Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions

Publication date: March 2017
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1861, Issue 3
Author(s): Shu-Jing Chang, Ying-Chi Chen, Chi-Hsun Yang, Soon-Cen Huang, Ho-Kai Huang, Chun-Chun Li, Hans I-Chen Harn, Wen-Tai Chiu
BackgroundFocal 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 Ca²⁺/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca²⁺ influx induces cascades of FA turnover in living cells.MethodsImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca²⁺ 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.ResultsVinculin 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.ConclusionsThese results suggest that Ca²⁺-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 Ca²⁺-mediated FA turnover.General significanceThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca²⁺.

Graphical abstract

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2j711re
via IFTTT