Beyond the tubulin code: Understanding how subunit diversity regulates the formation and function of microtubules

项目摘要： 细胞生物学中的一个基本问题是细胞如何构建功能和结构不同的微管（MT） 网络使用一组看似简单的蛋白质构建模块--微管蛋白异源二聚体。多年来， 细胞骨架领域的重点是MT结合蛋白和马达作为细胞骨架的主要调节因子的作用。 网络结构和功能。然而，现在很清楚，微管蛋白的构建模块并不那么简单。 而不是一个统一的轨道，MT表面是一个分子多样性的景观，是由遗传 和微管蛋白之间的翻译后差异。“微管蛋白编码”模型假定， 微管蛋白的固有无序羧基末端尾（CTT）结构域在MT处产生分子密码 这是由MT结合蛋白“读取”的表面。该提案的总体目标是建立 CTT与MT末端和晶格的构象多样性之间的机械联系，以及 了解这如何在细胞水平上调节复杂的MT网络功能。 这 结构 一项提案采用多系统、多尺度的方法来了解CTTs如何影响微管蛋白 和功能，以及这些如何导致细胞中MT网络功能的变化。我的实验室 建立了研究微管蛋白的专业知识，使用整合遗传模型，活细胞成像 和蛋白质生物化学我们在过去十年中的进步进一步加深了我们对如何实现可持续发展的理解- 微管蛋白CTTs调节MT网络，更广泛地说，微管蛋白异质性如何影响细胞和 发展过程。这个项目建立在我们的专业知识，以扩大目前的模型微管蛋白 代码，并提供更广泛的见解MT功能的机制。我们的目标包括：1）定义CTTs如何指导 MT的结构结束调节MT动力学，2）确定如何与不同氨基的微管蛋白的共混物 酸序列和翻译后修饰在MT网络水平上产生复杂的行为 在细胞中，3）定义CTT如何促进驱动蛋白运动沿着MT的方向性，以及4）建立一种新的 微管蛋白在缓冲细胞内阳离子浓度中的作用。我们的协同方法非常适合于 微管蛋白结构和功能的先进知识，将在广泛的背景下是重要的，提供 对微管网络如何调节和响应微管蛋白亚基水平变化的新见解， 以及它们如何影响不同的细胞环境。

Project Summary: A fundamental question in cell biology is how cells build functionally and structurally distinct microtubule (MT) networks using a seemingly simple set of protein building blocks -- -tubulin heterodimers. For many years, the cytoskeletal field has focused on the roles of MT-binding proteins and motors as the primary regulators of network structure and function. However, it is now clear that the -tubulin building blocks are not so simple. Rather than a uniform track, the MT surface is a molecularly diverse landscape that is generated by genetic and posttranslational differences between -tubulins. The `tubulin code' model posits that changes to the intrinsically disordered carboxy-terminal tail (CTT) domains of -tubulins create a molecular code at the MT surface that is “read” by MT-binding proteins. The overarching goals of this proposal are to establish mechanistic connections between CTTs and the conformational diversity of MT ends and lattices, and to understand how this regulates complex MT network functions at the cellular level. This structure proposal features a multi-system, multi-scale approach to understanding how CTTs impact tubulin and function, and how these lead to changes in the function of MT networks in cells. My lab has established expertise in investigating tubulin using approaches that integrate genetic models, live-cell imaging and protein biochemistry. Our progress over the past ten years has furthered our understanding of how - tubulin CTTs regulate MT networks, and, more broadly, how tubulin heterogeneity impacts cellular and developmental processes. This project builds upon our expertise to expand the current model of the tubulin code and give broader insights into mechanisms of MT function. Our goals include 1) define how CTTs guide the structure of MT ends to regulate MT dynamics, 2) determine how blends of -tubulins with different amino acid sequences and posttranslational modifications give rise to complex behaviors at the level of MT networks in cells, 3) define how CTTs promote the directionality of kinesin motility along MTs, and 4) establish a novel role for tubulins in buffering intracellular cation concentrations. Our synergistic approach is uniquely suited to advance knowledge of tubulin structure and function that will be important in a broad range of contexts, provide new insights into how microtubule networks regulate and respond to changes at the level of tubulin subunits, and how these impact different cellular contexts.