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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.

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Figure 1: Basic principle of the method described in this protocol.
Figure 2: Microscopy setup.
Figure 3: Vinculin dissociation at steady state.
Figure 4: Vinculin dissociation, associated with talin re-folding, after force release.
Figure 5
Figure 6: Examples of expected results.

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Acknowledgements

C.L.C. is supported by the Agence Nationale pour la Recherche (ANR-09-JCJC-0111 ADERACTIN). We thank the members of the 'Cytokeleton Dynamics and Motility' team for helpful discussions.

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C.C. and C.L.C. developed the microscopy assay, and acquired and analyzed the data presented in this protocol. B.F. cloned the cDNA and established the protocols to purify the proteins presented in this article. C.L.C. designed the experiments, supervised the project and wrote the manuscript.

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Correspondence to Christophe Le Clainche.

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The authors declare no competing financial interests.

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Ciobanasu, C., Faivre, B. & Le Clainche, C. Reconstituting actomyosin-dependent mechanosensitive protein complexes in vitro. Nat Protoc 10, 75–89 (2015). https://doi.org/10.1038/nprot.2014.200

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