David W Dumbauld1, Kristin E Michael1, Steven K Hanks2, Andrés J García1. 1. Woodruff School of Mechanical Engineering and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332. 2. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Abstract
BACKGROUND INFORMATION: FAK (focal adhesion kinase), an essential non-receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK-dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. RESULTS: Using FAK-null cells expressing wild-type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady-state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady-state strength by 30% compared with FAK-null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock-down of VCL abrogated the FAK-dependent differences in adhesive forces. FAK-dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr-397 and kinase domain Tyr-576/Tyr-577 sites were differentially required for FAK-mediated adhesive responses. CONCLUSIONS: We demonstrate that FAK reduces steady-state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.
BACKGROUND INFORMATION: FAK (focal adhesion kinase), an essential non-receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK-dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. RESULTS: Using FAK-null cells expressing wild-type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady-state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady-state strength by 30% compared with FAK-null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock-down of VCL abrogated the FAK-dependent differences in adhesive forces. FAK-dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr-397 and kinase domain Tyr-576/Tyr-577 sites were differentially required for FAK-mediated adhesive responses. CONCLUSIONS: We demonstrate that FAK reduces steady-state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.
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