RAB-5 Controls the Cortical Organization and Dynamics of PAR Proteins to Maintain C. elegans Early Embryonic Polarity Vincent Hyenne1*, Thierry Tremblay-Boudreault1, Ramraj Velmurugan1, Barth D. Grant3, Dinah Loerke4, Jean-Claude Labbe´ 1,2 1 Institute of Research in Immunology and Cancer (IRIC), Universite´ de Montre´al, Montre´al, Que´bec, Canada, 2 Department of Pathology and Cell Biology, Universite´ de Montre´al, Montre´al, Que´bec, Canada, 3 Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America, 4 Department of Physics and Astronomy, University of Denver, Denver, Colorado, United States of America Abstract In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C. elegans embryo, the mechanism controlling their asymmetric localization is not fully understood. Here we studied the role of endocytic regulators in embryonic polarization and asymmetric division. We found that depleting the early endosome regulator RAB-5 results in polarity-related phenotypes in the early embryo. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that PAR-6 is localized at the cell cortex in highly dynamic puncta and depleting RAB-5 decreased PAR-6 cortical dynamics during the polarity maintenance phase. Depletion of RAB-5 also increased PAR-6 association with clathrin heavy chain (CHC-1) and this increase depended on the presence of the GTPase dynamin, an upstream regulator of endocytosis. Interestingly, further analysis indicated that loss of RAB-5 leads to a disorganization of the actin cytoskeleton and that this occurs independently of dynamin activity. Our results indicate that RAB-5 promotes C. elegans embryonic polarity in both dynamin-dependent and -independent manners, by controlling PAR-6 localization and cortical dynamics through the regulation of its association with the cell cortex and the organization of the actin cytoskeleton. Citation: Hyenne V, Tremblay-Boudreault T, Velmurugan R, Grant BD, Loerke D, et al. (2012) RAB-5 Controls the Cortical Organization and Dynamics of PAR Proteins to Maintain C. elegans Early Embryonic Polarity. PLoS ONE 7(4): e35286. doi:10.1371/journal.pone.0035286 Editor: Sang-Chul Nam, Baylor University, United States of America Received February 1, 2012; Accepted March 14, 2012; Published April 24, 2012 Copyright: 2012 Hyenne 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: The Institute of Research in Immunology and Cancer is supported in part by the Canadian Center of Excellence in Commercialization and Research (CECR, http://www.nce-rce.gc.ca/ReportsPublications-RapportsPublications/CECR/Program-Guide-Programme_eng.asp), the Canada Foundation for Innovation (CFI, http://www.innovation.ca/en), and the Fonds de Recherche en Sante´ du Que´bec (FRSQ, http://www.frsq.gouv.qc.ca/fr/index.shtml). V.H. was a Research Fellow of the Terry Fox Foundation through an award from the National Cancer Institute of Canada (http://www.terryfox.org). B.D.G. was supported by National Institutes of Health (NIH) grant GM067237 (http://www.nih.gov). This work was supported by grant #019378 from the Canadian Cancer Society (http://www. cancer.ca/Research.aspx) to J.-C.L., who holds the Canada Research Chair in Cell Division and Differentiation (http://www.chairs-chaires.gc.ca/home-accueil-eng. aspx). 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: worm.machine@gmail.com Introduction Formation of polarized domains within a cell, through the asymmetric partition of proteins, lipids and RNAs, is required for a number of processes such as asymmetric cell division, cell migration, morphogenesis and maintenance of tissue architecture (reviewed in [1,2]). PAR proteins constitute an evolutionarily conserved molecular machinery that is essential for the formation and maintenance of such polarized domains [3]. Most PAR proteins display a cortical and polarized localization that is crucial for their function. The three proteins PAR-3, PAR-6 and aPKC/ PKC-3 (collectively referred to as anterior PAR proteins) can form a complex that localizes asymmetrically at the cortex of many different polarized cells. In the early C. elegans embryo, the anterior localization and the size of the domain occupied by the anterior PAR proteins are governed by actomyosin-dependent cortical flows, which initiate soon after fertilization and relocalize cortical components from the posterior to the anterior pole of the embryo [4]. Exclusion of the anterior PAR proteins from the posterior cortex by these cortical flows allows the recruitment of two other PAR proteins, PAR-1 and PAR-2, to the posterior cortex. Once localized, anterior and posterior PAR proteins mutually exclude one-another during the polarity maintenance phase, until the asymmetric division of the zygote. Although some anterior PAR proteins have been shown to interact with the membrane or with the underlying actin cytoskeleton in other cell types [2], the mechanisms that promote their asymmetric enrichment and dynamically regulate their cortical anchoring in C. elegans are unclear. In addition, the genes responsible to determine the proper location and size of the domain occupied by these proteins are largely unknown. Over the past years, several studies have shed light on the role of endocytic components in the control of cell polarity [5,6]. For instance, Rab5 is required for PAR protein cortical localization and for epithelial polarity in Drosophila [7]. While a recent study PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35286