Actin Organizes WASH Domains Figure 2. Automated quantification of the increase of endosomal WASH upon actin depolymerization. (A) 3T3 cells were loaded with fluorescent Tf until equilibrium, then treated with 10 mM nocodazole in the continuous presence of Tf for 1 h, then treated with 0.2 mM LatA or carrier in the presence of nocodazole and Tf for 10 min. Cells were then processed for immunofluorescence and observed as in Fig. 1. Scale bar: 10 mm (1 mm in inserts). The nocodazole treatment induces scattering of the endosomes thus allowing detection of the whole population of endosomes. The LatA treatment enlarges endosomes and increases the intensity of WASH on endosomes. (B) Image processing workflow (see also Methods). WASH and Tf images were processed as in Fig. 1. As the endosomes were scattered due to nocodazole treatment, both Tf-positive endosomes and WASH domains could be detected automatically after 3D segmentation, then each WASH domain was assigned to its proper endosome using a custom-made program (see Methods) and the WASH intensity was measured on each endosome. (C) Image stacks of cells treated as in Fig. 2A were segmented in 3D for both channels and analyzed (24 cells, 11872 endosomes for control; 44 cells, 11633 endosomes for LatA). The LatA treatment induces a shift of endosome sizes towards larger volumes (p,0.001, x2 test). (D) The average intensity (6 s.e.m.) of WASH domains increases with endosome size in both control and LatA-treated cells. Moreover, within a given endosome volume range, actin depolymerization induces an increase of WASH intensity as compared to non-treated cells. (E) The average ‘apparent concentration’ of WASH (6 s.e.m.) in domains increases when actin is depolymerized and is independent of endosome volume. All data were analyzed by a two way ANOVA using treatments and volume bins as parameters, and pairewise comparisons were performed using a Tukey test. *: p,0.001 compared with control within the same volume bin. doi:10.1371/journal.pone.0039774.g002 Figure 3. Actin depolymerization inhibits WASH recovery on endosomes after photobleaching. (A) Stable 3T3 cells expressing GFP- WASH were treated as in Fig. 2A. WASH domains were photobleached and signal recovery was monitored for 3 min. A representative sequence of recovery is displayed (8 sec between frames). Scale bar: 1 mm. (B) Recovery of 72 WASH domains for 16 control cells and 98 WASH domains for 13 LatA-treated cells were pooled from 2 independent experiments to calculate the average (6 s.e.m.) of WASH intensity at each time point. doi:10.1371/journal.pone.0039774.g003 PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39774