Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions
Abstract Background Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca 2+ /calpain-dependent proteolysis and disassembly. Here, we investigated how Ca 2+ influx induces cascades of FA turnover in living cells. Methods Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca 2+ ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. Results Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. Conclusions These results suggest that Ca 2+ -mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca 2+ -mediated FA turnover. General significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca 2+ . Highlights Degradation of focal adhesions by Ca 2+ /calpain-dependent proteolysis is demonstrated. Outer FA molecules are more sensitive to calpain-mediated proteolysis. The 3D architecture of FAs is related to the dynamics of FA molecule degradation. Graphical abstract [DISPLAY OMISSION]
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