Kinesin and +TIP-based microtubule steering

描述（由申请人提供）：微管细胞骨架的适当组织是许多细胞功能的基础，如神经元中的极化运输、上皮细胞中的营养运输和有丝分裂。虽然有丝分裂细胞中微管排列的控制机制已经被广泛研究，但分化细胞中微管排列的研究却少得多。果蝇神经元的树突可以作为一个系统来识别控制微管极性的机制，因为它们含有均匀极性的负端微管。在果蝇神经元中的体内实验已经产生了树突中微管排列的模型，其中在生长的微管正末端处的驱动蛋白-2和正尖端相互作用蛋白（+TIP）的复合物与在分支点处的静止微管相互作用，并且主动地将生长的正末端导向细胞体。这种微管操纵机制可以作为一个通用的模型，用于控制微管极性在不同的细胞类型，如上皮细胞。本提案的目标是使用体外重建，计算模拟和果蝇神经元的分析，以了解+TIP-驱动蛋白为基础的微管转向。本工作的第一个目的是利用纯化的蛋白质和微加工通道，建立一个新的实验系统，用于研究微管转向+TIP-驱动蛋白复合物在体外。这种体外重构将验证和扩展体内观察，并将提供用于定量该蛋白质复合物的活性的系统。第二个目标将是测量系统的特定组分之间的结合亲和力，以建立用于建模研究的定量生化参数。第三个目标将是开发计算模拟+TIP-驱动蛋白为基础的微管转向在体内和体外，使用从实验中产生的定量参数，然后使用体内实验测试预测的模型。模拟将结合微管和驱动蛋白马达的已知机械特性，并将提供对+TIPs承受持续微管弯曲所需的机械载荷的能力的见解。这些实验和计算研究将探索驱动蛋白马达和+TIP蛋白的新功能作用，这里开发的框架将为理解分化细胞中微管极性建立的普遍方面提供基础。许多人类神经退行性疾病与调控微管细胞骨架的基因突变有关，这强调了理解神经元中适当微管组织的重要性。 公共卫生相关性：基于微管的运输对于神经元的生长和维持至关重要，并且运输缺陷与神经退行性疾病如ALS、阿尔茨海默病和亨廷顿病有关。此外，轴突去除后，驱动蛋白-2已被证明是轴突从树突适当再生长所需的，这表明这里检查的微管转向机制可能在创伤神经元的修复中发挥作用。因此，这项工作的见解可以应用于增强人类损伤后的神经再生。

DESCRIPTION (provided by applicant): Proper organization of the microtubule cytoskeleton underlies many cellular functions such as polarized transport in neurons, nutrient transport in epithelial cells, and mitosis. While mechanisms that control microtubule alignment in mitotic cells have been extensively studied, alignment of microtubules in differentiated cells has been probed much less. Dendrites of Drosophila neurons can be used as a system to identify mechanisms that control microtubule polarity as they contain uniform-polarity minus-end-out microtubules. In vivo experiments in fly neurons have led to a model of microtubule alignment in dendrites in which a complex of kinesin-2 and plus-tip interacting proteins (+TIPs) at growing microtubule plus-ends interacts with stationary microtubules at branch points and actively directs the growing plus- end toward the cell body. This microtubule steering mechanism may serve as a general model for control of microtubule polarity in diverse cell types such as epithelial cells. The goal of this proposal is to use in vitro reconstitution, computational simulations, and analysis of Drosophila neurons to understand +TIP-kinesin based microtubule steering. The first aim of the work is to use purified proteins and microfabricated channels to develop a novel experimental system for studying microtubule steering by +TIP-kinesin complexes in vitro. This in vitro reconstitution will validate and extend in vivo observations, and will provide a system for quantifying the activity of this protein complex. The second aim will be to measure binding affinities between specific components of the system to establish quantitative biochemical parameters for modeling studies. The third aim will be to develop computational simulations of +TIP-kinesin based microtubule steering in vivo and in vitro, using quantitative parameters generated from the experiments, and then test predictions of the models using in vivo experiments. The simulations will incorporate known mechanical properties of microtubules and kinesin motors, and will provide insights into the ability of +TIPs, to withstand the mechanical loads necessary for sustained microtubule bending. These experimental and computational studies will explore novel functional roles for both kinesin motors and the +TIP proteins, and the framework developed here will provide a foundation for understanding universal aspects of microtubule polarity establishment in differentiated cells. The importance of understanding proper microtubule organization in neurons is underscored by the numerous human neurodegenerative diseases that are linked to mutations in genes involved in regulating the microtubule cytoskeleton. PUBLIC HEALTH RELEVANCE: Microtubule-based transport is crucial for the growth and maintenance of neurons and transport deficiencies are linked to neurodegenerative diseases such as ALS, Alzheimers and Huntington's disease. Furthermore, following axon removal, kinesin-2 has been shown to be required for proper regrowth of an axon from a dendrite, suggesting that the microtubule steering mechanism examined here may play a role in the repair of neurons from trauma. Thus, insights from this work could be applied to enhancing neural regeneration following injury in humans.