Actin Organizes WASH Domains Figure 6. Actin depolymerization induces coalescence of WASH domains. (A) Stable 3T3 cells expressing GFP-WASH were transfected with mCherry-Rab5Q79L to enlarge endosomes and treated with nocodozale and LatA as in Fig. 2A. Cells were imaged by spinning disk confocal microscopy (single planes). Scale bar: 10 mm (1 mm in inserts). WASH localizes to discrete domains at the surface of enlarged endosomes in control cells, but to a unique, crescent shaped structure upon actin depolymerization. (B) Confocal z-sections of the endosomes shown in the top insets in (A) were processed for 3D reconstruction (front and side views). doi:10.1371/journal.pone.0039774.g006 Rab5Q79L endosomes in control cells (Fig. 9A–B). Upon LatA treatment, Vps35 remained associated with the coalesced WASH domain. HRS, a component of the ESCRT-0 complex, also defined discrete domains on enlarged endosomes. However, these domains were distinct from the ones of WASH, and they did not coalesce upon actin depolymerization (Fig. 9C). These results suggest that domain coalescence upon actin depolymerization is a specific property of the domain containing both WASH and retromer complexes. Discussion Using LatA mediated actin depolymerization, we have observed two effects on the Arp2/3 activator WASH at the surface of endosomes. The first one is an increased WASH staining on endosomes and the second one is the coalescence of WASH domains at the surface of large endosomes. We are confident that these two effects are not due to artifactual antibody staining, as they were similarly observed using a GFP-WASH fusion protein. These effects were best seen when endosomes were scattered using microtubule depolymerization. However, the increased WASH staining and the crescent shaped WASH domain were also observed without nocodazole, suggesting that these alterations of WASH distribution solely rely on the actin cytoskeleton. Indeed, Arp2/3 depletion produced the same phenotype, thus ruling out non specific effects of LatA treatment. We attempted to confirm the endosomal WASH enrichment that we have observed upon actin depolymerization using established procedures for mem- brane cytosol biochemical fractionation assays [42,43]. However these attempts were not succesful. Several non-exclusive reasons might account for this discrepancy between microscopy and biochemical fractionations, such as the effect of dilution that displaces the equilibrium between these two pools or other exchanges between different membrane pools. To accurately quantify these effects on WASH distribution upon LatA treatment, we had to develop automated image analysis techniques to assign each WASH domain to its correct endosome in 3D. Specifically, our method overcomes the acute problem of the heterogeneity of size and shapes of endosomes. This method helped us to accumulate sufficient data to prove that the two effects on WASH distribution we report here are indeed general effects independent of the variable sizes and shapes of endosomes. We anticipate that these programs, available on request, will be useful for future endosomal studies, for instance to quantify protein recruitment onto endosomes and endosomal protein contents in vivo. Arp2/3 depletion phenocopies LatA treatment on WASH distribution, thus suggesting that WASH is controlled by the branched actin networks it generates through feedback mecha- PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39774