Single Molecule Study of Receptor Localization in Cellular Membranes

描述（由申请人提供）：细胞膜内的室室，特别是具有不同蛋白质和脂质组成的区域，涉及关键的膜功能，如细胞信号传导。这些区域也与病原体和毒素的进入以及阿尔茨海默病等疾病有关。在这个项目中，我们的目标是在单分子水平上研究受体蛋白在大肠杆菌K- 12模型系统中定位到特定区域的机制，该模型系统具有比真核生物更简单的膜结构。大肠杆菌内膜中单个荧光标记的丝氨酸化学感受器（Tsr）将以高达每秒1000帧的视频帧率成像，以确定“Tsr膜结构域”是如何组装和维持的。单分子成像的综合优势包括：能够观察到在大量情况下可能会丢失的罕见事件，多分子成像以及能够将分子定位到比罗利分辨率极限小得多的位置。因此，分子在其区域内的动力学以及它们与区域外的自由分子的进入和可能的交换可以被精确地监测。Tsr的成像将在正常细胞和突变细胞中进行，这些细胞在细胞处理程序中被遗传改变，预计会影响根据可测试的假设（例如，脂质组成，特定的伴侣蛋白相互作用和细菌细胞骨架）的定位。通过这些方法，我们期望对完整膜蛋白靶向特定位置或膜结构域的分子和物理机制有更深入的了解。

DESCRIPTION (provided by applicant): Compartments within cellular membranes, specifically regions with distinct protein and lipid compositions, have been implicated in critical membrane functions such as cellular signaling. These regions have also been implicated in pathogen and toxin entry, and in pathologies like Alzheimer's disease. In this project, we aim to investigate, at the single molecule level, the mechanism(s) by which receptor proteins are localized to specific regions in an Escherichia coli K- 12 model system, with simpler membrane architecture than what is found in eukaryotes. Individual fluorescently labeled serine chemoreceptors (Tsr) in the inner membrane of E. coli will be imaged at video frame rates up to 1000 frames per second to determine how the "Tsr membrane domain" is assembled and maintained. Single molecule imaging has the combined benefits of the ability to observe rare events that would be lost in bulk, multi-molecule imaging and the ability to localize molecules to positions much smaller than the Raleigh limit of resolution. As such, the dynamics of the molecules in their domain and their entrance and possible exchange with free molecules outside the domain can be accurately monitored. Imaging of Tsr will be carried out in normal and mutant cells that have been genetically altered in cellular processors that are predicted to affect localization in accordance with testable hypotheses (e. g., lipid composition, specific partner protein interactions, and the bacterial cytoskeleton). In these ways, we expect to gain a deeper understanding of molecular and physical mechanisms by which integral membrane proteins are targeted to specific locations or membrane domains. Project Narrative: All cells are surrounded by a membrane that contain a number of different molecules that are important for acquisition of nutrients, deposition of cell waste products, and a myriad of other interactions with its surroundings. These membrane molecules can be static or highly mobile depending upon their function. Understanding how cells control the dynamics of its membrane molecules is important for developing new strategies for combating processes like viral infection and preventing diseases such as Alzheimer's disease. New technological advances for high-speed single molecule detection developed in this project will also be directly applicable to many other important basic biological problems.