Becoming stable and strong: the interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation.
The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.
Institute of Bio- and Nanosystems, IBN-4, Biomechanics, Research Centre Jülich GmbH, Jülich, Germany.
Christoph
C
Möhl
Norbert
N
Kirchgessner
Claudia
C
Schäfer
Kevin
K
Küpper
Simone
S
Born
Gerold
G
Diez
Wolfgang H
WH
Goldmann
Rudolf
R
Merkel
Bernd
B
Hoffmann
D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D036681:Fluorescence Recovery After Photobleaching; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D016596:Vinculin / Q000235:genetics / Q000378:metabolism*
D016596:Vinculin