| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
The present project aimed to draw the route map of vesicle transport in the Drosophila photoreceptor cell. The map would provide a basis for the elucidation of the physiological role and the molecular mechanism of vesicle transport in the living neurons. Various kinds of Drosophila mutants that express the dominant negative versions of several Rab proteins were constructed and their defects in cell structure and protein transport were investigated. These analyses provided an overview of vesicle transport in the photoreceptor cell, and suggested that the mutation in RAB2, RAB5, and SHIBIRE would be useful to investigate the vesicle transport to dendrites, to axons, and in the synaptic termini, respectively.
To induce strong inhibitory effects of these mutant proteins, we developed a novel expression system for transport marker proteins. This system utilizes the Tet-on promoter, which is activated by the dietary tetracycline. Using this system, we could express the marker protein by feeding the flies with tetracycline under the sufficient expression of mutant Rab proteins with the heat-shock promoter. Furthermore, the marker protein for axon and synaptic termini (rat synaptophysin) was tagged with GFP to enable the real-time tracing of the transport vesicles. Using the fluorescence and electron microscopies, the analysis of vesicle transport pathways for synaptic termini is now in progress.
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