505 Research Article Introduction The plasma membrane of cells can assemble and disassemble actin filaments within seconds of adding ligands of receptors, such as those that bind EGF or thrombin (Peppelenbosch et al., 1993; Omann et al., 1987; Carson et al., 1986). In many cases this dynamic assembly and disassembly of actin has been linked to chemotaxis of cells such as Dictyostelium, neutrophils, T cells and macrophages (Goetzl et al., 2004; McRobbie and Newell, 1983). In this, and other processes where actin is nucleated and polymerized on membrane surfaces, the monomers are inserted at the membrane surface by a membrane-bound machinery (Tilney and Cardell, 1970). In many systems these rapid changes in actin are correlated with transient increases in phosphatidylinositol-(4,5)-bisphosphate (PIP2) (Yin and Janmey, 2003). Here, we address the rapid assembly of actin at the plasma membrane of mouse RAW 264.7 and J774A.1 macrophage cell lines (Defacque et al., 2000), and bone marrow primary macrophages. Preliminary evidence showed that the addition of serum to these cells led to a rapid accumulation of extracellular ATP that coincided with the actin assembly process. Extracellular nucleotides such as ATP, ADP and UTP bind to distinct members of a family of 15 cell-surface purinergic receptors and induce a spectrum of signaling events (Burnstock, 2006). We focused here on the P2X7 receptor (P2X7R), which is expressed by cells of the monocyte lineage, including macrophages, and is one of the best-characterized purinergic receptors (North, 2002). A prominent feature of P2X7R is its response to very high ATP levels (0.1-10 mM) where, apart from opening an ion channel pore, it might induce the opening of large pores that allow the passage of molecules of up to 900 Da into macrophages (Steinberg et al., 1987; Steinberg and Silverstein, 1987). Less appreciated, but important for the present, and the accompanying study (Kuehnel et al., 2009), is the finding that P2X7 receptor has an additional high-affinity site that responds to low concentrations (1-10 μM) of ATP (Klapperstück et al., 2001); such levels of ATP have been constitutively detected on the surface of many eukaryotic cells (Hayashi et al., 2004; Yegutkin et al., 2006b). Particularly relevant to the current study was the fact that the P2X7 receptor could be detected in a complex with 12 proteins, including actin, phosphoinositide 4-kinase and α-actinin (Kim et al., 2001). In conjunction with the parallel study focused on latex bead phagosomes (Kuehnel et al. 2009), our data collectively argue that the P2X7 receptor is part of a membrane-bound machinery that assembles actin. Results Serum induces actin assembly and releases extracellular ATP within seconds We modified a widely used quantitative assay for monitoring the rapid assembly and disassembly of actin at the plasma membrane in response to receptor activation; this is based on the amount of Rhodamine-phalloidin that binds to F-actin in fixed and permeabilized cells (Howard and Oresajo, 1985; Defacque et al., 2000). When medium containing fresh fetal bovine serum was added to either J774 or RAW macrophages, reproducible peaks of actin Eukaryotic plasma membranes assemble actin filaments within seconds of activation of many receptors, especially during chemotaxis. Here, serum or sphingosine-1-phosphate stimulation of J774 and RAW macrophages released ADP within seconds into the extracellular medium, along with an adenylate kinase activity that converted ADP to ATP. ATP then activated the P2X7 receptor (P2X7R) that was necessary for a peak of plasma-membrane actin assembly within 5 to 10 seconds in P2X7R-expressing J774, RAW and primary macrophages. Neither actin assembly nor characteristic P2X7R channel activity was seen in response to ATP in P2X7R-knockout macrophages, as detected by patch-clamp analysis. Since P2X7R has been shown previously to form a macromolecular complex with actin we propose that it is involved in the membrane assembly of actin. Our data reveal a surprisingly rapid and complex relay of signaling and externalization events that precede and control actin assembly induced by sphingosine-1- phosphate. The overall model we present is strongly supported by the data presented in the accompanying paper that focuses on latex bead phagosomes. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/4/505/DC1 Key words: ATP, P2X7, Sphingosine-1-P, Actin, Macrophages, Phagosomes Summary Sphingosine-1-phosphate receptors stimulate macrophage plasma-membrane actin assembly via ADP release, ATP synthesis and P2X7R activation Mark P. Kuehnel 1 , Miriam Reiss 1 , Paras K. Anand 1 , Irina Treede 1 , Daniela Holzer 1 , Eik Hoffmann 1,2 , Manuela Klapperstueck 3 , Thomas H. Steinberg 4 , Fritz Markwardt 3 and Gareth Griffiths 1, * 1 EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany 2 Institute for Biological Sciences, University of Rostock, Albert-Einstein-Str. 3, 18051 Rostock, Germany 3 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Magdeburger Str. 6, 06097 Halle, Germany 4 Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA *Author for correspondence (e-mail: griffiths@embl.de) Accepted 23 October 2008 Journal of Cell Science 122, 505-512 Published by The Company of Biologists 2009 doi:10.1242/jcs.034207 Journal of Cell Science