Elucidating how microtubule-microtubule interactions drive the dynamic reorganization of the microtubule cytoskeleton

微管是一种细胞聚合物，在细胞内部构建各种基本结构，它们是细胞骨架网络的主要组成部分。动态的微管排列和重排定义了细胞形状，为细胞内运输提供了轨道，并驱动细胞分裂和运动。因此，微管细胞骨架的主动重塑对细胞功能至关重要。近年来，微管及其相关蛋白的生化研究为单个微管聚合物动力学的分子机制提供了重要的见解。然而，控制单个微管如何相互作用以产生动态进化的细胞骨架网络结构的规则仍然很大程度上是未知的。该项目将采用多学科方法来阐明微管-微管相互作用如何编码微管网络的重塑，特别关注迁移细胞。重建和操纵细胞骨架整体动态结构的能力最终将允许控制细胞行为，以及生物启发活性材料的未来发展。该项目将为本科生和研究生提供多样化的跨学科培训，并将在当地学校和一个科学博物馆开展进一步的推广工作。本研究的目的是阐明微管-微管相互作用在微管细胞骨架动态重塑中的作用，特别关注细胞迁移背景下的微管网络组织。该假说认为，微管-微管相互作用的节点是网络重塑的焦点，为局部蛋白质活性提供编码的微域，并赋予网络增强对各种扰动的抵抗力。为了验证这一假设，该项目将结合细胞研究与体外重构方法和计算建模。最先进的成像技术将用于确定微管相互作用节点的特性，作为上皮细胞（LLC-PK1）和迁移细胞（B16黑色素瘤）片层中角度、蛋白质定位和微管动力学参数的函数。在体外，微管相互作用将在不同类别的微管相关蛋白（MAPs）存在下重建，这些微管相关蛋白靶向并调节微管末端动力学，稳定微管聚合物晶格，并诱导聚合物损伤和断裂。网络拓扑将使用微图案化技术进行控制；将利用微流体和激光切割施加生化和机械扰动；集合行为将使用计算模拟进行建模。利用计算机和体外方法获得的预测将通过观察细胞中的微管相互作用直接进行测试。总之，这些方法将揭示生物化学，力学和动力学在生理相关的背景下的相互作用。除了与理解细胞过程（如细胞运动和神经元生长锥引导）的直接相关性外，这里确定的机制在发育背景下将具有广泛的重要性，其中细胞骨架驱动的形态学变化通过分化过程定义了多细胞组织和器官结构。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Microtubules are cellular polymers that build a variety of essential structures inside of cells where they provide a major component of a cell’s cytoskeletal network. Dynamic microtubule arrangements and rearrangements define cell shape, provide tracks for intracellular transport, and drive cell division and motility. Thus, active remodeling of the microtubule cytoskeleton is vital for cellular function. In recent years, biochemical studies of microtubules and their associated proteins have provided important insight into the molecular mechanisms underlying the dynamics of individual microtubule polymers. However, the rules governing how individual microtubules interact to give rise to dynamically-evolving cytoskeletal network architectures are still largely unknown. This project will employ a multidisciplinary approach to elucidate how microtubule-microtubule interactions encode the remodeling of the microtubule network, specifically focusing on migrating cells. The ability to reconstitute and manipulate the dynamic architecture of cytoskeletal ensembles will ultimately allow the control of cellular behavior, as well as the future development of biologically-inspired active materials. The project will provide for a diverse interdisciplinary training of undergraduate and graduate students and additional outreach efforts will be carried out in local schools and a science museum.The goal of this research is to elucidate the role of microtubule-microtubule interactions in the dynamic remodeling of the microtubule cytoskeleton, with a particular focus on microtubule network organization in the context of cell migration. The hypothesis is that nodes of microtubule-microtubule interactions serve as focal points for network remodeling, providing encoded microdomains for localized protein activity, and endowing the network with enhanced resistance to a variety of perturbations. To test this hypothesis, this project will combine cellular studies with in vitro reconstitution approaches and computational modeling. State-of-the-art imaging will be used to determine the properties of microtubule interaction nodes, as a function of angle, protein localization and microtubule dynamics parameters in the lamella of epithelial (LLC-PK1) and migrating (B16 melanoma) cells. In vitro, microtubule interactions will be reconstituted in the presence of distinct classes of microtubule-associated-proteins (MAPs) that target and regulate microtubule end dynamics, stabilize the microtubule polymer lattice, and induce polymer damage and severing. The network topology will be controlled using micropatterning techniques; biochemical and mechanical perturbations will be exerted using microfluidics and laser severing; and ensemble behavior will be modeled using computational simulations. Predictions obtained using in silico and in vitro approaches will be directly tested by observations of microtubule interactions in cells. Together, these approaches will uncover the interplay of biochemistry, mechanics and dynamics in a physiologically-relevant context. In addition to the immediate relevance for understanding cellular processes such as cell motility and neuronal growth cone guidance, the mechanisms identified here will be broadly important in the developmental context, where cytoskeleton-driven morphological changes define multicellular tissue and organ structures through the process of differentiation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.