Probing Microtubule Function in Neuronal Development

神经元是多细胞生物中形态上最复杂的细胞类型，这种复杂性是 与神经系统信息处理的复杂性有着千丝万缕的联系。因此，神经元损伤 形态发生总是导致神经系统功能的改变， 疾病精确控制微管细胞骨架的组织是神经元的大多数（如果不是全部的话）方面的中心 从新生神经元的迁移，神经突延伸的周期， 收缩和分支到生长锥引导和突触形成。神经元的重要性 微管是反映在广泛的神经发育和神经退行性疾病， 与微管细胞骨架相关的蛋白质的遗传缺陷。人类遗传学与现代 测序技术鉴定了与经常发生的严重急性胰腺炎相关的相关基因中的许多突变。 皮质畸形这些包括微管蛋白基因本身的突变-所谓的微管蛋白病 - 以及神经元微管相关蛋白的突变，这些蛋白通常具有相似的 神经发育表型虽然通常认为这些突变破坏了发育， 我们不了解未成熟神经元通过发育中的皮层迁移的规则， MT在神经形态发生中的作用机制。在本申请中，我们建议使用新兴的 人类诱导多能干细胞（iPSC）技术与最先进基因组相结合 工程和我们在定量显微镜建模和解剖神经元细胞方面的专业知识 微管蛋白病样疾病的生物学和动态MT在神经形态发生中的功能。目标1： 重点关注DCX和微管蛋白突变导致皮质畸形，我们将分析DCX如何控制 神经元微管动力学和力学，以及神经元形态发生，基于我们最近的数据， DCX以独特的几何依赖性方式结合微管。在目标2中，我们采用新的光遗传学， 控制蛋白质与生长中的微管的相互作用，加上以秒和微米精度绘制的末端 微管加末端复合物如何促进神经元发育动力学。我们认为 定量和严格的理解的原则，管理MT功能在神经元发育， 神经发育疾病中出现的问题将对人类健康产生切实而重要的结果， 并且可以为未来的治疗方法奠定基础。

Neurons are the most morphologically complex cell type in multicellular organisms, and this complexity is intricately linked to the complexity of nervous system information processing. Thus, impairments in neuronal morphogenesis invariably result in alterations of nervous system function and often debilitating neurological disease. Precise control of microtubule cytoskeleton organization is central to most if not all aspects of neuron development and function ranging from the migration of newborn neurons, cycles of neurite elongation, retraction and branching to growth cone guidance and synapse formation. This importance of neuronal microtubules is reflected in the wide range of neurodevelopmental and neurodegenerative diseases linked to genetic defects in proteins associated with the microtubule cytoskeleton. Human genetics and modern sequencing techniques identified numerous mutations in related genes associated with frequently severe cortical malformations. These include both mutations of tubulin genes themselves – so-named tubulinopathies – as well as mutations in neuronal microtubule-associated proteins that often have a similar range of neurodevelopmental phenotypes. While it is generally assumed that these mutations disrupt the developmental migration of immature neurons through the developing cortex, we do not understand the rules governing MT function in neuromorphogenesis at a mechanistic level. In this application, we propose to use emerging human induced pluripotent stem cell (iPSC) technology in combination with state-of-the-art genome engineering and our established expertise in quantitative microscopy to model and dissect the neuronal cell biology of tubulinopathy-like diseases and the function of dynamic MTs in neuromorphogenesis. In Aim 1, we focus on DCX and tubulin mutations that cause cortical malformations, and we will analyze how DCX controls neuronal microtubule dynamics and mechanics, and neuronal morphogenesis based on our recent data that DCX binds microtubules in a unique geometry-dependent way. In Aim 2, we employ novel optogenetics to control protein interactions with growing microtubule plus ends with second and micrometer precision to map how microtubule plus end complexes contribute to neuronal development dynamics. We believe that quantitative and rigorous understanding of the principles that govern MT function in neuronal development and what goes wrong in neurodevelopmental disease will have tangible and important outcomes for human health, and can lay the foundation for future therapeutic approaches.