Mechanisms of TOR-dependent control of autophagy in Drosophila

项目总结 这个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小泡中的过程 被送到溶酶体进行降解。这个过程在细胞中扮演着几个不同的、至关重要的角色，作用于 回收老化或损坏的成分，以提供一种营养来源，以应对饥饿，并在一些 引发细胞死亡的病例。这些细胞功能是人们日益认识到自噬的影响的基础 研究范围广泛的人类疾病和衰老等正常生理过程。基本原理 关于自噬的问题仍有待解决，包括自噬的启动是如何由 营养物质和其他信号，自噬小泡如何成熟以适应降解，以及如何 自噬的速率维持在对细胞存活最有利的动态平衡水平。激动人心的前景 利用自噬作为一种治疗工具，有待于更好地理解这些基本原理。 拟议的研究将在果蝇系统中使用基于遗传、生化和成像的方法 在一个完整的生物体中帮助定义自噬调节的机制。我们以前的 研究确定了雷帕霉素靶点(TOR)的一些关键靶点和机制 途径抑制自噬，以响应有利的营养条件。目前的提案旨在 了解TOR信号如何与其他营养和发育信号整合，如cAMP- 依赖蛋白激酶A来控制自噬诱导的初始步骤；2)检验假设 描述TOR信号控制自噬小体和溶酶体融合的潜在机制 和随后的自溶酶体的酸化；以及3)破译反馈机制， 提供动态平衡控制，限制自噬活动的速率和水平。 这项提议中的实验是根据它们对关键问题产生高度影响的可能性而选择的 对自噬领域很重要。这项建议使用了最近开发的试剂，包括 几个自噬相关(ATG)基因的敲除、自噬活性的体内标记和新方法 在细胞克隆中进行基因操作。从果蝇自噬研究中获得的信息将提供 对以哺乳动物细胞培养为基础的研究的重要补充。

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.