INTRODUCTION Phagocytosis is a complicated rearrangement of the actin cytoskeleton that delivers extracellular particles into intracellular vacuoles called phagosomes. In macrophages, a particle opsonized with IgG can stimulate Fc receptor- mediated phagocytosis, in which pseudopodial extensions of the cell surface cover the particle and enclose it. Although it is established that these pseudopodia contain actin and many actin-associated proteins (Allen and Aderem, 1995), and that a functional actin cytoskeleton is necessary for phagocytosis (Allison et al., 1971), it is not yet known how the actin cytoskeleton is regulated to achieve this apparently coordinated combination of extension and closure. No single extant model explains all kinds of phagocytosis. According to the zipper model for phagocytosis, pseudopod advance over a particle is guided by interactions between opsonins and cognate receptors in the macrophage plasma membrane. Such zippering of a membrane along a particle surface, together with a membrane fusion process, has been considered sufficient to enclose particles completely (Greenberg and Silverstein, 1993; Swanson and Baer, 1995). However, in the analogous process of macropinocytosis, actin- rich pseudopodia extend and close into intracellular vesicles without any particle surface to guide them (Swanson and Watts, 1995). Moreover, some bacteria enter macrophages by a process resembling macropinocytosis (Alpuche-Aranda et al., 1994). Thus, without an additional mechanism for closing phagosomes, the zipper model cannot explain how pseudopodia close to form macropinosomes or spacious phagosomes. Recent studies indicate that phagocytosis requires two component activities of the actin cytoskeleton. Inhibitors of phosphoinositide 3-kinase (PI3-kinase) allow pseudopodia to extend onto an opsonized particle, but prevent them from closing into phagosomes, indicating that PI3-kinase regulates phagosome closure (Araki et al., 1996). Phagosome closure could occur by regulated actin polymerization, reorganized actin gel networks, altered membrane curvature or a localized contractile activity. Although myosins localize to phagosomes (Allen and Aderem, 1995; Stendahl et al., 1980) and evidently participate in the process (Ostap and Pollard, 1996), their contribution to phagocytosis remains undefined. Evans et al. (1993), measured forces generated by leukocytes during phagocytosis of yeast particles, and identified a contraction of the entire cell that followed pseudopod extension over a particle. This indicated that some contractile activity accompanies phagocytosis. Here we describe a localized 307 Journal of Cell Science 112, 307-316 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0075 Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface. Key words: Phagosome, Macrophage, Myosin, Contraction SUMMARY A contractile activity that closes phagosomes in macrophages Joel A. Swanson 1,2, *, Melissa T. Johnson 1 , Karen Beningo 2 , Penny Post 3 , Mark Mooseker 3 and Nobukazu Araki 1,4 1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA 2 Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA 3 Department of Biology, Yale University, New Haven, CT, USA 4 Department of Anatomy, Kagawa Medical University, Miki, Kagawa 791-0793, Japan * Author for correspondence at the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-0620 (e-mail: jswan@umich.edu) Accepted 7 November 1998; published on WWW 13 January 1999