Jonathan D. Zirin - US grants

[unreadable] 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 [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY ? TR&D3 Development of protein technologies for use in vivo in the Drosophila system has lagged behind development of molecular genetic technologies. In this project, we will develop protein-based technologies that complement and extend the type of studies that can be done in this exemplary model system. We will focus on development of proximity labeling, as facilitated by peroxidases or biotin ligases, as tools for in vivo proteomics analysis of specific subcellular compartments and signaling. We will additionally develop a robust pipeline for isolation of nanobodies that can be used in diverse applications, including for in vivo expression of fusion proteins that facilitate visualization and functional blocking. Collaborators with interests in using the technologies to study cell biology, development, transcription control, and neuronal networks provide appropriate assays for iterative testing and improvement. Altogether, this project will help fill an important gap in the Drosophila toolbox that can help provide a more complete picture of functions at the subcellular, cellular, organ, and whole animal levels that would not be achievable in cell systems or using only molecular genetic tools.