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. 2012 Jun 21;7(6):e39774. doi: 10.1371/journal.pone.0039774

Figure 7. Quantification of the coalescence of WASH domains upon actin depolymerization.

Figure 7

(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 (± s.e.m.) and their average apparent surface (± 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.