Actin Organizes WASH Domains Figure 1. Actin depolymerization increases the amount of endosomal WASH. (A) 3T3 cells were loaded with fluorescent Transferrin (Tf) until equilibrium, then treated with 0.2 mM Latrunculin A (LatA) for 10 min, 1 mM CytochalasinD (CytoD) for 30 min, or carrier in the continuous presence of Tf. Cells were processed for immunofluorescence using WASH antibody and observed by spinning disk confocal microscopy. A single plane is displayed. Scale bar: 10 mm (1 mm in inserts). LatA and CytoD treatments increase the fluorescence signal of WASH. (B) Since endosomes are clustered in the perinuclear region, the increase of WASH intensity was quantified on isolated endosomes from the cell periphery (see Methods). Both LatA and CytoD treatments induce a statistically significant increase of the WASH fluorescence signal (n refers to the number of endosomes, *: p,0.001, one way ANOVA followed by a Tukey pairwise comparison). doi:10.1371/journal.pone.0039774.g001 polymerization negatively regulates the surface density of WASH in its domain. To evaluate the dynamics of WASH when actin polymerization is impaired or not, we used Fluorescence Recovery After Photobleaching (FRAP) of GFP-WASH. WASH domains of similar size were bleached in control cells and in LatA treated cells. The bleached zone was large enough to encompass the whole endosome ensuring that fluorescence recovery was due to recruitment of cytosolic WASH and not to lateral diffusion (Movie S1). Recovery of fluorescence was monitored for 3 min (Movie S2 PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39774