Single Molecule Study of Receptor Localization in Cellular Membranes

细胞膜中受体定位的单分子研究

• 批准号: 7363582
• 负责人: Kenneth Ritchie
• 金额: $ 28.05万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7363582
• 关键词: Affect; Alzheimer' s Disease; Architecture; Arginine; Bacteria; Biological; Biological Models; Cell Wall; Cell membrane; Cells; Cellular Membrane; Chemoreceptors; Collaborations; Cytoskeleton; Cytosol; Deposition; Depth; Detection; Diffusion; Disease; Epithelial Cells; Escherichia coli; Eukaryota; Eukaryotic Cell; Event; Fluorescence; Goals; Gram-Negative Bacteria; Image; Image Analysis; Individual; Infection; Integral Membrane Protein; Label; Life; Lipids; Localized; Location; Mechanics; Membrane; Methods; Molecular; Monitor; Motion; Numbers; Nutrient; Pathology; Pathway interactions; Physiology; Polar Regions; Positioning Attribute; Process; Proteins; Publishing; Rate; Receptor Signaling; Research; Resolution; Serine; Signal Transduction; Speed; Standards of Weights and Measures; System; Time; Toxin; Twin Multiple Birth; Variant; Venus; Virus Diseases; Waste Products; bacterial genetics; cell envelope; intracellular protein transport; macromolecule; mutant; novel strategies; pathogen; periplasm; prevent; protein transport; receptor; serine receptor; single molecule; trafficking; translocase

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.

描述（由申请人提供）：细胞膜内的补体，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进入以及阿尔茨海默病等病理学有关。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定位于大肠杆菌K- 12模型系统中特定区域的机制，该系统具有比真核生物中发现的更简单的膜结构。荧光标记的丝氨酸化学感受器（Tsr）位于大肠杆菌的内膜。将以高达每秒1000帧的视频帧速率对大肠杆菌进行成像，以确定“Tsr膜结构域”如何组装和维持。单分子成像具有观察大量丢失的罕见事件的能力、多分子成像和将分子定位到比分辨率的罗利极限小得多的位置的能力的组合益处。因此，可以准确地监测分子在其结构域中的动力学以及它们的进入和与结构域外部的自由分子的可能交换。Tsr的成像将在正常和突变细胞中进行，这些细胞在细胞处理器中进行了遗传改变，这些细胞处理器根据可检验的假设（e.例如，在一个实施例中，脂质组成、特异性伴侣蛋白相互作用和细菌细胞骨架）。通过这些方式，我们期望能够更深入地了解整合膜蛋白靶向特定位置或膜结构域的分子和物理机制。 项目叙述：所有的细胞都被一层膜所包围，膜上含有许多不同的分子，这些分子对于获取营养、沉积细胞废物以及与周围环境的无数其他相互作用都很重要。这些膜分子可以是静止的或高度移动的，这取决于它们的功能。了解细胞如何控制其膜分子的动力学对于开发对抗病毒感染和预防阿尔茨海默病等疾病的新策略非常重要。本项目开发的高速单分子检测新技术也将直接应用于许多其他重要的基础生物学问题。