1. Volume 26, Issue 1, Pages 120-128 (January 2016)
Rab35 GTPase Triggers Switch-like Recruitment of the Lowe Syndrome Lipid Phosphatase OCRL on Newborn Endosomes 
Clothilde Cauvin, Morgane Rosendale, Neetu Gupta-Rossi, Murielle Rocancourt, Pierre Larraufie, Rémi Salomon, David Perrais, Arnaud Echard 
Current Biology 
Volume 26, Issue 1, Pages (January 2016)
DOI: /j.cub
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2. Current Biology 2016 26, 120-128DOI: (10.1016/j.cub.2015.11.040)
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3. Figure 1
Rab35 and OCRL Colocalize, and Their Depletion Redistributes CI-MPR to Peripheral Endosomes
(A) HeLa cells expressing fluorescently tagged Rab35 (green) and OCRL (red) were either pulsed for 5 min with labeled transferrin (Tf) or stained for endogenous CI-MPR (blue). White arrowheads indicate double colocalization Rab35/OCRL; yellow arrowheads indicate triple colocalization Rab35/OCRL and Tf or CI-MPR.
(B) Percentage of peripheral structures that show colocalization between indicated proteins (mean ± SD, n = 3, 600–800 structures per experiment).
(C) Steady-state distribution of CI-MPR (green) and EEA1 (red) in cells treated with indicated siRNAs. Arrowheads indicate colocalization or close association between CI-MPR and EEA1.
(D) Percentage of cells displaying peripheral CI-MPR after treatment with indicated siRNAs and transfected with plasmids encoding siRNA-resistant versions of indicated proteins (mean ± SD, n = 3, 100–200 cells per experiment).
(E) Cells expressing either GFP, GFP-Rab35 S22N, mCherry-EPI64B, control, or DENND1A shRNA-IRES-RFP. Top row: corresponding GFP or red signals. Bottom row: endogenous CI-MPR staining.
(F) Percentage of fluorescent cells with peripheral CI-MPR in cells described in (E) (mean ± SD, n = 3, 100–200 cells per experiment).
∗∗∗p < , χ2 test. Scale bars, 10 μm. See also Figure S1.
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4. Figure 2
Both Rab35 and OCRL Depletions Alter Endosomal Identity and CI-MPR Retrograde Trafficking from Endosomes to TGN
(A) Cells stably expressing CD8-CI-MPR were treated with indicated siRNAs and incubated at 4°C with anti-CD8 antibodies. After a 30-min chase at 37°C, cells were stained for CD8 antibodies, EEA1, and Golgi/TGN marker Rab6. Higher magnification around the Golgi region (i) and the cell periphery (ii) are displayed (merge).
(B) Percentage of cells with CD8-CI-MPR in endosomes only, in endosomes and in the perinuclear region, or in the perinuclear region only after indicated depletion (mean ± SD, n = 3, 190–230 cells per experiment).
(C) Rescue experiment with indicated plasmids after 120-min chase (mean ± SD, triplicates, 150–250 cells per experiment).
(D–F) Cells were treated with indicated siRNAs and stained for CI-MPR, EEA1, and dsRed-clathrin light chain (CLC) (D); EEA1, cortactin, and GFP-CI-MPR (E); or CI-MPR, EEA1 and GFP-Arp2/3 subunit p16Arc (F). Whole-cell (top left), three-color merged zoom (top right) and corresponding individual color channels (gray images, bottom) are displayed. Arrowheads indicate structures positive for all three markers.
(G) Percentage of colocalization between peripheral CI-MPR vesicles and cortactin or p16Arc in indicated treated cells (mean ± SD, n = 3, 250–550 structures per experiment).
∗∗p < 0.001; ∗∗∗p < , χ2 test. Scale bars, 10 μm. See also Figures S2 and S3.
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5. Figure 3 OCRL Association with Endosomes Depends on Rab35
(A) HeLa cells expressing GFP-OCRL after treatment with indicated siRNAs or coexpressing mCherry-EPI64B (asterisks) were stained for CI-MPR. High magnification of single channel (insets) and merged colors are displayed. Arrowheads indicate OCRL association with CI-MPR vesicles.
(B) Percentage of CI-MPR vesicles positive for OCRL in cells treated as indicated (mean ± SD, n ≥ 2, 300–1,000 structures per experiment).
∗∗∗p < , χ2 test. Scale bars, 10 μm. See also Figure S3.
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6. Figure 4
Rab35 Recruits OCRL with High Spatiotemporal Accuracy, Immediately after CCV Scission from the Plasma Membrane
(A) Top: snapshots from a time-lapse TIRF-microscopy movie of a HeLa cell expressing mCherry-Rab35 and GFP-OCRL, centered on the maximal OCRL fluorescence (time 0). Bottom left: average normalized fluorescence of 1,480 OCRL objects quantified in four cells, aligned to their maximum fluorescence (green trace). The corresponding fluorescence in the Rab35 channel is superimposed (red trace). Bottom right: histogram of the delay between Rab35 maximum and the OCRL maximum (74% of both maxima occur within less than 4 s). rel, relative.
(B) Same as in (A) for genome-edited HeLa cells expressing GFP-Rab35en and transfected with mCherry-OCRL. 4,850 objects quantified in five cells (70% of both maxima occur within less than 4 s).
(C) Top: examples of mCherry-Rab35 and mCherry-OCRL recruitment at the site of CCV formation detected with TfR-SEP using the ppH protocol (pink arrowhead indicates time of scission; time 0 indicates time of CCV detection). Bottom: average normalized recruitment profiles at endocytic sites of mCherry-Rab35 (red line) or mCherry-OCRL (green line) signals. Each curve indicates mean signal ± SEM, 1,600–1,800 vesicles detected using the ppH assay, for more than eight cells.
(D) Percentage of OCRL-positive CCVs (scissioned TfR-SEP vesicles) in indicated siRNA-treated cells (mean ± SD, n = 3, 350–450 vesicles per experiment).
(E and F) Mean signal ± SEM for GFP-DENND1A (black line, 750 vesicles, nine cells) (E) and for mCherry-EPI64B (blue line, 2,000 vesicles, seven cells) (F). Vesicles were detected using the ppH assay, as in (C). Rab35 recruitment profile from (C) is displayed for comparison.
(G) Summary of the recruitment kinetics of Rab35, DENND1A, and EPI64B on clathrin-coated structures with respect to CCP maturation, CCV formation (scission), and CCV uncoating.
(H) Proposed model for Rab35 activation, OCRL recruitment, and PtdIns(4,5)P2 homeostasis during the biogenesis of endosomes.
∗∗∗p < , χ2 test. See also Figure S4.
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