IMAGING OF ROD OUTER SEGMENT BY CRYO-ELECTRON TOMOGRAPHY

通过冷冻电子断层扫描对杆外段进行成像

• 批准号: 7208348
• 负责人: KRZYSZTOF PALCZEWSKI
• 金额: $ 19.31万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7208348
• 关键词: 3-Dimensional; ATP-Binding Cassette Transporters; Abbreviations; Address; Age; Age related macular degeneration; Biochemical; Biological; Biological Assay; Birds; Cells; Cellular Structures; Characteristics; Chemicals; Complex; Computer software; Condition; Data; Dehydration; Detection; Disease; Dose Fractionation; Electron Microscopy; Electrophysiology (science); Endoplasmic Reticulum; Environment; Eukaryotic Cell; Eye; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Genetic; Glutamic Acid; Higher Order Chromatin Structure; Human; Image; Imagery; Individual; Invasive; Knock-in Mouse; Knock-out; Laboratories; Light; Link; Macromolecular Complexes; Maps; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Biology; Molecular Weight; Morphologic artifacts; Morphology; Mus; Mutant Strains Mice; Mutation; Nature; Opsin; Organelles; Organism; Pathway interactions; Photons; Photoreceptors; Phototransduction; Preparation; Principal Investigator; Procedures; Process; Proteins; Research; Research Design; Research Personnel; Resolution; Retina; Retinal Cone; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Rod Outer Segments; Role; Sampling; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Space Perception; Staging; Staining method; Stains; Stimulus; Structure; Structure of retinal pigment epithelium; System; Techniques; Technology; Time; Transgenic Mice; Translating; Transmission Electron Microscopy; Vision; Visual Signal Transduction Pathway; Work; absorption; computerized data processing; concept; data acquisition; electron tomography; grasp; image processing; improved; innovation; insight; instrumentation; lecithin-retinol acyltransferase; macromolecule; member; molecular assembly/self assembly; mutant; neurotransmission; novel; photoreceptor cell outer segment; programs; protein function; reconstitution; reconstruction; rho; rod outer segment disc; sample fixation; stoichiometry; two-dimensional

DESCRIPTION (provided by applicant): The venue for visual signal transduction, the process whereby a light stimulus is translated biochemically into an electrical signal, is the outer segment of photoreceptor cells. This system has been studied extensively and therefore a wealth of information is available. For instance, we know many of the players involved in signaling and their respective roles, and the high-resolution crystal structures of some of the signaling proteins. Despite the intense efforts and the abundance of information, a complete picture of the signaling process is still unavailable. Gaps in our understanding include critical pieces of information such as the spatial organization and the stoichiometry of proteins involved in signaling and the nature of the complexes that are formed by those proteins. Moreover, little is known about how the morphology of the outer segments is maintained and what causes their degeneration in disease and with age. In this study, we propose to develop cryo-electron tomography (cryo-ET) to study the structure of rod outer segments (ROS). Specifically we will characterize: 1) the structure of mouse ROS using cryo-ET; (2) the higher-order organization of ROS containing opsin (LRAT mice) or P23H mutant Rho associated with adRP; and (3) a high molecular weight protein that functions as a pillar holding apart membranes by a specific distance. These studies will add significantly to our understanding of the normal and disease stage organization of the rod outer segments.

描述(申请人提供)：视觉信号转导的场所，光刺激被生物化学转化为电信号的过程，是光感受器细胞的外层部分。这个系统经过了广泛的研究，因此有大量的信息可用。例如，我们知道许多参与信号转导的参与者及其各自的角色，以及一些信号转导蛋白的高分辨率晶体结构。尽管进行了紧张的努力和丰富的信息，但仍无法获得信号传递过程的完整图景。我们理解的空白包括一些关键的信息，如参与信号传递的蛋白质的空间组织和化学计量，以及由这些蛋白质形成的复合体的性质。此外，对于外节的形态是如何维持的，以及是什么导致它们在疾病中和随着年龄的推移而退化，人们知之甚少。在这项研究中，我们建议发展冷冻电子断层扫描(CRYO-ET)来研究棒状外段(ROS)的结构。具体地说，我们将描述：1)使用冷冻-ET技术的小鼠ROS的结构；(2)含有视蛋白的ROS(LRAT小鼠)或与adrp相关的P23H突变体Rho的高阶组织；以及(3)一种高分子量蛋白质，其功能是以特定距离将膜分开。这些研究将大大增加我们对杆状外节的正常和疾病阶段组织的理解。