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Identifying constitutive and context-specific molecular-tension-sensitive protein recruitment within focal adhesions.

Developmental cell (2023-03-17)
Arnold Tao, Andrew S LaCroix, T Curtis Shoyer, Vidya Venkatraman, Karen L Xu, Bradley Feiger, Brenton D Hoffman
RESUMEN

Mechanosensitive processes often rely on adhesion structures to strengthen, or mature, in response to applied loads. However, a limited understanding of how the molecular tensions that are experienced by a particular protein affect the recruitment of other proteins represents a major obstacle in the way of deciphering molecular mechanisms that underlie mechanosensitive processes. Here, we describe an imaging-based technique, termed fluorescence-tension co-localization (FTC), for studying molecular-tension-sensitive protein recruitment inside cells. Guided by discrete time Markov chain simulations of protein recruitment, we integrate immunofluorescence labeling, molecular tension sensors, and machine learning to determine the sensitivity, specificity, and context dependence of molecular-tension-sensitive protein recruitment. The application of FTC to the mechanical linker protein vinculin in mouse embryonic fibroblasts reveals constitutive and context-specific molecular-tension-sensitive protein recruitment that varies with adhesion maturation. FTC overcomes limitations associated with the alteration of numerous proteins during the manipulation of cell contractility, providing molecularly specific insights into tension-sensitive protein recruitment.

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Sigma-Aldrich
Anti-vinculina monoclonal antibody produced in mouse, clone hVIN-1, ascites fluid
Sigma-Aldrich
Monoclonal Anti-Talin antibody produced in mouse, clone 8d4, ascites fluid
Sigma-Aldrich
Anti-FHL2 antibody produced in rabbit, Ab2, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution