Regulation of Focal Adhesions by Flightless I Involves Inhibition of Paxillin Phosphorylation via a Rac1-Dependent Pathway  Zlatko Kopecki, Geraldine M. O'Neill, Ruth M. Arkell, Allison J. Cowin  Journal of Investigative Dermatology  Volume 131, Issue 7, Pages 1450-1459 (July 2011) DOI: 10.1038/jid.2011.69 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flightless I (Flii) overexpression stabilizes actin filaments and increases focal adhesion formation. (a) Flii+/−, wild-type (WT), and FliiTg/Tg fibroblasts coimmunostained with phalloidin (green) and paxillin (red) at 180minutes post-seeding. Scale bar=25μm. (b) Quantification of F-actin in FliiTg/Tg fibroblasts compared with WT fibroblasts at 15 and 60minutes and at 24hours. (c, d) FliiTg/Tg fibroblasts have significantly increased number of longer focal adhesions compared with both Flii+/− and WT fibroblasts. (e) Representative image of WT cells showing focal adhesion and focal complex structures. (f) Flii+/− and WT fibroblasts stained with paxillin show focal complex formation at 90minutes post-seeding. (g) FliiTg/Tg fibroblasts have significantly reduced percentage of focal complex positive cells compared with Flii+/− and WT fibroblasts at 90minutes post-seeding. n=100 cells. Scale bar=10μm in a and 1μm in e and f. Mean±SEM. *P<0.05. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Flightless I (Flii) overexpressing fibroblasts have reduced paxillin phosphorylation and increased α-actinin expression. (a) Paxillin (red) and p-paxillin (green) immunostained Flii+/−, wild-type (WT), and FliiTg/Tg fibroblasts plated at 180minutes post-seeding. (b) Distribution of the ratios of p-paxillin at individual focal adhesions (n=100 from >5 different cells); p-pax=phosphopaxillin, pax=total paxillin. (c) Western blot analysis of p-paxillin, total paxillin, and β-tubulin levels. (d) Graph showing the ratio of p-paxillin/total paxillin. Mean±SEM. *P<0.05. (e) Western blot analysis of talin, vinculin, α-actinin, and β-tubulin levels. (f) α-Actinin staining of Flii+/−, WT, and FliiTg/Tg fibroblasts plated on fibronectin and laminin substrates illustrates increased expression of α-actinin in FliiTg/Tg fibroblasts. Scale bar=10μm. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Enhanced fibrillar adhesion formation in Flightless I (Flii) overexpressing fibroblasts. Cells plated onto collagen and laminin-coated coverslips were fixed after 180minutes and coimmunostained for F-actin (green) and fibronectin (red) (a) and tensin (green) and paxillin (red) (b). The boxed region in the FliiTg/Tg merged cell images is magnified at higher magnification (× 100) below. Arrows indicate F-actin (green) and fibronectin (red) (in a) and paxillin (red) and tensin (green) (in b) in positive fibrillar adhesions. WT, wild type. Scale bar=10μm. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Reduced activation of Rho GTPases affects the spreading of Flightless I (Flii) overexpressing fibroblasts. (a) Spreading of Flii+/−, wild-type (WT), and FliiTg/Tg fibroblasts stained with phalloidin reveals impaired spreading of FliiTg/Tg fibroblasts. (b) Total and active levels of RhoA, Rac1, and Cdc42 in cell lysates of sub-confluent Flii+/−, WT, and FliiTg/Tg fibroblasts as determined by pull down activation assay and western blotting. FliiTg/Tg fibroblasts have decreased activation of Rac1 and Cdc42 GTPases required for focal complex formation and turnover of adhesion structures. (c, d) Overexpression of constitutively active Rac1 GTPase in FliiTg/Tg fibroblasts induces lamellipodia formation (arrow) and significantly increases cell spreading. Scale bar=10μm. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Overexpression of Flightless I (Flii) results in downregulation of adhesion signaling pathways. (a) Western blot analysis of p130Cas, pp130Cas, Src, and β-tubulin protein levels. (b) Band densitometry illustrating total p130Cas (p130Cas+pp130Cas) expression and (c) the ratio of pp130Cas and p130Cas protein levels. FliiTg/Tg fibroblasts show significantly increased total p130Cas expression compared with Flii+/− fibroblasts but decreased levels of active pp130Cas compared with both Flii+/− and wild-type (WT) fibroblasts. (d) Graphical representation of Src protein expression determined by densitometry. FliiTg/Tg fibroblasts show decreased levels of Src proteins compared with both Flii+/− and WT fibroblasts. The data show the mean of three independent repeats. Mean±SEM. *P<0.05. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of Flightless I (Flii) on paxillin activation and signaling in wounds in vivo. (a–f and k) Representative 3 day images of full thickness incisional wounds, (g–i) immunofluorescent staining of p-paxillin and (j) p-paxillin. FliiTg/Tg mice wounds show reduced paxillin staining compared with Flii+/− and WT counterparts, n=6. Scale bar=500μm (d–f) and 30μm (g–i). (l) Western blot showing decreased expression of Src and p130Cas in wounds of FliiTg/Tg mice. (m) Three-dimensional floating collagen gel contraction assay at 24hours. Magnification × 6.5, dotted line=well size and original gel size. (n) Flii+/− fibroblasts have significantly reduced collagen gel contraction compared with both wild-type (WT) and FliiTg/Tg fibroblasts at 24, 48, and 72hours. n=5. Mean±SEM. *P<0.05. Journal of Investigative Dermatology 2011 131, 1450-1459DOI: (10.1038/jid.2011.69) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions