Illuminating the process of rod outer segment morphogenesis

阐明视杆细胞外节形态发生的过程

• 批准号: 8323403
• 负责人: Yoshikazu Imanishi
• 金额: $ 33.91万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8323403
• 关键词: Animal Model; Bardet-Biedl Syndrome; C-terminal; Cells; Chimera organism; Chimeric Proteins; Cyclic GMP; Disease; Genes; Goals; Image; Knock-out; Knockout Mice; Label; Lead; Life; Light; Link; Maintenance; Mediating; Membrane; Membrane Proteins; Methods; Microscopy; Monitor; Morphogenesis; Movement; Neurons; Pathway interactions; Peptide Signal Sequences; Photoreceptors; Phototransduction; Process; Protein Biosynthesis; Proteins; Retinal; Retinitis Pigmentosa; Rhodopsin; Rod Outer Segments; Role; Signal Transduction; Structure; Tadpoles; Time; Usher Syndrome; Xenopus; Xenopus laevis; abstracting; ciliopathy; melanopsin; novel; peripherin; photoreceptor disc; protein transport; retinal rods; scaffold; trafficking; two-photon; ultraviolet irradiation

Abstract: Three membrane proteins in the rod photoreceptors, rhodopsin, peripherin/rds and cGMP-gated channels, localize to different sub-membrane compartments of the outer segments. This differential compartmentalization is essential for phototransduction signaling, as well as the morphogenesis and maintenance of the rod outer segments. Since photoreceptor outer segments do not have machinery for protein synthesis, proteins need to be synthesized and transported from the photoreceptor inner segments by an intraflagellar transport mechanism. Deficiencies in such transport have been implicated in a broad spectrum of diseases collectively called "retinal ciliopathy". Details of how photoreceptor outer segment morphogenesis is regulated have been controversial, however increasing evidence appears to support a direct association between intraflagellar transport malfunction and retinal ciliopathy. We have developed a new method to fluorescently label newly synthesized proteins in rod photoreceptors and track their movements via confocal and two-photon microscopy in living Xenopus laevis tadpoles. By using this method, we propose: Aim1. Elucidate the process of photoreceptor disk membrane morphogenesis. ; Aim2. Dissect the functions of signal sequences located at the C-terminal regions of photoreceptor outer segment specific proteins. In Aim1, we will study the process of disk morphogenesis by using rhodopsin and peripherin/rds fused to a novel fluorescent protein Dendra2. Dendra2 is a photoactivatable protein which turns from green to red upon irradiation by UV or intense blue light, and is able to label newly synthesized proteins in green. In Aim2, we will differentiate the trafficking pathways mediated by the C-terminal region of three proteins--rhodopsin, peripherin/rds and cGMP-gated channel ¿- subunit. Toward this goal, we will use various Dendra2 fusion proteins and live tadpole imaging. In another aim, we will elucidate the yet unsolved and controversial role of rhodopsin in the process of outer segment morphogenesis. We propose: Aim3. Determine the structural role of rhodopsin in the integrity of the outer segment. In this aim, we will use a newly established animal model which expresses melanopsin instead of rhodopsin to form the outer segments. We will then determine if the specific structure of rhodopsin is contributing to the integrity of the outer segments by using rhodopsin-melanopsin chimeras expressed in Xenopus rod photoreceptors. The outer segment is not observed in rhodopsin knockout mice, and rhodopsin mislocalization is observed in a broad spectrum of retinal ciliopathies such as Bardet Biedl Syndrome, Usher Syndrome, and other non-syndromic retinitis pigmentosa. Understanding the process and mechanism of outer segment morphogenesis could reveal novel pathways which involve the products of ciliopathy causative genes.

摘要： 视杆细胞光感受器中的三种膜蛋白，视紫红质，外周蛋白/RDS和 cGMP门控通道，定位于细胞膜的不同亚膜区室， 外部片段。这种差异化的划分对于以下方面至关重要： 光转导信号，以及形态发生和维持的 杆外部节段。由于光感受器外节没有机械 对于蛋白质合成，蛋白质需要合成并从 光感受器内节的鞭毛内运输机制。 这种运输的缺陷与多种疾病有关 统称为“视网膜睫状体病”。光感受器外节 形态发生是受调控的一直存在争议，然而越来越多的证据表明， 似乎支持鞭毛内运输功能障碍 和视网膜睫状体病。我们开发了一种新的荧光标记方法 合成的蛋白质在杆光感受器和跟踪他们的运动通过共聚焦 和双光子显微镜在活的非洲爪蟾蝌蚪。通过使用该 方法，我们建议：目标1。阐明感光盘的过程 膜形态发生目标2.剖析信号序列的功能 位于感光细胞外节特异性的C-末端区域 proteins.在Aim 1中，我们将使用 视紫红质和peripherin/rds融合到一种新的荧光蛋白Dendra 2。 Dendra 2是一种光活化蛋白，在照射后从绿色变为红色 通过紫外线或强烈的蓝光，并且能够以绿色标记新合成的蛋白质。 在Aim 2中，我们将区分由C-末端介导的运输途径， 三种蛋白质的区域--视紫红质、外周蛋白/rds和cGMP门控通道-- 亚单位为了实现这一目标，我们将使用各种Dendra 2融合蛋白， 蝌蚪成像在另一个目的，我们将阐明尚未解决和有争议的 视紫红质在外节形态发生过程中的作用。我们建议： 目标3。确定视紫红质的结构作用的完整性外 片段为此，我们将使用一种新建立的动物模型， 表达黑视蛋白而不是视紫红质以形成外节。我们将 然后确定视紫红质的特定结构是否有助于 通过使用在非洲爪蟾中表达的视紫红质-黑视蛋白嵌合体， 杆状光感受器在视紫红质敲除小鼠中未观察到外节， 在广泛的视网膜睫状体病中观察到视紫红质定位错误 例如BardetBiedl综合征、Usher综合征和其它非综合征性视网膜炎 色素沉着。了解外部段的过程和机制 形态发生可以揭示涉及纤毛病变产物的新途径 致病基因