Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite Fiona Angrisano1,2, David T. Riglar1,2., Angelika Sturm3., Jennifer C. Volz1,2.¤, Michael J. Delves4, Elizabeth S. Zuccala1,2, Lynne Turnbull5, Chaitali Dekiwadia6, Maya A. Olshina1,2, Danushka S. Marapana1, Wilson Wong 1 , Vanessa Mollard4, Clare H. Bradin1, Christopher J. Tonkin1,2, Peter W. Gunning7, Stuart A. Ralph 6 , Cynthia B. Whitchurch5, Robert E. Sinden3, Alan F. Cowman1,2, Geoffrey I. McFadden4, Jake Baum1,2* 1 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 2 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia, 3 School of Botany University of Melbourne, Parkville, Victoria, Australia, 4 Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London, United Kingdom, 5 The ithree Institute, University of Technology Sydney, Sydney, New South Wales, Australia, 6 Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia, 7 Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia Abstract Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu. Citation: Angrisano F, Riglar DT, Sturm A, Volz JC, Delves MJ, et al. (2012) Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite. PLoS ONE 7(2): e32188. doi:10.1371/journal.pone.0032188 Editor: Thomas J. Templeton, Weill Cornell Medical College, United States of America Received November 17, 2011; Accepted January 23, 2012; Published February 28, 2012 Copyright: 2012 Angrisano 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 made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. Funding for the research came from the National Health and Medical Research Council of Australia (NHMRC Project Grants 516747 JB & CJT and 637341 JB & GIM). DTR is supported by a Pratt Foundation PhD scholarship through the University of Melbourne; JCV was initially supported by a HFSP long-term fellowship; CJT is supported by an NHMRC Career Development Award (575523); LT is supported by a Chancellor’s Postdoctoral Fellowship from the University of Technology, Sydney; PWG was a Principal Research Fellow of the NHMRC; SAR is supported by an Australian Research Council (ARC) Future Fellowship (FT0990350); CBW is supported by a Senior Research Fellowship from the NHMRC; AFC is an Australia Fellow of the NHMRC. GIM is an ARC Federation Fellow with support from the NHMRC; JB is supported by an ARC Future Fellowship (FT100100112). 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: jake@wehi.edu.au . These authors contributed equally to this work. ¤ Current address: The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom Introduction Malaria constitutes a huge health and economic burden on humanity [1]. The disease is caused by obligate intracellular parasites from the genus Plasmodium, a group of protozoa whose developmental lifecycle is completed between mosquito and human hosts. During this complex journey the parasites navigate a variety of tissues and infects several distinct cell types [2,3,4]. Three motile and/or invasive forms define this journey: ookinete, sporozoite and merozoite. The ookinete traverses the mosquito midgut [4]. The sporozoite establishes salivary gland infection in the mosquito along with subsequent transmission to the human host and infection of the liver [3]. Finally, the merozoite, the smallest form, infects circulating erythrocytes in the bloodstream and is responsible for initiating all pathology associated with malaria disease [2]. Despite gross morphological differences and disparate environmental niches, each of these developmental forms retain the classical cytoskeletal architecture and organelle repertoire (with the exception of the PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32188