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A light-inducible protein trapping system for studying cellular dynamics in Drosophila

用于研究果蝇细胞动力学的光诱导蛋白捕获系统

基本信息

批准号：
9387859
负责人：
Chun Han
金额：
$ 18.73万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9387859
关键词：
Adaptor Signaling Protein Affect Afferent Neurons Alpha Cell Animal Model Animals Behavior Biological Assay Biological Models Biological Process CRISPR/Cas technology Cells Communities Complex Darkness Dendrites Development Diffuse Disease Drosophila genus Drosophila inturned protein Effectiveness Engineering Environment Epithelial Cells Etiology Exhibits Family Future Gene Expression Gene Expression Regulation Goals Growth Human Development Image Individual Intracellular Membranes Knowledge Larva Light Location Measures Mediating Membrane Methods Molecular Morphogenesis Morphology Neurodegenerative Disorders Neurons Outcome Pattern Problem Solving Process Protein Inhibition Proteins Publishing RNA Interference Reagent Regulation Research Resources Role System Tauopathies Techniques Testing Thinness Time cell growth regulation cell type design gene function genome editing human disease in vivo innovation knockout gene light gated loss of function nervous system disorder novel optogenetics prevent protein degradation spatiotemporal success temporal measurement tool trafficking

项目摘要

PROJECT SUMMARY/ABSTRACT Spatiotemporal regulation of cellular dynamics is fundamental to animal development. A thorough understanding of developmental mechanisms requires approaches that permit in vivo perturbation of endogenous proteins in a spatially and temporally controlled manner. Such approaches are particularly important for understanding neuronal morphogenesis and the etiology of neurological disorders, as neurons usually occupy broad spatial domains and exhibit diverse growth dynamics at different dendritic and axonal branches. However, existing techniques for manipulating endogenous gene function in animal models, such as gene knockout, RNAi, and protein degradation, affect the whole cell and require time to take effect, and therefore lack the spatial and temporal resolution needed for dissecting dynamic growth behaviors of neurons at the subcellular level. We propose to develop an optogenetic system in Drosophila to enable rapid inhibition of endogenous proteins in precisely defined regions of cells. This will be achieved by light-inducible trapping of GFP-tagged endogenous proteins in large protein clusters. Such a strategy should be effective for inhibiting proteins whose functions require specific subcellular locations. Our system will be tested in sensory neurons and epidermal epithelial cells of Drosophila larvae, two cell types that are relevant to a broad range of human diseases. Several endogenously tagged proteins of diverse size, subcellular localization, and function will be first tested in a protein trapping assay. To validate the effectiveness of light-induced protein inhibition, the roles of Rab5 and Fry in dendrite morphogenesis of Drosophila sensory neurons will be investigated using GFP- tagged endogenous proteins. Rab5 and Fry are important for dendritic patterning. However, it is unknown whether they control dendritic growth by locally regulating dendritic dynamics or by globally modulating gene expression. By locally inhibiting Rab5 and Fry in individual dendritic branches, it will be determined whether they regulate local dendritic dynamics. The primary goal of this project is to establish the first Drosophila light- inducible loss-of-function system for investigating the local and moment-to-moment function of endogenous proteins in vivo. The increasing number of endogenous proteins tagged by GFP in Drosophila and the convenience of CRISPR/Cas9-mediated genome editing make our approach applicable to the study of a wide variety of proteins, biological processes, and human diseases. Our approach should also be applicable in other model organisms.

项目总结/文摘