Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments Emiko Ohashi., Kenji Tanabe*., Yuji Henmi, Kumi Mesaki, Yuka Kobayashi, Kohji Takei Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Abstract Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs. Citation: Ohashi E, Tanabe K, Henmi Y, Mesaki K, Kobayashi Y, et al. (2011) Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments. PLoS ONE 6(5): e19942. doi:10.1371/journal.pone.0019942 Editor: Diane Bassham, Iowa State University, United States of America Received February 8, 2011; Accepted April 6, 2011; Published May 20, 2011 Copyright: 2011 Ohashi 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 from the Ministry of Education, Science, Sports, Culture and Technology of Japan, (23113721) http://kaken.nii.ac.jp/ en/p/23113721, (15079206) http://kaken.nii.ac.jp/en/p/15079206; and by the Okayama Medical Foundation, http://omf.umin.ac.jp/. 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: kt0911@gmail.com . These authors contributed equally to this work. Introduction Early endosomes (EEs) are highly dynamic compartments that act as entry portals, sorting stations, and signaling platforms [1,2,3]. They sort molecules and direct them into the appropriate pathway. Degradative molecules are sorted into particular membrane domains and this process is followed by maturation along with acidification and formation of intraluminar vesicles, referred to as multivesicular bodies (MVBs) [2,4]. Finally, MVBs/late endosomes (LEs) fuse with lysosomes where protein degradation occurs. However, recycling molecules are directly transported to the plasma membrane (PM) by vesicular transport [5,6,7,8] or indirectly by recycling endosomes (REs) via large tubules [9]. Much progress has been made in understanding MVB biogenesis [10]. However, the process of membrane remodeling for the recycling pathway, including tubula- tion and segregation activities, remains to be elucidated. Membrane remodeling is induced by lipid-interacting proteins, lipid-modifying enzymes, and cytoskeletons and their related proteins [11,12,13,14,15,16,17]. Of these, recent evidence has indicated that actin plays essential roles in endosome biogenesis [18,19,20]. The role of actin in intracellular trafficking is well known for endocytosis, phagocytosis, and bacterial motility. In endocytosis, actin may provide a motile force to assist the fission activity of dynamin GTPase [21]. Actin functions in short-range movements through actin-rich regions [22,23] and may be involved in endosome movement [24], cargo transport [25,26], and endosome morphology [27]. Recent studies have shown that several actin- related proteins are required for endosomal actin reorganization. These include myosin1B [27], N-WASP [28], cortactin [26], CART, an Hrs/actinin-4/BERP/myosin V protein complex [29], Annexin A2, Spire1, and Arp2/3 [20]. As actin polymerization is a good candidate for inducing a motile force for membrane fission, understanding actin regulation of intracellular transport is sure to be a key step for further elucidating membrane trafficking. In this report, to determine the role of actin filaments in relation to EEs, we investigated the inhibitory effects of actin dynamics on both the transport from EEs and endosome morphology. We found that inhibition of actin dynamics induced the enlargement of EEs with several distinct vacuoles and inhibited their transportation ability. Moreover, cortactin, an actin-nucleating factor, was found to be required for segregation in EEs. Thus, actin and cortactin are required for efficient transport of endosomes toward the perinuclear region. We propose that actin and cortactin play essential roles in segregation relating to the recycling and degradative pathways and in transport toward the perinuclear region in coordination with microtubules. Results Actin dynamics regulate transport beyond EEs We initially investigated whether actin plays an essential role in transport from EEs. Internalized transferrin (Tfn) signals were partially, but clearly colocalized with actin 10 min after internal- ization (Fig. 1A), indicating that actin localizes in EEs, as has been reported previously [24,30]. Next, to investigate the significance of actin filaments in EEs, we used latrunculinB (LatB) as an actin depolymerizing agent [31]. For visualization of transport from EEs, we used fluorescence-labeled Tfn and EGF as tracers for the recycling and degradative pathways, respectively. HeLa cells were bound to these ligands on ice, washed, and incubated at 37uC for PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19942