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.

Public Health Relevance

The ultimate understanding of human disease requires knowledge of how endogenous proteins act in time and space to control cellular dynamics in the native in vivo environment. This project will develop a novel method for manipulating endogenous proteins in a spatially and temporally controlled manner in a model organism, in the hope to apply it to diverse animal models for finely dissecting broad developmental and disease mechanisms. By investigating Rab5 and Fry with the new method, this study will likely yield results that can serve as a new basis for understanding neurodevelopmental and neurodegenerative diseases related to these two proteins.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21OD023824-01A1
Application #
9387859
Study Section
Molecular Neurogenetics Study Section (MNG)
Program Officer
Zou, Sige
Project Start
2017-08-15
Project End
2019-07-31
Budget Start
2017-08-15
Budget End
2018-07-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Cornell University
Department
Miscellaneous
Type
Organized Research Units
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850