Chemical and Mechanical Interactions in Microtubules

当今生物学的一个主要挑战是整合我们对生物分子的知识来解释复杂的细胞行为。这一挑战是科学和工程领域更广泛的挑战的一部分，即从纳米尺度到微米尺度的整合。这种整合的一个主要例子是微管，其纳米尺寸的组分，α和β微管蛋白单体蛋白质的异二聚体，自组装形成直径为25 nm和几微米长的管。一旦形成，微管介导重要的亚细胞货物的运输，包括线粒体，膜结合的分泌和其他囊泡，和染色体。 因此，了解微管组装和拆卸的基本机制对于了解细胞在生长、有丝分裂和对外界信号的反应过程中如何重组细胞质至关重要。微管表现出一种非常不寻常和复杂的自组装行为，称为"动态不稳定性"，即单个微管在生长和缩短的交替阶段之间随机切换。已经提出了各种理论来解释动态不稳定性的分子起源，但它们都不能解释微管尖端的力学和结构。所需要的是一个计算框架，将化学动力学和热力学（与微管蛋白的添加或损失和GTP水解）与力学（与微管蛋白的构象变化）相结合。鉴于微管动力学中机械力的重要性，确定施加在细胞质中微管上的外力的来源和大小变得至关重要。微管在细胞质中通常是高度弯曲的，并且已经表明肌动球蛋白的收缩性可以引起微管弯曲。然而，目前尚不清楚这是否是主要机制，或者其他机制是否也可能对弯曲有显著影响。初步研究表明，微管聚合导致弯曲，即使当正端不接触细胞的前缘。 这些研究还表明，未弯曲，这在文献中没有被考虑之前，可能会导致解聚和肌动球蛋白收缩。微管动力学的机械调节可能是调节微管进入细胞质的皮质或外周区域的主要手段，这反过来又可以是定向运动或极化（矢量定向）细胞生长期间细胞形状和极性的决定因素。 该项目有三个具体目标：1，了解微管动态不稳定性的机械化学基础; 2，了解活细胞中微管弯曲和不弯曲的机制; 3，发展细胞系统生物学的教育和推广计划。 这些问题将使用综合的理论，计算和实验方法来解决。机械化学计算建模将提供具体的定量预测，这些预测将直接针对实验结果进行测试，包括活细胞中单个微管的GFP荧光成像和纳米级受控力的应用（使用高精度磁珠力应用系统）。为了处理微管动力学固有的复杂性，包括其化学和机械组成部分，这种综合系统方法是必要的。这个项目将促进我们对化学动力学，热力学和力学如何相互作用以介导微管组装和拆卸的理解，从而形成了一个基于理论的计算框架，可用于理解微管相关蛋白如何控制微管行为。它还将促进我们对机械力如何施加于活细胞中的微管以及微管如何自行产生的理解。拟议活动产生的更广泛影响：作为这个项目的一部分，Odde博士将开发一个"细胞生物学合作建模倡议"，通过该倡议，细胞生物学家将与明尼苏达大学BMEn 5351的工程大四学生和研究生团队合作。细胞工程"课程，将开发细胞过程的计算机模拟。此外，Odde博士将继续为来自代表性不足群体的生物医学工程专业学生开展他成功的"未来教师计划"。

A major challenge in biology today is to integrate our knowledge of biomolecules to explain complex cell behavior. This challenge is part of the broader challenge in science and engineering of integrating from the nanoscale to the microscale. A prime example of such integration is the microtubule, whose nanometer-sized components, heterodimers of alpha and beta tubulin monomer proteins, self-assemble to form tubes that are 25 nm in diameter and several micrometers long. Once formed, microtubules mediate the transport of vital subcellular cargoes, including mitochondria, membrane-bound secretory and other vesicles, and chromosomes. Understanding the fundamental mechanisms of microtubule assembly and disassembly is therefore crucial to understanding how cells reorganize their cytoplasm during cellular growth, mitosis, and responses to external signals.Microtubules exhibit a highly unusual and complex self-assembly behavior known as "dynamic instability," where individual microtubules switch stochastically between alternate phases of growth and shortening. Various theories have been offered to explain the molecular origin of dynamic instability, but they all fail to explain the mechanics and structure of the microtubule tip. What is required is a computational framework that integrates chemical kinetics and thermodynamics (associated with tubulin addition or loss and GTP hydrolysis) with mechanics (associated with conformational changes in tubulin). Given the importance of mechanical force in microtubule dynamics, it then becomes essential to identify the origin and magnitude of external forces exerted on, and by, microtubules in the cytoplasm. Microtubules are often highly curved in the cytoplasm, and it has been shown that actomyosin contractility can cause microtubule bending. However, it is not clear whether this is the dominant mechanism, or whether other mechanisms might also contribute significantly to bending. Preliminary studies suggest that microtubule polymerization causes bending, even when the plus end is not in contact with the leading edge of the cell. These studies also suggest that unbending, which has not been considered before in the literature, may result from depolymerization and from actomyosin contractility. Mechanical regulation of microtubule dynamics could be a major means of regulating microtubule access to the cortical or peripheral regions of the cell's cytoplasm, which in turn can be a determinant of cell shape and polarity during directed movement or polarized (vectorially directed) cell growth. This project has three specific aims: 1, to understand the mechanochemical basis of microtubule dynamic instability; 2, to understand the mechanisms of microtubule bending and unbending in living cells; and 3, to develop educational and outreach programs in cellular systems biology. These questions will be addressed using an integrated theoretical, computational and experimental approach. The mechanochemical computational modeling will provide specific, quantitative predictions that will be tested directly against experimental results, including GFP fluorescence imaging of single microtubules in living cells and application of controlled forces on the nanonewton scale (using a high precision magnetic bead force application system). This integrated systems approach is necessary in order to deal with the inherent complexity of microtubule dynamics, including both its chemical and mechanical components.Intellectual merit of the proposed activity: this project will advance our understanding of how chemical kinetics, thermodynamics and mechanics interact to mediate microtubule assembly and disassembly, leading to a theoretically based computational framework that can be used to understand how microtubule-associated proteins control microtubule behavior. It will also advance our understanding of how mechanical forces are imposed on and self-generated by microtubules in living cells.Broader impacts resulting from the proposed activity: as part of this project, Dr. Odde will develop a "Collaborative Modeling in Cell Biology Initiative" through which cell biologists will collaborate with with teams of engineering seniors and graduate students in the University of Minnesota BMEn 5351 "Cell Engineering" course who will develop computer simulations of cellular processes. In addition, Dr. Odde will continue to run his successful "Future Faculty Program" for biomedical engineering students from underrepresented groups.