The role of intracellular vesicle transport on the structural and functional development of neuron.

本项目旨在绘制果蝇感光细胞中囊泡运输的路线图。该图谱的建立为进一步阐明囊泡在神经元中的生理作用和分子机制提供了基础。构建了表达多种Rab蛋白显性负性版本的果蝇突变体，并研究了它们在细胞结构和蛋白质转运方面的缺陷。这些分析提供了光感受器细胞中的囊泡运输的概述，并建议在RAB 2，RAB 5和SHIBIRE中的突变将是有用的研究囊泡运输到树突，轴突，和在突触末端，分别。为了诱导这些突变蛋白的强烈抑制作用，我们开发了一种新的表达系统的运输标记蛋白。该系统利用Tet-on启动子，其由膳食四环素激活。利用该系统，我们可以通过给果蝇喂食四环素，在具有热激启动子的突变体Rab蛋白的充分表达下表达标记蛋白。此外，轴突和突触末端的标记蛋白（大鼠突触素）与GFP标记，使运输囊泡的实时跟踪。利用荧光显微镜和电子显微镜，突触末端的囊泡运输途径的分析正在进行中。

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.