Using Drosophila Neurons to Identify Mechanisms that Control Microtubule Polarity

使用果蝇神经元识别控制微管极性的机制

• 批准号: 8461178
• 负责人: Melissa Rolls
• 金额: $ 26.68万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461178
• 关键词: Area; Axon; Biological Assay; Biological Models; Cell Polarity; Cells; Centrosome; Cytoskeleton; Dendrites; Distal; Drosophila genus; Exhibits; Foundations; Genetic; Golgi Apparatus; Growth; Health; Image; Kinesin; Maintenance; Microtubule Proteins; Microtubule-Organizing Center; Microtubules; Minus End of the Microtubule; Mitochondria; Molecular; Motor; Motor Activity; Motor Neuron Disease; Neuromuscular Junction; Neurons; Organelles; Pathway interactions; Play; Plus End of the Microtubule; Positioning Attribute; Proteins; RNA Interference; Reading; Relative (related person); Role; Signal Transduction; Site; Source; Synapses; System; Testing; Tubulin; Williams Syndrome; Work; base; in vivo; insight; mutant; nervous system disorder; neuronal cell body; neuronal transport; novel; polarized cell; presynaptic; synaptic function; tool; trafficking

DESCRIPTION (provided by applicant): Neurons are extremely polarized cells, and this polarity is crucial for their function. Dendrites receive signals and axons send them. One of the most basic differences between axons and dendrites, that could be the foundation for their important functional differences, is polarity of the microtubule (MT) cytoskeleton. As MTs have intrinsic polarity that is read by motor proteins, MT polarity is likely to be extremely important for polarized neuronal trafficking. However, mechanisms that control neuronal MT polarity are poorly understood. We will use a simple Drosophila model system to study this problem. In all systems axonal MTs are oriented with plus ends distal to the cell body (plus-end-out). Dendrites are distinguished by the presence of minus-end-out MTs. In cultured mammalian neurons, dendritic MTs have mixed polarity. But in vivo in Drosophila, and perhaps in mammalian neurons, dendritic MTs have essentially uniform polarity that is opposite of axons (minus-end-out). In this proposal we will focus on two particularly understudied aspects of neuronal MT polarity: establishment of a uniform minus-end-out dendritic MT array, and the organization of MTs in branched regions of axons. As no mechanistic studies on uniform minus-end-out dendritic MTs had been performed, we began our studies with close observation of dendritic MTs in vivo. This allowed us to hypothesize that MT growth must be directed in dendrites to maintain uniform polarity. We have now confirmed this hypothesis and identified KIF3 as a key player in directed MT growth that is required for minus-end-out polarity. In this proposal we will build upon this novel idea of directed MT growth by identifying proteins that allow KIF3 to interact with growing MTs and by determining where in dendrites it acts. In addition to continuing to study maintenance of dendritic MT polarity, we will investigate how minus- end-out polarity is established by focusing on the minus ends. It is not known whether dendritic MT minus ends are focused at a known microtubule organizing center (MTOC), for example the Golgi complex. We will investigate the role of known MTOCs by removing them from dendrites and assaying MT organization. We will also identify the pathways that generate minus ends in dendrites: nucleation only, or severing existing microtubules. Identifying the pathway responsible for making minus ends is crucial for understanding how a minus-end-out MT array is generated and controlled. Having established assays to study neuronal microtubule polarity in vivo, we will extend our analysis to a region of the cell which we have not yet examined: the distal branched region of axons. Precise MT organization in distal axons could be extremely important for synaptic function. The proposed studies will provide major insight into mechanisms that control the tracks for long-range neuronal transport. By focusing on poorly studied dendrites and distal axons we will have maximum impact.

描述（由申请人提供）：神经元是极化的细胞，这种极性对其功能至关重要。树突接收信号，轴突发送。轴突和树突之间最基本的差异之一，这可能是其重要功能差异的基础，是微管（MT）细胞骨架的极性。由于MT具有由运动蛋白读取的内在极性，因此MT极性对于极化神经元运输可能非常重要。但是，控制神经元MT极性的机制知之甚少。我们将使用简单的果蝇模型系统来研究此问题。 在所有系统中，轴突MT均在细胞体远端（加末端）端加的末端定向。树突的区分是存在减去端的MT。在培养的哺乳动物神经元中，树突状MT具有混合极性。但是在果蝇中的体内，也许在哺乳动物的神经元中，树突状MT具有与轴突相反的基本均匀极性（负末端）。在此提案中，我们将重点关注神经元MT极性的两个特别研究的方面：建立一个均匀的负端的树突状MT阵列，以及在轴突的分支区域内的MTS组织。 由于尚未对均匀的减去树突状MT进行均匀的机械研究，因此我们在体内对树突状MT的仔细观察开始了研究。这使我们能够假设必须在树突中定向MT生长以保持极性均匀。现在，我们已经证实了这一假设，并确定KIF3是负极性极性所需的指向MT增长的关键参与者。在这项建议中，我们将通过识别允许KIF3与成长的MT相互作用并确定在树突中的位置来基于这种定向MT生长的新思想。 除了继续研究树突状MT极性的维持外，我们还将研究如何通过关注减去端来确定减去最终的极性。尚不清楚树突状MT负末端是否集中在已知的微管组织中心（MTOC），例如高尔基体配合物。我们将通过将其从树突中删除并分析MT组织来研究已知的MTOC的作用。我们还将确定在树突中产生负端的途径：仅成核或切断现有微管。确定负责减去末端的途径对于理解如何生成和控制减去端的MT阵列至关重要。 在研究体内神经元微管极性的建立测定中，我们将把分析扩展到尚未检查的细胞区域：轴突的远端分支区域。远端轴突中的精确MT组织对于突触功能可能非常重要。 拟议的研究将为控制远程神经元转运的轨道的机制提供重大洞察力。通过专注于研究良好的树突和远端轴突，我们将产生最大的影响。