Molecular motors and neuronal microtubule polarity

分子马达和神经元微管极性

• 批准号: 9367009
• 负责人: JILL C WILDONGER
• 金额: $ 29.87万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9367009
• 关键词: Address; Affect; Axon; Binding Proteins; CRISPR/Cas technology; Centrosome; Charcot-Marie-Tooth Disease; Complex; Coupled; Cytoskeleton; Data; Dendrites; Disease; Dynein ATPase; Equilibrium; GRIP1 gene; Genetic; Genetic Screening; Genome engineering; Golgi Apparatus; Growth; Hereditary Spastic Paraplegia; Human; Human Pathology; Image; In Vitro; Intracellular Transport; Invaded; Invertebrates; Investigation; Kinesin; Learning; Light; Mediating; Microtubules; Modeling; Molecular Motors; Motor; Motor Activity; Mutagenesis; Mutation; Neurites; Neurons; Organelles; Pathology; Phosphorylation; Play; Process; Proteins; Publishing; Regulation; Signal Transduction; Structure; Study models; System; Testing; Time; Transport Vesicles; Vertebrates; Vesicle; Vesicle Transport Pathway; Walking; Work; base; fly; gamma Tubulin; genetic approach; human disease; imaging approach; in vivo; insight; novel; scaffold; single molecule; trafficking

The microtubule cytoskeleton is essential to neuronal activity. Microtubules have an intrinsic polarity that motors read out to localize cargo, and differences in microtubule orientation between axons and dendrites are a defining feature of a polarized neuron (microtubule polarity is uniform in axons and mixed in dendrites). Despite the importance of microtubule organization to neuronal function, the mechanisms that create and maintain polarized microtubule arrays in axons and dendrites are poorly understood. While existing models focus largely on the motor-mediated translocation of microtubules into and out of neurites, there is now strong evidence from both vertebrates and invertebrates that local, non-centrosomal microtubule nucleation affects microtubule polarity in axons and dendrites, signaling the need for new models and a better understanding of local nucleation mechanisms. Our proposal addresses three fundamental, outstanding questions. How is nucleation machinery localized by molecular motors to specific compartments (Aim 1)? How is local γ-tubulin- mediated microtubule nucleation regulated to maintain the unique polarities of axonal and dendritic cytoskeletons, and how does local microtubule growth affect intracellular transport (Aim 2)? Using a fly model, we exploit cutting-edge genome engineering and live imaging approaches to dissect novel mechanisms of motor-based transport and local nucleation in vivo. There are two known platforms for γ-tubulin-mediated microtubule nucleation in neurons: Golgi outposts (dendrites only) and augmin (dendrites and axons). In Aim 1, we delineate a novel mechanism of polarized transport in which the coordinated and spatially regulated activities of kinesin-1 and dynein localize Golgi outposts to dendrites. In Aim 2, we determine how the localization of γ-tubulin to Golgi outposts or augmin (or novel nucleation centers) regulates microtubule polarity in axons and dendrites, and the effects of local microtubule growth on the transport of vesicles and organelles. To identify novel regulators of microtubule nucleation and microtubule polarity, we are leveraging our in vivo system in a forward genetic screen to gain new insights into the poorly understood mechanisms controlling local nucleation and microtubule polarity. Our studies will create a new mechanistic framework for understanding how polarized transport of Golgi outposts and local microtubule nucleation maintains neuronal polarity and supports intracellular trafficking. Multiple human disorders are associated with deficits in microtubule-based trafficking, and with mutations in kinesin-1 and dynein, and our investigations may shed light on the pathology of these diseases.

微管细胞骨架对神经元活动是必不可少的。微管具有内在极性， 马达读出定位货物，轴突和树突之间微管方向的差异是 极化神经元的定义特征（微管极性在轴突中是均匀的，在树突中是混合的）。 尽管微管组织对神经元功能的重要性，但产生和 维持轴突和树突中的极化微管阵列的研究知之甚少。虽然现有的模型 主要集中在运动介导的微管进出神经突的易位，现在有很强的 来自脊椎动物和无脊椎动物的证据表明局部非中心体微管成核影响 轴突和树突中的微管极性，这表明需要新的模型和更好地理解 局部成核机制我们的建议涉及三个根本的、悬而未决的问题。怎么样 成核机制本地化的分子马达，以特定的隔间（目标1）？局部γ-微管蛋白- 介导的微管成核调节，以维持轴突和树突的独特极性 细胞骨架，以及局部微管生长如何影响细胞内转运（目的2）？使用苍蝇模型， 我们利用尖端的基因组工程和活体成像方法来剖析 基于马达的运输和体内局部成核。存在两种已知的γ-微管蛋白介导的细胞内表达平台。 神经元中的微管成核：高尔基体前哨（仅树突）和Augmin（树突和轴突）。在目标1中， 我们描述了一种新的极化运输机制，其中协调和空间调节 驱动蛋白-1和动力蛋白的活性将高尔基体前哨定位于树突。在目标2中，我们确定 γ-微管蛋白定位于高尔基体前哨或augmin（或新的成核中心）调节微管极性 在轴突和树突，以及对小泡和细胞器的运输局部微管生长的影响。 为了鉴定微管成核和微管极性的新型调节剂，我们正在利用我们的体内 系统在向前遗传筛选，以获得新的见解知之甚少的机制控制 局部成核和微管极性。我们的研究将建立一个新的机制框架， 了解高尔基体前哨和局部微管成核的极化运输如何维持神经元 极性和支持细胞内运输。多种人类疾病与以下缺陷有关： 基于微管的运输，以及驱动蛋白-1和动力蛋白的突变，我们的调查可能会摆脱 了解这些疾病的病理。