Journal
JOURNAL OF CELL SCIENCE
Volume 128, Issue 18, Pages 3435-3443Publisher
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jcs.172031
Keywords
Vinculin; Focal adhesion; Mechanotransduction; Tyrosine phosphorylation; Cell stiffness; Traction; FRAP; Actin pulldown
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Funding
- Deutscher Akademischer Austauschdienst (DAAD) [50021371]
- Deutsche Forschungsgemeinschaft (DFG) [FA336-6]
- National Institutes of Health (NIH) [BRP:HL65960]
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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.
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