Role of dynamin in photoreceptor disc morphogenesis

项目摘要/摘要 光感受器外段是一种改良的初级纤毛，含有不断更新的盘状堆叠 膜。每天大约有10%的外层部分被替换，外层形成新的磁盘 在远端外节端，节基和旧的圆盘脱落。椎间盘形态发生始于外翻 纤毛质膜，紧随其后的是膜变平和侧向生长，最后是 完全或部分阀瓣外壳(分别为杆状和锥形)。完整的磁盘存储模块包括唯一的 将盘膜与周围外段血浆分离的膜断裂事件 薄膜。这一事件的分子基础仍然完全未知。在其他人的内吞过程中 在真核细胞中，膜的断裂是由动力蛋白完成的，它将膜泡释放到细胞内 细胞质。因此，本提案的目标1探讨了动力素在杆盘断裂过程中的作用。 目标2试图确定执行这一重要功能的蛋白质的关键。我的研究策略将 利用缺乏特定动力蛋白亚型的基因敲除小鼠的超微结构分析，各种方法 研究亚细胞蛋白定位，有针对性地寻找存在于高密度脂蛋白中的内胞蛋白 纯化的杆状外段制备及同位素标记质量法绝对蛋白质定量 光谱分析。这些方法的可行性在初步实验中得到了验证。总而言之， 拟议的研究将使我们能够更好地了解椎间盘形态发生是如何在分子上进行的。 水平，这与理解这些迷人细胞的基本生物学和揭示 人类遗传性致盲疾病的病理生理机制。

PROJECT SUMMARY/ABSTRACT The photoreceptor outer segment is a modified primary cilium containing constantly renewing stack of disc membranes. Around 10% of the outer segment is replaced every day, with new discs formed at the outer segment base and old discs shed at the distal outer segment end. Disc morphogenesis starts with evagination of the ciliary plasma membrane, is followed by membrane flattening and lateral outgrowth, and concludes with complete or partial disc enclosure (in rods and cones, respectively). Complete disc enclosure involves a unique membrane scission event separating the disc membrane from the surrounding outer segment plasma membrane. The molecular basis for this event remains entirely unknown. During endocytosis in other eukaryotic cells, membrane scission is performed by dynamins, which release membrane vesicles into the cytoplasm. Therefore, Aim 1 of this proposal explores the role of dynamins in the process of rod disc scission and Aim 2 seeks to identify the key of proteins performing this important function. My research strategy will utilize ultrastructural analysis of knockout mice lacking specific dynamin isoforms, various approaches to studying subcellular protein localization, targeted proteomic search for endocytic proteins present in highly purified rod outer segment preparations and absolute protein quantification using isotope-labeled mass spectrometry. The feasibility of these approaches is validated in preliminary experiments. Taken together, the proposed studies will allow us to better understand how disc morphogenesis is performed on the molecular level, which is highly relevant to understanding the basic biology of these fascinating cells and uncovering the pathophysiological mechanisms of inherited blinding diseases in human patients.