way ANOVA with Sidak ' s multiple comparisons test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice (Gpr56fl/fl; Cx3cr1cre/+) and controls (Gpr56+/+; Cx3cr1cre/+). Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA. The yellow squares indicated the AAV2 non-infected areas and the dotted outlines indicated the AAV2 infected areas. Scale bar, 200 µm.(B) Confocal images of vGluT2+ synapses in the AAV2 non-infected areas and infected areas of the dLGN. Scale bar, 20 µm. (C) Quantification of vGluT2+ synapses in the non-infected areas and infected areas of microglial GPR56 conditional knockout mice and controls. N = 4 for biological replicates, mean ± SD, * p<0.05, **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.


Figure 2RNAPII - RBP - RNA complex was immunoprecipitated by anti - RNAPII antibody or control antibody ( cont ) , and subjected to immunoblotting . The input lanes contained 0 . 4 % of lysates used in the immunoprecipitation experiments . Generation of tRIP - seq libraries of RNAPII - RBP - RNA complexes . After UV crosslinking , protein - RNA complexes were immunoprecipitated with anti - RNAPII ( 1st IP ) . RNA of the isolated RNAPII - RBP - RNA complexes was partially digested with RNase on beads , and RBP - RNA complexes dissociated from the bead were further immunoprecipitated with anti - FUS or anti - U1C antibody bound to the other beads ( 2nd IP ) . The immunoprecipitated FUS - RNA or U1C - RNA complex was treated with TEX , and then subjected to tRIP - seq library generation . The bottom panels show PCR - amplification of tRIP - seq libraries generated from RNAPII - FUS - RNA complex ( left panel ) and RNAPII - U1C - RNA complex ( right panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in the immunoprecipitation experiments.Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking, protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex (left panel) and RNAPII-U1C-RNA complex (right panel).


Figure 4PolyA - seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage . Cells were treated with siRNA against Fus ( siFus ) and / or U1 antisense morpholino ( U1AS ) . Control cells ( Cont ) were treated with both control siRNA and control antisense morpholino . A . Correlation of changes in polyA site usage between U1 - inhibition and Fus - silencing . Fold change in the RPM of a polyA site between siFus and Cont ( siFus / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . B . Correlation of changes in polyA site usage between U1 - inhibition alone and the combination of Fus - silencing and U1 - inhibition . Fold change in the RPM of a polyA site between siFus + U1AS and Cont ( siFus + U1AS / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . A slope of 1 . 0 indicates that Fus - silencing has no additive effect on U1 - inhibition .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4PolyA-seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. B. Correlation of changes in polyA site usage between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on U1-inhibition.


Figure 6Localization of FUS in R495X _ N2A cells ( R495X ) , and wild - type N2A cells ( WT ) . Cells were fractionated to separate nucleus and cytoplasm . Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies . Interactions between FUS and U1 snRNP in the R495X _ N2A cells ( R495X ) and wild - type N2A cells ( WT ) . Total cell lysates were immunoprecipitated with anti - U1 - 70K antibody or a control antibody ( cont ) , and immunoblotted with indicated antibodies . Effects of the R495X mutation on four categories of polyA sites . Four categories of polyA sites are comprised of ( 1 ) FUS - repressed ( rep ) / U1 - repressed sites , ( 2 ) FUS - activated ( act ) / U1 - repressed sites , ( 3 ) FUS - repressed / U1 - activated sites , and ( 4 ) FUS - activated / U1 - activated sites , according to Fig 4A . Fold change of each polyA site usage by the R495X mutation ( R495X / WT ) is shown in violin plots . Bars indicate the median and 95 % confidential intervals for each category . * * * p < 0 . 001 , * * * * p < 0 . 0001 , based on the Steel - Dwass test for comparing all 6 pairs of categories .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Localization of FUS in R495X_N2A cells (R495X), and wild-type N2A cells (WT). Cells were fractionated to separate nucleus and cytoplasm. Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies.Interactions between FUS and U1 snRNP in the R495X_N2A cells (R495X) and wild-type N2A cells (WT). Total cell lysates were immunoprecipitated with anti-U1-70K antibody or a control antibody (cont), and immunoblotted with indicated antibodies.Effects of the R495X mutation on four categories of polyA sites. Four categories of polyA sites are comprised of (1) FUS-repressed (rep) /U1-repressed sites, (2) FUS-activated (act) /U1-repressed sites, (3) FUS-repressed/U1-activated sites, and (4) FUS-activated/U1-activated sites, according to Fig 4A. Fold change of each polyA site usage by the R495X mutation (R495X/WT) is shown in violin plots. Bars indicate the median and 95% confidential intervals for each category. *** p < 0.001, **** p < 0.0001, based on the Steel-Dwass test for comparing all 6 pairs of categories.


