title: Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions.
abstract: Actin-myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30-40% higher than serum-free cultures. Inhibition of myosin light chain kinase or Rho-kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum-induced enhancements in strengthening. Blebbistatin-induced inhibition of myosin II reduced serum-induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin-null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin-dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum-induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK-dependent, vinculin-containing focal adhesion assembly.
authors.affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0363, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Dumbauld
authors.forename: Heungsoo
authors.initials: H
authors.lastname: Shin
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Gallant
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Michael
authors.forename: Harish
authors.initials: H
authors.lastname: Radhakrishna
authors.forename: Andrés J
authors.initials: AJ
authors.lastname: García
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D002465:Cell Movement; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D022001:Focal Adhesions / Q000201:enzymology*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011485:Protein Binding; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000172:deficiency / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D016023:Integrins; D018994:Myosin Light Chains; D016608:Talin; D016596:Vinculin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054460:rho-Associated Kinases; D009218:Myosins