Like polarized epithelial cells, neurons are divided into two functionally distinct compartments and the axon and dendrites of a neuron performs distinctly different functions. Considerable work has established the existence of additional distinct sets of proteins that are selectively partitioned into these compartments. It is obvious that localization of these proteins is critical for proper functioning of the cell and the flow of information in the nervous system. Intuitively such a process should require a sorting system to identify the proteins for a certain intracellular destinations like the dendritic compartment, a transport machine to carry them, and finally, a mechanism that will retain the proteins in their respective place of action. This is an important problem in contemporary Cellular Neurobiology in which mechanistic understanding is quite limited. The goal of this collaborative project is to identify genes needed for preferential sorting of proteins and vesicles to the dendritic compartment of neurons. In order to achieve the stated objective, a live assay for dendritic sorting and transport will be developed in Drosophila using the green fluorescent protein (GFP) tagged tranferrin receptor (TfR) and the Drosophila homologues (ARD, ALS) of the a-subunit of nicotinic acetylcholine receptor (nAChR) proteins. These GFP tagged proteins will be expressed in the embryonic and larval nervous system and their subcellular localization will be characterized in both the live and fixed tissue preparations. An epifluorescence video microscopic set up as well as the confocal microscopic techniques will be used to observe this sorting process. Once established the assay will be used to characterize various known mutants of the fly genome and also to screen for new mutants that will affect the dendritic targeting of TfR/ARD/ALS-GFP proteins. It is expected that, at the end of a three-year period, these experiments will yield a list of candidate proteins involved in the dendritic sorting of TfR, ARD and ALS in Drosophila. In addition, it will also establish a genetically testable cellular assay system for dendritic protein sorting in live animal models. The proposed project will be executed in collaboration with Dr. Krishanu Ray at TIFR, India, and the research will be done primarily in India as an extension of NIH grant # ROl GM 35252.
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