Figure 2 ( A ) Representative confocal photomicrographs showing coronal slices from 8W hGFAP ‐ tTA ; H2B ‐ GFP mice with and without doxycycline treatment , upon immunofluorescent staining with the stem cell marker Nestin ( red ) . DAPI was used for nuclear counterstain . Cross indicates slice directions : M = medial , D = dorsal , V = ventral , L = lateral . Scale bar = 20 µm . ( B ) Quantification of Nestin expression in apical and basal cells in doxycycline - treated and untreated animals . N = 3 ( - Doxy ) , N = 4 ( + Doxy ) . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± SEM . * and # indicate significance : between apical and basal ( * ) and between doxycycline treatments ( # ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by one - way ANOVA with Tukey ' s multiple comparisons test ( B ) ( C ) Same as ( A ) , with addition of the primary cilia marker AC3 ( yellow ) . Arrows indicate Nestin - positive ( white ) and GFP - positive ( green ) cells with primary cilium . Scale bar = 10 µm . ( D ) Quantification of ciliated GFP + NSCs in apical and basal cell layer as percentage of total apical / basal GFP + cells . N = 7 . ( E ) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker ; N = 7 . Data information : N number refers to biological replicates ; for each biological replicate , at least three technical replicates were performed . Data is represented as mean ± SEM . * and # indicate significance : between apical and basal ( * ) ; * / # p < 0 . 05 , * * p < 0 . 01 , by two - tailed unpaired student ' s t - test ( D ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative confocal photomicrographs showing coronal slices from 8W hGFAP‐tTA;H2B‐GFP mice with and without doxycycline treatment, upon immunofluorescent staining with the stem cell marker Nestin (red). DAPI was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm. (B) Quantification of Nestin expression in apical and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy). Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) and between doxycycline treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B)(C) Same as (A), with addition of the primary cilia marker AC3 (yellow). Arrows indicate Nestin-positive (white) and GFP-positive (green) cells with primary cilium. Scale bar = 10 µm. (D) Quantification of ciliated GFP+ NSCs in apical and basal cell layer as percentage of total apical/basal GFP+ cells. N = 7. (E) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker; N = 7. Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) ; */#p<0.05, **p<0.01, by two-tailed unpaired student's t-test (D).


Figure 3 ( A ) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP - H2BGFP mice , which had been administered doxycycline ( Doxy ) as indicated . Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red , respectively . DAPI counterstaining of the nuclei is blue . Scale bar indicates 20 µm . ( B , C ) Quantitative analyses of the number of apical and basal cells expressing GFP ( G + ) or not ( G - ) ; N = 8 ( B ) , and Ki67 immunoreactivity in these populations ; N ≥ 4 ( C ) . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations . * p < 0 . 05 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP-H2BGFP mice, which had been administered doxycycline (Doxy) as indicated. Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red, respectively. DAPI counterstaining of the nuclei is blue. Scale bar indicates 20 µm.(B, C) Quantitative analyses of the number of apical and basal cells expressing GFP (G+) or not (G-); N = 8 (B), and Ki67 immunoreactivity in these populations; N ≥ 4 (C). Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.


Figure 5 ( B ) Representative micrographs of the OB of AAV - GFP - injected animals , at 14 days ( 14D ) and 6 weeks ( 6W ) after virus injection . Scale bar indicates 20 µm . ( C ) Quantification of GFP + and / or DCX + cells in the OB , as percentage of total DAPI cells N ≥ 5 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons test . Representative confocal images of coronal slices of the core of the olfactory bulb ( OB ) obtained from RiboTag mice injected with AVV - hGFAP - mycCRE particles . In ( E ) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated . Scale bars = 20 µm . ( F ) Quantification of RiboTag - HA ( HA ) + and / or DCX + cells in the OB in N ≥ 2 . Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * indicates significance : * p < 0 . 05 , determined by two - way ANOVA with Sidak ' s multiple comparisons test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Representative micrographs of the OB of AAV-GFP-injected animals, at 14 days (14D) and 6 weeks (6W) after virus injection. Scale bar indicates 20 µm. (C) Quantification of GFP+ and/or DCX+ cells in the OB, as percentage of total DAPI cells N ≥ 5. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.Representative confocal images of coronal slices of the core of the olfactory bulb (OB) obtained from RiboTag mice injected with AVV-hGFAP-mycCRE particles. In (E) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated. Scale bars = 20 µm. (F) Quantification of RiboTag-HA (HA)+ and/or DCX+ cells in the OB in N ≥ 2. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.


