Using Drosophila Neurons to Identify Mechanisms that Control Microtubule Polarity

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 具有由运动蛋白读取的内在极性，因此 MT 极性可能对于极化神经元运输极其重要。然而，人们对控制神经元 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 均以远离细胞体的正端（正端向外）定向。树突的特征是存在负端 MT。在培养的哺乳动物神经元中，树突状微管具有混合极性。但在果蝇体内，也许在哺乳动物神经元中，树突状微管具有与轴突相反的基本一致的极性（负端）。在本提案中，我们将重点关注神经元 MT 极性的两个特别未充分研究的方面：建立统一的负端树突 MT 阵列，以及轴突分支区域中 MT 的组织。 由于尚未对均匀负端树突状 MT 进行机理研究，因此我们从对体内树突状 MT 的密切观察开始我们的研究。这使我们能够假设 MT 生长必须定向于树突以保持均匀的极性。我们现在已经证实了这一假设，并确定 KIF3 是负端极性所需的定向 MT 生长的关键参与者。在本提案中，我们将基于定向 MT 生长的新想法，通过识别允许 KIF3 与生长中的 MT 相互作用的蛋白质，并确定它在树突中的作用位置。 除了继续研究树突 MT 极性的维持之外，我们还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 MT 负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 MTOC 并分析 MT 组织来研究已知 MTOC 的作用。我们还将确定在树突中产生负端的途径：仅成核，或切断现有的微管。识别产生负端的途径对于理解负端输出 MT 阵列是如何生成和控制的至关重要。 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检查的细胞区域：轴突的远端分支区域。远端轴突中精确的 MT 组织对于突触功能极其重要。 拟议的研究将为控制长程神经元运输轨迹的机制提供重要见解。通过关注研究不足的树突和远端轴突，我们将产生最大的影响。公共健康相关性：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，神经元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简单但极其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等神经系统疾病奠定基础，因为这些疾病是由微管组织或运输的扰动引起的。

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.