title: Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions.
abstract: 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 Ca2+/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2+influx induces cascades of FA turnover in living cells.
  
  
  
  Images 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.
  
  
  
  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.
  
  
  
  These 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.
  
  
  
  This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2+.
authors.affiliation: Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Shu-Jing
authors.initials: SJ
authors.lastname: Chang
authors.affiliation: Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Ying-Chi
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Chi-Hsun
authors.initials: CH
authors.lastname: Yang
authors.affiliation: Department of Obstetrics and Gynecology, Chi Mei Medical Center, Liouying Campus, Tainan 736, Taiwan.
authors.forename: Soon-Cen
authors.initials: SC
authors.lastname: Huang
authors.affiliation: Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Ho-Kai
authors.initials: HK
authors.lastname: Huang
authors.affiliation: Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Chun-Chun
authors.initials: CC
authors.lastname: Li
authors.affiliation: Institute of Basic Medical Sciences, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Hans I-Chen
authors.initials: HI
authors.lastname: Harn
authors.affiliation: Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan; Institute of Basic Medical Sciences, National Cheng Kung University, Tainan 701, Taiwan; Medical Device Innovation Center, National Cheng Kung University, Tainan 701, Taiwan. Electronic address: wtchiu@mail.ncku.edu.tw.
authors.forename: Wen-Tai
authors.initials: WT
authors.lastname: Chiu
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D051419:Paxillin / Q000378:metabolism; D059748:Proteolysis; D015398:Signal Transduction / Q000502:physiology; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism
chemical_list: D051419:Paxillin; D016608:Talin; D016596:Vinculin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002154:Calpain; D002118:Calcium
keywords: 3D architecture; Ca(2+); Calpain; Focal adhesion; Turnover