Actin Organizes WASH Domains Figure 9. The domains formed by the Vps subcomplex of the retromer, but not HRS domains, coalesce like WASH domains. (A) Stable 3T3 cells expressing GFP-Vps35 were treated as in Fig. 6A, then processed for immunofluorescence using WASH antibody and observed by epifluorescence microscopy followed by deconvolution (single planes). Scale bar: 10 mm. (B) corresponds to the insets displayed in (A). Scale bar: 1 mm. Vps35 colocalizes with WASH and behaves similarly to WASH upon actin depolymerization. (C) 3T3 cells were transfected with GFP-Rab5Q79L and DsRed-HRS, and treated as above. Cells were fixed and observed by spinning disk confocal microscopy followed by deconvolution (single planes). Scale bar: 10 mm (1 mm in insets). HRS localizes to discrete domains at the surface of enlarged endosomes, whether or not actin polymerization is prevented. doi:10.1371/journal.pone.0039774.g009 once, retransfected 2 days later and analyzed after 2 more days. Antibodies and constructs were previously described in [7], except pAb targeting EEA1 (Cell Signaling Technology, Beverly, MA), Ds-Red-HRS plasmid (gift of Dominique Lallemand, Institut Curie, Paris) and GFP-Rab5Q79L plasmid (gift of Philippe Chavrier, Institut Curie, Paris). Transferrin (Tf) uptake was performed as described [7]. For drug treatment, nocodazole (Sigma, St. Gallen, Switzerland), Latrunculin A (Cayman Chem- icals, Ann Harbor, MI) and Cytochalasin D (Merck, Darmstadt, PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39774