A System for FLIM Microscopy

描述(申请人提供)：蛋白质相互作用和构象变化控制着细胞功能的各个方面，而蛋白质相互作用紊乱或构象变化是许多人类疾病的基础，从炎症的开始到慢性神经退行性疾病和肾脏疾病。能够准确测量蛋白质相互作用和构象变化的机制将允许在理解基本细胞功能的基础上发展疾病机制和治疗方法的新概念。时间相关单光子计数荧光寿命成像显微镜(TCSPC-Flim)是一种可以测量蛋白质相互作用和构象变化的方法；这种方法改进了以前的许多方法，马萨诸塞州总医院对这项技术的需求在过去一年里急剧增加，因此现在的需求远远超过了可获得性。这项共享仪器基金的目标是在马萨诸塞州总医院的肾脏单位建立一个电影显微镜系统，作为研究人类疾病和基础细胞生物学中蛋白质功能的共享研究资源。这个系统还将促进研究人员之间的互动，这些研究人员对相同的生物过程感兴趣。四个主要项目将首先使用该系统用于薄膜显微镜，以解决蛋白质功能的不同方面。在项目1中，PI将使用FLIM来监测内核膜和外核膜上组装成白三烯膜合成复合体的蛋白质之间的相互作用，以启动包括哮喘和动脉粥样硬化在内的疾病的炎症。在项目2中，M.阿明·阿瑙特博士将利用FLIM的能力来监测细胞激活过程中表面黏附分子的构象变化。在项目3中，Sanja Sever博士将使用Flim监测蛋白质动力素的蛋白质寡聚，并探讨其在肾足细胞功能和疾病中的作用。最后，在项目4中，Bradley Hyman博士将开发和测试新的工具和方法来使用和分析FLIM，特别是在研究控制阿尔茨海默病中淀粉样β蛋白(Abeta)生成的伽马分泌酶复合体的蛋白质相互作用方面。还将研究在帕金森氏症中发挥作用的蛋白质相互作用，从而最大限度地发挥显微镜的能力。

DESCRIPTION (provided by applicant): 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.