Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding. 10.7554/eLife.31326.028 Figure 8—source data 1. Quantification of RABGEF1 recruitment to damaged mitochondria during mitophagy. 10.7554/eLife.31326.029 Figure 8—source data 2. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin. 10.7554/eLife.31326.030 Figure 8—source data 3. Binding affinities of recombinant GST-mRABGEF1 with ubiquitin or phosphorylated ubiquitin.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Expressing, Immunostaining, Western Blot, Recombinant, Ubiquitin Proteomics, Pull Down Assay, Binding Assay

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) The indicated cells were treated with DMSO or valinomycin for 3 hr followed by immunostaining. Bars, 10 μm. Graphs for quantification of RABGEF1 recruitment to mitochondria were shown below the images. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ±SE and over 100 cells were counted in each of three separate wells. ( B ) WT and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr. GFP-mRABGEF1 signals were enhanced by immunostaining with anti-GFP antibody. Bars, 10 μm. ( C ) Total cell lysates in ( B ) were analyzed by immunoblotting. * and # denote ubiquitinated forms and truncated forms, respectively. 10.7554/eLife.31326.031 Figure 8—figure supplement 1—source data 1. This excel file contains quantification of RABGEF1 (WT and Y26A/A58D mutant) recruitment to mitochondria in HCT116 (WT and TBC1D15/17 DKO) cells.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Immunostaining, Stable Transfection, Expressing, Western Blot, Mutagenesis

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) GFP-mRABGEF1 was transiently expressed in siRNA-treated HeLa cells. The cells were then treated with valinomycin for 3 hr followed by immunostaining. Bars, 20 μm. ( B ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HeLa cells. ( C ) Quantification of mitochondrial recruitment of GFP-mRABGEF1 in HCT116 cells. None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. The error bars represent mean ± SE and over 100 cells were counted in each of three separate wells. 10.7554/eLife.31326.032 Figure 8—figure supplement 2—source data 2. Quantification of RABGEF1 recruitment to mitochondria in HeLa cells treated with the indicated siRNA during mitophagy. 10.7554/eLife.31326.033 Figure 8—figure supplement 2—source data 3. Quantification of RABGEF1 recruitment to mitochondria in HCT116 cells treated with the indicated siRNA during mitophagy.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Immunostaining

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet: ( A ) WT and RABGEF1-mAID HCT116 cells were treated with or without IAA for 16 hr. Total cell lysates were analyzed by immunoblotting. ( B ) Quantification of Parkin recruitment to mitochondria in WT and RABGEF1-mAID HCT116 cells after 3 hr of valinomycin treatment. Partial and complete denote that YFP-Parkin signals were overlapped with some of and all mitochondria, respectively. ( C ) YFP-Parkin stably expressing WT and RABGEF1-mAID HCT116 cells pre-treated with IAA were treated with valinomycin for the indicated times. Total cell lysates were analyzed by immunoblotting. ( D ) WT and RABGEF1-mAID HCT116 cells stably expressing YFP-Parkin and mt-mKeima were treated with IAA for 16 hr followed by DMSO or OAQ for 6 hr and subjected to FACS analysis. Plots are representative of n = 3 experiments. ( E ) Quantification of mitophagy in ( D ). Error bars represent mean ±SE of three independent experiments. Statistical differences were determined by student’s t-test. *p<0.05. 10.7554/eLife.31326.035 Figure 9—source data 1. Quantification of YFP-Parkin recruitment to mitochondria in RABGEF1-mAID HCT116 and the corresponding WT cells during mitophagy. 10.7554/eLife.31326.036 Figure 9—source data 2. Quantification of mitophagy using mt-mKeima and FACS analysis.

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Western Blot, Stable Transfection, Expressing

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GFP (ab6556; Abcam, Cambridge, MA), mouse anti-MFN2 (ab56889; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-LC3B (L7543; Sigma, St. Louis, MO), mouse anti-MT-CO2 (ab110258; Abcam), mouse anti-Actin (MAB1501R; Millipore, Bedford, MA), mouse anti-RAB7 (ab50533; Abcam), rabbit anti- RABGEF1 (NBP1-49938; NOVUS BIOLOGICALS, Littleron, CO), mouse anti-CCZ1 (sc-514290; Santa Cruz Biotechnology), mouse anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), and rabbit anti-S65 phosphorylated ubiquitin (described previously [ ]).

