Actin Organizes WASH Domains Figure 8. Direct observation of WASH domain coalescence in live cells upon actin depolymerization. Stable 3T3 cells expressing GFP- WASH and transiently expressing mCherry-Rab5Q79L were treated with 10 mM nocodazole for 1 h, then imaged by live spinning disk confocal microscopy. 12 planes separated by an increment of 0.4 mm were acquired at each time point, and z-projected. Elapsed time is in seconds. Scale bar: 2 mm. 0.2 mM LatA was added at the beginning of the movie. Arrowheads show individual WASH domains that fuse sequentially. doi:10.1371/journal.pone.0039774.g008 3 complex and its activator have intrinsic built-in dynamics rather than a specific biochemical pathway mediating this feedback regulation. One possible molecular scenario, explaining how the regulation of WASH dynamics may arise from its own activity, is depicted in Fig. 10. Active WASH molecules at the surface of endosomes activate Arp2/3 complexes, which generate branched actin networks. If the interaction between WASH and Arp2/3 is maintained long enough while the branched junction moves backwards, because of filament elongation, then one might understand how WASH, and by extension any Arp2/3 activator, can be actively detached from the membrane where it has been activated. Indeed, in vitro evidence exists for a transient tripartite interaction implicating the actin network, the Arp2/3 complex and its activator N-WASP [46]. In the cell, WASH molecules from the lamellipodium were also found by speckle microscopy to undergo a retrograde movement like the Arp2/3 complex at the branched junction [47]. We have also observed that branched actin networks control the lateral distribution of WASH domains at the surface of endosomes, since actin depolymerization induces coalescence of the WASH domains. This spontaneous coalescence of WASH domains is reminiscent of the behavior of lipidic domains at the surface of artificial vesicles [48,49]. The WASH complex directly binds to lipids through its FAM21 subunit, which has a broad specificity for negatively charged lipids [7,24]. This interaction however seems insufficient to account for membrane recruitment of the WASH complex, since recent experiments showed the cargo selective complex of the retromer was required to recruit the WASH complex to the endosomal surface through an interaction between FAM21 and Vps35 [10,41]. The ability of the WASH complex to interact with lipids might promote lipid reorganization after it has been recruited to the endosomal surface through the retromer. Once WASH is at the surface of the endosome, it is possible that it recognizes specific lipids and hence that WASH behavior reflects the behavior of the underlying lipids. These ideas need to be confirmed by the identification of the precise lipids that the WASH complex recognizes at the surface of endosomes and the development of specific probes thereof. A crucial step in the dissection of these mechanisms will be the ability to reconstitute these phenomena in vitro with purified machineries at the surface of model membranes. Our hypothesis to explain WASH coalescence is centered on a platform containing specific lipids clustered by the WASH complex. The WASH platform would have an intrinsic coherence that is independent of actin, and a long-range organization that depends on the branched actin network it is associated with. The short-range coherence accounts for the coalescence of two platforms when they meet at the surface of endosomes and for the fact that WASH molecules remain in clusters without actin. Without such a short-range coherence, we would expect WASH molecules diffusing all over the surface of endosomes, which we did not observe with or without actin. This short-range coherence might arise from WASH induced lipid repartitioning and domain formation, as previously described for some viral or bacterial lipid binding proteins [50,51]. The long-range organization provided by the branched actin network is required to understand why the platform cannot grow above an upper limit and prevent coalescence of WASH platforms when they encounter at the surface of endosomes. One can imagine that the WASH mediated branched actin network can grow only until a definite shell-like architecture that prevents further addition of WASH-lipids units is reached. This long-range organization argues for an active role of the actin cytoskeleton in lateral compartmentalization of endoso- mal membranes, which is crucial for sorting processes. The WASH complex might promote the scission of transport intermediates by several mechanisms, which collaborate. First, it recruits dynamin, directly or indirectly through the cortactin- positive branched actin network it generates [7]. Second, it probably favors the so-called line tension driven mechanism of membrane scission through an ability to organize a proteo-lipidic platform. Lipid repartitioning and domain formation is not only favored by protein binding to lipids, but also by membrane curvature, which is high in the tubule concentrating cargoes [51,52,53]. Line tension, a force that develops at the interface between lipid domains, favors constriction and ultimately scission of the membrane tubule [48,49,52,54]. The actin cytoskeleton was recently demonstrated to perform lipid reorganization and dynamin-independent scission of membrane tubules induced by Shiga toxin in HeLa cells [55]. The WASH complex thus has the required properties to coordinate these two mechanisms for the scission of endosomal transport intermediates. Materials and Methods Cell treatments Flip-In 3T3 cells (Invitrogen) and 3T3 GFP-WASH stable cells were handled and transfected as previously described [7] with the following modifications for Arp2/3 depletion: 40 nM of On- Target Plus siRNA (p34 duplex #1: sense AGGAAGCGCU- GUCGACCGA; p34 duplex #2: sense GGUAAUGAGUUG- CAGGUAA Dharmacon, Lafayette, Colorado) were transfected PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39774