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Cilium-associated structures in rod cells

杆状细胞中的纤毛相关结构

基本信息

批准号：
10133075
负责人：
THEODORE G WENSEL
金额：
$ 38.8万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10133075
关键词：
Animal Model Bardet-Biedl Syndrome Carrier Proteins Cell Death Cellular Structures Cilia Color Computer software Cryo-electron tomography Deficiency Diseases Development Disease Electron Microscopy Electrons Environment Event Eye diseases Fiber Fluorescence Fluorescence Microscopy Functional disorder Genes Genetic Genetic Techniques Goals Hereditary Disease Imaging technology Lead Light Link Membrane Membrane Proteins Microtubules Molecular Mus Nerve Degeneration Noise Pathology Pharmaceutical Preparations Play Proteins Resolution Retina Retinal Cone Retinal Degeneration Retinal Photoreceptors Rod Role Route Secondary to Sensory Signal Transduction Structure Testing Tissues Tomogram Triplet Multiple Birth Vertebrate Photoreceptors appendage ciliopathy experimental study imaging approach innovation kinetosome molecular scale mouse model nanometer resolution novel novel therapeutic intervention particle protein complex protein function restoration retinal rods three dimensional structure trafficking

项目摘要

Rod and cone photoreceptors of the vertebrate retina detect light using their outer segments, highly specialized forms of primary cilia. Primary ciliary throughout the body play important roles in sensing the cellular environment, and genetic deletions in their molecular components, known as ciliopathies, lead to devastating congenital diseases, including blinding forms of retinal degeneration. The goal of this project is to develop a thorough understanding of the structural and molecular basis of primary cilium function, with a focus on the rod sensory cilium, and to understand the molecular mechanisms of rod cell death in ciliopathies. We have developed and applied innovative molecular-scale imaging approaches using fluorescence and electron microscopy to this problem, and now propose to introduce additional improvements in the imaging technology and to use them to test hypotheses about normal ciliary structures and mechanisms, and about pathophysiological mechanisms in animal models of retinal ciliopathies. Specific Aims: 1. Use cryo-electron tomography (cryo-ET) and recent developments in sub-tomogram averaging to determine the three- dimensional structure to nanometer resolution of repeating structures of the rod cell connecting cilium and basal body, including microtubule doublets and triplets, microtubule inner proteins, “Y-shaped links”, transition fibers and appendages. Our goal is to apply recent developments in hardware and software to rod cells in both wild type retinas and in animal models of retinal degeneration. 2. Use superresolution fluorescence to test hypotheses about trafficking of specific proteins and about the roles of IFT (intraflagellar transport) particles and the BBSome (a coat-forming protein complex implicated in the blinding ciliopathy, Bardet-Biedl syndrome) in ciliary trafficking in rods. Two-color superresolution fluorescence and quantitative interaction analysis will be used to assess putative interactions between IFT proteins or BBS proteins and outer segment membrane proteins, as well as well as proteins normally excluded from the outer segment which mis-accumulate there in BBS-deficient mice. These experiments will test the hypothesis that specific membrane proteins are actively trafficked through the connecting cilium membrane through their association with IFT particles, whereas others are transported via alternative routes and excluded proteins are actively removed by the BBSome. 3. Use mouse models to test the hypotheses that CEP290 is a major component of the “Y-shaped links” extending from the ciliary axoneme to the membrane, using superresolution fluorescence, conventional TEM, and cryo- electron tomography with timed gene disruption or gene restoration at different developmental stages to distinguish initiating as opposed to secondary events in the development of the pathophysiology of ciliopathies associated with this protein

脊椎动物视网膜的杆状和锥状光感受器利用它们高度特化的外节来探测光线