Journal
NATURE PROTOCOLS
Volume 10, Issue 1, Pages 75-89Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.200
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- Agence Nationale pour la Recherche (ADERACTIN) [ANR-09-JCJC-0111]
- Agence Nationale de la Recherche (ANR) [ANR-09-JCJC-0111] Funding Source: Agence Nationale de la Recherche (ANR)
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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-mu m-diameter disc-shaped islands, which are regularly spaced by 35 mu m 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 alpha-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.
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