Becoming stable and strong: The interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation

Abstract

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.

Vinculin Facilitates Cell Invasion into Three-dimensional Collagen Matrices

“…The strength of cellmatrix adhesions is mainly regulated through integrins whose cytoplasmic tail is linked to the actomyosin cytoskeleton through binding of focal adhesion proteins such as vinculin. In this context, vinculin has been described to act as a mechanocoupling protein (8,9,30). Here, we found that the adhesion strength of FN-coated beads is 3-fold lower in MEFvin Ϫ/Ϫ cells compared with MEFvin wt/wt cells, consistent with the notion that the mechano-coupling function of vinculin is important for firm adhesion to the ECM.…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…In the kerotinocytes, vinculin phosphorylation at Tyr 1065 was transient and was correlated with increased vinculin exchange dynamics during focal adhesion growth (39). Focal adhesion maturation was correlated with a reduction in vinculin phosphorylation and associated with an increase in cellular adhesive traction force.…”

“…The function of vinculin phosphorylation at Tyr 1065 has been previously investigated in cultured migrating human epidermal keratinocytes and a mouse embryonic fibroblast cell line using vinculin phosphorylation site mutants (39,40,50). In the kerotinocytes, vinculin phosphorylation at Tyr 1065 was transient and was correlated with increased vinculin exchange dynamics during focal adhesion growth (39).…”

“…In vitro studies as well as in vivo studies in several cell types have described both tyrosine and serine phosphorylation sites on the vinculin molecule (37,38). There is evidence that phosphorylation at the Tyr 1065 site near the C terminus of the tail domain affects cell traction and spreading and the dynamics of the incorporation of vinculin into focal adhesions (37,39,40). We previously found that vinculin undergoes phosphorylation at tyrosine 1065 concurrently with force development during the stimulation of tracheal smooth muscle tissues with acetylcholine and that the phosphorylation of vinculin at this site remains elevated for the duration of the contraction (10).…”

The key feature for early migratory processes

“…25,26 Additionally, experiments on migrating keratinocytes showed a correlation between the maturation state of FAs and the amplitudes of applied forces. 13 Other studies discuss additional mechanisms for assembly and maturation of nascent adhesions. They postulate a key role of actin polymerization at adhesion sites for accumulation of α-actinin and myosin II during FA maturation.…”