Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Knock-Out, Staining, Expressing, Marker, Transduction, Sequencing, Isolation

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: All images show formaldehyde‐fixed HeLa cells. HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7. Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red). Co‐staining of endogenous RAB7 (green) and the trans ‐Golgi network marker TGN46 (red). Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red). Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red). Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells. GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red. Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Staining, Marker, Western Blot, Microscopy

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: A Mitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations. B RAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes. C, D RAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20. E RAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Purification, Magnetic Beads, Marker, Transduction, Staining, Variant Assay, Infection, Labeling, Live Cell Imaging, Microscopy

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: A, B Parental HeLa cells and clonal VPS35 KO cells were infected with lentiviruses expressing inactive GFP‐RAB7‐T22N (A) or constitutively active GFP‐RAB7‐Q67L (B) and co‐stained for endogenous TOM20 (red). Co‐localization was analyzed across two independent experiments. C RILP effector assay comparing VPS35 KO cells and VPS35 KO cells that had been infected with a lentivirus encoding untagged human VPS35. D Immunofluorescent staining showing that lentivirally expressed VPS29‐myc restores vesicular VPS29. E RILP effector assay with lysates from parental HeLa cells and VPS35 KO cells and lysates from VPS35 KO cells that were spiked with recombinant VPS35 produced in bacteria. Note that re‐addition of VPS35 has no effect on the amount of active RAB7 binding to the GST‐RILP beads. F Quantitative proteomic interactome analysis of GFP‐RAB7 isolated from parental and VPS35 KO cell lines. The SILAC ratios (from two independent experiments with swapped isotope labeling) for the indicated proteins suggest that RAB7 is more active in the knockout cells, as the RAB7 effector RILP is increased in the RAB7 IP from KO cells while the RAB chaperones GDI1, GDI2, and PRA1 are strongly decreased. G HeLa cells were fixed in cold methanol and stained for endogenous RAB7a and LAMP2. The same cell and same Z ‐position was then imaged at low‐laser settings (left) and at high‐laser setting on a confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Infection, Expressing, Staining, Recombinant, Produced, Binding Assay, Isolation, Labeling, Knock-Out, Microscopy

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: HeLa cells fixed in methanol were co‐stained for the indicated endogenous proteins. Green signal is Alexa 488, and red signal is Alexa 594. Co‐staining of endogenous VPS35 and the late endosome marker LBPA (left) and the late endosome/lysosome marker LAMP2. Note that VPS35 seems to be less associated with LBPA‐positive domains than with LAMP2‐decorated entities. 3D reconstruction of PFA‐fixed single cells expressing GFP‐RAB5 and co‐stained for endogenous LAMP2 (Alexa 405, blue) and endogenous VPS35 or FAM21 (Alexa 594, red). HeLa cells transfected with siRNA targeting VPS35 and RAB7a were co‐stained for the endogenous glucose transporter GLUT1 (Alexa 594, red) and the lysosomal marker LAMP1 (Alexa 488, green). Efficiency of the siRNA was tested by Western blotting. Co‐localization was analyzed over two independent experiments with 10 images each. Data information: All scale bars = 10 μm besides lower panel in (A), where scale bar = 5 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Staining, Marker, Expressing, Transfection, Western Blot

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: HeLa cells were co‐transfected with CRISPR/Cas9 constructs targeting the indicated genes and a puromycin resistance marker. Five days after puromycin selection of transfected cells, all cell lines were stained for endogenous RAB7a and the lysosomal marker LAMP2, and co‐localization was quantified across 12 images acquired in two independent experiments. The cell lines described above for panel (A) were lysed, and RAB7a activity was assayed with the GST‐RILP activity assay. Western blotting was used to confirm the efficiency of the CRISPR/CAS9 targeting approach. RILP‐bound RAB7a was quantified over three independent experiments. Methanol‐fixed HeLa cells and VPS35 and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Methanol‐fixed parental HeLa cells and TBC1D5 KO cells were stained for endogenous VPS35 (green) and LAMP2 (red). Methanol‐fixed parental HeLa and TBC1D5 KO cells were co‐stained for endogenous RAB7a (green) and endogenous VPS35 (red), and co‐localization was quantified over two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Transfection, CRISPR, Construct, Marker, Selection, Staining, Activity Assay, Western Blot

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: GFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35. PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Construct, Mutagenesis, Binding Assay, Transduction, Activity Assay, Expressing, Staining

