Light-dependent Transducin Movement in Retinal Rods.

视网膜杆中的光依赖性转导蛋白运动。

基本信息

批准号：
8065978
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
项目类别：
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/8065978
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

项目摘要

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）：对光转导后的光适应和恢复机制的分析是国家眼睛和视力研究计划（http://www.nei.nih.gov/strategicplanning/np_retinal）中概述的目标之一。 ASP）。视网膜视杆细胞响应光照而发生的最引人注目的分子事件之一是视杆细胞 G 蛋白（转导蛋白）的迁移。在黑暗中，转导蛋白集中在视杆细胞的外节 (OS)。光来说，它从操作系统迁移到内节（IS）、核层和突触末端。这种现象的意义和机制仍然存在争议。基于对小鼠和果蝇的遗传学研究，许多视觉科学家假设转导蛋白的运动需要主动的运输机制。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。在该模型中，转导蛋白重定位的光依赖性和方向性通过以下简单机制来解释。激活后，杆转导蛋白 (i) 解离成 Galpha 和 Gbeta-gamma，(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操作系统中释放和随后的重新定位唯一必要且充分的两个事件。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（防止光损伤）。具体目标 1 将设计和测试杆转导蛋白 α 亚基的突变形式，该亚基由于与膜、Gbeta-gamma 和 LGN（杆内室中假定的结合伴侣）亲和力改变而无法易位。这些 Galpha 突变体将首先在转染的哺乳动物细胞中进行验证，然后使用病毒转化的小鼠棒进行原位验证。具体目标 2 是在体内表达这些显性突变体（转基因小鼠），以检查其对光适应和视杆细胞存活（视网膜变性）的影响。一系列电生理学、生物化学和组织学分析将研究视杆细胞的光适应和长期存活。该项目将实现两个目标：建立光依赖性杆状转导蛋白重新分布的分子机制并了解这种现象的生理意义。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概念框架。公共健康相关性：许多蛋白质响应外部刺激而在细胞内重新定位。该项目研究光感受器杆中异源三聚体G蛋白的光依赖性细胞内运动的基本原理和意义。拟议的实验将确定 G 蛋白重新定位的能力对于光适应和长期感光细胞的重要性。这些新见解对于理解目前无法治愈的致盲疾病非常重要，对于更广泛的神经元变性也很重要。