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.
Force-sensing is a critical aspect of cell growth, morphology and development. We will study in molecular detail how two families of stretch-activated membrane channels contribute to osmotic stress survival in model eukaryotic and prokaryotic systems and to virulence in selected human pathogens.
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