Detection of fluorescent protein mechanical switching in cellulo.

Autor:	Shoyer TC; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA., Collins KL; Department of Chemistry, Duke University, Durham NC 27708, USA., Ham TR; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA., Blanchard AT; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA., Malavade JN; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA., Johns BA; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA., West JL; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA; Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA., Hoffman BD; Department of Biomedical Engineering, Duke University, Durham NC 27708, USA. Electronic address: brenton.hoffman@duke.edu.
Jazyk:	angličtina
Zdroj:	Cell reports methods [Cell Rep Methods] 2024 Jul 15; Vol. 4 (7), pp. 100815. Date of Electronic Publication: 2024 Jul 09.
DOI:	10.1016/j.crmeth.2024.100815
Abstrakt:	The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, determining the mechanisms by which forces affect protein function inside cells remains challenging. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated whether force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo within a synthetic actin crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cell force generation, external stiffness, and force-sensitive bond dynamics of the biosensor. This work describes a framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu