Biophysical, Structural and Functional Analysis of Mechanosensitive Channels

描述(由申请人提供)：机械感觉是一种普遍存在的现象，是细胞生长、形态和发育的关键方面，但在分子和细胞水平上却知之甚少。这里提出的实验旨在从分子细节上揭示细胞和细胞器如何利用机械敏感(MS)通道来响应周围环境的突然变化，从而导致渗透压的变化。在这种情况下，水可能会涌入细胞，导致膜张力，如果不加以控制，可能会导致细胞死亡。这项研究的目的是了解膜结合的机械传感器如何被渗透休克等环境提示激活，以及张力敏感通道如何调节保护性的生理反应。细菌MS通道的大(MSCL)和小(MSCS)电导为机械响应蛋白的结构和生物物理分析以及生命所有领域的MS通道的生理功能提供了一个范例。为了了解它们是如何发挥作用的，人们设计了许多不同的实验。一个关键的目标是通过观察单个细胞受到不同的渗透性下激波的速率和幅度，在细胞水平上了解MS通道的功能。一种新开发的微流体系统将被用来对单个细胞和细胞器施加渗透环境的快速变化，并定量地测量MS通道的数量和类型与渗透下击存活的关系。这些生理学研究将得到结构研究的补充，旨在提供对MS通道构象的更丰富的了解，通道结构的各种元素如何对功能起作用，并对细胞对下行冲击的反应进行定量建模。在模拟细菌和植物系统上进行的工作将为研究MS通道在细菌和原生动物人类病原体的生命周期和毒力中的结构和功能奠定基础，因此与人类健康直接相关。

DESCRIPTION (provided by applicant): Mechanosensation is a ubiquitous phenomenon and a critical aspect of cell growth, morphology and development, yet it is poorly understood at the molecular and cellular levels. Experiments proposed here aim to reveal in molecular detail how cells and organelles employ mechanosensitive (MS) channels to respond to sudden changes in the surrounding environment, resulting in changes in osmotic pressure. Under these circumstances, water can rush into the cell resulting in a membrane tension that, if unchecked, can result in cell death. The goal of this research is to understand how membrane-bound mechanosensors are activated by environmental cues such as osmotic shock and how tension-sensitive channels mediate a protective physiological response. Bacterial MS channels of large (MscL) and small (MscS) conductance serve as a paradigm for the structural and biophysical analysis of mechanoresponsive proteins and for the physiological functions of MS channels across all domains of life. To develop an understanding of how they function, a number of different experiments have been designed. One key objective is to understand the function of MS channels at the cellular level by watching individual cells subjected to different rates and amplitudes of osmotic downshock. A newly developed microfluidics system will be used to impose rapid changes in osmotic environment on individual cells and organelles and to quantitatively measure the relationship between the number and type of MS channels and osmotic downshock survival. These physiological studies will be complemented by structural studies aimed at providing a richer understanding of MS channel conformations, how various elements of channel structure contribute to function and to quantitatively model the response of cells to downshock. The work performed on model bacterial and plant systems will form the foundation for investigation into the structure and function of MS channels in the life cycle and virulence of bacterial and protozoan human pathogens, and are therefore directly relevant to human health.