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High-throughput screen for autophagy induction in cultured Drosophila myocytes

培养果蝇肌细胞自噬诱导的高通量筛选

基本信息

批准号：
7609107
负责人：
JONATHAN D ZIRIN
金额：
$ 5.01万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2010-03-31
项目状态：
已结题

项目摘要

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

描述（由申请人提供）：自噬是真核细胞中一种进化上保守的机制，用于细胞器的大量降解。在酵母中，这个过程通过在饥饿期间回收营养物质来帮助维持细胞活力。高等生物在多种细胞、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，自噬基因 Beclin 1 在大部分散发性乳腺癌和卵巢癌中被删除。尽管基本的细胞表型已得到很好的表征，并且一些保守的分子成分也已被鉴定，但人们对自噬的调节知之甚少。胰岛素和 Tor 信号传导在抑制自噬诱导中发挥着关键作用。然而，这些途径和自噬机制之间的分子联系仍然是个谜。我的目标是利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组成部分。具体目的和实验设计如下：（1）建立培养果蝇肌细胞自噬诱导和检测的方法。将在用不同水平的类固醇激素和/或雷帕霉素处理的细胞中监测Atg8-GFP和lysotracker的表达和定位。治疗将针对后续筛选进行优化。 (2) 对培养细胞中参与自噬诱导的基因进行基于RNAi的筛选。表达 Atg8-GFP 的细胞将浸泡在双链 RNA 中以敲低特定基因。激素和/或雷帕霉素处理后，将通过 Atg8-GFP 定位和表达来测定自噬体形成。 (3) 使用 Perrimon 实验室开发的高质量转基因 RNAi 文库对肌肉和脂肪体中的筛选命中进行体内验证。使用 Gal4/UAS 系统，针对选定命中的 RNAi 发夹将在幼虫肌肉和脂肪体中特异性表达，并通过 Atg8-GFP 和 lysotracker 分析自噬体的形成。这些研究将为果蝇的自噬提供全面的全基因组分析。鉴定参与果蝇肌肉细胞自噬的基因将为了解人类肌肉萎缩和表现出过度自噬的肌肉疾病的病理学提供见解

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles.

DOI：
10.1016/j.ydbio.2013.08.029
发表时间：
2013-11-15
期刊：
DEVELOPMENTAL BIOLOGY
影响因子：
2.7
作者：
Zirin, Jonathan;Cheng, Daojun;Dhanyasi, Nagaraju;Cho, Julio;Dura, Jean-Maurice;VijayRaghavan, Krishnaswamy;Perrimon, Norbert
通讯作者：
Perrimon, Norbert

Primary cell cultures from Drosophila gastrula embryos.

来自果蝇原肠胚胚胎的原代细胞培养物。