Figure 7 ( A , B ) Representative micrographs of the V - SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 ( AAV Hes1 - sh ) or a scrambled sequence ( AAV sc - sh ) . Coronal sections were stained with either notch intracellular domain ( NICD , A ) or Ki67 to visualize cycling cells ( B ) . DAPI was used for nuclear counterstain , scale bars indicate 20 µm . Arrows indicate apical GFP + ( white ) and basal GFP - ( yellow ) cells displaying NICD staining . Quantification of fold change in NICD - levels ; N = 3 ( C ) , Ki67 + cells ; N = 5 ( D ) Data information : N number refers to biological replicates ; for each biological replicate at least three technical replicates were performed . Bars represent mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by two - way ANOVA with Sidak ' s multiple comparisons

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A, B) Representative micrographs of the V-SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 (AAV Hes1-sh) or a scrambled sequence (AAV sc-sh). Coronal sections were stained with either notch intracellular domain (NICD, A) or Ki67 to visualize cycling cells (B). DAPI was used for nuclear counterstain, scale bars indicate 20 µm. Arrows indicate apical GFP+ (white) and basal GFP- (yellow) cells displaying NICD staining. Quantification of fold change in NICD-levels; N = 3 (C), Ki67+ cells; N = 5 (D) Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test


Figure 1A , Misreading ( A ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes ( n = 8 misreading , mutant A1555G mitohybrid ribosomes ( n = 8 misreading , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . Mistranslation was determined by assessing the misreading at His245 ( CAC codon ) of the F - luc gene that was replaced by the near - cognate codon CGC or the non - cognate codon AGA , both encoding for Arg . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity . read - through ( B ) were measured by dual luciferase gain - of - function assays using wild - type mitohybrid ribosomes n = 4 read - through ) , mutant A1555G mitohybrid ribosomes n = 4 read - through ) , E . coli wild - type ribosomes ( n = 9 ) , E . coli mutant A127Y ribosomes ( n = 9 ) , M . smegmatis merodiploid wild - type ribosomes ( n = 9 ) , and M . smegmatis merodiploid mutant S152Y ribosomes ( n = 9 ) . To determine read - through the Asp357 ( GAC codon ) of the F - luc gene was replaced by the nonsense TGA stop codon . Results are derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity . C Ratio of 35S - Cysteine and 35S - Methionine incorporation by in - vitro translation using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , and MT - CO1 mRNA . Following immunoprecipitation , the MT - CO1 band was quantified by autoradiography , wild - type mitohybrid ribosomes were set as 1 ( n = 3 ) , error bars indicate SEM ; * * * * p < 0 . 001 ( Student ' s t - test ) . D Ratio of 35S - Cysteine and 35S - Methionine incorporation by in - vitro translation in the presence of tobramycin using wild - type mitohybrid ribosomes , mutant A1555G mitohybrid ribosomes , and MT - CO1 mRNA ; the MT - CO1 band was quantified by autoradiography . E Misreading of wild - type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin . Misreading was assessed by dual luciferase assay , and results derived by calculating mutant hFluc / hRluc activity related to wild - type hFluc / hRluc activity . F In - vitro translation of MT - CO1 - TGA mRNA and MT - CO1 - AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin ( 0 - 20 µM ) . Autoradiography of immunoprecipitated MT - CO1 proteins ; top : 35S - Met labelling , bottom : 14C - Lys labelling . In - organello translated 35S - Met labelled MT - CO1 was used as a marker . * MT - CO1 , * * MT - CO1 extended by read - through , MT - CO1 AGA - polyA and MT - CO1 TGA - polyA constructs used for in - vitro translation are schematized . G Ratio of 14C - Lys and 35S - Met labelled immunoprecipitated MT - CO1 proteins . In - vitro translation using MT - CO1 - TGA mRNA , MT - CO1 - AGA mRNA , and mutant A1555G mitohybrid ribosomes in the presence of tobramycin ( 0 - 20 µM ) . The corresponding MT - CO1 band was quantified by autoradiography .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes n=4 read-through), mutant A1555G mitohybrid ribosomes n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test).D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography.E Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized.G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.