Techniques: Sequencing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot, Recombinant, Software, Microscopy

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: ( A ) A schematic showing conserved functions of Rab5/RabGEF1 in endocytosis and autophagosome closure. (B) Rabgef1 expression in developing whole retina or flow-sorted rod and S-cone-like photoreceptors (identified from RNA-seq data in [ , ]). Nrl -GFP mice enabled purification of rod photoreceptors (Rods), whereas Nrl -GFP mice crossed with the cone-only Nrl -/- mice allowed sorting of S-cone-like photoreceptors . (C) In situ hybridization profile of postnatal (P)21 control and Rabgef1 -/- (KO) retina. Red/white punctate dots represent single Rabgef1 mRNA molecules. Arrow indicates high Rabgef1 transcripts in photoreceptor layer. Scale bar = 20 μm. ONL, outer nuclear layer; INL, inner nuclear layer. (D) Immunoblot analysis of P4 –P28 control and Rabgef1 -/- P14 retinal lysates probed with anti-RabGEF1 antibody. The total protein loading control is included in . (E) Immunohistochemistry of P14 control and Rabgef1 -/- retinal sections using anti-RabGEF1 antibody. Arrows indicate high RabGEF1 expression in photoreceptor inner segments and synaptic terminals. Only non-specific background staining (punctate dots), also observed in control, is detected in Rabgef1 -/- retinal sections. DAPI was used for visualizing nuclei. Scale bar = 20 μm.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Expressing, RNA Sequencing Assay, Purification, In Situ Hybridization, Western Blot, Immunohistochemistry, Staining

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) H&E staining of developing control and Rabgef1 -/- retina, showing near complete ablation of photoreceptors (ONL) by postnatal day 45. Scale bar = 50 μm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B, C) ERG stimulus intensity-amplitude functions in the control and KO mice reared in a 12h light/12h dark cycle and measured at postnatal day 15 and 21 (P15, P21), respectively. (D) ERG stimulus intensity-amplitude functions in control and Rabgef1 -/- mice born and raised in the dark. Dark-reared Rabgef1 -/- mice have similar ERG stimulus intensity-amplitude functions as animals reared in cyclic light. Asterisks indicate p-value < 0.05 (*), <0.01 (**) and <0.001 (***) as determined by a t-test using Prizm software. In panels B and C, n = 7–10 per genotype and in D, 2–3 per genotype. Error bars indicate SD.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Staining, Software

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) Immunohistochemistry of P15 retinas using antibodies against proteins of the phototransduction cascade and proteins required for outer segment integrity. Retinal sections were counterstained with DAPI. ONL, outer nuclear layer; OS, outer segments. Scale bar = 20 μm. (B) Immunoblots of proteins from retinal lysates through development, from P6—P14. Rabgef1 -/- retinal lysate protein levels appear reduced at all developmental stages compared to littermate controls. γ-tubulin and Histone H3 are used as loading controls. 40 μg of protein loaded per lane.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Immunohistochemistry, Western Blot

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) Transmission electron micrographs of control and Rabgef1 -/- retinas at around eye opening (P14). White box highlights accumulation of amorphous, electron-dense structures in Rabgef1 -/- photoreceptor inner segment regions. (B) Magnification of white boxed area in panel A and an image at the same magnification but in P22 Rabgef1 -/- retina (C) , demonstrating the accumulation of electron-dense deposits in photoreceptor cytoplasm (black arrowheads).

