Photoreceptor disk membrane morphogenesis

 描述（由申请人提供）：光感受器外节是光转导的部位。每个外节的光转导盘膜不断更新，导致大量的膜从感光器内节运输到感光器外节，并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，并且更新是光感受器细胞生物学和疾病的中心。竞争的假设，从不同的EM观察结果，已在文献中提出，以说明这些磁盘膜是如何形成的。有人认为，不同的结果是由于不同的组织固定方法。然而，除了组织保存的方法，不同的研究集中在不同的物种，而且，一天中的时间没有被考虑（然而，有证据表明，在磁盘膜生长的每日周期）。拟议的研究将推进我们的知识新生杆外段磁盘的组织，从而磁盘形态发生的机制，通过解决已发表的工作中的差距，并通过应用新的技术来解决这个问题。我们将研究老鼠和猴子的视网膜。对于小鼠，我们将通过使用高压冷冻/冷冻置换将基底盘膜的保存方法提高到更高的水平，从而消除化学初级固定引入的潜在伪影。此外，我们将在小鼠和猴子的基底杆盘进行EM系列断层扫描，以获得纳米级的，3D分辨率的组织。这种方法提供的上级分辨率，以及3D图像，将使膜连接和连续性的完整分析，已无法通过迄今为止使用的方法。这种新的方法，将使我们能够专注于定义杆外节中新形成的基底盘的根本重要组织。在对WT小鼠的研究之后，我们将使用具有缺陷性椎间盘形态发生的三种突变小鼠系进行可比分析。对这些小鼠异常椎间盘的EM断层扫描分析将提供对正常过程的深入了解，并增加我们对这些小鼠模型的视网膜色素变性、视锥-视杆营养不良和黄斑变性形式的潜在机制的理解。总的来说，该提案将应用最先进的EM技术和3D图像分析，这将为光感受器生物学和疾病中最重要的细胞生物学问题之一提供重大进展。

 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.