Photoreceptor disk membrane morphogenesis

 描述(申请人提供)：光感受器外段是光转导的部位。每个外节的感光盘膜不断更新，导致大量的膜从感光细胞内段转运到感光细胞外段，最终到达视网膜色素上皮细胞。外节盘膜的形成是膜重塑的主要过程，而更新是光感受器细胞生物学和疾病的中心。文献中已经提出了不同的EM观察所产生的相互竞争的假设，以解释这些盘膜是如何形成的。有人认为，不同的结果是由于不同的组织固定方法所致。然而，除了不同的组织保存方法外，不同的研究侧重于不同的物种，而且还没有考虑一天中的不同时间(然而，有证据表明盘膜的生长存在每日循环)。这项拟议的研究将通过解决已发表的工作中的空白，并通过应用新技术来解决这一问题，从而促进我们对新生杆状外节段磁盘的组织结构以及磁盘形态发生机制的了解。我们将研究老鼠和猴子的视网膜。对于小鼠，我们将采用高压冷冻/冷冻替代的方法将基底膜的保存方法提高到更高的水平，从而避免化学初步固定带来的潜在伪影。此外，我们将对小鼠和猴子的基底节视盘进行EM序列断层扫描，以获得其组织的纳米级、3D分辨率。这种方法提供的超高分辨率，加上3D图像，将能够对膜连接和连续性进行完整的分析，这是迄今为止使用的方法所无法实现的。这一新的方法将使我们能够专注于确定杆状外节中新形成的基底盘的基本重要组织。在对WT小鼠进行研究之后，我们将使用三个具有盘形态发生缺陷的突变小鼠品系进行可比性分析。对这些小鼠异常视盘的电子显微镜断层扫描分析将提供对正常过程的洞察，并增加我们对这些小鼠所建立的视网膜色素变性、视锥-视杆细胞营养不良和黄斑变性的潜在机制的理解。总体而言，这项提案将应用最先进的EM技术和3D图像分析，这将在光感受器生物学和疾病中最基本的细胞生物学问题之一方面取得重大进展。

 DESCRIPTION (provided by applicant): The photoreceptor outer segment is the site of phototransduction. The phototransductive disk membranes of each outer segment are continually renewed, resulting in a large amount of membrane trafficking from the photoreceptor inner segment to the photoreceptor outer segment, and, eventually, to the retinal pigmented epithelial cells. The formation of outer segment disk membranes is a major process of membrane remodeling, and the renewal is central to photoreceptor cell biology and disease. Competing hypotheses, resulting from differing EM observations, have been presented in the literature to account for how these disk membranes are formed. It has been argued that the different results are due to different methods of tissue fixation. However, in addition to differen methods of tissue preservation, the different studies have focused on different species, and, moreover, time of day has not been considered (yet, there is evidence of a daily cycle in disk membrane growth). The proposed research will advance our knowledge of the organization of nascent rod outer segment disks, and thus mechanisms of disk morphogenesis, by addressing the gaps in published work, and by applying novel technologies to this problem. We will study mouse and monkey retinas. With mouse, we will take the method of preservation of the basal disk membranes to a higher level by using high-pressure freezing/freeze substitution, and thus obviate potential artifacts introduced by chemical primary fixation. Further, we will perform EM serial tomography of the basal rod disks in mouse and monkey, in order to obtain nano-scale, 3D resolution of their organization. The superior resolution afforded by this method, together with the 3D imagery, will enable a complete analysis of membrane connections and continuities that has been unavailable by the methods used to date. This novel approach, will allow us to focus on defining the fundamentally important organization of newly formed basal disks in rod outer segments. Following the studies on WT mouse, we will perform comparable analyses, using three lines of mutant mice that possess defective disk morphogenesis. EM tomographic analyses of the aberrant disks in these mice will provide insight into the normal process, as well as increase our understanding of mechanisms underlying the forms of retinitis pigmentosa, cone-rod dystrophy, and macular degeneration that these mice model. Overall, this proposal will apply state-of-the-art EM technology and 3D image analyses that will provide a major advance in one of the most essential cell biological problems in photoreceptor biology and disease.