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Transmission Assay

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) Immunoblot analyses of Rabbit-IgG and anti-RabGEF1 pulldown (PD) protein complexes (from 40 μg retinal lysate) with antibodies against RabGEF1 and Rabaptin5. Input was 3% of the protein from the control P21 mouse retinal lysate. Rabbit-IgG served as a negative control. (B) Mass spectrometry analysis of RabGEF1 co-immunoprecipitated proteins from P21 control and Rabgef1 -/- retinas. RabGEF1 binds to Rabaptin-5 in a 1:2 ratio respectively (n = 2). (C) Immunohistochemistry with anti-EEA1 antibody on control and Rabgef1 -/- retinal slices. Retinal sections were counterstained with DAPI. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar = 20 μm. Boxed areas indicate regions of retina that were quantified, also shown at larger scale in . (D) Quantitative measurements of EEA1-positive endosomes at P10, 12 and 14 control and Rabgef1 -/- individual z-stack images of the inner segment regions. ( E) Immunoblot of control and Rabgef1 -/- P14 retinal lysates probed with anti-EEA1. Anti-beta-actin was used as a loading control. There is no significant difference between EEA1 protein amounts in control and Rabgef1 -/- retinal lysates Asterisks indicate p-value < 0.05 (*) and < 0.01 (**) as determined by t-test using Prizm software. In panels D and E, n = 3 biological replicates. Error bars indicate SD.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Western Blot, Negative Control, Mass Spectrometry, Immunoprecipitation, Immunohistochemistry, Software

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) Immunohistochemistry of control and Rabgef1 -/- retinas using anti-LC3A/B antibody. Retinal sections were counterstained with DAPI. Scale bar = 20 μm. ONL, outer nuclear layer; IS, inner segment. (B) Low magnification images of anti-LC3A/B staining in P17 retina. Scale bar = 20 μm. INL, inner nuclear layer; GCL, ganglion cell layer. (C) Immunoblot of P12 and 14 control and Rabgef1 -/- retinal lysates probed with antibody to LC3A/B (upper panel). Lower arrowhead indicates increase in the LC3A/B-II lipidated form in Rabgef1 -/- retinas. For quantification of LC3A/B-II level, P12 and P14 band intensities were combined and normalized to γ-tubulin (lower panel). (D) Left panels show low-magnification images of anti-p62 staining in P17 retina. Scale bar = 50 μm. Right panels show the same sections imaged at high magnification using Airyscan (AS*). Scale bar = 20 μm. (E) Transmission electron micrographs of Rabgef1 -/- photoreceptor inner segments at P17 immunolabeled with anti-p62/immunogold or immunogold without primary antibody. Arrowheads indicate anti-p62 immunolabeling in electron-dense deposits. Scale bar = 500 nm. (F) Immunoblot of P14 control and Rabgef1 -/- retinal lysates probed with antibody to p62. β-actin was used as a loading control.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Immunohistochemistry, Staining, Western Blot, Transmission Assay, Immunolabeling

Journal: PLoS Genetics

Article Title: Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

doi: 10.1371/journal.pgen.1009259

Figure Lengend Snippet: (A) Heatmap of differentially expressed genes in pairwise comparisons of age-matched control (WT) and Rabgef1 -/- (KO) retina. The annotation block indicates whether differentially expressed genes are part of Mitochondria (Mitocarta), Phototransduction, Oxidative stress response pathway, and/or Endocytosis pathways. (B) Functional enrichment plot of differentially expressed genes for each time point. Colored shapes represent the source of pathways or gene groups: Gene Ontology Biological Processes (BP; gold square), Gene Ontology Cellular Component (CC; grey diamond), KEGG (purple triangle), or Reactome (blue circle). Only top 10 most significant (by p-value) pathways are plotted. (C) Early changes in Rabgef1 -/- retina at P6. Volcano plots show differential expression of phototransduction and OXPHOS genes at P6, soon after rod photoreceptor birth. Red and blue labels in volcano plots denote gene over- and under- expression with significance, respectively. (D) Volcano plots showing continued downregulation of phototransduction and OXPHOS genes in Rabgef1 -/- retina at P10. (E) Volcano plots showing significant upregulation of lysosomal and early endosome related genes in Rabgef1 -/- retina at P14. (F) A possible model of photoreceptor cell death in mouse retina lacking RabGEF1, illustrating the centrality of its function in endocytosis and autophagy during normal development and homeostasis.

Article Snippet: Briefly, 5μg Rabex5/RabGEF1 antibody (Sigma-Aldrich, St. Louis, MO) was covalently conjugated to 1 mg of Dynabeads and incubated overnight with retina extracts at 4°C.

