A dynamic podosome-like structure of epithelial cells $ Laura Spinardi, a, * Jens Rietdorf, b Lucio Nitsch, c Maria Bono, d Carlo Tacchetti, d Michael Way, e and Pier Carlo Marchisio a a DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute and University Vita-Salute San Raffaele, 20132 Milan, Italy b European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany c C.E.O.S. Department of Biology and Pathology, Cellular and Molecular L. Califano, University of Napoli Federico II, 80131 Naples, Italy d Department of Experimental Medicine, Anatomy Section, University of Genova, Genoa, Italy e Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3PX, UK Received 25 July 2003, revised version received 22 December 2003 Abstract Focal contacts and hemidesmosomes are cell – matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin a3h1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of h4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and h4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion. D 2004 Elsevier Inc. All rights reserved. Keywords: Epithelial adhesion; Integrin a6h4; Hemidesmosome; Podosome; Motility Introduction The interaction of epithelial cells with laminin at the basement membrane is critical for the maintenance of cell polarity and for the regulation of adhesion, migration, and invasion [1]. In epithelial cells, the adhesion of the basement membrane to the extracellular matrix (ECM) is mediated by several different types of adhesion structures. Focal contacts represent the major site of actin filament attachment to the ECM contact surface in vitro [2,3]. Focal contacts are composed of integrin receptors and associated cytoplasmic cytoskeletal proteins, including a-actinin, pax- illin, talin, tensin, vinculin and several protein kinases [2– 5]. Hemidesmosomes are another type of adhesion com- plex that provide a link between intermediate filament networks and the basement membrane [6–8]. They are composed of transmembrane receptors, such as the integrin a6h4 and the bullous pemphigoid antigen 180 (BPA180), which are connected to cytoplasmic proteins, including bullous pemphigoid antigen 230 (BPA230) and plectin [9,10]. Cells of mesenchymal origin, such as macrophages and osteoclasts, assemble unique, and highly dynamic types of adhesion structures called podosomes [11–18]. Podosomes have been recently reviewed by Linder and Apfelbacher [19]. Podosomes are also assembled by many transformed cells including some human leukemia cells [20–23]. Podo- somes are columnar arrays of actin filaments surrounding a 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.01.007 $ Supplementary data for this article can be found at doi:10.1016/ S0014-4827(04)00016-3. * Corresponding author. DIBIT, Department of Biological and Technological Research, Stem Cell Research Institute, San Raffaele Scientific Institute and University Vita-Salute San Raffaele, Via Olgettina 58-20132 Milano, Italy. Fax: +39-02-21-56-02-20. E-mail address: spinardi.laura@hsr.it (L. Spinardi). www.elsevier.com/locate/yexcr Experimental Cell Research 295 (2004) 360 – 374