Light-dependent Transducin Movement in Retinal Rods.

视网膜杆中的光依赖性转导蛋白运动。

• 批准号: 8264353
• 负责人: Vladlen Z Slepak
• 金额: $ 36.12万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264353
• 关键词: Active Biological Transport; Afferent Neurons; Affinity; Animals; Arrestins; Binding; Biochemical; Cell Survival; Cells; Cessation of life; Cyclic GMP; Cytoprotection; DNA Sequence Rearrangement; Darkness; Data; Degenerative Disorder; Dependence; Diffusion; Disease; Dissociation; Distal; Drosophila genus; Electroretinography; Employee Strikes; Enzymes; Event; Eye; Future; GRK1 gene; GTP-Binding Proteins; GTPase-Activating Proteins; Gated Ion Channel; Gene Transfer; Genetic; Goals; Guanosine Triphosphate; Guanylate Cyclase; Health; Heterotrimeric GTP-Binding Proteins; Hydrolysis; In Situ; In Vitro; Investigation; Knowledge; Laboratories; Left; Light; Light Adaptations; Light Cell; Lighting; Longitudinal Studies; Mammalian Cell; Mediating; Membrane; Modeling; Molecular; Molecular Motors; Movement; Mus; Mutation; Nerve Degeneration; Noise; Nuclear; Pathway interactions; Phagocytosis; Phosphorylation; Photons; Photoreceptors; Phototransduction; Physiological; Physiology; Presynaptic Terminals; Process; Proteins; Recovery; Research; Research Personnel; Rest; Retinal; Retinal Degeneration; Rhodopsin; Rod Outer Segments; Role; Scientist; Series; Signal Transduction; Signaling Protein; Stimulus; Structure of retinal pigment epithelium; Subfamily lentivirinae; Testing; Transducin; Transgenic Mice; Transgenic Organisms; Translating; Vertebrate Photoreceptors; Vision; Vision research; Work; alpha Subunit Transducin; base; design; in vivo; insight; light intensity; migration; mouse model; mutant; phosphoric diester hydrolase; polarized cell; prevent; protein protein interaction; research study; response; restoration; retinal rods; rhodopsin kinase

DESCRIPTION (provided by applicant): Analysis of the mechanisms underlying light adaptation and recovery following phototransduction is one of the goals outlined in the National Plan for Eye and Vision Research (http://www.nei.nih.gov/strategicplanning /np_retinal.asp). One of the most striking molecular events that occurs in retinal rods in response to illumination is the migration of rod G protein, transducin. In darkness, transducin concentrates in the outer segments (OS) of rods. In light, it migrates from the OS to the inner segment (IS), nuclear layer and the synaptic terminals. The significance and the mechanism responsible for this phenomenon are a subject of debate. Based on genetic studies in mice and drosophila, a number of vision scientists hypothesized that transducin movement requires active transport machinery. However, this laboratory showed that movement of transducin does not require ATP and therefore must be driven by diffusion rather than molecular motors. In this model, the light-dependence and directionality of transducin relocalization is explained by the following simple mechanism. Upon activation, rod transducin (i) dissociates into Galpha and Gbeta-gamma, (ii) the subunits become soluble and disperse throughout the cell. Dissociation of the subunits and their subsequent solubilization are the only two events necessary and sufficient for the release of transducin from the OS and subsequent relocalization. The proposed research will use this model to establish whether transducin translocation is indeed necessary for light adaptation, and whether it contributes to cell survival (protection from light damage). Specific Aim 1 will design and test mutant forms of rod transducin alpha subunit that cannot translocate by virtue of the altered affinity to the membranes, Gbeta-gamma, and LGN, a putative binding partner in the inner compartments of the rods. These Galpha mutants will be first validated in transfected mammalian cells and then in situ using virally transformed mouse rods. Specific Aim 2 is to express these dominant mutants in vivo (transgenic mice) in order to examine the effect on light adaptation and rod survival (retinal degeneration). A series of electrophysiological, biochemical and histological analyses will investigate light adaptation and long- term survival of the rods. This project will achieve two goals: establish the molecular mechanism of light-dependent rod transducin redistribution and understand physiologic significance of this phenomenon. This new basic knowledge will provide a conceptual framework for designing new strategies to prevent and treat retinal degenerative diseases. PUBLIC HEALTH RELEVANCE: Many proteins re-localize within cells in response to external stimuli. This project investigates the basic principles and significance of light-dependent intracellular movements of a heterotrimeric G protein in photoreceptor rods. The proposed experiments will determine how the ability of the G protein to relocalize is important for light adaptation and long-term photoreceptor cells. The new insights are important for understanding currently incurable blinding diseases, and is also important for broader aspects of neuronal degeneration.

描述（由申请人提供）：分析光转导后的光适应和恢复机制是国家眼与视觉研究计划（http://www.nei.nih.gov/strategicplanning /np_retinal.asp）概述的目标之一。在视网膜杆状体中发生的最引人注目的分子事件之一是杆状体G蛋白（转导蛋白）的迁移。在黑暗中，转导蛋白集中在杆状细胞的外节（OS）。在光线下，它从OS迁移到内节段（IS）、核层和突触终末。造成这一现象的意义和机制是一个有争议的话题。基于对小鼠和果蝇的遗传研究，许多视觉科学家假设转导蛋白的运动需要主动运输机制。然而，该实验室表明，转导蛋白的运动不需要ATP，因此必须由扩散而不是分子马达驱动。在这个模型中，转导蛋白重定位的光依赖性和方向性可以用以下简单的机制来解释。激活后，杆状转导蛋白(i)解离成α α和β - γ， （ii）亚基变得可溶并分散在整个细胞中。亚基的解离及其随后的溶解是转导蛋白从OS释放和随后的再定位所必需和充分的两个事件。拟议的研究将使用该模型来确定转导蛋白易位是否确实是光适应所必需的，以及它是否有助于细胞存活（免受光损伤）。特异性目标1将设计和测试杆状细胞转导蛋白α亚基的突变形式，该亚基由于与膜、gβ - γ和LGN（杆状细胞内室的一种假定的结合伙伴）的亲和力改变而不能易位。这些Galpha突变体将首先在转染的哺乳动物细胞中验证，然后在原位使用病毒转化的小鼠棒。特异性目的2是在体内表达这些显性突变体（转基因小鼠），以研究对光适应和杆存活（视网膜变性）的影响。一系列的电生理、生化和组织学分析将探讨视杆细胞的光适应和长期存活。本项目将实现两个目标：建立光敏杆转导蛋白重分布的分子机制，了解这一现象的生理意义。这一新的基础知识将为设计预防和治疗视网膜退行性疾病的新策略提供一个概念框架。公共卫生相关性：许多蛋白质在响应外部刺激时在细胞内重新定位。本项目研究了光感受器棒中异三聚体G蛋白依赖光的细胞内运动的基本原理和意义。所提出的实验将确定G蛋白重新定位的能力对光适应和长期光感受器细胞的重要性。这些新发现对于理解目前无法治愈的致盲性疾病非常重要，对神经元变性的更广泛方面也很重要。