Techniques: Blocking Assay, Functional Assay, Expressing

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: Cultured hippocampal Pclo wt/wt or Pclo gt/gt neurons were stained for different endosome proteins. Subsequently, their intensity was measured in the cell soma ( A–D ) or along dendrites ( E–H ) marked by MAP2. No difference in the staining intensity of GFP-2x-FYVE ( A ), EEA1 ( B ) or Rab5 ( C ) is detectable at the somata of Pclo wt/wt vs Pclo gt/gt neurons (GFP-2x-FYVE: Pclo wt/wt = 1 ± 0.08, n = 16 soma; Pclo gt/gt = 1.14 ± 0.32, n = 10 soma; three independent experiments; EEA1: Pclo wt/wt = 1 ± 0.05, n = 34 soma; Pclo gt/gt = 0.94 ± 0.16, n = 19 soma; p=0.6559; five independent experiments; Rab5: Pclo wt/wt = 1 ± 0.07, n = 18 soma; Pclo gt/gt = 0.92 ± 0.06, n = 18 soma; p=0.4158; three independent experiments). ( D ) Rabex5 intensity is decreased at somata of Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.07, n = 16 soma; Pclo gt/gt = 0.65 ± 0.06, n = 15 soma; two independent experiments). ( E ) Intensity of GFP-2x-FYVE (GFP) organelles along MAP2 positive dendrites is not different between Pclo wt/wt and Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.02, n = 451 puncta; Pclo gt/gt = 0.94 ± 0.02, n = 400; two independent experiments). ( F ) In Pclo gt/gt neurons, the intensity of EEA1-positive organelles along MAP2 positive dendrites is increased ( Pclo wt/wt = 1 ± 0.03, n = 462 puncta; Pclo gt/gt = 1.28 ± 0.04, n = 358; two independent experiments). ( G and H ) Intensity of Rab5 ( G ) along Pclo gt/gt dendrites is increased compared to Pclo wt/wt dendrites, however intensity of Rabex5 ( H ) is not different (Rab5: Pclo wt/wt = 1 ± 0.02, n = 405 puncta; Pclo gt/gt = 1.43 ± 0.05, n = 372; three independent experiments; Rabex5: Pclo wt/wt = 1 ± 0.03, n = 232 puncta; Pclo gt/gt = 1.06 ± 0.04, n = 278; two independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM. Student`s t –test. *** denotes p<0.001.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101004, RRID: AB_1210382 ), Synapsin (1:200; rabbit; abcam, Cambridge, UK; Cat# ab64581, RRID: AB_1281135 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:500; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:500; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:200; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), GFP (1:500; chicken; Thermo Scientific, Waltham, USA; Cat# A10262, RRID: AB_2534023 ), GFP (1:500; mouse; Roche, Basel, Switzerland; Cat# 11814460001, RRID: AB_390913 ), Rabex5 (1:100; rabbit; Thermo scientific, Waltham, USA; Cat# PA5-21117, RRID: AB_11157010 ) MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID: AB_571049 ), Piccolo (1:500; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:500, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ).

Techniques: Cell Culture, Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Schematic of early endocytic trafficking steps. After pinching off from the plasma membrane early endocytic vesicles undergo consecutive maturation steps. The lipid PI3P is generated and a stable complex consisting of Rab5 and its GEF Rabex5 is formed creating a pool of active Rab5. This step is necessary to recruit EEA1 and form early endosomes. ( B ) Images depicting Rab5 and EEA1 intensities at GFP-2x-FYVE organelles along axons in Pclo gt/gt vs Pclo wt/wt neurons. ( C–F ) Quantification of B. ( C ) The levels of Rab5 at PI3P-positive organelles are decreased ( Pclo wt/wt = 1 ± 0.02, n = 1645 puncta; Pclo gt/gt = 0.81 ± 0.02, n = 1233 puncta; six independent experiments). ( D ) The amount of EEA1 at endosome membranes is reduced ( Pclo wt/wt = 1 ± 0.04, n = 1634 puncta; Pclo gt/gt = 0.37 ± 0.02, n = 1169 puncta; six independent experiments). ( E ) Quantification of double positive compartments along axons. The fraction of GFP-2x-FYVE/Rab5 is not altered ( Pclo gt/gt = 0.97 ± 0.33, n = 5 independent experiments). ( F ) The relative percentage of GFP-2x-FYVE/Rab5/EEA1 positive vesicles is decreased in Pclo gt/gt neurons (GFP-2x-FYVE/Rab5/EEA1: Pclo gt/gt = 0.28 ± 0.10, n = 6 independent experiments). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t -test. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001 and **** denotes p<0.0001. 10.7554/eLife.46629.016 Figure 5—source data 1. This spreadsheet contains the normalized values used to generate the bar plots shown in .

