Rothenberg Katheryn E, Puranam Ishaan, Hoffman Brenton D
Department of Biomedical Engineering, Duke University.
Department of Biomedical Engineering, Duke University;
J Vis Exp. 2018 Nov 2(141). doi: 10.3791/58619.
Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the Förster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.
细胞通过一种称为机械转导的过程将机械刺激转化为生物化学可检测的信号，从而感知并响应其环境中的物理线索。机械转导的一个关键步骤是在外部和内部环境之间传递力。为了传递力，必须通过一系列蛋白质 - 蛋白质相互作用形成持续、不间断的物理连接。对于给定的蛋白质 - 蛋白质相互作用，机械负荷可能对相互作用没有影响，导致相互作用更快解离，甚至稳定相互作用。了解分子负荷如何决定活细胞中蛋白质的周转可以提供有关蛋白质机械状态的有价值信息，进而阐明其在机械转导中的作用。现有的测量力敏蛋白动力学的技术要么缺乏对蛋白质负荷的直接测量，要么依赖于在细胞外环境中进行的测量。在这里，我们描述了一种用于荧光共振能量转移 - 光漂白后荧光恢复（FRET - FRAP）技术的方案，该技术能够测量活细胞内力敏蛋白的动力学。这项技术可能适用于任何基于FRET的张力传感器，有助于研究各种亚细胞结构和不同细胞类型中的力敏蛋白动力学。
