title: Reconstituting actomyosin-dependent mechanosensitive protein complexes in vitro.
abstract: In many mechanosensitive biological processes, actin-binding proteins (ABPs) sense the force generated by the actomyosin cytoskeleton and respond by recruiting effector proteins. We developed an in vitro assay, with pure proteins, to observe the force-dependent binding of a protein to a cryptic binding site buried in the stretchable domain of an ABP. Here we describe the protocol to study the actomyosin-dependent binding of vinculin to the ABP talin. In this assay, talin is immobilized in 5-μm-diameter disc-shaped islands, which are regularly spaced by 35 μm and micropatterned on a glass coverslip. In response to the force generated by an actomyosin network, talin extension reveals cryptic vinculin-binding sites (VBSs). To follow this reaction, fluorescent proteins are visualized by total internal refection fluorescence (TIRF) microscopy. EGFP-vinculin fluorescence in talin-coated discs reveals the binding of vinculin to stretched talin. Actomyosin structures are visualized by the fluorescence of Alexa Fluor 594-labeled actin. This protocol describes the purification of the proteins, the preparation of the chamber in which talin is coated on a micropatterned surface, and the biochemical conditions to study several kinetic parameters of the actomyosin-dependent binding of vinculin to talin. A stable actomyosin network is used to measure the steady-state dissociation of vinculin from talin under constant force. In the presence of α-actinin-1, actomyosin cables undergo cycles of force application and release, allowing the measurement of vinculin dissociation associated with talin re-folding. Expression and purification of the proteins requires at least 3 weeks. The assay can be completed within 1 d.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France.
authors.forename: Corina
authors.initials: C
authors.lastname: Ciobanasu
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Faivre
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Le Clainche
mesh_terms: D000205:Actomyosin / Q000378:metabolism*; D001696:Biomechanical Phenomena; D066298:In Vitro Techniques; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism*; D009930:Organic Chemicals; D011485:Protein Binding; D016608:Talin / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism*
chemical_list: C417664:Alexa594; D046912:Multiprotein Complexes; D009930:Organic Chemicals; D016608:Talin; D016596:Vinculin; D000205:Actomyosin