Disk membrane synthesis in rod and cone photoreceptors

脊椎动物的视网膜包含两种主要类型的光感受器：杆和锥。在这些细胞中，光探测发生在一个称为外节（OS）的复杂结构中，这是一种经过修饰的感觉纤毛。在视杆细胞中，OS由数千个被质膜包围的膜盘组成，并通过连接纤毛与细胞体分离。圆盘含有高浓度的视紫红质。视杆细胞的底部形成含有视紫红质的视杆细胞膜。每个视杆细胞膜与其他视杆细胞膜分离;新的视杆细胞膜逐渐向视网膜色素上皮细胞转移，并在那里脱落。相反，锥盘膜彼此连续。这一非凡过程背后的机制仍在调查中，有两个原则竞争模型，一些基本前提正在辩论中。主要的障碍是缺乏工具，以及缺乏对分子参与者的洞察力。我们已经开发了研究盘膜生物合成的新技术，并将在以下目的中使用这些技术：*1。视锥细胞膜蛋白合成与定位之研究。锥体OS膜组织相对未表征。在大多数物种中，操作系统是圆锥形的，并且磁盘不密封。它们被认为是一个卷曲的膜系统，在圆盘和质膜之间没有明确的边界;然而，已知边缘蛋白是分离的。我们将确定1）视锥“质膜”蛋白是否类似地被限制于等同于视杆OS质膜的OS膜的亚结构域或在整个视锥OS盘中发现，和（2）如果是，它们是否可以自由扩散到等同的亚结构域，和（3）类似地确定视锥盘边缘蛋白是否能够扩散遍及所有OS盘边缘。如果锥盘边缘或质膜蛋白的限制，我们将进一步测试它们是否可以用来测量锥盘合成，这是以前只在杆使用视紫红质。我们已经开发了用于在转基因X的视杆和视锥中诱导表达GFP或表位标记的蛋白的构建体。光滑。我们将通过热休克诱导表达。* 2：涉及Risin-1/prcad-21的盘合成机制的研究。Prominin-1和prcad-21是唯一已知的与视杆基盘特异性相关的蛋白质。它们可能在磁盘合成中发挥作用，并且已知彼此相互作用。 我们将通过抗体标记（包括电子显微镜）和CRISPR/cas9敲除技术在X.光感受器我们将使用可诱导的视紫红质-GFP表达和/或视紫红质糖基化的抑制来确定敲低盘合成速率的后果。我们将通过电子显微镜和其他膜结构域标志物的诱导表达来确定敲低对OS和盘形态的影响。

The vertebrate retina contains photoreceptors of two principle types: rods and cones. Within these cells, photodetection occurs in an elaborate structure called the outer segment (OS), a modified sensory cilium. In rods, the OS consists of thousands of membranous discs surrounded by a plasma membrane and separated from the cell body by a connecting cilium. The disks contain high concentrations of rhodopsin. Rhodopsin-containing rod disc membranes are created at the base of the rod OS. Each disc is isolated from the others; new discs are displaced gradually towards the retinal pigment epithelium, where they are shed. In contrast, cone disc membranes are continuous with each other. The mechanisms underlying this remarkable process are still under investigation, with two principle competing models, and some basic premises under debate. The chief obstacles are lack of tools, and lack of insight into molecular players. We have developed new techniques for studying disc membrane biosynthesis, and will use these techniques in the following Aims:***1. Investigation of membrane protein synthesis and localization in cone receptors. Cone OS membrane organization is relatively uncharacterized. The OS is conical in most species, and the disks are not sealed. They are thought to be a convoluted membrane system with no clear boundary between the disk and plasma membrane; however, rim proteins are known to be segregated. We will determine whether 1) cone "plasma membrane" proteins are similarly constrained to a subdomain of OS membrane equivalent to the rod OS plasma membrane or are found throughout the cone OS disks and (2) if yes, whether they can freely diffuse to equivalent subdomains, and (3) similarly determine whether cone disk rim proteins are capable of diffusing throughout all OS disk rims. If cone disk rim or plasma membrane proteins are constrained, we will further test whether they can be used to measure cone disk synthesis, which has been previously performed only in rods using rhodopsin. We have developed constructs for inducible expression of GFP- or epitope-tagged proteins in both rods and cones of transgenic X. laevis. We will induce expression via heat shock.***2: Investigation of disk synthesis mechanisms involving prominin-1/prcad-21. Prominin-1 and prcad-21 are the only known proteins associated specifically with rod basal disks. They likely play a role in disk synthesis, and are known to interact with each other. We will examine these proteins by antibody labeling (including electron microscopy) and CRISPR/cas9 knockout techniques in X. laevis photoreceptors. We will determine the consequences of knockdown on disk synthesis rate using inducible rhodopsin-GFP expression and/or inhibition of rhodopsin glycosylation. We will determine the consequences of knockdown on OS and disk morphology by electron microscopy and inducible expression of other membrane domain markers.********