Detection of Fluorescent Protein Mechanical Switching in Cellulo.
Detection of Fluorescent Protein Mechanical Switching in Cellulo.
复制标题
纤维素中荧光蛋白机械开关的检测。
DOI：
10.1101/2024.01.10.575065
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Hoffman,BrentonD
中科院分区：
文献类型：
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作者：
Shoyer,TCurtis;Collins,KasieL;Ham,TrevorR;Blanchard,AaronT;Malavade,JuileeN;West,JenniferL;Hoffman,BrentonD
细胞感知和响应机械力的能力在许多生理和病理过程中至关重要。然而，确定力影响细胞内蛋白质功能的机制仍然具有挑战性。受荧光蛋白（FP）进行荧光可逆机械开关的体外演示的启发，我们研究了FP功能的力敏变化是否可以在细胞中可视化。指导FP机械开关的计算模型，我们开发了一种形式主义，其检测在福斯特共振能量转移（FRET）为基础的生物传感器，并证明其occurrencin cellulowithin一个合成的肌动蛋白交联剂和机械接头蛋白纽蛋白。我们发现thatin细胞力学开关是可逆的，并通过操纵细胞力的产生，外部刚度和力敏感的键动力学的生物传感器改变。这项工作描述了一个评估FP机械稳定性的框架，并提供了一种探测细胞内力敏感蛋白功能的方法。
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 byin vitrodemonstrations 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 occurrencein cellulowithin a synthetic actin crosslinker and the mechanical linker protein vinculin. We find thatin cellulomechanical 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.