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Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

ABSTRACT: The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, 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-mediated 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 wild-type 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-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.

SUBMITTER: Auernheimer V

PROVIDER: S-EPMC4582403 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

Auernheimer Vera V Lautscham Lena A LA Leidenberger Maria M Friedrich Oliver O Kappes Barbara B Fabry Ben B Goldmann Wolfgang H WH

Journal of cell science 20150803 18

The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, 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-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse em ...[more]

PMID: 26240176

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Project description:Focal adhesions are dynamic constructs at the leading edge of migrating cells, linking them to the extracellular matrix and enabling force sensing and transmission. The lifecycle of a focal adhesion is a highly coordinated process involving spatial and temporal variations of protein composition, interaction, and cellular tension. The assembly of focal adhesions requires the recruitment and activation of vinculin. Vinculin is present in the cytoplasm in an autoinhibited conformation in which its tail is held pincerlike by its head domains, further stabilized by two high-affinity head-tail interfaces. Vinculin has binding sites for talin and F-actin, but effective binding requires vinculin activation to release its head-tail associations. In migrating cells, it has been shown that the locations of vinculin activation coincide with areas of high cellular tension, and that the highest recorded tensions across vinculin are associated with adhesion assembly. Here, we use a structure-based model to investigate vinculin activation by talin modulated by tensile force generated by transient associations with F-actin. We show that vinculin activation may proceed from an intermediate state stabilized by partial talin-vinculin association. There is a low-force regime and a high-force regime where vinculin activation is dominated by two different pathways with distinct responses to force. Specifically, at zero or low forces, vinculin activation requires substantial destabilization of the main head-tail interface, which is rigid and undergoes very limited fluctuations, despite the other being relatively flexible. This pathway is not significantly affected by force; instead, higher forces favor an alternative pathway, which seeks to release the vinculin tail from its pincerlike head domains before destabilizing the head-tail interfaces. This pathway has a force-sensitive activation barrier and is significantly accelerated by force. Experimental data of vinculin during various stages of the focal adhesion lifecycle are consistent with the proposed force-regulated activation pathway.

Project description:The ability of cells to impart forces and deformations on their surroundings underlies cell migration and extracellular matrix (ECM) remodeling and is thus an essential aspect of complex, metazoan life. Previous work has resulted in a refined understanding, commonly termed the molecular clutch model, of how cells adhering to flat surfaces such as a microscope coverslip transmit cytoskeletally generated forces to their surroundings. Comparatively less is known about how cells adhere to and exert forces in soft, three-dimensional (3D), and structurally heterogeneous ECM environments such as occur in vivo. We used time-lapse 3D imaging and quantitative image analysis to determine how the actin cytoskeleton is mechanically coupled to the surrounding matrix for primary dermal fibroblasts embedded in a 3D fibrin matrix. Under these circumstances, the cytoskeletal architecture is dominated by contractile actin bundles attached at their ends to large, stable, integrin-based adhesions. Time-lapse imaging reveals that α-actinin-1 puncta within actomyosin bundles move more quickly than the paxillin-rich adhesion plaques, which in turn move more quickly than the local matrix, an observation reminiscent of the molecular clutch model. However, closer examination did not reveal a continuous rearward flow of the actin cytoskeleton over slower moving adhesions. Instead, we found that a subset of stress fibers continuously elongated at their attachment points to integrin adhesions, providing stable, yet structurally dynamic coupling to the ECM. Analytical modeling and numerical simulation provide a plausible physical explanation for this result and support a picture in which cells respond to the effective stiffness of local matrix attachment points. The resulting dynamic equilibrium can explain how cells maintain stable, contractile connections to discrete points within ECM during cell migration, and provides a plausible means by which fibroblasts contract provisional matrices during wound healing.

Dataset Information

Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

Publications

Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

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