Introduction Gross alterations in cytoskeletal architecture are tightly linked to increased cell motility, invasion, metastasis and angiogenesis. The remodeling of actin cytoskeleton structures is commonly accompanied by the rearrangement of focal substrate adhesions, and rapid focal adhesion turnover is consistent with elevated migration rates (for a review, see Kaverina et al., 2002b). While most quiescent cultured cells develop predominantly spear-shaped, stable focal contacts, which serve to anchor actin stress fibers to the sites of cell- matrix adhesion (reviewed in Schoenwaelder and Burridge, 1999; Sastry and Burridge, 2000), motile and spreading cells in addition form transient focal complexes in their cell periphery (Rottner et al., 1999; Smilenov et al., 1999). Monocyte-derived cells, like leukocytes, macrophages and osteoclasts, by contrast, form highly dynamic, dot- or donut- shaped adhesion sites that are generally referred to as podosomes. Transformation of cells by Rous sarcoma virus (RSV) induces podosomes in fibroblasts (Provenzano et al., 1998; Hakak et al., 2000; Mizutani et al., 2002) and epithelial cells (Tarone et al., 1985), and causes a dramatic rearrangement of the actin cytoskeleton and associated focal adhesion sites (Tarone et al., 1985; Marchisio et al., 1988). This transformation is mediated by a single tyrosine kinase, Src, which localizes to the actin cytoskeleton at the ends of stress fiber bundles in focal contacts (Fincham et al., 2000; Marchisio et al., 1988). Focal adhesions and podosomes share major structural components including α-actinin, vinculin, zyxin and paxillin (Duong and Rodan, 2000; Chellaiah et al., 2001). However, podosomes also harbor the actin polymerization machinery components Arp2/3 and Wiskott-Aldrich Syndrome protein (WASp), and may thus generally form at sites where rapid polymerization/depolymerization of actin filaments occurs (Schafer et al., 1998). Differentiation of monocyte-derived precursor cells into osteoclasts by oseteoclast differentiation factor RANKL-ODF (Matsuzaki et al., 1998) induces high levels of integrin αvβ3 expression (Pfaff and Jurdic, 2001). In addition, αvβ3 localizes to the sites of podosome initiation and the interaction with the non-receptor tyrosine kinase Pyk2 is required for the reinforced distribution of podosomes (Pfaff and Jurdic, 2001). Expression of αvβ3 integrins is strongly restricted to a few cell types, namely activated endothelial cells, vascular smooth muscle cells (VSMCs), tumor cells, and osteoclasts (Westlin, 2001). In agreement with these findings, we have shown recently that VSMCs can also form podosomes in vitro (Gimona et al., 2003). In response to phorbol ester, the pattern of adhesions in cultured rat vascular smooth muscle cells is partially modulated from spear-shaped focal adhesions to rosette-like podosomes. This process can be blocked by specific inhibitors of PKC and Src (Hai et al., 2002; Brandt et al., 2002) and is 4915 Phorbol ester triggers the conversion of focal adhesions into podosomes in A7r5 smooth muscle cells. Here we followed the dynamics of podosome formation using dual fluorescence live video and confocal microscopy, as well as interference reflection and evanescent wave microscopy. We show that podosomes form at the outer region of stress fiber bundles, at specialized sites where they are embedded in adhesion plaques at the basal surface of the plasma membrane, and that cortactin resides constitutively at these microdomains. We further demonstrate that the formation of podosomes requires Arp2/3-dependent actin polymerization at the stress fiber-focal adhesion interface. Concentration of Arp2/3 coincides with podosome formation and precedes the engagement of SM22 and alpha-actinin, while the focal adhesion components zyxin and vinculin redistribute only at later stages of podosome development. We thus suggest that the genesis of podosomes includes two steps, one requiring the early de novo polymerization of actin filaments, and a second, late phase characterized by the recruitment of focal adhesion components. Moreover, we provide evidence for the existence of an as yet unidentified region in close proximity to the focal adhesion-stress fiber interface, which marks the site of actin cytoskeleton remodeling and is a novel site of Arp2/3-dependent F-actin polymerization. Movies available online Key words: Podosomes, Arp2/3, Smooth muscle, Dynamics, Cytoskeleton Summary Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de- novo actin polymerization at discrete microdomains Irina Kaverina 1 , Theresia E. B. Stradal 2 and Mario Gimona 1, * 1 Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences; Billrothstrasse 11, A-5020 Salzburg, Austria 2 Gesellschaft für Biotechnologische Forschung (GBF), Department of Cell Biology, D-38124 Braunschweig, Germany *Author for correspondence (e-mail: mgimona@imb.oeaw.ac.at) Accepted 5 August 2003 Journal of Cell Science 116, 4915-4924 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00818 Research Article