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 Ca2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2 + influx induces cascades of FA turnover in living cells. Methods 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. 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 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 significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2 +.
| Original language | English |
|---|---|
| Pages (from-to) | 624-635 |
| Number of pages | 12 |
| Journal | Biochimica et Biophysica Acta - General Subjects |
| Volume | 1861 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2017 Mar 1 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Biochemistry
- Molecular Biology
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Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions. / Chang, Shu Jing; Chen, Ying Chi; Yang, Chi Hsun; Huang, Soon Cen; Huang, Ho Kai; Li, Chun Chun; Harn, Hans I.Chen; Chiu, Wen Tai.
In: Biochimica et Biophysica Acta - General Subjects, Vol. 1861, No. 3, 01.03.2017, p. 624-635.Research output: Contribution to journal › Article
TY - JOUR
T1 - Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions
AU - Chang, Shu Jing
AU - Chen, Ying Chi
AU - Yang, Chi Hsun
AU - Huang, Soon Cen
AU - Huang, Ho Kai
AU - Li, Chun Chun
AU - Harn, Hans I.Chen
AU - Chiu, Wen Tai
PY - 2017/3/1
Y1 - 2017/3/1
N2 - 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 Ca2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2 + influx induces cascades of FA turnover in living cells. Methods 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. 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 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 significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2 +.
AB - 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 Ca2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2 + influx induces cascades of FA turnover in living cells. Methods 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. 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 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 significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2 +.
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U2 - 10.1016/j.bbagen.2017.01.002
DO - 10.1016/j.bbagen.2017.01.002
M3 - Article
C2 - 28063985
AN - SCOPUS:85008958055
VL - 1861
SP - 624
EP - 635
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0006-3002
IS - 3
ER -