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: 8651497
• 负责人: Melissa Rolls
• 金额: $ 28.01万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8651497
• 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; 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; in vivo imaging; 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(申请人提供)：神经元是极端极化的细胞，这种极化对它们的功能至关重要。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固有极性，MT的极性对于极化神经元的运输可能是极其重要的。然而，控制神经元MT极性的机制却知之甚少。我们将使用一个简单的果蝇模型系统来研究这个问题。在所有的系统中，轴突MTs的方向是细胞体远侧的正端(正端向外)。树枝晶的区别在于存在负端向外的MT。在培养的哺乳动物神经元中，树突状MT具有混合的极性。但在果蝇体内，或许在哺乳动物神经元中，树突状MT具有与轴突相反的基本一致的极性(负端向外)。在这项提案中，我们将重点关注神经元MT极性的两个特别未被研究的方面：建立统一的负端向外树突MT阵列，以及在轴突的分支区域中MT的组织。由于还没有对均匀的负端向外树枝状MTS进行机理研究，我们的研究始于对树枝状MTS的体内密切观察。这使得我们可以假设，MT的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，并确认KIF3是MT定向生长的关键角色，这是负末端输出极性所必需的。在这项提案中，我们将通过识别允许KIF3与生长中的MT相互作用的蛋白质，并通过确定它在树突中的作用位置，来建立这种定向MT生长的新想法。除了继续研究树突MT极性的维持外，我们还将调查负末端是如何通过关注负端来建立的。目前尚不清楚树突状MT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。我们将通过将已知的MTOCs从树突中移除并分析MT组织来研究它们的作用。我们还将确定在树突中产生负端的途径：仅成核，或切断现有的微管。识别负责产生负端的路径对于理解负端向外MT阵列是如何产生和控制至关重要的。在建立了在体内研究神经元微管极性的方法后，我们将把我们的分析扩展到细胞的一个我们还没有检查过的区域：轴突的远端分支区域。远端轴突中MT的精确组织对突触功能可能是极其重要的。拟议的研究将为控制远程神经元运输轨迹的机制提供重要的洞察力。通过专注于研究较少的树突和远端轴突，我们将产生最大的影响。