A Conserved Role for SNX9-Family Members in the Regulation of Phagosome Maturation during Engulfment of Apoptotic Cells Johann Almendinger 1,4 * . , Kimon Doukoumetzidis 1. , Jason M. Kinchen 2 , Andres Kaech 3 , Kodi S. Ravichandran 2 , Michael O. Hengartner 1 1 Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland, 2 Center for Cell Clearance and the Department of Microbiology, University of Virginia, Charlottesville, Virginia, United States of America, 3 Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland, 4 Molecular Life Sciences PhD Program, Life Science Zurich Graduate School, UZH/ETHZ, Zurich, Switzerland Abstract Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi- cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 ( lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution. Citation: Almendinger J, Doukoumetzidis K, Kinchen JM, Kaech A, Ravichandran KS, et al. (2011) A Conserved Role for SNX9-Family Members in the Regulation of Phagosome Maturation during Engulfment of Apoptotic Cells. PLoS ONE 6(4): e18325. doi:10.1371/journal.pone.0018325 Editor: Janine Santos, University of Medicine and Dentistry of New Jersey, United States of America Received January 6, 2011; Accepted March 2, 2011; Published April 8, 2011 Copyright: ß 2011 Almendinger 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 the Kanton of Zurich, the Swiss National Science Foundation and the Ernst Hadorn Foundation (MOH), grants from the National Institutes of Health (KSR) and a Scientist Development Grant from the American Heart Association (JMK). 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: johann.almendinger@imls.uzh.ch . These authors contributed equally to this work. Introduction Apoptotic cell clearance is an important process during development, tissue homeostasis and wound healing [1,2]. The nematode C. elegans is a useful in vivo model to study programmed cell death and the clearance of apoptotic cells, as large numbers of cells die during embryonic development and during oogenesis in the adult germ line [3]. During clearance of apoptotic cells, two partially redundant pathways comprised of CED-1/MEGF10/ LRP1, CED-6/GULP and CED-7/ABCA1, and of MIG-2/ RhoG, UNC-73/Trio, CED-2/CrkII, CED-5/DOCK180 and CED-12/ELMO respectively, regulate the small GTPase CED- 10/Rac, which in turn directs the actin polymerization and membrane extension required to engulf the dying cell [4]. CED- 10/Rac activity during corpse engulfment is also regulated by ABL-1/Abl and its target ABI-1/Abi [5], the RacGAP SRGP-1 [6] as well as through a non-canonical Wnt pathway [7]. Following internalization, the two small GTPases RAB-5 and RAB-7, together with many additional factors, sequentially control the maturation of the nascent phagosome, ultimately leading to lysosome fusion and corpse degradation [8–15] for review see add [16,17]. How RAB-5 is recruited to early phagosomes is the subject of intensive study. We previously described an evolutionary conserved mechanism for the recruitment of RAB-5 to the early phagosome involving the large GTPase DYN-1/Dynamin and the phophatidylinositol-3 kinase VPS-34 [18,10]. Zhou and colleagues recently extended these observations by demonstrating that the sorting nexin LST-4 also functions at this step, interacting physically with DYN-1 and promoting the fusion of the maturing phagosomes with endosomes and lysosomes [19]. In this study, we confirm these observations and extend our understanding of LST-4 function through a careful genetic and cell biological dissection of the early steps in phagosome maturation. We also show that a mammalian homologue of PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18325