Mechanisms of TOR-dependent control of autophagy in Drosophila

项目摘要 该项目的长期目标是了解自噬的调节和生理作用， 蛋白质、细胞器和大量细胞质被隔离在自噬囊泡内的过程， 递送至溶酶体进行降解。这个过程在细胞中扮演着几个不同的重要角色， 回收老化或损坏的组件，以提供营养源以应对饥饿，并且在一些实施例中， 启动细胞死亡的病例。这些细胞功能是自噬作用日益受到重视的基础 对广泛的人类疾病和正常的生理过程，如衰老。基本 关于自噬的问题仍然有待解决，包括自噬的启动是如何被调节的。 营养物质和其他信号，自噬囊泡如何成熟以能够降解，以及如何 自噬的速率维持在细胞存活的最佳稳态水平。令人兴奋的前景 利用自噬作为一种治疗工具需要更好地理解这些基本原理。 拟议中的研究将在果蝇系统中使用遗传、生物化学和基于成像的方法 以帮助在完整的有机体中定义自噬调节机制。我们以前的 研究确定了雷帕霉素靶点（TOR）的许多关键靶点和机制 在有利的营养条件下，自噬通路抑制自噬。目前的建议旨在 理解1）TOR信号传导如何与其他营养和发育线索（如cAMP）整合， 依赖性蛋白激酶A来控制自噬诱导的初始步骤; 2）测试假设， 描述了TOR信号控制自噬体与溶酶体融合的潜在机制 以及随后的自体溶酶体酸化;以及3）破译反馈机制， 提供限制自噬活性的速率和水平的稳态控制。 本提案中的实验之所以被选中，是因为它们可能对关键问题产生很大影响 对自噬领域很重要。该提案利用最近开发的试剂，包括 几种自噬相关（Atg）基因的敲除、自噬活性的体内标记物和新方法 细胞克隆的基因操作。从果蝇自噬研究中获得的信息将提供 这是对基于哺乳动物细胞培养的研究的重要的全动物补充。

PROJECT SUMMARY The long term goal of this project is to understand the regulation and physiological roles of autophagy, a process by which proteins, organelles and bulk cytoplasm are sequestered within autophagic vesicles and delivered to the lysosome for degradation. This process plays several distinct, vital roles in the cell, acting to recycle aged or damaged components, to provide a source of nutrients in response to starvation, and in some cases to initiate cell death. These cellular functions underlie a growing appreciation for the impact of autophagy on a broad range of human illnesses and on normal physiological processes such as aging. Fundamental questions regarding autophagy remain to be addressed, including how autophagy initiation is regulated by nutrients and other signals, how autophagic vesicles mature to become competent for degradation, and how rates of autophagy are maintained at homeostatic levels optimal for cell survival. The exciting prospect of harnessing autophagy as a therapeutic tool awaits better understanding of these basic principals. The proposed studies will use genetic, biochemical and imaging-based approaches in the Drosophila system to help define mechanisms of autophagy regulation in the context of in an intact organism. Our previous studies identified a number of key targets and mechanisms through which the Target of Rapamycin (TOR) pathway inhibits autophagy in response to favorable nutrient conditions. The current proposal seeks to understand 1) how TOR signaling is integrated with other nutrient and developmental cues such as cAMP- dependent protein kinase A to control the initial steps of autophagy induction; 2) to test hypotheses that describe potential mechanisms by which TOR signaling controls the fusion of autophagosomes with lysosomes and the subsequent acidification of the autolysosome; and 3) to decipher the feedback mechanisms that provide homeostatic control limiting the rates and levels of autophagic activity. Experiments in this proposal were selected for their likelihood of having a high impact on key questions important to the field of autophagy. This proposal makes use of recently developed reagents including knockouts of several autophagy-related (Atg) genes, in vivo markers of autophagic activity, and novel methods of genetic manipulation in cell clones. Information gained from studies of autophagy in Drosophila will provide an important whole-animal complement to mammalian cell culture-based studies.