Phosphatidylinositol 3-Phosphate in the Regulation of Autophagic Membrane Remodeling

项目摘要/摘要 自噬是一种基本的细胞过程，介导溶酶体依赖的蛋白质，细胞器， 以及细胞内的病原体。自噬功能障碍与许多疾病有关，包括癌症， 神经变性、糖尿病和慢性感染。更好地阐明其分子机制 自噬可能会激发治疗这些疾病的新方法。自噬开始发生在 内质网(ER)亚区富含磷脂酰肌醇3-磷酸(PI3P)。自噬是 当Vps34耗尽或失活时被阻断，磷脂酰肌醇(PI)3-激酶负责PI3P 自噬过程中的合成。然而，PI3P如何在膜重塑事件中起到至关重要的作用 自噬小体的生物发生仍然难以捉摸。关于PI3P的合成和周转情况也有很多未知之处 在自噬起始点精心策划的。谢博士最近发现军团菌PI 3-激酶MavQ 在内质网上产生PI3P，并驱动膜重塑。他还发现，MavQ与 军团菌PI 3-磷酸酶SidP时空调控ER亚区PI3P水平，诱导 囊泡/小管萌发。这种病理过程与自噬的启动有惊人的相似之处 促使谢博士剖析PI3P为自噬细胞膜带来的分子和物理因素 改建。此外，目前解决这一问题的技术障碍基本上可以通过使用 MavQ和SIDP作为工具。在谢博士提出的研究中，Aim 1将开发光遗传方法来控制 在细胞中产生PI3P，并利用活细胞确定PI3P如何调节自噬细胞膜重构 成像和系统性遗传扰动。AIM 2将结合体外生物物理分析、光遗传学 控制和定量成像，以阐明PI3P结构域的形成如何导致膜重构。目标 3将在体外重建反应-扩散系统，并使用该系统来确定PI3-激酶和 磷酸酶驱动富含PI3P的膜亚域的形成。谢博士的职业目标是成为 在细胞生物物理学和膜生物学方面处于领先地位，专注于生理和病理过程 涉及膜重塑。颁奖期间的培训将使他准备好领导一家独立的 研究小组使用细胞生物学、生化和生物物理方法来了解 自噬细胞膜重塑的基础。德克萨斯大学西南医学中心提供良好的环境 以帮助谢博士提出的研究和职业发展。谢长廷还成立了一个顾问委员会 由具有互补研究专长的顶尖科学家组成。在他们的指导下，谢博士将 接受必要的研究培训，如准备和使用各种模型膜系统，以及 在获奖期间进一步发展专业技能。这些都将大大促进谢博士的 转变为一名独立的调查员，并确保他未来的成功。

PROJECT SUMMARY/ABSTRACT Autophagy is a fundamental cellular process mediating lysosome-dependent degradation of proteins, organelles, and intracellular pathogens. Dysfunction of autophagy is associated with many diseases, including cancer, neurodegeneration, diabetes, and chronic infections. Better elucidation of the molecular mechanisms for autophagy may inspire new therapeutic approaches to these diseases. Autophagy initiation occurs at endoplasmic reticulum (ER) subdomains enriched with phosphatidylinositol 3-phosphate (PI3P). Autophagy is blocked when depleting or inactivating VPS34, the phosphatidylinositol (PI) 3-kinase responsible for PI3P synthesis during autophagy. Nevertheless, how PI3P contributes to membrane remodeling events crucial for autophagosome biogenesis remains elusive. Much is also unknown about how PI3P synthesis and turnover are orchestrated at autophagy initiation sites. Dr. Hsieh recently discovered that the Legionella PI 3-kinase MavQ generates PI3P on the ER and drives membrane remodeling. He also found that MavQ is coupled with the Legionella PI 3-phosphatase SidP to spatiotemporally modulate PI3P levels at ER subdomains, inducing vesicle/tubule budding. The striking similarity between this pathological process and autophagy initiation prompts Dr. Hsieh to dissect the molecular and physical factors that PI3P brings for autophagic membrane remodeling. Moreover, the current technical hurdles in tackling this question can mostly be cleared by using MavQ and SidP as tools. In Dr. Hsieh’s proposed research, Aim 1 will develop optogenetic methods to control PI3P generation in the cell and determine how PI3P regulates autophagic membrane remodeling using live-cell imaging and systematic genetic perturbations. Aim 2 will combine in vitro biophysical assays, optogenetic control, and quantitative imaging to elucidate how PI3P domain formation leads to membrane remodeling. Aim 3 will reconstitute a reaction-diffusion system in vitro and use this system to determine how PI 3-kinases and phosphatases drive the formation of PI3P-enriched membrane subdomains. Dr. Hsieh’s career goal is to become a leader in cell biophysics and membrane biology, focusing on physiological and pathological processes involving membrane remodeling. Training during the award period will prepare him to lead an independent research group using cell biological, biochemical, and biophysical approaches to understand the mechanistic basis of autophagic membrane remodeling. UT Southwestern Medical Center provides an excellent environment to aid Dr. Hsieh’s proposed research and career development. Dr. Hsieh has also set up an advisory committee consisting of leading scientists with complementary research expertise. Under their guidance, Dr. Hsieh will receive the necessary research training, such as preparing and using various model membrane systems, and further develop professional skills during the award period. These will significantly facilitate Dr. Hsieh's transition into an independent investigator and ensure his future success.