Genetic Analysis of Dendritic Targeting

描述(由申请人提供) 像极化的上皮细胞一样，神经元在功能上分为两个 神经元的不同隔室和轴突和树突执行 有着截然不同的功能。大量的工作已经证实了 另外一组不同的蛋白质被选择性地分割成 这些隔间。很明显，这些蛋白质的定位是 对于细胞的正常运行和信息在 神经系统。直觉上，这样的过程应该需要一个分类系统来 识别特定细胞内目的地的蛋白质，如 树枝状的隔间，一个运载它们的运输机，最后，一个 将蛋白质保留在各自作用位置的机制。 这是当代细胞神经生物学中的一个重要问题 机械性的理解是相当有限的。此协作项目的目标是 项目是识别蛋白质优先排序所需的基因，并 囊泡到达神经元的树突室。为了实现这一目标 所述的目标，树突分选和运输的活检测将是 利用标记的绿色荧光蛋白(GFP)在果蝇中开发 转铁蛋白受体(TFR)和果蝇同系物(ARD，ALS) 烟碱型乙酰胆碱受体(NAChR)蛋白的A亚单位。这些GFP 标记蛋白将在胚胎和幼虫神经系统中表达 它们的亚细胞定位将在活体和 固定组织制剂。还建立了一套荧光视频显微镜 由于将使用共聚焦显微镜技术来观察这种分选 进程。一旦确定，该分析将被用于表征各种已知的 苍蝇基因组的突变体，并筛选将影响 TfR/ARD/ALS-GFP蛋白的树突状靶向。预计，在 在为期三年的实验结束后，这些实验将产生一份清单 参与TFR、ARD和ALS树突状分选的候选蛋白质 果蝇。此外，它还将建立一种可遗传测试的细胞 活体动物模型树突状细胞蛋白分选检测系统。建议数 该项目将与印度TIFR的Krishanu Ray博士合作执行， 这项研究将主要在印度进行，作为#年国家卫生研究院补助金的延期。 罗尔通用汽车35252。

DESCRIPTION (provided by applicant) 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.