578 Research Article Introduction Cells use different strategies to internalise particles and solutes, including pinocytosis, receptor-mediated endocytosis and phagocytosis. Phagocytosis is a universal cell function that exploits a ubiquitous and mostly conserved cell machinery to couple receptor-dependent binding of particulate material (>0.5 m in diameter) to its internalisation. Although primitive organisms use phagocytosis primarily for acquisition of nutrients, phagocytosis in metazoans occurs in specialised phagocytic cells, such as macrophages, dendritic cells and neutrophils. (Aderem, 2002; Aderem and Underhill, 1999; Castellano et al., 2001; Greenberg and Grinstein, 2002; Underhill and Ozinsky, 2002). The molecular mechanisms underlying phagocytosis are extremely complex and not precisely defined, but recent studies have described many aspects of the process. Phagocytosis is initiated by binding of specific ligands on the particles to their cognate receptors, such as Fc, mannose and complement receptors, which trigger intracellular signals. These signalling cascades lead to the polymerisation and rearrangement of filamentous actin (F-actin) beneath the particle and coordinate the tractional forces that internalise the particles (Aderem and Underhill, 1999; Castellano et al., 2001). Different receptors generate different signalling cascades, which have distinct effects on the actin cytoskeleton, and different biological responses (Allen and Aderem, 1996; Caron and Hall, 1998; Kuiper et al., 2008). For example, Fc receptor (FcR)-mediated phagocytosis requires the Cdc42-Rac-Rho signalling pathway to modify the actin cytoskeleton, whereas complement receptor only requires Rho GTPase activity for the F-actin rearrangement (Caron and Hall, 1998). With the help of the actin cytoskeleton, particles get engulfed and form the phagosomes, which harbour a number of characterised and uncharacterised polypeptides, including phagocytic receptors, cytoskeleton proteins [e.g. actin and actin-binding proteins (ABPs)], signalling molecules (e.g. protein kinase C) and membrane trafficking proteins (e.g. Rab 5 and Rab 7). As for the signalling events, different receptors influence the protein composition of phagosomes (Hoffmann et al., 2010). In addition, with ongoing maturation, the protein and lipid composition of the phagosome alters (Desjardins et al., 1994a; Haas, 2007). In the past decade, several proteomic studies have set out to determine the protein composition of phagosomes (Desjardins et al., 1994a; Hoffmann et al., 2010; Griffiths and Mayorga, 2007; Martinez-Solano et al., 2006; Morrissette et al., 1999; Slomianny et al., 2006), but most of these studies did not focus on the individual functions of these proteins. Like many other proteins, ABPs are known to be present on the phagosomes, but their role in the biogenesis of phagosomes is still poorly understood. Among the ABPs present on phagosomes are proteins of the annexin family (Diakonova et al., 1997). Annexins are type II (non-EF hand) Ca 2+ -binding proteins, which bind to negatively charged phospholipids in the presence of Ca 2+ . Annexins comprise four or eight 70-amino-acid repeats and a variable N-terminus, which is believed to be responsible for their different activities (Moss, 1992). Previous studies suggested that annexins participate in a broad range of intracellular processes, including membrane dynamics, membrane cytoskeleton interactions and vesicle trafficking (Futter and White, 2007; Gerke et al., 2005; Moss, 1992). Annexin A1, formerly known as lipocortin 1, was initially identified as an anti-inflammatory protein that is glucocorticoid regulated and secreted atypically from cells (D’Acquisto et al., 2008). Intracellularly, annexin A1 is predominantly a cytosolic Summary Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin- binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca 2+ and facilitates the F-actin–LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation (‘actin flashing’). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation. Key words: Annexin A1, F-actin, LBP, Phagocytosis Accepted 19 October 2010 Journal of Cell Science 124, 578-588 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.076208 Annexin A1 is a new functional linker between actin filaments and phagosomes during phagocytosis Devang M. Patel 1 , Syed Furquan Ahmad 1 , Dieter G. Weiss 1 , Volker Gerke 2 and Sergei A. Kuznetsov 1, * 1 Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein Straße 3, Rostock 18059, Germany 2 Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, University of Münster, Von-Esmarch-Straße 56, Münster 48149, Germany *Author for correspondence (sergei.kuznetsov@uni-rostock.de) Journal of Cell Science