Autophagosome closure by the ESCRT machinery

通过 ESRT 机制封闭自噬体

• 批准号: 10383918
• 负责人: HONG-GANG WANG
• 金额: $ 7.5万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-09-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10383918
• 关键词: ATP phosphohydrolase; Aging; Autophagocytosis; Autophagosome; Biogenesis; Biological Assay; Caenorhabditis elegans; Cell Membrane Permeability; Complex; Data; Defect; Drosophila genus; Endosomes; Event; Filament; Goals; Heart Diseases; Homeostasis; Immunity; Left; Libraries; Ligands; Lipids; Liver diseases; Lysosomes; Malignant Neoplasms; Mammals; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Monitor; Neck; Nerve Degeneration; Nuclear Envelope; Nutrient; Pathogenesis; Peptides; Phenotype; Phosphatidylethanolamine; Process; Proteins; Recycling; Reporter; Role; Small Interfering RNA; Sorting - Cell Movement; Starvation; Stress; Structure; Testing; Vesicle; base; human disease; novel; polymerization; recruit; response; screening; seal; tetramethylrhodamine

Project Summary/Abstract During macroautophagy (hereafter referred to as autophagy), a crescent-shaped membrane known as the phagophore elongates to engulf damaged cargo and seals to generate a double-membrane vesicle called the autophagosome. While much of the core machinery regulating autophagy induction and phagophore nucleation and elongation has been identified, the machinery for phagophore closure is largely unknown. The goal of this application is to define the molecular mechanism by which the phagophore seals to generate the completed autophagosome. Phagophore closure is a critical step in autophagy that is required for lysosomal fusion and efficient degradation and recycling of autophagic cargo. The sealing of phagophores has misguidedly been described as a membrane fusion event when the process of generating the inner and outer autophagosomal membrane actually requires membrane scission. Notably, the topology of phagophore closure is similar to ESCRT (endosomal sorting complexes required for transport)-mediated scission, in which the ESCRT machinery mediates membrane fission from within the membrane neck. While ESCRT defects accumulate immature autophagosomal structures in C. elegans, Drosophila and mammals, a molecular understanding for the phenotype remains unclear. Exploiting the topology of LC3 during autophagosome biogenesis, we established a novel HaloTag-LC3 based assay to differentiate phagophores, autophagosomes and autolysosomes. Using this assay, we have identified ESCRT-III as a candidate regulator of phagophore closure. We hypothesize that phagophore-associated factors assemble a novel adaptor for the recruitment and assembly of ESCRT-III filaments for phagophore closure. We will test our hypotheses in the following Specific Aims: (1) to define the core ESCRT machinery required for phagophore closure; (2) to identify the upstream factor(s) that serve as an autophagy-specific adaptor for ESCRT-III assembly during phagophore closure.

项目总结/摘要 在巨自噬（以下简称自噬）过程中，一种称为自噬的新月形膜形成。 吞噬细胞伸长以吞噬受损的货物和密封物， 自噬体虽然许多调节自噬诱导和吞噬细胞的核心机制 尽管已经鉴定了成核和伸长，但吞噬细胞闭合的机制在很大程度上是未知的。的 本申请的目的是定义吞噬体密封以产生 完成自噬体。吞噬细胞关闭是自噬的关键步骤， 自噬货物的融合、有效降解和回收。吞噬细胞的封闭 被描述为一个膜融合事件时，产生的内部和外部的过程， 自噬体膜实际上需要膜断裂。值得注意的是，吞噬细胞闭合的拓扑结构 类似于ESCRT（运输所需的内体分选复合物）介导的断裂，其中 ESCRT机制从膜颈内介导膜分裂。虽然ESCRT缺陷 在C.线虫、果蝇和哺乳动物， 对表型的理解仍不清楚。利用自噬体中LC 3的拓扑结构 生物发生，我们建立了一种新的HaloTag-LC 3为基础的分析，以区分吞噬细胞，自噬体 和自体溶酶体。使用该测定，我们已经确定ESCRT-III作为噬菌体的候选调节剂 结束我们假设吞噬细胞相关因子组装了一个新的适配器，用于招募和 用于吞噬细胞闭合的ESCRT-III细丝的组装。我们将在以下具体案例中检验我们的假设： 目的：（1）确定吞噬细胞关闭所需的核心ESCRT机制;（2）确定上游的ESCRT机制。 在吞噬细胞关闭期间作为ESCRT-III组装的自噬特异性衔接子的因子。