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中从纳米尺度到微尺度整合的更广泛挑战的一部分。微管是这种整合的一个主要例子，它的纳米级组件，即α和β微管蛋白单体蛋白的异源二聚体，自组装形成直径25纳米、长几微米的管。一旦形成，微管调节重要的亚细胞货物的运输，包括线粒体、膜结合的分泌物和其他囊泡以及染色体。因此，了解微管组装和分解的基本机制对于了解细胞在细胞生长、有丝分裂和对外部信号的反应过程中如何重组细胞质是至关重要的。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”，即单个微管在生长和缩短的交替阶段之间随机切换。人们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。所需要的是一个将化学动力学和热力学(与微管蛋白的添加或丢失以及GTP的水解相关)与力学(与微管蛋白的构象变化相关)相结合的计算框架。鉴于机械力在微管动力学中的重要性，因此必须确定外力的来源和大小，这些外力施加在细胞质中的微管上，并由其产生。微管通常在细胞质中高度弯曲，已有研究表明肌动蛋白的收缩能力可导致微管弯曲。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做出重大贡献。初步研究表明，微管聚合会导致弯曲，即使正端没有接触细胞的前沿。这些研究还表明，在以前的文献中没有考虑到的不弯曲，可能是由于解聚和肌动球蛋白的收缩引起的。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围区域的主要手段，而细胞质又可以决定定向运动或极化(定向)细胞生长过程中细胞的形状和极性。该项目有三个具体目标：1，了解微管动态不稳定性的机械力化学基础；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。这些问题将采用理论、计算和实验相结合的方法加以解决。机械力化学计算模型将提供具体的、定量的预测，并将直接与实验结果进行测试，包括活细胞中单个微管的GFP荧光成像和在纳米尺度上施加受控力(使用高精度的磁珠力应用系统)。为了处理微管动力学固有的复杂性，包括其化学成分和机械成分，这种集成的系统方法是必要的。拟议活动的智力优点：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来调节微管组装和拆卸的理解，导致一个基于理论的计算框架，可以用来理解微管相关蛋白如何控制微管行为。这也将提高我们对活细胞中微管是如何施加机械力并由其自身产生的了解。拟议活动产生的更广泛的影响：作为该项目的一部分，Odde博士将开发一项“细胞生物学协作建模计划”，通过该计划，细胞生物学家将与明尼苏达大学BMEn 5351“细胞工程”课程的工程大四学生和研究生团队合作，他们将开发细胞过程的计算机模拟。此外，奥德博士还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.