Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043-1057

Weizmann Institute of Science, Rhovot, Central District, Israel
The Journal of Cell Biology (Impact Factor: 9.83). 01/2008; 179(5):1043-57. DOI: 10.1083/jcb.200703036
Source: PubMed


Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

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    • "Paxillin and vinculin are multi-domain adapter proteins that are key regulators of focal-adhesion formation and cell adhesion [24] [25]. To elucidate in greater detail the mechanism by which NAC restores cell adhesion upon exposure to oxidative stress, "
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    • "constitutively open conformation (Marg et al., 2010). Constitutively active T12 mutants also displayed slower exchange dynamics in focal adhesions but a similarly increased immobile fraction compared to phospho-mimicking vinculin (Cohen et al., 2006; Humphries et al., 2007). Interestingly, the force transmission in T12 mutants, in phospho-mimicking vinculin mutants and in rescue cells was similar, suggesting that the vinculin present in the focal adhesions of wild-type cells is mostly in an open, active conformation, which is in agreement with data from FRET-based experiments (Chen et al., 2005). "
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    ABSTRACT: The focal adhesion protein vinculin connects the actin cytoskeleton via talin and integrin with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP, and actin-binding assays. Compared to cells expressing wildtype or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with non-phosphorylatable phenylalanine. Replacing tyrosines with phospho-mimicking glutamic acid restored cellular tractions, stiffness, adhesion strength as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins. © 2015. Published by The Company of Biologists Ltd.
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    • "Regarding the class of pores with diameter minus of 0.1 µm, MIP analysis allowed us to conclude that their presence was not related to the use of salt in the manufacturing process but to the evaporation of the solvent used for melt PCL in the 3 different kinds of samples (Figure 3C). Since biocompatibility, in terms of cell surface adhesion characterization, is a process also involving cytoskeletal reorganization of actin and vinculin363738our in vitro tests, for the above three different type of scaffold, were performed using NIH/3T3 cells culture and, laser scanning confocal microscopy, to collect preliminary data on cell morphology and cell-scaffold interaction. Confocal microscopy qualitative analysis showed in Figure 4that the 3 different surfaces were all biocompatible. "

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