Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes Emmanuel Derivery.¤, Emmanue` le Helfer., Ve´ ronique Henriot, Alexis Gautreau* Laboratoire d’Enzymologie et Biochimie Structurales, CNRS UPR3082, Gif-sur-Yvette, France Abstract Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed. Citation: Derivery E, Helfer E, Henriot V, Gautreau A (2012) Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes. PLoS ONE 7(6): e39774. doi:10.1371/journal.pone.0039774 Editor: James Keen, Thomas Jefferson University, United States of America Received October 20, 2011; Accepted May 30, 2012; Published June 21, 2012 Copyright: 2012 Derivery et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants (ANR-08-BLAN-0012-03, ANR-08-PCVI-0010-03, and ANR-09-JCJC-0040) from Agence Nationale pour la Recherche (http://www.agence-nationale-recherche.fr/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexis.gautreau@lebs.cnrs-gif.fr ¤ Current address: Department of Biochemistry, University Sciences II, Geneva, Switzerland . These authors contributed equally to this work. Introduction The Arp2/3 complex is a major actin nucleator that generates branched actin networks at different cellular locations [1]. Recent evidence suggest a division of labor between distinct Arp2/3 activators [2,3,4,5]. WAVE proteins activate the Arp2/3 complex at the plasma membrane and generate lamellipodia. N-WASP generates a burst of actin polymerization at coated pits during clathrin mediated endocytosis. WHAMM regulates Golgi mor- phology and ER to Golgi transport [6]. We and others recently reported that WASH is found at the surface of vesicles of the endosomal/lysosomal system, yet with a marked enrichment in sorting endosomes [7,8,9,10,11,12]. Consistently, WASH regu- lates endosome morphology and WASH depletion affects the major endosomal routes, including recycling, degradation and retrograde pathways [7,8,9,10]. WASH has also been implicated in removal of v-ATPases from lysosomes [11]. The division of labor between Arp2/3 activators is, however, not absolute. For example, N-WASP was also detected at the rear of endosomes that are propelled in the cytoplasm through actin comet tails in special cases, when cells were stimulated by phorbol esters or when the lipid phosphatidylinositol(4,5)bis-phosphate was overproduced [13,14]. However, in normal circumstances, endosomes move along microtubules and WASH appears as the major Arp2/3 activator at their surface [7]. Regulation of actin polymerization at the surface of endosomes also implicates Annexin A2 [15] and diaphanous related formins [16,17]. Actin polymerization at the surface of endosomes was previously involved in the regulation of vesicle fusion [18,19,20,21] and in the biogenesis of multivesicular bodies [15]. WASH dependent branched actin networks, specifically labeled with the branch marker, cortactin [22], were shown to define specific domains of the endosomal membrane [7,8,9,23]. These domains might be due in part to the lipid binding ability of the multiprotein complex that WASH forms [7,24]. These actin rich endosomal domains were found to be important for cargo sorting [25]. Branched actin networks at the surface of endosomes are important in at least two steps. First, they stabilize a membrane tubule, while it clusters cargoes [23]. Then, they promote the scission of the transport intermediate through an incompletely understood mechanism that involves dynamin [7]. The latter step appears analogous to the collaboration between dynamin and the N-WASP dependent branched actin network during scission of the clathrin coated pit [26,27]. The identity of the endosomal transport intermediate, contain- ing specific cargoes, is conferred by its content in Rab GTPases that defines its target destination [28,29]. Lateral compartmental- ization of cargoes and Rab proteins relies on a dynamic network of lipid-protein interactions [30,31]. For example, Rab5 domains are PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39774