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: HeLa, SNX5/6 double KO, and TBC1D5 KO cells were fixed in PFA and stained for CI‐MPR (red) and the trans ‐Golgi network marker TGN46. Note that knockout of SNX5/6 results in dispersal of CI‐MPR from the TGN. HeLa, VPS29, and TBC1D5 KO cells were fixed in PFA and stained for GLUT1 (green) and LAMP2 (red). Co‐localization between GLUT1 and LAMP2 was quantified over ten images for each condition. HeLa cells were transfected with a pool of three distinct CRISPR/Cas9 plasmids targeting the RAB7a gene at three sites together with a puromycin resistance plasmid. Following puromycin selection and 5 days of incubation, the RAB7a KO cells were transduced with lentiviruses expressing the indicated GFP‐RAB7 proteins. A Western blot for RAB7 confirms high knockout efficiency and demonstrates that the GFP‐RAB7 variants are expressed at low endogenous levels. Parental HeLa cells and clonal VPS29 and VPS35 KO cells transduced with GFP‐LC3b were starved in EBSS for 4 h without (EBSS) or with addition of bafilomycin A1 to the EBSS (EBSS + Bafi) to assess autophagic flux. Autophagic flux was calculated from the increase in lipidated GFP‐LC3 in the EBSS + bafilomycin samples compared to the EBSS‐only sample. The quantification was done across four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Staining, Marker, Knock-Out, Transfection, CRISPR, Plasmid Preparation, Selection, Incubation, Transduction, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: Control of RAB 7 activity and localization through the retromer‐TBC1D5 complex enables RAB 7‐dependent mitophagy

doi: 10.15252/embj.201797128

Figure Lengend Snippet: All images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments. RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments. Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P < 0.05 in a t ‐test of the respective condition compared to the control cells. Source data are available online for this figure.

Article Snippet: Further antibodies used were as follows: mouse anti‐GAPDH (10494‐1‐AP), anti‐VARP (24034‐1‐AP), and anti‐TBC1D5 (17078‐1‐AP) from Proteintech; SNX6 from Sigma‐Aldrich (S6324); rat anti‐myc (clone 9E1) and rat anti‐GFP (clone 3H9) from Chromotek; and mouse anti‐LAMP1 (clone H4A3) and mouse anti‐LAMP2 (clone H4B4) from the DSHB.

Techniques: Transduction, Construct, Staining, Isolation, Western Blot, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Calmodulin enhances mTORC1 signaling by preventing TSC2-Rheb binding

doi: 10.1016/j.jbc.2024.108122

Figure Lengend Snippet: Ca 2+ /CaM affects TSC2 localization without altering TSC2 phosphorylation . A , HEK293 cells were starved of serum (−FBS) for 3 h, pretreated with DMSO or BAPTA-AM (50 μM) or W-7 (30 μM) for the last 1 h (BAPTA-AM) or 5 min (W-7) and then stimulated with or without (−) carbachol (100 μM) for 15 min (+CCh). Cell lysates were analyzed by Western blotting with the indicated antibodies. The images of phospho-T389-S6K1 and S6K1 panels in DMSO and BAPTA-AM lanes are reused from Fig. 6 C . B , Quantitation of the relative intensity of phospho-T1462-TSC2 to total TSC2 of (A), in which the carbachol stimulation (DMSO) was set to 1. Graphs represent mean ± SD of three independent experiments. One-way ANOVA with Tukey’s test. C , HEK293 cells were starved of serum (−FBS) for 3 h, and then treated with carbachol (100 μM) or insulin (100 nM) for 15 min. The cells were then immunostained with anti-TSC2 and anti-LAMP2 antibodies. Merged images of TSC2 ( green ), LAMP2 ( magenta ) and nuclei staining with Hoechst 33342 ( blue ) are also shown. Scale bar, 10 μm. D , colocalizations of TSC2 with LAMP2 were quantified, and Pearson’s correlation coefficient is shown. Data from four independent experiments are shown in blue triangles and were used for statistical analysis. Bars represent averages. The values of Pearson’s correlation coefficient of five individual images in each experiment are also shown in gray circles . One-way ANOVA with Tukey’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For immunofluorescence, TSC2 (D93F12, Cell Signaling Technology #4308) and LAMP2 (H4B4, Proteintech 65053-1-Ig) were used as primary antibodies.

Techniques: Phospho-proteomics, Western Blot, Quantitation Assay, Staining