Actin Organizes WASH Domains Figure 5. Arp2/3 depletion induces the appearance of elongated WASH domains. (A) Stable 3T3 cells expressing GFP-WASH were depleted of the Arp2/3 complex and treated with nocodazole as in Fig. 4B. Scale bar: 1 mm. GFP-WASH forms a crescent at the surface of large EEA1 positive endosomes upon Arp2/3 depletion. (B) 3T3 cells were depleted of the Arp2/3 complex and processed for immunofluorescence using WASH and p16Arc antibodies. Scale bar: 10 mm. WASH crescents are also observed with endogenous WASH in the absence of nocodazole. (C) Stable 3T3 cells expressing GFP-WASH depleted for the Arp2/3 complex and imaged by live spinning disk confocal microscopy after Tf loading. Elapsed time is in seconds. Scale bar: 2 mm. Arp2/3 depletion inhibits Tf uptake and WASH forms crescents at the surface of large endosomes that are dimly stained by Tf. doi:10.1371/journal.pone.0039774.g005 of WASH domains. The Vps35 subunit of the so-called ‘cargo selective subcomplex’ of the retromer was recently shown to bind directly to the FAM21 subunit of the WASH complex and to be required for the endosomal recruitment of the WASH complex [10,41]. We thus thought to study whether retromer domains similarly coalesce upon actin depolymerization. We established a stable 3T3 cell line which expresses GFP-Vps35 and found that Vps35 and WASH colocalize in discrete domains at the surface of PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39774