Figure 2A Autoradiography of in - organello mitochondrial translation products derived from HEK293 wild - type cells in the presence of tobramycin , incorporation of 35S - Met and 35S - Cys in MT - CO1 protein . Following in - organello translation proteins were immunoprecipitated and analyzed by autoradiography . Lane 1 , 3 : controls ; lanes 2 , 4 : in - organello translation in the presence of 1000 µM tobramycin . MT - CO1 bands were quantified and ratio of 35S - Cys / 35S - Met calculated , the 35S - Cys / 35S - Met ratio in the absence of tobramycin was set as 1 ( n = 3 ) , error bars indicate SEM . B Ratios of 35S - Cys / 35S - Met labelled MT - CO1 and MT - CO2 proteins synthesized in in - organello translation and effect of tobramycin . Following in - organello translation in the presence of tobramycin ( 1000 µM , 2000 µM ) proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 and MT - CO2 bands were quantified and ratio of 35S - Cys / 35S - Met incorporation calculated ( n = 3 ) , error bars indicate SEM ; * p < 0 . 05 , ns , not significant ( Student ' s t - test ) . Ratio of 35S - Cys and 35S - Met labelled MT - CO1 protein synthesized in in - organello translation using mitochondria from HEK293 MRPS5 wild - type cells and HEK MRPS5 mutant V336Y cells . Following in - organello translation , proteins were immunoprecipitated and analyzed by autoradiography . MT - CO1 bands quantified and ratios calculated . The ratio of the MRPS5 wild - type was set as 1 ( n = 8 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) . D Autoradiography of in - organello mitochondrial translation products derived from HEK293 MRPS5 wild - type cells in the presence of tobramycin ( 0 - 750 µM ) . Proteins were 35S - Met labeled and loaded either directly on SDS - PAGE ( total , top ) or following poly - lysine immunoprecipitation ( bottom ) . * MT - CO1 and * * MT - CO1 with poly - lysine extension . E Quantification of MT - CO1 proteins in HEK293 MRPS5 wild - type cells and HEK293 MRPS5 mutant V336Y cells . In - organello mitochondrial translation in the presence of 35S - Met and 750 µM tobramycin . Proteins were loaded either directly on SDS - PAGE ( total ) or following poly - lysine immunoprecipitation and analyzed by autoradiography . For total protein samples the MT - CO1 band was quantified , for poly - lysine immunoprecipitated samples the extended MT - CO1 band was quantified and the ratio of extended MT - CO1 / total MT - CO1 calculated . The ratio of MRPS5 wild - type was set as 1 ( MRPS5 wild - type , n = 5 clones , MRPS5 V336Y , n = 7 clones ) , error bars indicate SD ; * * * p < 0 . 0001 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.B Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).


Figure 3A Generation time ( n = 8 clones , ± SD ) . B Whole cell in - vivo mitochondrial translation as determined by 35S - Met incorporation ( n = 4 clones , ± SEM ) . C Autoradiography of in - organello translation using 35S - Met labelling .

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Figure 3A Generation time (n=8 clones, ± SD).B Whole cell in-vivo mitochondrial translation as determined by 35S-Met incorporation (n=4 clones, ± SEM).C Autoradiography of in-organello translation using 35S-Met labelling.


Figure 4Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . A OCR in brain mitochondria from 19 - months mutant mice ( black circle ) compared with OCR from wild - type mice ( white circle ) . The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown . Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . Baseline OCR , effect of age ( p < 0 . 0001 ) and genotype ( p < 0 . 05 ) . Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . C State 3 OCR ( after addition of ADP ) , effect of age ( p < 0 . 001 ) and genotype ( p < 0 . 0001 ) . Oxygen consumption rate ( OCR ) was measured in freshly isolated mitochondria from wild - type and mutant mouse cortex . D State 3 uncoupled OCR ( state 3u after addition of FCCP ) , effect of genotype ( p < 0 . 0001 ) and age - genotype interaction ( p < 0 . 0001 ) . E Ratio of mitochondrial complex IV and complex II activities . F ATP levels . G Superoxide anion radicals .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O B-PANEL_START O O O O

