Genetic Analysis of Dendritic Targeting

树突靶向的遗传分析

• 批准号: 6691093
• 负责人: Lawrence S. Goldstein
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-14 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691093
• 关键词: Drosophilidae; India; acetylcholine; arthropod genetics; axon; cell sorting; chimeric proteins; confocal scanning microscopy; cooperative study; dendrites; epithelium; gene targeting; green fluorescent proteins; intracellular transport; mutant; neural information processing; neurons; neurophysiology; nicotinic receptors; protein localization; synapses; tissue /cell culture; transferrin receptor; video microscopy

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.

描述（由申请人提供） 像极化上皮细胞一样，神经元在功能上分为两个 神经元的不同隔室以及轴突和树突进行 截然不同的功能。大量工作已经建立了存在 选择性分配到的其他不同不同的蛋白质集 这些隔间。显然，这些蛋白质的定位是 对于细胞正确运行和信息流的至关重要 神经系统。直观地，这样的过程应需要一个分类系统才能 识别某个细胞内目的地（例如）的蛋白质 树突隔室，运输它们的运输机，最后是 将保留蛋白质在各自作用地点的机制。 这是当代细胞神经生物学中的一个重要问题，其中 机械理解非常有限。这个合作的目标 项目是确定优先分类蛋白质所需的基因和 囊泡到神经元的树突室。为了实现 陈述的目标，用于树突状和运输的现场测定法 使用标记的绿色荧光蛋白（GFP）在果蝇中开发 tranferrin受体（TFR）和果蝇同源物（ARD，ALS） 烟碱乙酰胆碱受体（NACHR）蛋白的A-亚基。这些GFP 标记的蛋白质将在胚胎和幼虫神经系统中表达 它们的亚细胞本地化将在现场和 固定组织制剂。还建立了落叶视频显微镜 由于共聚焦微观技术将用于观察此分类 过程。一旦建立，该测定将用于表征各种已知的 苍蝇基因组的突变体，并筛选出会影响的新突变体 TFR/ARD/ALS-GFP蛋白的树突状靶向。预计，在 在三年期结束时，这些实验将产生 参与TFR，ARD和ALS的树突状排序的候选蛋白 果蝇。此外，它还将建立一个可遗传测试的细胞 活动物模型中的树突状蛋白质分类的测定系统。提议 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NIH赠款＃的扩展 ROL GM 35252。