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

Cornell University, Ithaca NY

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Abstract

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.