Figure 4Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. A OCR in brain mitochondria from 19-months mutant mice (black circle) compared with OCR from wild-type mice (white circle). The sequential injection of mitochondrial substrates and inhibitors is indicated by dotted lines Values corresponding to the different respiratory states are shown.Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. Baseline OCR, effect of age (p<0.0001) and genotype (p<0.05).Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. C State 3 OCR (after addition of ADP), effect of age (p<0.001) and genotype (p<0.0001).Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. D State 3 uncoupled OCR (state 3u after addition of FCCP), effect of genotype (p<0.0001) and age-genotype interaction (p<0.0001).E Ratio of mitochondrial complex IV and complex II activities.F ATP levels.G Superoxide anion radicals.


Figure 5A Average distance to center as measure of anxiety in the open field ( ANOVA : genotype F1 , 24 = 16 . 24 p = 0 . 0005 , age F2 , 24 = 5 . 217 p = 0 . 0131 ) . Mutants show increased anxiety levels at 9 - and 14 - months . B Estimated frequency of rearing activity as measure of exploratory drive in the open field ( ANOVA : genotype F1 , 24 = 39 . 40 p < 0 . 0001 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . Mutants showed little exploratory activity at 9 - and 14 - months . C Light - dark transition test , % time spent in the bright compartment ( ANOVA : genotype F1 , 16 = 7 . 393 p = 0 . 0152 ) . Reduced scores at 9 - months old animals indicate increased anxiety . D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage ( ANOVA : phase F1 , 18 = 617 . 2 p < 0 . 0001 ) . Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9 - months old animals . E Learning in the hippocampus - dependent IntelliCage serial reversal task ( ANOVA : day F2 , 56 = 26 . 71 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance . F Learning in the hippocampus - dependent IntelliCage chaining task ( ANOVA : day F2 , 54 = 450 . 1 p < 0 . 0001 ) . Scores provided no evidence for a mutation effect on learning rate or performance . G Water - maze place navigation , swim path during acquisition and reversal ( ANOVA : day F4 , 104 = 21 . 14 p < 0 . 0001 , genotype F1 , 26 = 13 . 11 p = 0 . 0013 , age x genotype F2 , 26 = 8 . 204 p = 0 . 0017 ) . All groups learned : controls showed reduced performance with increasing age , mutants showed poor performance already at 9 - and 14 - months . H Number of wall approaches as indicator of poor coping with the test and non - spatial strategy choice ( ANOVA : genotype F1 , 26 = 18 . 31 p = 0 . 0002 , age F2 , 26 = 4 . 486 p = 0 . 0212 , age x genotype F2 , 26 = 7 . 068 p = 0 . 0035 ) . Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9 - and 14 - months . Water - maze probe trial , spatial retention score for target zones 1 - 3 comprising 5 , 10 and 10 % of the pool surface versus remaining 75 % of surface ( ANOVA : zone F3 , 78 = 36 . 04 p < 0 . 0001 , genotype x zone F3 , 78 = 7 . 807 p = 0 . 0001 ) . Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9 - and 14 - months .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Average distance to center as measure of anxiety in the open field (ANOVA: genotype F1,24=16.24 p=0.0005, age F2,24=5.217 p=0.0131). Mutants show increased anxiety levels at 9- and 14-months.B Estimated frequency of rearing activity as measure of exploratory drive in the open field (ANOVA: genotype F1, 24=39.40 p<0.0001, age x genotype F2,26=8.204 p=0.0017). Mutants showed little exploratory activity at 9- and 14-months.C Light-dark transition test, % time spent in the bright compartment (ANOVA: genotype F1,16=7.393 p=0.0152). Reduced scores at 9-months old animals indicate increased anxiety.D Exploratory corner visits in dark and light phase during adaptation to the IntelliCage (ANOVA: phase F1,18=617.2 p<0.0001). Corner visits during the dark phase as a correlate of exploratory activity showed premature reduction in 9-months old animals.E Learning in the hippocampus-dependent IntelliCage serial reversal task (ANOVA: day F2,56=26.71 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.F Learning in the hippocampus-dependent IntelliCage chaining task (ANOVA: day F2,54=450.1 p<0.0001). Scores provided no evidence for a mutation effect on learning rate or performance.G Water-maze place navigation, swim path during acquisition and reversal (ANOVA: day F4,104=21.14 p<0.0001, genotype F1,26=13.11 p=0.0013, age x genotype F2,26=8.204 p=0.0017). All groups learned: controls showed reduced performance with increasing age, mutants showed poor performance already at 9- and 14-months.H Number of wall approaches as indicator of poor coping with the test and non-spatial strategy choice (ANOVA: genotype F1,26=18.31 p=0.0002, age F2,26=4.486 p=0.0212, age x genotype F2,26=7.068 p=0.0035). Number of wall approaches increased with age in controls and was prematurely increased in mutants at 9- and 14-months.Water-maze probe trial, spatial retention score for target zones 1-3 comprising 5, 10 and 10% of the pool surface versus remaining 75% of surface (ANOVA: zone F3,78=36.04 p<0.0001, genotype x zone F3,78=7.807 p=0.0001). Spatial selectivity declined with age in controls and was prematurely reduced in mutants at 9- and 14-months.


