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: 7790177
• 负责人: Melissa Rolls
• 金额: $ 25.27万
• 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/7790177
• 关键词: Area; Axon; Biological Assay; Biological Models; Cell Polarity; Cells; Centrosome; Cytoskeleton; Dendrites; Distal; Drosophila genus; Exhibits; Foundations; Genetic; Golgi Apparatus; Growth; 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; public health relevance; 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. PUBLIC HEALTH RELEVANCE: Microtubules are the tracks for long-range cellular transport, and they are particularly important for the function of elongated neuronal cells, which have specific arrangements of microtubules in axons and dendrites. We will identify molecular mechanisms required for organization of microtubules using Drosophila neurons as a simple, but extremely powerful, model system. Our results will form a foundation for understanding neurological diseases ranging from motor neuron disease to Williams syndrome, as they result from perturbations in microtubule organization or trafficking.

描述（由申请人提供）：神经元是极端极化的细胞，这种极性对其功能至关重要。树突接收信号，轴突发送信号。轴突和树突之间最基本的差异之一是微管（MT）细胞骨架的极性，这可能是它们重要功能差异的基础。由于MT具有由马达蛋白读取的内在极性，MT极性对于极化神经元运输可能是极其重要的。然而，控制神经元MT极性的机制知之甚少。我们将使用一个简单的果蝇模型系统来研究这个问题。 在所有系统中，轴突MT的正端朝向细胞体的远端（正端向外）。树突的区别在于存在负端出MT。在培养的哺乳动物神经元中，树突状MT具有混合极性。但在果蝇体内，也许在哺乳动物神经元中，树突状MT具有与轴突相反的基本上一致的极性（负端出）。在这个建议中，我们将集中在两个特别是欠研究的神经元MT极性方面：建立一个统一的负端出树突状MT阵列，并在轴突的分支区域的MT的组织。 由于没有机制的研究，均匀负端出树突状MT已经进行，我们开始了我们的研究与密切观察树突状MT在体内。这使我们能够假设MT生长必须定向在树突中以保持均匀的极性。我们现在已经证实了这一假设，并确定KIF3是负端出极性所需的定向MT生长的关键参与者。在这项提案中，我们将建立在这个新的想法，定向MT的增长，通过识别蛋白质，使KIF3与不断增长的MT相互作用，并确定在树突它的行为。 除了继续研究树突MT极性的维持外，我们将研究如何通过关注负端来建立负端输出极性。目前尚不清楚树突状MT负末端是否集中在已知的微管组织中心（MTOC），例如高尔基复合体。我们将通过从树突中去除已知的MTOC并测定MT组织来研究它们的作用。我们还将确定在树突中产生负末端的途径：仅成核，或切断现有的微管。确定负责制造负末端的途径对于理解负末端MT阵列是如何产生和控制的至关重要。 在建立了体内研究神经元微管极性的测定方法后，我们将把我们的分析扩展到我们尚未研究过的细胞区域：轴突的远端分支区域。远端轴突中精确的MT组织对于突触功能可能是极其重要的。 拟议的研究将提供主要的洞察机制，控制远程神经元运输的轨道。通过集中研究缺乏研究的树突和远端轴突，我们将产生最大的影响。公共卫生关系：微管是长距离细胞运输的轨道，它们对于细长神经元细胞的功能特别重要，这些细胞在轴突和树突中具有特定的微管排列。我们将使用果蝇神经元作为一个简单但非常强大的模型系统来确定微管组织所需的分子机制。我们的研究结果将为理解从运动神经元疾病到威廉姆斯综合征的神经系统疾病奠定基础，因为它们是由微管组织或运输的扰动引起的。