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: 8142025
• 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/8142025
• 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; ciliopathy; melanopsin; novel; peripherin; photoreceptor disc; protein transport; public health relevance; retinal rods; scaffold; trafficking; two-photon; ultraviolet irradiation

DESCRIPTION (provided by applicant): 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: Aim 1. Elucidate the process of photoreceptor disk membrane morphogenesis. ; Aim 2. Dissect the functions of signal sequences located at the C-terminal regions of photoreceptor outer segment specific proteins. In Aim 1, 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 Aim 2, 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: Aim 3. 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. PUBLIC HEALTH RELEVANCE: Light is sensed by a part of the photoreceptor neuron called the "outer segment," which disappears or gets shorter in blinding diseases called "ciliopathies". To better understand the process of ciliopathies, we study in two ways how the outer segment is formed. First, we visualize directly the process of outer segment formation by microscopy. Second, we will determine the structural role of the key protein, rhodopsin, in building the outer segment.

描述（由申请人提供）：视杆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，定位于外节的不同亚膜室。这种差异区隔化对于光导信号传导以及杆外段的形态发生和维持是必不可少的。由于光感受器外节段没有蛋白质合成机制，蛋白质需要通过鞭毛内运输机制从光感受器内节段合成和运输。这种运输的缺陷与一系列统称为“视网膜纤毛病”的疾病有关。关于光感受器外段形态发生如何调控的细节一直存在争议；然而，越来越多的证据似乎支持鞭毛内运输功能障碍与视网膜纤毛病之间的直接联系。我们开发了一种新的方法来荧光标记新合成的蛋白质杆状光感受器，并通过共聚焦和双光子显微镜跟踪它们的运动在非洲爪蟾蝌蚪活体。利用这种方法，我们提出：目标1。阐明感光盘膜的形态形成过程。;目标2。剖析位于光感受器外段特异性蛋白c端区域的信号序列的功能。在Aim 1中，我们将通过将视紫红质和外周蛋白/rds融合到一种新的荧光蛋白Dendra2中来研究圆盘形态发生的过程。Dendra2是一种光激活蛋白，在紫外线或强蓝光照射下由绿色变为红色，并能将新合成的蛋白质标记为绿色。在Aim 2中，我们将区分三种蛋白质（视紫红质、外周蛋白/rds和cgmp门控通道¿-亚基）的c端区域介导的运输途径。为了实现这一目标，我们将使用各种Dendra2融合蛋白和活体蝌蚪成像。另一方面，我们将阐明视紫红质在外节形态发生过程中尚未解决和有争议的作用。我们建议：目标3。确定视紫红质在外段完整性中的结构作用。为此，我们将使用一种新建立的表达黑视素而不是视紫红质的动物模型来形成外节段。然后，我们将通过在非洲爪蟾杆光感受器中表达的视紫红质-黑视质嵌合体来确定视紫红质的特定结构是否有助于外段的完整性。在视紫红质敲除小鼠中未观察到外段，在广泛的视网膜纤毛病（如Bardet Biedl综合征、Usher综合征和其他非综合征性视网膜色素变性）中观察到视紫红质错误定位。了解纤毛外段形态发生的过程和机制，有助于揭示纤毛病致病基因产物的新途径。