Figure 6Noise exposure and ABR measurements . Exposure of mice to broadband noise ( 2 - 10 kHz ) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds . Thresholds in Mrps5V338Y / V338Y mutant mice ( black bars ) were significantly higher after exposure than thresholds in littermates ( open bars ) , ( p < 0 . 0001 , two - way ANOVA for combined 108 and 110 dB data ) . Frequencies of 12 , 24 and 32 kHz were selected for measurement to cover the mid - frequency to high - frequency regions of the cochlea . n = 6 for baseline littermates ; n = 6 for baseline mutants ; n = 3 for all noise exposures .

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Figure 6Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures.


Figure 1 ( A ) Fed rat liver lysosomes were extracted with 1 % of the indicated detergents and then subjected to sucrose gradient centrifugation . Aliquots collected from top to bottom were subjected to SDS - PAGE and immunoblotted for LAMP ‐ 2A and flotillin ‐ 1 . Stars indicate the aliquots of the gradient of translucent appearance ( highly enriched in lipids ) . ( B ) The distribution of LAMP ‐ 2A through the sucrose gradient after extraction with 1 % Triton X ‐ 114 was calculated from the densitometric quantification of immunoblots as the one shown here . Values are expressed as percentage of the total lysosomal levels and are mean + s . e . of four experiments .

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Figure 1(A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS-PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids).(B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.


Figure 2 ( A ) Lysosomes from livers of fed ( Fed ) or 24 h starved rats ( Stv ) were extracted with 1 % Triton X ‐ 114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent ‐ resistant ( DR ) fraction , and anything below this fraction was pooled , as the solubilized fraction ( Sol ) ( B ) . Lanes 5 - 6 are one ‐ tenth of the amount of lysosomes loaded in the gradient . ( C ) Distribution of LAMP ‐ 2A ( L ‐ 2A ) and flotillin ‐ 1 ( Flot ‐ 1 ) in fed rats liver lysosomes extracted with 1 % Triton X ‐ 114 at 4 or 37 ° C . DR fractions were pooled as in ( B ) , and the aliquots below this region were divided in three fractions ( a soluble ( Sol ; aliquot 15 ) and two intermediate fractions ( light : aliquots 11 - 12 ; heavy : aliquots 13 - 14 ) . ( D ) Fed ratliverlysosomes were incubated with MBCD or filipin , as described under Material and methods , and then processed as in ( C ) . The mean value of the densitometric quantification of the LAMP ‐ from two experiments is shown at the bottom in ( C ) and ( D ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient.(C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).(D) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).


Figure 3 ( A ) Lysosomalmembranes from fed ratlivers and their corresponding 1 % Triton X ‐ 114 ‐ resistant regions , isolated as described under Material and methods , were subjected to one ( left ) ‐ or two ‐ dimensional ( right ) electrophoresis and stained with Sypro Ruby . ( B ) Immunoblot for the indicated proteins of the detergent resistant ( DR ) , intermediate ( Int ) and solubilized ( Sol ) fractions of lysosomal membranes obtained as in Figure 2 .

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Figure 3(A) Lysosomalmembranes from fed ratlivers and their corresponding 1% Triton X‐114‐resistant regions, isolated as described under Material and methods, were subjected to one (left)‐ or two‐dimensional (right) electrophoresis and stained with Sypro Ruby.(B) Immunoblot for the indicated proteins of the detergent resistant (DR), intermediate (Int) and solubilized (Sol) fractions of lysosomal membranes obtained as in Figure 2.