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101004, RRID: AB_1210382 ), Synapsin (1:200; rabbit; abcam, Cambridge, UK; Cat# ab64581, RRID: AB_1281135 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:500; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:500; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:200; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), GFP (1:500; chicken; Thermo Scientific, Waltham, USA; Cat# A10262, RRID: AB_2534023 ), GFP (1:500; mouse; Roche, Basel, Switzerland; Cat# 11814460001, RRID: AB_390913 ), Rabex5 (1:100; rabbit; Thermo scientific, Waltham, USA; Cat# PA5-21117, RRID: AB_11157010 ) MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID: AB_571049 ), Piccolo (1:500; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:500, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ).

Techniques: Clinical Proteomics, Membrane, Generated

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A ) Immunocytochemical staining of hippocampal neurons for Rabex5. ( B ) Quantification of ( A ). Rabex5 is present at Pclo wt/wt and Pclo gt/gt synapses, no difference in the intensities is detectable ( Pclo wt/wt = 1 ± 0.02, n = 2397 synapses; Pclo gt/gt = 0.96 ± 0.02, n = 1802 synapses; seven independent experiments). ( C ) Images depicting Rabex5 intensities at PI3P-positive organelles. ( D ) Quantification of ( C ). Less Rabex5 is present at PI3P-positive membranes in Pclo gt/gt neurons ( Pclo wt/wt = 1 ± 0.04, n = 728 puncta; Pclo gt/gt = 0.80 ± 0.03, n = 652 puncta; six independent experiments). ( E ) Quantification of double positive compartments along axons. The fraction of GFP-2x-FYVE/Rabex5 double positive vesicles is increased in Pclo gt/gt neurons ( Pclo gt/gt = 1.49 ± 0.33, n = 5 independent experiments). ( F ) The relative percentage of GFP-2x-FYVE-Rabex5-Rab5 triple positive compartments is decreased in Pclo gt/gt neurons ( Pclo gt/gt = 0.57 ± 0.14, n = 5 independent experiments). Scale bar represents 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, Student`s t-test.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101004, RRID: AB_1210382 ), Synapsin (1:200; rabbit; abcam, Cambridge, UK; Cat# ab64581, RRID: AB_1281135 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:500; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:500; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:200; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), GFP (1:500; chicken; Thermo Scientific, Waltham, USA; Cat# A10262, RRID: AB_2534023 ), GFP (1:500; mouse; Roche, Basel, Switzerland; Cat# 11814460001, RRID: AB_390913 ), Rabex5 (1:100; rabbit; Thermo scientific, Waltham, USA; Cat# PA5-21117, RRID: AB_11157010 ) MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID: AB_571049 ), Piccolo (1:500; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:500, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ).

