4572 Research Article Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation Anika Steffen1, Markus Ladwein2, Georgi A. Dimchev1, Anke Hein1, Lisa Schwenkmezger1, Stefan Arens3, Kathrin I. Ladwein2, J. Margit Holleboom1,2, Florian Schur4, J. Victor Small4, Janett Schwarz5, Ralf Gerhard5, Jan Faix6, Theresia E. B. Stradal2,3, Cord Brakebusch7 and Klemens Rottner1,2,* 1Institute of Genetics, University of Bonn, Karlrobert-Kreiten Strasse 13, D-53115 Bonn, Germany 2Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, D-38124 Braunschweig, Germany 3Institute for Molecular Cell Biology, University of Mu¨nster, Schlossplatz 5, D-48149 Mu¨nster, Germany 4Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria 5Institute for Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany 6Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany 7Biomedical Institute, BRIC, University of Copenhagen, DK-2200 Copenhagen, Denmark *Author for correspondence ([email protected]) Accepted 9 July 2013 Journal of Cell Science 126, 4572–4588 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.118232 Summary Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac – although not essential – can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Journal of Cell Science Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration. Key words: Actin, Rac1, Migration, Adhesion, Lamellipodia, Filopodia, Chemotaxis, CAAX Introduction complex expression is stably suppressed (Wu et al., 2012) or ES- Lamellipodia initiate the migration of various cell types, ranging cell-derived fibroblastoid cells genetically deficient for the Arp2/ from fibroblasts and epithelial cells to different types of 3 complex subunit ArpC3 (Suraneni et al., 2012) fail to form leukocytes (Small et al., 2002; Renkawitz and Sixt, 2010). lamellipodia, as do cells depleted of WAVE complex Lamellipodia are composed of networks of actin filaments, components by RNAi (Innocenti et al., 2004; Steffen et al., directly connected in part by branch junctions generated by the 2004; Steffen et al., 2006; Nicholson-Dykstra and Higgs, 2008), Arp2/3 complex (Yang and Svitkina, 2011; Vinzenz et al., 2012). confirming the importance of WAVE to Arp2/3 complex The Arp2/3 complex is activated at the interface between the signaling for lamellipodial protrusion. However, the impact of plasma membrane and growing actin network (Miyoshi et al., lamellipodium suppression on migration efficiency remained 2006; Iwasa and Mullins, 2007; Lai et al., 2008) by Scar/WAVE controversial (Suraneni et al., 2012; Wu et al., 2012). and associated proteins (Stradal et al., 2001; Stradal and Scita, The family of Rho GTPases comprises 20 members in 2006), now called the WAVE complex (Chen et al., 2010; mammals (Heasman and Ridley, 2008; Ladwein and Rottner, Derivery and Gautreau, 2010), that accumulates at the tips of 2008), overexpression of most of which drives major lamellipodia (Hahne et al., 2001; Steffen et al., 2004). The reorganizations of the actin cytoskeleton (Aspenstro¨m et al., pentameric WAVE complex can be directly targeted and 2004). Although Rac proteins such as Rac1 are known to induce activated by the Rho GTPase Rac1 (Ismail et al., 2009; formation of lamellipodia and membrane ruffles (Ridley et al., Lebensohn and Kirschner, 2009). Fibroblasts in which Arp2/3 1992; Nobes and Hall, 1995), other Rho proteins such as the Rac- related RhoG or Cdc42 can also trigger these structures (Nobes and Hall, 1995; Aspenstro¨m et al., 2004). Analyses of migratory This is an Open Access article distributed under the terms of the Creative Commons Attribution performance of primary fibroblasts depleted of Rho proteins by License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. RNAi have recently indicated multiple, redundant pathways Rac function in protrusion and migration 4573 operating downstream of Cdc42, Rac proteins and RhoG Conditional Rac1 alleles in fibroblasts were previously deleted (Monypenny et al., 2009). Although biochemical connections by Cre recombinase delivered through adenovirus (Guo et al., established potential convergence of signaling on Rac, both 2006) or a cell-permeable HIV–TAT fusion (Vidali et al., 2006). downstream of Cdc42 (Baird et al., 2005; Nishimura et al., 2005) Interestingly, although Vidali and colleagues found only and RhoG (Katoh and Negishi, 2003), it remained unclear moderate effects on migration efficiency, cell spreading and whether Cdc42 or RhoG could also induce cytoskeletal focal adhesion formation, Guo et al. concluded Rac1 to be rearrangements by bypassing Rac GTPases. For instance, essential for focal adhesion and stress fiber assembly (Guo et al., although a direct interaction of the WAVE complex with 2006). More recently, modest defects in migration, adhesion site RhoG-GTP had previously been proposed (Ridley, 2006), and stress fiber formation were also observed in primary binding of this GTPase to the WAVE complex was reported to fibroblasts isolated after tamoxifen-mediated Rac1 deletion in be rather weak and nucleotide independent (Meller et al., 2008). mice (Liu et al., 2009). In spite of reasonably efficient Rac1 gene Furthermore, it could not be formally excluded that Cdc42 can deletion in these populations, no individual clones and thus drive lamellipodium formation independent of Rac. permanent Rac12/2 cell lines were developed, precluding faithful Journal of Cell Science Fig. 1. Rac1-deficient MEFs are unable to form lamellipodia and ruffles. (A) Genotyping of Rac1fl/fl cells and individual Rac12/2 clones for genotypes as indicated. (B) Western blot of Rac1fl/fl and individual Rac12/2 clones with Rac1/3 antibody (upper panel) and tubulin antibody (lower panel). (C,D) Phalloidin staining of Rac1fl/fl (C) and Rac12/2 MEFs (D). (E–L) Growth factor stimulation. Rac1fl/fl MEFs (E–H) and Rac12/2 MEFs (I–L) were either starved (E,I), or stimulated with PDGF (F,J), EGF (G,K) or HGF (H,L). (M) Quantification of growth factor stimulations. Data were collected from three independent experiments and are expressed as means 6 s.e.m.; n5total number of cells analyzed. 4574 Journal of Cell Science 126 (20) analysis of the consequences of Rac1 gene deletion at the single lamellipodium formation (Bergert et al., 2012). Filopodium cell level. The Rac-deficient cell lines developed for our study formation was also commonly observed (see below), indicating avoid the possibility that variability in phenotypes could derive that distinct protrusion types, lamellipodia, filopodia and blebs from incomplete Rac1 removal. may be interdependent, to certain extents under specific conditions, yet are mechanistically separable. Results To explore Rac functions in stimulated actin cytoskeletal Generation of permanent Rac12/2 fibroblast cell lines rearrangements, we employed treatments with various growth Rac1 was deleted in Rac1fl/fl mouse embryonic fibroblasts factors. Rac1fl/fl cells responded within minutes to PDGF, EGF (MEFs) by Cre recombinase. Individual clones were isolated and HGF addition with the formation of prominent dorsal ruffles and genotyped for the presence of excised and floxed alleles. (Fig. 1F–H) but few peripheral ruffles (unpublished data). In Rac1 alleles harboring the respective deletion in exon 3 was contrast, dorsal ruffle formation was entirely abolished in Rac1- detected in all clones obtained after isolation and further deficient fibroblasts (Fig. 1J–L). The frequency of dorsal ruffle expansion (more than a dozen; for a selection of clones see formation in Rac1 control cells was highest after HGF treatment Fig. 1A). Loss of Rac1 protein was also confirmed by western (68%), whereas 33% and 35% of Rac1 control cells showed blotting (Fig. 1B), employing an antibody that