Figure 4LAMP ‐ 2A localizes in cholesterol ‐ and GM ‐ 1 ‐ enriched intracellular membrane domains . Mouse fibroblasts grown on coverslips in the presence ( + ) or absence ( − ) of serum were fixed and labeled with filipin ( red ) ( A ) or incubated with Texas ‐ Red ‐ cholera toxin B subunit to label GM ‐ 1 ‐ enriched membrane regions ( red ) and then fixed . ( B ) Both groups of cells were then subjected to indirect immunofluorescence for LAMP ‐ 2A ( green ) . Where indicated , cells were treated with MBCD ( 25 mM ) for 30 min before fixation . The merged images of both fluorophores are shown on the right . Insets show vesicles at higher magnification . Bar : 10 μm . The quantification of the colocalization of the two fluorophores is shown in ( C ) . Values are expressed as the mean + s . e . of percentage of colocalization in 20 - 40 cells .

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Figure 4LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed.(B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm.The quantification of the colocalization of the two fluorophores is shown in (C). Values are expressed as the mean+s.e. of percentage of colocalization in 20-40 cells.


Figure 5 ( A , B ) Regions of the lysosomalmembrane resistant to 1 % Triton X ‐ 114 recovered from the sucrose gradients were fixed and processed for electron microscopy ( A ) or visualized without fixation by electron microscopy ( B ) . Arrows point to the membrane bilayer . ( C ) Intact ratliverlysosomes ( top ) and DR regions of the lysosomal membranes ( bottom ) were fixed and subjected to immunogold labeling for LAMP ‐ 2A and LAMP ‐ 2B , and a gold ‐ conjugated secondary antibody as described under Material and methods . Top right panels show ' clusters ' of gold particles ( black arrows ) in individual lysosomes at higher magnification . Graph shows the labeling density ( gold particles per μm ) in the indicated fractions for both proteins . Values are mean + s . e . from the quantification of 15 different micrographs . ( D ) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP ‐ 2A and flotillin ‐ 1 . Representative lysosomes are shown . Far right panel : zoomed ‐ in image of the distribution of LAMP ‐ 2A in regions containing ( black arrows ) and not containing ( white arrows ) flotillin ‐ 1 . Bars : 0 . 2 μm .

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Figure 5(A, B) Regions of the lysosomalmembrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer.(C) Intact ratliverlysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show 'clusters' of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs.(D) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP‐2A and flotillin‐1. Representative lysosomes are shown. Far right panel: zoomed‐in image of the distribution of LAMP‐2A in regions containing (black arrows) and not containing (white arrows) flotillin‐1. Bars: 0.2 μm.


Figure 6 ( A ) Fed ratliverlysosomes were incubated in an isotonic buffer without additions , or in the presence of ratlivercytosol ( 25 μg ) , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ( GAPDH ) ( 25 μg ) , GST ‐ hsc70 ( 10 μg / ml ) , ATP ( 5 mM ) or the indicated combinations for 15 min at 37 ° C . Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel . At the end of the incubation , lysosomes were subjected to Triton X ‐ 114 extraction , apyrase density gradient centrifugation and immunoblot as in Figure 2C . Graph shows changes in LAMP ‐ 2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3 - 6 experiments as the one shown here . Because all the incubations were performed in the presence of protease inhibitors , total levels of LAMP ‐ 2A in lysosomes remained constant throughout the incubation . ( B ) Lysosomes from livers of fed rats , untreated or treated with paraquat ( + PQ ) , were processed as in ( A ) . Graph shows changes in LAMP ‐ 2A distribution induced by the PQ treatment , expressed as times the value in untreated animals and are the mean of three experiments . ( C ) Lysosomes with high ( CMA + ) and low ( CMA − ) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in ( A ) . Top left : Levels of two CMA components ( LAMP ‐ 2A and hsc70 ) and of an unrelated lysosomalmembrane protein ( LAMP ‐ 1 ) in each of the four groups of lysosomes . Bottom left : Percentage of total lysosomal LAMP ‐ 2A present in each fraction , determined by densitometric quantification of the immunoblots .

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Figure 6(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation.(B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments.(C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.