Techniques: Staining

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet: ( A and B ) Images of Pclo wt/wt ( A ) and Pclo gt/gt ( B ) neurons stained for Synaptophysin and Rab5 with and without TTX treatment. ( C ) Quantification of ( A and B ). Synaptic Rab5 levels do not significantly alter in Pclo wt/wt and Pclo gt/gt synapses upon TTX treatment ( Pclo wt/wt = 1 ± 0.05, n = 391 synapses; Pclo wt/wt (TTX) = 0.73 ± 0.05, n = 482 synapses; Pclo gt/gt = 1.14 ± 0.09, n = 400 synapses; Pclo gt/gt (TTX) = 0.90 ± 0.04, n = 372 synapses; two independent experiments). ( D and E ) Images of Pclo wt/wt ( D ) and Pclo gt/gt ( E ) neurons stained for Synaptophysin and EEA1 with and without TTX treatment. ( F ) Quantification of ( D and E ). In Pclo wt/wt synapses EEA1 levels drop upon TTX treatment ( Pclo wt/wt = 1 ± 0.03, n = 1653 synapse; Pclo wt/wt (TTX) = 0.72 ± 0.03, n = 1887 synapses; four independent experiments). In contrast, EEA1 levels slightly increase in Pclo gt/gt synapses due to TTX treatment ( Pclo gt/gt = 0.68 ± 0.04, n = 918; Pclo gt/gt (TTX) = 0.78 ± 0.03, n = 1352; four independent experiments). ( G and H ) Pclo wt/wt ( G ) and Pclo gt/gt ( H ) neurons stained for Synaptophysin and Rabex5 with and without TTX treatment. ( I ) Quantification of ( G and H ). Rabex5 level at synapses slightly decrease in Pclo wt/wt and Pclo gt/gt neurons upon TTX treatment ( Pclo wt/wt = 1 ± 0.02, n = 2124 puncta; Pclo wt/wt (TTX) = 0.85 ± 0.02, n = 2058 puncta; Pclo gt/gt = 0.93 ± 0.02, n = 1889, Pclo gt/gt (TTX) = 0.77 ± 0.02, n = 2305; six independent experiments). Scale bars represent 10 μm. Error bars in bar graph represent 95% confidence intervals. Numbers given represent mean ± SEM, ANOVA with Tukey multi comparison test. * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001 and **** denotes p<0.0001.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101004, RRID: AB_1210382 ), Synapsin (1:200; rabbit; abcam, Cambridge, UK; Cat# ab64581, RRID: AB_1281135 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:500; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:500; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:200; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), GFP (1:500; chicken; Thermo Scientific, Waltham, USA; Cat# A10262, RRID: AB_2534023 ), GFP (1:500; mouse; Roche, Basel, Switzerland; Cat# 11814460001, RRID: AB_390913 ), Rabex5 (1:100; rabbit; Thermo scientific, Waltham, USA; Cat# PA5-21117, RRID: AB_11157010 ) MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID: AB_571049 ), Piccolo (1:500; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:500, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ).

Techniques: Staining, Comparison

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; mouse; synaptic systems, Göttingen, Germany; Cat# 101011, RRID: AB_887824 ), Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101004, RRID: AB_1210382 ), Synapsin (1:200; rabbit; abcam, Cambridge, UK; Cat# ab64581, RRID: AB_1281135 ), VGlut1 (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 135304, RRID: AB_887878 ), Rab5 (1:500; mouse; synaptic systems, Göttingen, Germany; Cat# 108011, RRID: AB_887773 ), Rab7 (1:500; rabbit; abcam, Cambridge, UK; Cat# ab137029, RRID: AB_2629474 ), EEA1 (1:200; rabbit; cell signaling, Danvers, USA; Cat# 3288S, RRID: AB_2096811 ), GFP (1:500; chicken; Thermo Scientific, Waltham, USA; Cat# A10262, RRID: AB_2534023 ), GFP (1:500; mouse; Roche, Basel, Switzerland; Cat# 11814460001, RRID: AB_390913 ), Rabex5 (1:100; rabbit; Thermo scientific, Waltham, USA; Cat# PA5-21117, RRID: AB_11157010 ) MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID: AB_571049 ), Piccolo (1:500; rabbit; synaptic systems, Göttingen, Germany; Cat# 142002, RRID: AB_887759 ), Piccolo (1:1000; rabbit; abcam, Cambridge, UK; Cat# ab20664, RRID: AB_777267 ), Piccolo (1:500, guinea pig, synaptic systems, Göttingen, Germany; Cat# 142104, RRID: AB_2619831 ).

Techniques: Recombinant, Subcloning, Plasmid Preparation, Sequencing

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Rabex5 (rabbit polyclonal) , Thermo scientific , RRID: AB_11157010 , (1:100).

Techniques: Recombinant, Subcloning, Plasmid Preparation, Sequencing

Journal: eLife

Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

doi: 10.7554/eLife.31326

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-RABGEF1 (polyclonal) , NOVUS BIOLOGICALS , NBP1-49938 AB_10012128 , 1:500 (WB).

Techniques: Sequencing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot, Recombinant, Software, Microscopy