IMAGING OF ROD OUTER SEGMENT BY CRYO-ELECTRON TOMOGRAPHY

描述（由申请人提供）：视觉信号转导的场所，即光刺激被生物化学转化为电信号的过程，是感光细胞的外部部分。对这一系统进行了广泛的研究，因此可以获得丰富的资料。例如，我们知道许多参与信号传导的参与者和他们各自的角色，以及一些信号蛋白的高分辨率晶体结构。尽管付出了巨大的努力并获得了丰富的信息，但仍然无法获得信号传递过程的完整图景。我们在理解上的差距包括一些关键的信息，比如与信号传导有关的蛋白质的空间组织和化学计量学，以及由这些蛋白质形成的复合物的性质。此外，对于外节的形态是如何维持的，以及是什么导致它们在疾病和年龄中退化，人们知之甚少。在这项研究中，我们提出发展低温电子断层扫描（cryo-ET）来研究棒材外段（ROS）的结构。具体来说，我们将描述：1)使用cryo-ET小鼠ROS的结构；(2)含视蛋白ROS的高阶组织（LRAT小鼠）或与adRP相关的P23H突变体Rho；(3)一种高分子量的蛋白质，其功能是将膜分开一定距离。这些研究将大大增加我们的理解正常和疾病阶段的组织杆外段。

DESCRIPTION (provided by applicant): The venue for visual signal transduction, the process whereby a light stimulus is translated biochemically into an electrical signal, is the outer segment of photoreceptor cells. This system has been studied extensively and therefore a wealth of information is available. For instance, we know many of the players involved in signaling and their respective roles, and the high-resolution crystal structures of some of the signaling proteins. Despite the intense efforts and the abundance of information, a complete picture of the signaling process is still unavailable. Gaps in our understanding include critical pieces of information such as the spatial organization and the stoichiometry of proteins involved in signaling and the nature of the complexes that are formed by those proteins. Moreover, little is known about how the morphology of the outer segments is maintained and what causes their degeneration in disease and with age. In this study, we propose to develop cryo-electron tomography (cryo-ET) to study the structure of rod outer segments (ROS). Specifically we will characterize: 1) the structure of mouse ROS using cryo-ET; (2) the higher-order organization of ROS containing opsin (LRAT mice) or P23H mutant Rho associated with adRP; and (3) a high molecular weight protein that functions as a pillar holding apart membranes by a specific distance. These studies will add significantly to our understanding of the normal and disease stage organization of the rod outer segments.