Molecular Mechanisms of Neurotransmitter Release: Analysis of in Vivo Protein Interactions in Synaptic Vesicle Trafficking

由于神经系统的细胞和分子很小，研究单个神经元内突触功能的分子机制是具有挑战性的。然而，精密显微镜和成像方法的发展使克服这一障碍成为可能。在这个项目中，荧光标记蛋白质的体内成像将被用来检测蛋白质-蛋白质相互作用和电信号在神经元之间传递的分子机制。这些研究将在果蝇身上进行，因为果蝇突触传递的功能机制与脊椎动物相似。该项目集成了两种强大的方法来研究电信号如何在神经元之间传递：神经系统中的电信号分析和荧光标记蛋白质的成像。表达在突触起作用的适当的荧光标记蛋白质对的果蝇菌株将被分析在单个活细胞中的结合相互作用。相互作用将使用荧光共振能量转移(FRET)来检测。对转基因果蝇品系的突触传递的电生理分析将被用来确认它们的功能正常。这一项目将产生广泛的影响。它将让一群来自不同文化的年轻研究人员参与一个需要高水平智力互动的合作研究和培训项目。该项目将为所有阶段的科学家提供机会，从高级研究助理到本科生，并将促进女性和少数民族在科学领域的发展。迄今为止产生的许多研究材料已经在世界各地的实验室传播用于相关的研究应用。最后，在这个项目中开发的材料和方法被整合到一门高级细胞和分子神经科学实验室课程中，为本科生提供深入接触当前神经科学研究方法的机会。

Investigating the molecular mechanisms of synaptic function within a single neuron is challenging because of the small size of the cells and molecules of the nervous system. However the development of sophisticated microscopy and imaging methods are making it possible to overcome this obstacle. In this project, in vivo imaging of fluorescently labeled proteins will be employed to examine the protein-protein interactions and molecular mechanisms by which electrical signals are transmitted among neurons. The studies will be done in Drosophila melanogaster, because the functional mechanisms of synaptic transmission in the fruit fly are similar to those of vertebrates. The project integrates two powerful approaches to examine how electrical signals are transmitted among neurons: analysis of electrical signaling in the nervous system and imaging of fluorescently labeled proteins. Drosophila strains expressing appropriate pairs of fluorescently labeled versions of proteins that function at synapses will be analyzed for binding interactions in single living cells. The interactions will be detected using Fluorescence Resonance Energy Transfer (FRET). Electrophysiological analysis of synaptic transmission in the transgenic fly strains will be used to confirm that they are functioning normally.This project will have a broad impact. It will engage a group of young investigators from several cultures in a collaborative research and training program requiring a high level of intellectual interaction. The project will provide opportunities for scientists at all stages, from senior research associate to undergraduate, and will promote the development of women and minorities in science. Many research materials generated thus far have already been disseminated for related research applications in laboratories throughout the world. Finally, the materials and methods developed in this project are integrated into an advanced cellular and molecular neuroscience laboratory course providing undergraduates with intensive exposure to current neuroscience research methods.