Actin Organizes WASH Domains Figure 7. Quantification of the coalescence of WASH domains upon actin depolymerization. (A) Image processing workflow (see also Methods). Confocal slices were acquired for GFP-WASH and mCherry-Rab5Q79L channels and processed as in Fig. 2 B, except that the wavelet filter was substituted by an ‘Adaptative blind’ deconvolution step. (B) Image stacks were segmented in 3D for both channels (23 cells, 2179 endosomes for control; 22 cells, 1221 endosomes for LatA). The average number of WASH domains (6 s.e.m.) and their average apparent surface (6 s.e.m.) were plotted as a function of endosome volume. The average number of WASH domains increases with endosome volume (p,0.001 Kruskal-Wallis one way analysis of variance on ranks). Moreover, upon actin depolymerization, the number of WASH domains in large endosomes decreases (*: p,0.05 compared with control within the same volume bin, Kruskal-Wallis test followed by a Dunn pairwise comparison). Concomitantly, the surface occupied by WASH domains increases (* p,0.001 compared with control within the same volume bin, two way ANOVA followed by a Tukey pairewise comparison). Altogether, these data suggest that WASH domains coalesce upon actin depolymerization. doi:10.1371/journal.pone.0039774.g007 nisms. Indeed, both treatments induce an accumulation of WASH on endosomes. Moreover, FRAP experiments on GFP-WASH have revealed that LatA treatment ‘freezes’ the dynamic exchange of WASH between cytosolic and endosomal pools. The lack of WASH recovery after photobleaching indicates that cytosolic WASH is less recruited to endosomes upon LatA treatment. There are two possible interpretations to explain this effect. Either the recruitment of WASH from the cytosol requires actin – however, it is difficult to envision in this case why the endosomal pool of WASH increases upon LatA treatment – or, it is the detachment of WASH from endosomes that requires actin. We favor this hypothesis, which is consistent with the increased endosomal pool of WASH upon actin depolymerization. This hypothesis is less intuitive as to why there is less recovery of WASH after photobleaching. It is likely that defective recovery of GFP-WASH in this hypothesis is due to the constant occupancy of endosomal binding sites by bleached WASH, which do not detach to return to the cytosol. Such a feedback mechanism of actin networks on other Arp2/3 activators has been observed in different systems. Actin depoly- merization increases the membrane pool of WAVE complex and freezes its dynamics in neutrophils [44]. Actin depolymerization also freezes the dynamics of N-WASP in the actin tails of Vaccinia virus [45]. For these reasons, we favor a model in which the Arp2/ PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39774