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：视杆细胞和视锥细胞。在这些细胞内，光探测发生在一种叫做外段（OS）的复杂结构中，这是一种改良的感觉纤毛。在杆状细胞中，OS由数千个被质膜包围的膜盘组成，并通过连接的纤毛与细胞体分离。圆盘含有高浓度的视紫红质。含有紫红质的杆状圆盘膜在杆状OS的基部形成。每个圆盘与其他圆盘隔离；新椎间盘逐渐向视网膜色素上皮移位，并脱落。相反，锥盘膜彼此是连续的。这一引人注目的过程背后的机制仍在调查中，有两种主要的相互竞争的模型，一些基本前提也在争论中。主要的障碍是缺乏工具，以及缺乏对分子参与者的了解。我们开发了研究盘状膜生物合成的新技术，并将这些技术用于以下目的：***1。锥细胞受体膜蛋白合成及定位的研究。锥形OS膜组织是相对不具特征的。大多数物种的操作系统是圆锥形的，磁盘不是密封的。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)锥体“质膜”蛋白是否同样局限于与棒状OS质膜等效的OS膜子域，或者在整个锥体OS磁盘中发现(2)如果是，它们是否可以自由扩散到等效的子域，以及(3)类似地确定锥体磁盘边缘蛋白是否能够扩散到所有OS磁盘边缘。如果锥盘边缘或质膜蛋白受到限制，我们将进一步测试它们是否可以用于测量锥盘合成，这在以前只在视紫红质中进行过。我们已经开发了用于诱导表达GFP-或表位标记的蛋白的构建体在转基因X. laevis的杆和锥。我们将通过热休克诱导表达。***2：涉及prominent -1/prcad-21的圆盘合成机制研究。pronin -1和prcad-21是唯一已知的与棒状基底盘特异性相关的蛋白。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敲除技术来检测这些蛋白。我们将通过诱导视紫红质- gfp表达和/或抑制视紫红质糖基化来确定敲除对磁盘合成率的影响。我们将通过电子显微镜和其他膜结构域标记物的诱导表达来确定敲除对OS和磁盘形态的影响。********

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.********