generated at the surface of early endosomes through the recruitment of several Rab5 effectors, which oligomerize, bind to lipids and further activate Rab5 [32,33,34,35]. These dynamic proteo-lipidic domains contrast with membrane rafts that depend on strong hydrophobic lipid-lipid interactions [36]. Such dynamic proteo-lipidic domains were proposed to depend on cytoskeletal elements, among other factors, for their cohesion [28]. Here we investigated the role of actin polymerization at the surface of endosomes by monitoring the WASH marker, which defines an endosomal domain. Using quantitative image analysis, we identified two specific feedback regulations of the WASH domain by the branched actin network it nucleates: actin filaments control the lateral organization of WASH domains and the dynamic exchange of WASH molecules between the endosomal domain and the cytosolic pool. The potential role of these feedback regulations in the mechanism of endosomal scission is discussed. Results Actin dynamics promote dynamic exchange of the WASH complex between endosomal and cytosolic pools To analyze the role of actin dynamics in the physiology of endosomal WASH domains, 3T3 cells were loaded with fluores- cent transferrin (Tf) to label sorting and recycling endosomes, then treated with actin depolymerizing drugs in the presence of Tf. We used Latrunculin A (LatA), which sequesters monomeric actin and prevents it from polymerizing [37], and Cytochalasin D (CytoD), which caps filament barbed ends [38]. Both treatments seemed to induce an increase of the WASH fluorescence signal on endosomes as compared to the control (Fig. 1A). We thus decided to quantify the WASH signal on endosomes in presence or absence of actin. Endosomes were detected using the Tf channel and the fluorescence intensity of the corresponding WASH domains was measured (see Methods). The high degree of endosome clustering in the perinuclear region restricted this observation to the few endosomes that are scattered in the cytoplasm. This preliminary analysis showed that both treatments induce a statistically significant increase of the WASH signal per endosome (Fig. 1B). However the effect of CytoD is less pronounced than that of LatA, consistent with the fact that actin disassembly by CytoD is less complete than the one induced by LatA. These results suggest that actin dynamics play a role in the recruitment of WASH onto endosomes. To confirm this result more quantitatively, we needed an accurate statistical analysis to measure precisely the WASH signal on each endosome. This obviously required the accumulation of data over thousands of endosomes, due to their high heterogeneity in size and shape. We thus additionally treated the cells with nocodazole, which depolymerizes microtubules and therefore prevents endosome clustering around the nucleus. In these conditions, we were able to resolve almost all endosomes. In these conditions as well, the WASH signal appeared brighter in LatA- treated cells than in control cells (Fig. 2A). We verified that branched actin networks were depolymerized upon LatA treat- ment (Fig. S1). Upon LatA treatment, endosomes appeared larger, consistently with previous observations [9,15]. We thus wondered if the increase of WASH intensity was a simple consequence of endosome enlargement. We developed an automated procedure to detect Tf-positive endosomes and WASH domains from confocal z-stacks of cells, which allowed us to measure the intensity of each WASH domain as well as the volume of each endosome (Fig. 2B). We then faced the problem of assigning Actin Organizes WASH Domains automatically each WASH domain to its respective endosome. Classical object-based colocalization methods associate objects segmented in two different channels when the distance between their fluorescence centroids is below a threshold limit, usually fixed to the resolution of the microscope [39]. However, classical object- based methods perform poorly in our case, especially on LatA enlarged endosomes, because the cytoplasm is crowded with endosomes and because WASH localizes to domains, which may be far from the centroid of large endosomes. Increasing the threshold did not alleviate the problem and still yielded false assignations. We thus developed a novel object-based colocaliza- tion method based on a local, rather than global, threshold to account for the large heterogeneity of endosome size and shape. The principle of this method is the following (see Methods for further details): 1) a yellow stack composed of common pixels is generated from the segmented images of both green and red channels; 2) this stack is then segmented and each yellow region is assigned to its correct red and green regions, from which the association between the red and the green regions is deduced. Although a common pixel criteria is not usually a good marker of colocalization because of the limited resolution of the microscope, the probability to have more than one endosome per diffraction limited volume is strongly diminished in our case due to the use of nocodazole to scatter compartments. This method resulted in reliable 3D-assignations between endosomes and WASH domains, independently on their size and shape, and thus allowed us to quantitatively address the relationship between WASH intensity and endosome volume in the presence or not of LatA. This analysis confirmed quantitatively that LatA treatment significantly shifts the distribution of endosome volumes, with a reduced number of normal sizes and the appearance of large endosomes above 1 mm3 (Fig. 2C, p,0.001, x2 test). Moreover, this analysis showed that in the control situation, WASH domain intensity increases with the volume of the endosome (Fig. 2D). This is a ,2-fold effect, when the smallest endosomes are compared to the largest ones. However, the LatA treatment induced a 3-fold increase of the WASH intensity, whenever endosomes of similar size are compared (Fig. 2D). As a proxy for WASH surface density, we calculated the apparent concentration of WASH by dividing the intensity of each WASH domain by the volume it occupies. This ‘apparent concentration’ of WASH is remarkably constant over the whole range of endosome sizes, but increases about 2-fold when actin is depolymerized by LatA (Fig. 2E). All the above observations were statistically significant (p,0.001, two way ANOVA followed by a Tukey Test for pairwise comparisons). Importantly, these quantitative results were also observed when CytoD was used instead of LatA to depolymerize actin (Fig. S2). This increased WASH recruitment at the surface of endosomes was observed with endogenous WASH analyzed by immunoflu- orescence. One can argue that this effect of LatA is due to a better accessibility of WASH to antibodies, when actin networks are depolymerized. We therefore performed the same experiment in a stable 3T3 cell line that expresses GFP-WASH. This GFP-WASH construct rescues the defect associated with depletion of endog- enous WASH [7]. The stable cell line expresses GFP-WASH at a level similar to that of endogenous WASH, because GFP-WASH replaces the endogenous WASH in its multiprotein complex (Fig. S3A). We observed similar behavior for GFP-WASH and its endogenous counterpart (Fig. S3B–D), ruling out the possible artifact of a better accessibility effect after LatA treatment. Altogether, these image analyses suggested two conclusions: (i) large endosomes have larger WASH domains than smaller ones, but with a similar surface density of WASH, (ii) in contrast, actin PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39774