Abstract
Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.
Highlights
Two key proteins implicated in the physical connections between integrins and F-actin are the actin-binding proteins talin and vinculin
Role of Vinculin head-tail interaction (HTI) in Formation of a Ternary Complex with 1 Integrin and Talin in Cells—To investigate the role of vinculin conformation in assembly of a ternary complex of talin, vinculin, and 1 integrin in cells, we examined these protein-protein interactions in the context of an ectopic recruitment assay
We examined the effect of HTI mutations on the dynamics of vinculin and its focal adhesion ligands by Fluorescence Recovery after Photobleaching (FRAP) of green fluorescent protein (GFP) fusion proteins
Summary
Whereas weakly structured fragments of talin show enhanced affinity for Vhd compared with intact talin in vitro [25], well folded and measurably more stable domains of talin undergo structural rearrangement associated with VBS exposure only in the presence of Vhd1 [26]. It remains controversial as to whether VBS peptides or weakly structured talin fragments reflect bona fide conformations for talin or merely mimic an end state induced by vinculin binding. Conformational changes in vinculin have been directly visualized and linked to exposure of its actin- and vinexin-binding sites in focal adhesions of live cells [27]. We have examined the functional relevance of HTI to the formation of a talin-vinculin complex in living cells by testing the extent to which HTI regulates the recruitment of vinculin to talin or talinintegrin clusters in cells. We conclude that regulation of HTI can determine the lifetime of specific vinculin-based functional complexes within the longer lived architecture of a mature focal adhesion
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