Protein Interactions and changes in conformation govern all aspects of cellular function, and disordered protein interactions or changes in conformation lay at the basis of many human diseases ranging from the initiation of inflammation to chronic neurodegenerative and kidney diseases. Mechanisms that can accurately measure protein interactions and changes in conformation will allow the development of novel concepts of disease mechanisms and therapeutic approaches based on the understanding of basic cell functions. Time-correlated single photon counting fluorescent lifetime imaging microscopy (TCSPC-FLIM) is an approach that allows measurement of protein interactions and changes in conformation;this approach improves on much of the previous approaches, and the demand for this technology at Massachusetts General Hospital has dramatically increased over the last year, so that demand now far outpaces accessibility. The goal of this Shared Instrument Grant is to establish a System for FLIM Microscopy in the Renal Unit at Massachusetts General Hospital that will serve as a shared research resource for the study of protein function in human diseases and in basic cell biology. This system will also catalyze the interaction between investigators sharing interest in the same biological processes. Four major projects will initially use The System for FLIM Microscopy to address different aspects of protein function. In Project 1, The PI will use FLIM to monitor the interactions of proteins assembled into the leukotriene membrane synthetic complex on the inner and outer nuclear membranes to initiate inflammation in diseases that include asthma and atherosclerosis. In Project 2, Dr. M. Amin Arnaout, will exploit the ability of FLIM to monitor conformational changes in surface adhesion molecules during cell activation. In Project 3 Dr. Sanja Sever, will use FLIM to monitor protein oligomerization of the protein dynamin and probe its role in function and diseases of kidney podocytes. Finally, in Project 4, Dr. Bradley Hyman will develop and test novel tools and approaches for use and analysis of FLIM, particularly in the study of the protein interactions of gamma secretase complex that controls amyloid beta (Abeta) generation in Alzheimer's disease. Protein interactions that play a role in Parkinson's disease will also be studied, thus maximizing the capabilities of the microscope.
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