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First published online June 5, 2007
doi: 10.1242/10.1242/jcs.003020

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The retromer complex and clathrin define an early endosomal retrograde exit site

¹ Laboratoire Trafic et Signalisation, UMR144 Curie/CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
² Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Building 18T, Room 101, Bethesda, MD 20892, USA
³ Laboratoire Structure et Compartiments Membranaires, UMR144 Curie/CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France

View larger version (112K): [in this window] [in a new window]   Fig. 1. (A-C) Clathrin, AP1 and retromer localization on STxB-containing EE/RE. STxB-HRP was bound to HeLa cells kept on ice that were then incubated for 45 minutes at 19.5°C. Cells were prepared for whole-mount analysis, followed by immunogold-labeling with antibodies against (A,B) clathrin or (C) the AP1 subunit -adaptin (10-nm gold particles), and in all cases to Vps26 (15-nm gold particles). STxB-HRP-containing membranes appeared electron-dense owing to the polymerization product. Note that some structures are double-labeled for clathrin-AP1 and Vps26 (arrows), whereas others are stained for clathrin or -adaptin only (arrowheads). Bars, 200 nm.

View larger version (125K): [in this window] [in a new window]   Fig. 2. Retromer protein Vps26 on STxB-containing endosomes. (A) Cy3-STxB was bound to HeLa cells kept on ice that were then transferred to 37°C for 10 minutes, fixed and labeled for the indicated proteins. Bar, 10 µm. (B) Magnification of boxed areas in A. Insets show detailed view of an STxB-containing tubular structure. (B') Pair-wise overlays of images shown in B. Note that TfR overlaps with vacuolar parts of STxB structures (arrows), whereas Vps26 overlaps with tubular parts (arrowheads). (B,B') Bar, 5 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Live cell imaging of retrograde STxB sorting on Tf-containing EE/RE. (A) HeLa cells were incubated with Alexa Fluor-488-labeled STxB (green) and Alexa Fluor-568-labeled human Tf (red) for 60 minutes at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy. Shown is one cell with STxB and Tf accumulated in early endosomes. Merging images indicate overlapping localization (yellow). Images of the right panels are magnifications of the boxed areas on the left. The series show an STxB-containing tubule moving away from an early endosome (green arrows), while Tf remains associated only with the endosome (red arrows). (B) HeLa cells transiently expressing Vps29-GFP (green) were incubated with Cy3-labeled STxB (red) for 60 minutes at 19.5°C. After transferring cells to 37°C, they were imaged by time-lapse microscopy. Images of the right panels are magnifications of the boxed areas on the left. The series show an STxB-containing tubule moving away from an early endosome (red arrows) and Vps29-GFP associated to the same tubules (green arrows). Time after the start of imaging is shown in seconds. Bars, 10 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Sulfation analysis of retrograde transport in Vps26 siRNA-treated cells. (A,B) Western blot (A) and sufation analysis (B) performed on HeLa cells transfected with siRNAs targeting Vps26. Three different siRNA sequences (Seq.1, Seq.2, Seq.3) have been used, which all induced the downregulation of Vps26 expression. Loss of Vps26 expression correlated with inhibition of Shiga toxin transport to TGN/Golgi membranes. Results are given as the mean ± s.e.m. of three determinations. (C) Dose-response experiment with siRNA number 3. A correlation was observed between the expression levels of Vps26 (as determined by western blotting) and retrograde Shiga toxin trafficking (as determined by sulfation analysis). Linear-regression analysis shows that both variables are linked with a probability higher than 99%.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Immunofluorescence analysis of STxB transport in Vps26 siRNA-treated cells. (A,B) Cy3-labeled STxB (red) was incubated for 45 minutes at 37°C with (A) mock-transfected or (B) Vps26 siRNA-transfected cells. After fixation, cells were stained for Vps26 (green) and CTR 433 (blue), a marker of the Golgi apparatus. In control cells, STxB accumulated in Golgi membranes, whereas it failed to do so in Vps26 siRNA-treated cells. There, it accumulated in peripheral structures. Bars, 10 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 6. (A-C) Endosomal STxB accumulation in Vps26 siRNA-treated cells. Cy3-STxB (red) was incubated with Vps26-depleted cells for 45 minutes at (A,B) 37°C or (C) 19.5°C. After fixation, cells were labeled for Vps26 (not shown) and TfR or EEA1 (green), as indicated. (A'-C') Magnified views of boxed areas of A-C. Additional magnification views are shown as insets in A',B',C'. (A"-C") The orientation of the shifts between STxB and TfR at 37°C (A"), STxB and EEA1 at 37°C (B"), and STxB and TfR at 19.5°C (C") are shown for the boxed areas in A-C, respectively. Centers of STxB-positive structures are marked `x' (red), centers of TfR or EEA1 positive structures are marked `+' (green). Colocalized structures are encircled, structures with shifts are marked by arrows. Dotted circles indicate ambiguous situations. Note that at 37°C, STxB-positive structures are neighboring to TfR- or EEA1-positive structures, whereas at 19.5°C, exact superposition is observed for STxB and TfR. Bars, 10 µm (A-C) and 5 µm (A'-C').

View larger version (102K): [in this window] [in a new window]   Fig. 7. Ultrastructural localization of STxB in Vps26 and clathrin RNAi conditions. (A-E) STxB was incubated for 45 minutes at 37°C with (A) mock transfected cells, (B,C) Vps26-depleted and (D,E) clathrin-depleted cells. After fixation and cryosectionning, preparations were labeled with anti-STxB antibody (A-E, 15-nm gold particles) and with anti-TfR antibody (C-E, 10-nm gold particles). In C, arrows indicate clusters of TfR-specific labeling, arrowheads indicate clusters of STxB-specific labeling. In D, the star indicates a tubule in which both TfR and STxB can be detected, arrowhead indicates the vacuolar endosome from which the tubule emanates. Note that in Vps26-depleted cells, STxB is found throughout the cytoplasm, whereas it is often localized close to multivesicular endosomes in clathrin-depleted cells. Bars, 200 nm.

View larger version (83K): [in this window] [in a new window]   Fig. 8. STxB-containing endosomal tubules appear to persist longer in Vps26 siRNA-treated cells. (A,B) Vps26-depleted HeLa cells were incubated for 45 minutes at 37°C with (A) Alexa Fluor-488-labeled STxB alone (green), or (B) Alexa Fluor-488-labeled STxB (green) together with Alexa Fluor-568-labeled human Tf (red) and then imaged by time-lapse microscopy. In B, merged image indicates overlapping localization (yellow). (B',B") magnified images of the boxed areas in B. In addition to colocalization, the series in B' show regions in the endosomes that contain either only STxB (green arrows) or only Tf (red arrows). The series in B" show endosomal tubules that contain only STxB. Time after the start of imaging is shown in seconds. Bars, 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 9. Working model for retrograde sorting on the early endosome. (A) In control cells, clathrin organizes microdomains with which Shiga-toxin associates (step 1). We hypothesize that microdomain construction induces initial membrane-curvature changes leading to tube formation. A retromer-dependent process then allows stabilizing and processing of membrane curvatures, leading to the formation of retrograde transport intermediates (step 2). (B) In clathrin-depleted cells, the early endosomal retrograde sorting domain cannot form any more. Under these conditions, retrograde transport to TGN/Golgi membranes is inhibited and Shiga toxin distributes throughout the early endosomal pathway, perfectly overlapping with TfR. (C) In Vps26-depleted cells, retrograde transport intermediates cannot form and STxB remains blocked in retrograde tubules that are devoid of TfR. For further details see text.

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