Actin Organizes WASH Domains Figure 4. Depletion of the Arp2/3 complex increases the amount of endosomal WASH. (A) Stable 3T3 cells expressing GFP-WASH were depleted from the Arp2/3 complex using siRNAs and analyzed by Western Blot. (B) siRNA-transfected cells were treated with 10 mM nocodazole for 1 h, then processed for immunofluorescence using antibodies recognizing EEA1 and the p16Arc subunit of the Arp2/3 complex, and observed by spinning disk confocal microscopy (single planes). Scale bar: 10 mm. Arp2/3 depletion increases endosomal WASH staining. (C–D) Image stacks (36 cells, 6046 endosomes for Ctrl siRNA; 31 cells, 3889 endosomes for p34Arc #1 siRNA; 26 cells, 3470 endosomes for p34Arc #2 siRNA) were processed and presented as in Fig. 2D–E after normalization of endosomes volumes. Upon Arp2/3 complex depletion, the intensity and ‘apparent concentration’ of GFP-WASH domains increases but does not depend on endosome volume. *: p,0.001 compared with control within the same volume bin, two way ANOVA using treatments and volume bins as parameters, followed by pairwise comparisons using Tukey test. doi:10.1371/journal.pone.0039774.g004 and Fig. 3A). To quantify the recovery, we tracked the bleached WASH domain, since its position fluctuates over time, and verified that the tracked centroid of WASH fluorescence remained over time onto the same endosome surface, which was visualized using the unbleached signal of Tf. More than 50 recovery curves were averaged in both cases (Fig. 3B). In control cells, WASH recovers to ,60% of the initial fluorescence during the first minute. This plateau at 60% indicates that 40% of WASH molecules are immobile at this time scale. In sharp contrast, when actin is depolymerized, WASH recovers to only 20% after 3 minutes, corresponding to 80% immobile WASH. This result suggests that the exchange of WASH between cytosolic and endosome-bound pools strongly depends on the actin cytoskeleton. However, LatA depolymerizes all actin networks, whether the Arp2/3 complex had nucleated them or not. We thus depleted the Arp2/3 complex to specifically impair the generation of branched actin networks. Transfection of 3T3 cells with siRNAs targeting p34Arc induced a strong decrease in the protein levels of p34Arc and p16Arc, suggesting depletion of the entire complex (Fig. 4A). Arp2/3 depletion reduced considerably Tf internalization, in line with the implication of the N-WASP-Arp2/3 pathway in clathrin- mediated endocytosis [26,27]. We therefore used EEA1, which allowed us to detect early endosomes similarly in Arp2/3 depleted and non depleted cells. Arp2/3 depletion led to an increased intensity of GFP-WASH staining on EEA1 positive endosomes (Fig. 4B). We were able to quantify WASH on the whole population of early endosomes, upon microtubule depolymeriza- tion using the method described above. As for pharmalogical inhibition of actin polymerization, Arp2/3 depletion by two independent siRNAs led to a significant increase of GFP-WASH recruitment at the surface of endosomes, when endosomes of similar size were compared (Fig. 4C and D). Together, these experiments suggest that the dynamics of branched actin networks at the surface of endosomes increases the dynamics of WASH, the multiprotein complex that controls the formation of these same branched actin networks. Actin depolymerization promotes lateral coalescence of WASH domains at the surface of large endosomes We often observed elongated GFP-WASH-positive structures on large endosomes upon Arp2/3 depletion (Fig. 5A). Such structures were also observed with endogenous WASH upon Arp2/3 depletion (Fig. 5B). The appearance of such elongated WASH structures upon actin depolymerization was previously noticed and attributed to endosome tubulation [9]. To verify whether these elongated structures were indeed membrane tubules, we imaged GFP-WASH in Arp2/3 depleted cells using time-lapse spinning disk confocal microscopy. In this experiment, we added fluorescent Tf in the medium, which provided us with a weak endosomal signal, because its internalization is impaired by Arp2/3 depletion. Strikingly, WASH positive structures on dimly Tf-stained endosomes did not exhibit the fluctuations expected for fine membrane tubules, but appeared rather as crescent-shaped domains at the surface of large endosomes (Fig. 5C, Movie S3). The same observation was made with a LatA treatment following Tf internalization (Movie S4, see also Fig. 1A). These WASH crescents on large endosomes contrast with the several discrete WASH domains detected when endosomes are enlarged by homotypic fusion through the expression of the active mutant of Rab5, Rab5Q79L [7,40]. These observations suggested the idea that actin depolymerization induces coalescence of WASH domains. To investigate this possibility, we examined the effect of LatA on endosomes, which were already enlarged by transient transfection with mCherry-Rab5Q79L. Confocal examination of LatA treated cells indeed revealed numerous examples of large endosomes, in which the number of WASH domains is reduced and their surface increased compared to control cells (Fig. 6A). 3D reconstructions confirmed this impression obtained from single confocal planes (Fig. 6B and Movie S5). We modified slightly our image analysis program to obtain the number of WASH domains per endosome and their apparent surface area (Fig. 7A). In the control situation, the number of WASH domains indeed increases with the endosome volume, from 1 for the smallest endosomes to about 5 for the largest ones (Fig. 7B). This result is in line with the observation that WASH localizes to one domain on small EEA1 endosomes, but to two domains on larger ones in HeLa cells [8]. The apparent surface that each WASH domain occupies saturates at about 0.8 mm2 for endosomes larger than 1 mm 3 . When LatA is applied, the increase in the number of WASH domains as a function of endosome volume is strongly diminished. For the largest endosomes, it culminates at 2 WASH domains per endosome, compared to 5 in the control situation. Concomitantly, the apparent surface that each WASH domain occupies increases steadily to 2.5 mm2 for the largest endosomes. All the above observations were statistically significant (see legend of figure 7). Altogether, these image analyses suggested two conclusions: (i) large endosomes have more WASH domains than smaller ones; (ii) actin polymerization constrains the surface area of WASH domains below an upper limit. To directly demonstrate that actin depolymerization induces coalescence of WASH domains, we sought to image the process after LatA addition. To alleviate the problem of out of plane WASH domains on enlarged endosomes, we acquired 12 z-planes by spinning disk confocal microscopy every 30 seconds, and performed a maximum intensity z-projection of the planes (Fig. 8 and Movie S6). This experiment showed that actin depolymer- ization induces WASH domain coalescence in a sequential manner whenever they meet. We initially reported that WASH belongs to a stable multi- protein complex [7]. We thus investigated how another subunit of the WASH complex, namely FAM21, behaves upon LatA treatment. As expected, FAM21 colocalized with WASH both in control and LatA-treated cells (Fig. S4). This suggests that actin depolymerization does induce the coalescence of the whole WASH complex at the endosome surface and thus does not interfere with the stability of the complex. We next examined whether coalescence upon actin depolymer- ization is a general behavior of endosomal domains or a specificity PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39774