Figure 7 ( A ) Degradation of long ‐ lived proteins in mouse fibroblasts ( NIH3T3 cells ) treated or not with 25 mM MBCD . Where indicated , 15 mM NH4Cl / 100 μM leupeptin ( NH4Cl + Leup ) were added into the medium . Values are the mean + s . e . of three experiments with triplicate samples . ( B ) Effect of MBCD on total rates of protein degradation , measured as in ( A ) , in wild ‐ type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP ‐ 2A ( LAMP ‐ 2A ( − ) ) . The ratio of degradation in MBCD ‐ treated and untreated cells is shown for cells maintained with or without 10 mM 3 ‐ methyladenine ( 3MA ) . ( C ) Proteolysis of [ 14C ] GAPDH or a pool of [ 3H ] labeled cytosolic proteins ( pool proteins ) by isolated rat liver lysosomes , treated or not with MBCD . Broken : lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins . Values are expressed as percentage of the degradation by untreated lysosomes and are the mean + s . e . of three experiments with triplicate samples . ( D ) Lysosomal - mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol ( 50 μM ) . Top : Distribution of LAMP ‐ 2A after Triton X ‐ 114 extraction and sucrose density gradient centrifugation . Bottom : Ability of intact or disrupted lysosomal - mitochondrial fractions to proteolize [ 14C ] GAPDH . Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean ± s . e . of triplicate samples .

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Figure 7(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.


Figure 8 ( A ) Lysosomes from mouse fibroblasts transfected with wild ‐ type hLAMP ‐ 2A and maintained in the presence ( Serum + ) or absence ( serum − ) of serum were collected by centrifugation and subjected to Triton X ‐ 114 extraction , sucrose density gradient centrifugation and immunobloting for human ( hL2A ) or mouse ( mL2A ) LAMP ‐ 2A . Lane 1 : homogenate . ( B ) Amino - acid sequence of the transmembrane and cytosolic region of hLAMP - 2A and the mutations performed . Replaced amino acids are shadowed . ( C ) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP ‐ 2A and maintained in the presence of serum were processed as in ( A ) . ( D ) The percentage of total lysosomal hLAMP ‐ 2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here . Values , corrected for the amount of mLAMP ‐ 2A in each fraction , are mean + s . e . of 3 - 4 experiments . ( E ) Changes in hLAMP ‐ 2A distribution induced by serum removal . Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments . Changes in the three nonDR regions are shown together ( no DR ) . ( F ) Proteolysis of a pool of 3 [ H ] labeled cytosolic proteins by intact ( left ) or disrupted ( right ) lysosomes isolated from untransfected mouse fibroblasts ( none ) or fibroblasts transfected with wild ‐ type ( WT ) or the indicated mutant forms of hLAMP ‐ 2A . Values are the mean + s . e . of two experiments with triplicate samples . ( G ) Part of the lysosomes from ( F ) was treated with MBCD before the incubation with the radiolabeled proteins . Values are expressed as percentage of degradation by untreated lysosomes and are the mean + s . e . of two experiments with triplicate samples .

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Figure 8(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.(B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed.(C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A).(D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3-4 experiments.(E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR).(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.(G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.


Figure 9 ( A ) The Triton X ‐ 114 ‐ resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous ( 10 - 80 % ) sucrose density gradient , SDS - PAGE and immunoblot for LAMP ‐ 2A . The molecular weight range of each fraction is as follows : fraction 1 ( 100 kDa ) ; fraction 2 ( 100 - 200 kDa ) ; fraction 3 ( 200 - 400 kDa ) ; fraction 4 ( 400 - 600 kDa ) ; fraction 5 ( > 600 kDa ) . ( B ) Fed rat liver lysosomes were incubated with 1 mM CaCl2 ( + Ca2 + ) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors ( PI ) . Samples were subjected to Triton X ‐ 114 extraction , sucrose density gradients centrifugation and LAMP ‐ 2A immunoblot . ( C ) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS - PAGE and immunobloted with antibodies against the cytosolic ( Top ; L ‐ 2A ) and luminal ( Bottom ; L ‐ 2 ) regions of LAMP ‐ 2A . Arrow : LAMP ‐ 2A proteolytic fragment ( Cuervo and Dice , 2000a ) . ( D , E ) Rat liver lysosomes incubated or not with 1 mM CaCl2 ( + Ca2 + ) were subjected to Triton X ‐ 114 extraction and sucrose density gradients centrifugation . Levels of cathepsin A ( D ) and carboxypeptidase A activity ( E ) were measured in each fraction by immunoblot or using a standardized colorimetric assay , respectively . Values in ( E ) are expressed as times the activity in untreated membranes and are mean + s . e . of two experiments .

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Figure 9(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.