2980 Research Article Introduction Actins are a family of highly conserved cytoskeletal proteins that play fundamental roles in nearly all aspects of eukaryotic cell biology. In vertebrates, six actin isoforms, i.e. two striated muscle [α-skeletal (α-SKA) and α-cardiac (α-CAA)], two smooth muscle (α- and γ-SMA) and two cytoplasmic (β- and γ-CYA) actins are encoded by distinct genes (Vandekerckhove and Weber, 1978). Muscle actins are tissue specific and organized in contractile units, whereas β- and γ-CYA are ubiquitous and essential for cell survival (Harborth et al., 2001). All actin isoforms exhibit highly conserved primary amino acid sequences with the main differences located at the extreme N-terminus. In particular, the two cytoplasmic actins (CYAs) differ only by four amino acids located at positions 1, 2, 3 and 9. Contrary to what is known for muscle actins (Chaponnier and Gabbiani, 2004; Lambrechts et al., 2004), little is known about the possible specific roles of β- and γ-CYA, probably because of embryonic lethality when CYAs are absent or mutated (Shawlot et al., 1998). To avoid the early lethal phase, a conditional-knockout approach using the Cre-loxP system has been recently employed to specifically ablate the expression of γ-CYA in skeletal muscle (Sonnemann et al., 2006). This represses the isoform expression in the costamere and results in a progressive myopathy (Sonnemann et al., 2006). Distinct localizations of β-(ACTB) and γ-CYA (ACTG1) mRNAs in several cell types (Hill and Gunning, 1993; Watanabe et al., 1998;Bassell et al., 1998) have suggested different spatioregulation for the two isoforms. A crucial step to explore the specific biological roles of the two CYAs is documenting their subcellular localization. A few studies using polyclonal antibodies (Abs) both in cultured cells (Otey et al., 1986; Schevzov et al., 2005) and in vivo (Hofer et al., 1997; Yao et al., 1995) have previously attempted to investigate whether CYAs are sorted into different intracellular compartments; these studies are, however, limited by the cross-reactivity of γ-CYA Abs with α- and γ-SMA (Otey et al., 1986; Schevzov et al., 2005). Although β-actin monoclonal antibodies (mAbs) have been produced for many years (Gimona et al., 1994), the unavailability of a specific γ-CYA Ab has precluded the precise comparative appreciation of the sorting of the two isoforms. The ability of cells to divide, move, generate contractile force and maintain shape is based upon specialized actin-containing structures. Numerous actin-binding proteins (ABPs) exert tight control over actin-filament stability and function. Among the ABPs, some link actin filaments in tight bundles and stress fibers, or in branched networks, and others anchor filaments to membranes (Winder and Ayscough, 2005). Specific CYA sorting in these different cellular structures has not been reported. Using two newly developed mAbs, we investigated the subcellular localization of β- and γ-CYAs in quiescent cells and in models of spreading, migration, division and contraction. Moreover, we used selective inhibitors of actin nucleation pathways to study which pathway controls the distinct organization of β- and γ-CYAs. Finally, we explored the effects of silencing each of these isoforms using specific siRNAs. Our results bring new data in support of the assumption that β- and γ-CYAs play different functional roles. Results Characterization of anti-CYA antibodies Mice were injected with the N-terminal nonapeptides of β- and γ- CYA. The β- and γ-CYA mAbs were selected by ELISA screenings using the isoform synthetic peptides and their specificity was confirmed by western blots (Fig. 1). Using newly generated monoclonal antibodies, we have compared the distribution of β- and γ-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas β-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, γ-actin displays a more versatile organization, according to cell activities. In moving cells, γ-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, γ-actin is also recruited into stress fibers. β-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, γ-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, β- and γ-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of β- and γ-actin organization that support their functional diversity. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/16/2980/DC1 Key words: Actin isoforms, Actin network, Cytoskeleton Summary β- and γ-cytoplasmic actins display distinct distribution and functional diversity Vera Dugina 1 , Ingrid Zwaenepoel 2, *, Giulio Gabbiani 2 , Sophie Clément 2 and Christine Chaponnier 2,‡ 1 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia 2 Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, CMU, Geneva, Switzerland *Present address: Centre National de la Recherche, Scientifique (CNRS), UMR 144, Institut Curie, Paris, France ‡ Author for correspondence (christine.chaponnier@unige.ch) Accepted 26 May 2009 Journal of Cell Science 122, 2980-2988 Published by The Company of Biologists 2009 doi:10.1242/jcs.041970 JCS ePress online publication date 28 July 2009