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感光器：视杆细胞和视锥细胞。在这些细胞中，光检测发生在称为外节(OS)的精细结构中，这是一种改良的感觉纤毛。在杆状结构中，OS由数千个被质膜包围的膜盘组成，并由连接的纤毛与细胞体分开。视盘含有高浓度的视紫红质。含有视紫红质的杆状盘膜在杆状OS的底部形成。每一个视盘都是相互隔离的；新的视盘逐渐向视网膜色素上皮移位，并在那里脱落。相反，锥形盘膜是彼此连续的。这一非凡过程背后的机制仍在调查中，两种相互竞争的原则模式和一些基本前提正在辩论中。主要的障碍是缺乏工具，以及对分子参与者缺乏洞察力。我们已经开发了研究视盘细胞膜生物合成的新技术，并将在以下方面使用这些技术：*1.研究视锥细胞受体中膜蛋白的合成和定位。锥形OS膜的组织结构相对较不典型。大多数物种的操作系统是圆锥形的，圆盘不是密封的。它们被认为是一个缠绕的膜系统，在圆盘和质膜之间没有明确的边界；然而，已知RIM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在OS膜的一个等同于视杆OS质膜的亚域上，或者是否在视锥OS盘中被发现，以及(2)如果是，它们是否可以自由扩散到相同的亚域，以及(3)类似地确定视锥盘缘蛋白是否能够扩散到所有OS盘缘。如果视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视盘合成，这是以前仅在使用视紫红质的视杆中进行的。我们已经开发出在转基因莱氏X.laevis的杆状和圆锥体中诱导表达GFP或表位标记蛋白的结构。我们将通过热休克诱导表达。*2：涉及Argin-1/prcad-21的盘状合成机制的研究。Prominin-1和prcad-21是仅有的与视杆基底盘相关的已知蛋白质。它们可能在盘状合成中发挥作用，并且已知它们相互作用。我们将通过抗体标记(包括电子显微镜)和CRISPR/Cas9基因敲除技术在莱氏X.laevis光感受器中检测这些蛋白。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基因敲除对视盘合成率的影响。我们将通过电子显微镜和其他膜结构域标记的诱导表达来确定击倒OS和磁盘形态的后果。

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.********