Role of dynamin in photoreceptor disc morphogenesis

动力在感光盘形态发生中的作用

• 批准号: 10584570
• 负责人: Christin Hanke-Gogokhia
• 金额: $ 7.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10584570
• 关键词: Address; Affect; Biogenesis; Biological Process; Biology; Blindness; Breeding; Cell membrane; Cells; Cellular biology; Characteristics; Cilia; Complex; Cytoplasm; Data; Databases; Development; Disease; Distal; Dynamin; Dynamin I; Dynamin III; Electron Microscopy; Endocytosis; Eukaryotic Cell; Event; Fellowship; Future; Genetic; Human; Inherited; Isotope Labeling; Knock-out; Knockout Mice; Lateral; Libraries; Light; LoxP-flanked allele; Mass Spectrum Analysis; Membrane; Molecular; Morphogenesis; Mus; Mutation; Neurons; Organelles; Pathology; Patients; Photoreceptors; Preparation; Process; Protein Isoforms; Proteins; Proteomics; Research; Research Personnel; Retina; Retinal Cone; Retinal Degeneration; Rod; Rod Outer Segments; Role; Specificity; Stains; Structure; Synaptic Vesicles; Techniques; Testing; Therapeutic; Training; Vertebrate Photoreceptors; Vesicle; Vision research; Work; base; conditional knockout; experimental study; fascinate; inhibitor; mouse model; personalized medicine; pharmacologic; photoreceptor degeneration; photoreceptor disc; recruit; retinal rods; reuptake; subretinal injection; tissue processing; transcriptome sequencing

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.

项目总结/摘要 光感受器外节是一种变性的初级纤毛，含有不断更新的盘状结构 膜。每天大约10%的外节被替换，外侧形成新的圆盘 节基部和旧盘在远端外节末端脱落。椎间盘形态发生始于外翻 睫状体质膜，其次是膜扁平和横向生长，并得出结论， 完全或部分圆盘封闭（分别在杆和锥中）。完整的磁盘存储涉及一个独特的 膜断裂事件将盘膜与周围的外节血浆分离 膜的这一事件的分子基础仍然完全未知。在其他细胞的内吞过程中， 在真核细胞中，膜断裂是由发动蛋白完成的，发动蛋白将膜囊泡释放到细胞中。 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 利用缺乏特异性发动蛋白同种型的敲除小鼠的超微结构分析， 研究亚细胞蛋白定位，对高度存在的内吞蛋白进行靶向蛋白质组学搜索 纯化的视杆外节制备物和使用同位素标记质量的绝对蛋白质定量 光谱法初步实验验证了这些方法的可行性。总的来说， 提出的研究将使我们更好地了解椎间盘形态发生是如何在分子水平上进行的。 水平，这是高度相关的了解这些迷人的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。