Illuminating the process of rod outer segment morphogenesis

描述(申请人提供)：视紫红质、外周蛋白/rds和cGMP门控通道中的视紫红质、外周蛋白/rds和cGMP门控通道中的三种膜蛋白定位于外节的不同亚膜间隔。这种不同的区划对于光转导信号以及杆状外节的形态发生和维持都是必不可少的。由于光感受器外节没有蛋白质合成的机制，蛋白质需要通过鞭毛内转运机制从光感受器内节段合成和运输。这种转运的缺陷与一系列统称为“视网膜纤毛病”的疾病有关。关于光感受器外节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障碍与视网膜纤毛病变之间的直接联系。我们开发了一种新的方法来荧光标记新合成的蛋白质在杆状光感受器中，并通过共聚焦显微镜和双光子显微镜跟踪它们在活的非洲爪哇蝌蚪中的运动。通过这种方法，我们提出：目的1.阐明光感受器视盘膜的形态发生过程。目的2.剖析位于光感受器外节段特异蛋白C末端的信号序列的功能。在目标1中，我们将利用视紫红质和外周蛋白/RDS融合到一个新的荧光蛋白Dendra2上来研究视盘形态发生的过程。Dendra2是一种可光激活的蛋白质，在紫外光或强蓝光照射下从绿色变为红色，并能够将新合成的蛋白质标记为绿色。在目标2中，我们将区分由视紫红质、外周蛋白/RDS和cGMP门控通道？亚基三种蛋白的C-末端区域介导的转运途径。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。在另一个目的中，我们将阐明视紫红质在外段形态发生过程中尚未解决的和有争议的作用。我们建议：目的3.确定视紫红质在外节完整性中的结构作用。为了达到这个目的，我们将使用一种新建立的动物模型，它表达黑素而不是视紫红质来形成外节。然后，我们将通过利用非洲爪哇视紫红质-黑色素嵌合体表达的光感受器来确定视紫红质的特定结构是否有助于外节的完整性。在视紫红质基因敲除的小鼠中没有观察到视紫红质外段，视紫红质错误定位在广泛的视网膜纤毛疾病中观察到，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。了解纤毛外段形态发生的过程和机制，可以揭示纤毛病变致病基因产物的新途径。 与公共健康相关：光是由光感受器神经元的一部分感受到的，这一部分被称为“外节”，在被称为“睫毛病变”的致盲疾病中，它消失或变短。为了更好地了解纤毛病变的过程，我们从两个方面研究了外段是如何形成的。首先，我们用显微镜直观地显示了外节的形成过程。其次，我们将确定关键蛋白视紫红质在构建外段中的结构作用。

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.