Using Drosophila Neurons to Identify Mechanisms that Control Microtubule Polarity

描述（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区别之一是微管（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固有极性，因此MT极性可能对极化神经元运输非常重要。然而，控制神经元MT极性的机制尚不清楚。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的正端指向细胞体的远端（正端向外）。在培养的哺乳动物神经元中，树突的MTs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mt本质上具有与轴突相反的统一极性（负端向外）。在本提案中，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：建立一个统一的负端-外树突MT阵列，以及MT在轴突分支区域的组织。由于没有对均匀负端外树突状mt的机制进行研究，我们开始了我们的研究，密切观察树突状mt在体内。这允许我们假设MT生长必须在树突中定向以保持均匀的极性。我们现在已经证实了这一假设，并确定KIF3是定向MT生长的关键参与者，这是负端输出极性所必需的。在本提案中，我们将通过鉴定允许KIF3与生长的MT相互作用的蛋白质并确定其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，我们还将通过关注负端来研究负端外向极性是如何建立的。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的MTOCs并分析其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。确定负责制造负端的途径对于理解负端输出MT阵列是如何产生和控制的至关重要。已经建立了在体内研究神经元微管极性的方法，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。在远端轴突中精确的MT组织对突触功能可能非常重要。所提出的研究将为控制远距离神经元转运的机制提供重要的见解。通过专注于研究不足的树突和远端轴突，我们将有最大的影响。公共卫生相关性：微管是远距离细胞运输的轨道，它们对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组织微管所需的分子机制。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，因为它们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.