ERM proteins and cell morphology in the retina

该实验室的长期目标是研究调节光感受器外节发育和维持的机制。*身体的每种细胞类型必须获得并保持与其功能相适应的特定形态。我们的实验室专注于光感受器，即眼睛的感光细胞，以及它们专门的感觉末梢，即外部部分。外部部分是扩张和修饰的纤毛，包含成堆的扁平的膜盘，承载着光学感光颜料。为了建立和更新一个外节，需要反复地外翻质膜，每次都会产生一个新的盘。然而，驱动和控制磁盘扩张的机制尚不清楚。同时，微绒毛样突起，称为帽状突起，垂直于视盘延伸，环绕外节底部。肾盏突起的目的尚不清楚，但它们的破坏与光感受器退化有关。*在这项资助中，我们研究了肌动蛋白细胞骨架和质膜如何合作，以创造光感受器外段的复杂形态。重要的是，花盏突起由肌动蛋白细丝的核心束支撑，但外节圆盘外旋，没有任何细胞骨架。然而，肌动蛋白存在于新形成的视盘附近，并且是视盘启动所必需的。因此，肌动蛋白细胞骨架对肾盏突和外节都是必需的，但方式不同。由于OS盘没有肌动蛋白核心，我们认为它们是通过起泡形成的，这是一个细胞过程，通过肌动球蛋白收缩导致细胞内压力增加和膜从细胞骨架中释放。相反，微绒毛样CPS通过其肌动蛋白核心的延伸而形成。ERM(Ezrin，Radixin，Moesin)蛋白作为细胞骨架和膜之间的连接物，在起泡和微绒毛形成过程中发挥着广泛的作用。我们假设ERM蛋白Ezrin锚定了肾盏突起，但在外节的底部周期性地被抑制，以允许盘外翻。*我们使用斑马鱼作为我们的模型系统来研究光感受器，因为它们有许多实验优势，视网膜发达和视力良好。在这里，我们将利用体外和体内的分析方法，通过三个主要问题来研究ERM蛋白Ezrin和肌动蛋白细胞骨架是如何促进外节的生长的：1)起泡是否驱动光感受器外节盘的外翻？2)盏突是否限制OS盘的生长并稳定OS？3)肌动蛋白和Ezrin在稳定盏突起和外段盘逃逸中有哪些具体功能？*影响：本研究将加深我们对膜-细胞骨架相互作用如何控制复杂细胞形态的理解。

The long-term objective of the lab is to study the mechanisms regulating development and maintenance of the photoreceptor outer segment.*** Each cell type of the body must acquire and maintain a specific morphology adapted to its function. Our lab focuses on photoreceptors, the light-sensing cells of the eye, and their specialized sensory endings, the outer segments. Outer segments are expanded and modified cilia containing stacks of flattened membranous disks laden with opsin photopigments. To build and renew an outer segment requires repeated evagination of the plasma membrane, each time giving rise to a new disk. However, the mechanisms driving and controlling disk expansion are unclear. At the same time, microvilli-like protrusions, called calyceal processes, extend perpendicular to the disks and surround the base of the outer segment. The purpose of calyceal processes is unknown, but their disruption is associated with photoreceptor degeneration. *** In this grant, we look at how the actin cytoskeleton and plasma membrane co-operate to create the complex morphology of the photoreceptor outer segment. Importantly, calyceal processes are supported by a core bundle of actin filaments, but outer segment disks evaginate without any cytoskeleton. Actin is however present adjacent to newly forming disks and is required for disk initiation. Therefore, the actin cytoskeleton is necessary for both calyceal processes and outer segments, but in distinct ways. As OS disks do not have an actin core, we propose that they form through blebbing, a cellular process whereby actomyosin contraction leads to increased intracellular pressure and release of the membrane from the cytoskeleton. In contrast, the microvilli-like CPs form by extension of their actin core. ERM (Ezrin, Radixin, Moesin) proteins act as linkers between the cytoskeleton and membrane and have well-described roles in both blebbing and microvilli formation. We hypothesize that the ERM protein Ezrin anchors calyceal processes, but at the base of the outer segment is periodically inhibited to allow for disk evagination.*** We use zebrafish as our model system to study photoreceptors because of their many experimental advantages, well-developed retina and excellent vision. Here, we will take advantage of both in vitro and in vivo assays to examine how the ERM protein Ezrin and the actin cytoskeleton promote growth of the outer segment, by asking three main questions:1) Does blebbing drive evagination of photoreceptor outer segment disks? 2) Do calyceal processes limit growth of OS disks and stabilize the OS? 3) What are the specific functions of actin and Ezrin in calyceal process stabilization and outer segment disk evagination?***Impact: This research will improve our understanding of how membrane-cytoskeletal interactions control the morphologesis of complex cells.