title: 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.
authors.affiliation: Department of Physics, Biophysics Group, University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Auernheimer
authors.affiliation: Department of Physics, Biophysics Group, University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Lena A
authors.initials: LA
authors.lastname: Lautscham
authors.affiliation: Institute of Medical Biotechnology, Department of Chemical and Biological Engineering University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Leidenberger
authors.affiliation: Institute of Medical Biotechnology, Department of Chemical and Biological Engineering University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Friedrich
authors.affiliation: Institute of Medical Biotechnology, Department of Chemical and Biological Engineering University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kappes
authors.affiliation: Department of Physics, Biophysics Group, University of Erlangen-Nuremberg, 91052 Erlangen, Germany.
authors.forename: Ben
authors.initials: B
authors.lastname: Fabry
authors.affiliation: Department of Physics, Biophysics Group, University of Erlangen-Nuremberg, 91052 Erlangen, Germany wgoldmann@biomed.uni-erlangen.de.
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Goldmann
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology*; D004735:Energy Transfer; D005347:Fibroblasts / Q000502:physiology / Q000648:ultrastructure; D016023:Integrins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D017354:Point Mutation; D017433:Protein Structure, Secondary; D016596:Vinculin / Q000502:physiology*; D019061:src-Family Kinases / Q000502:physiology
chemical_list: D016023:Integrins; D016596:Vinculin; D019061:src-Family Kinases
keywords: Actin pulldown; Cell stiffness; FRAP; Focal adhesion; Mechanotransduction; Traction; Tyrosine phosphorylation; Vinculin