High-throughput screen for autophagy induction in cultured Drosophila myocytes

描述(申请人提供)：自噬是真核细胞中细胞器大量降解的一种进化保守的机制。在酵母中，这一过程通过在饥饿期间循环营养物质来帮助维持细胞的活力。高等生物在广泛的细胞、发育和疾病环境中动员自噬，包括程序性细胞死亡、对入侵病原体的反应以及几种人类肌肉和神经退行性疾病。此外，在很大比例的散发性乳腺癌和卵巢癌中，自噬基因Beclin 1被缺失。虽然基本的细胞表型已经很好地描述了，一些保守的分子成分已经被确定，但关于自噬的调控还知之甚少。胰岛素和Tor信号在抑制自噬诱导中起着关键作用。然而，这些途径和自噬机制之间的分子联系是一个谜。我的目标是使用原代细胞培养系统和果蝇强大的遗传学来识别自噬途径的新成分。具体目标和实验设计如下：(1)建立果蝇心肌细胞自噬诱导和检测方法。在用不同水平的类固醇激素和/或雷帕霉素处理的细胞中，将监测ATG8-GFP和溶酶追踪器的表达和定位。这些处理将针对后续筛选进行优化。(2)在培养细胞中进行基于RNAi的自噬诱导基因筛选。表达Atg8-GFP的细胞将沐浴在双链RNA中，以敲除特定的基因。在激素和/或雷帕霉素处理后，将通过Atg8-GFP的定位和表达来检测自噬小体的形成。(3)使用Perrimon实验室开发的高质量转基因RNAi文库，在体内验证肌肉和脂肪体的屏幕命中。针对选定的HITS的RNAi发夹将使用Gal4/UAS系统在幼虫肌肉和脂肪体中特异性表达，并通过Atg8-GFP和LysoTracker检测自噬小体的形成。这些研究将为果蝇的自噬提供全面的全基因组分析。识别参与果蝇肌肉细胞自噬的基因将有助于深入了解人类肌肉萎缩和表现出过度自噬的肌肉疾病的病理机制

DESCRIPTION (provided by applicant): Autophagy is an evolutionarily conserved mechanism in eukaryotic cells for the bulk degradation of organelles. In yeast, this process helps to maintain cell viability by recycling nutrients during periods of starvation. Higher organisms mobilize autophagy in a wide variety of cellular, developmental, and disease contexts, including programmed cell death, the response to invading pathogens, and several human muscular and neurodegenerative disorders. Furthermore, the autophagy gene Beclin 1 is deleted in a large percentage of sporadic breast and ovarian cancers. Although the basic cellular phenotype is well characterized, and some conserved molecular components have been identified, there is little known about the regulation of autophagy. Insulin and Tor signaling play critical roles in suppressing autophagy induction. However, the molecular link between these pathways and the autophagic machinery is a mystery. My goal is to use a primary cell culture system, and the powerful genetics of Drosophila, to identify new components of the autophagy pathway. The specific aims and experimental design are as follows: (1) Establish an assay for the induction and detection of autophagy in cultured Drosophila myocytes. Atg8- GFP and lysotracker expression and localization will be monitored in cells treated with varying levels of steroid hormones and/or rapamycin. The treatments will be optimized for the subsequent screen. (2) Conduct an RNAi-based screen for genes involved in autophagy induction in the cultured cells. Cells expressing Atg8-GFP will be bathed in double-stranded RNAs to knockdown specific genes. Following hormone and/or rapamycin treatment, autophagosome formation will be assayed by Atg8-GFP localization and expression. (3) in vivo validation of screen hits in both muscle and fat body using a high quality transgenic RNAi library developed in the Perrimon lab. RNAi hairpins targeting selected hits will be expressed specifically in larval muscle and fat-body using the Gal4/UAS system, and the formation of autophagosomes assayed by Atg8- GFP and lysotracker. These studies will provide a comprehensive, genome-wide analysis of autophagy in Drosophila. Identification of genes that participate in Drosophila muscle cell autophagy will provide insights into the pathology of human muscle wasting and muscular disorders that exhibit excessive autophagy