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

项目总结： 细胞生物学中的一个基本问题是细胞如何在功能和结构上形成不同的微管。 使用一组看似简单的蛋白质构建块---微管蛋白异源二聚体的网络。多年来, 细胞骨架领域一直关注MT结合蛋白和马达作为主要调节因子的作用。 网络结构和功能。然而，现在很清楚的是，-微管蛋白构建块并不是那么简单。 MT表面不是一个统一的轨迹，而是一个分子多样性的景观，它是由基因 以及-微管蛋白之间的翻译后差异。“微管蛋白代码”模型假定对 -微管蛋白内在无序的羧基末端尾部结构域在MT处创造了一个分子密码 被MT结合蛋白“读取”的表面。这项提案的首要目标是建立 CTTS与MT末端和晶格的构象多样性之间的机械联系，以及 了解这如何在蜂窝级别调节复杂的MT网络功能。 这 结构 Proposal采用多系统、多尺度的方法来了解CTTS对微管的影响 和功能，以及这些如何导致细胞内MT网络功能的变化。我的实验室有 在使用遗传模型、活细胞成像等方法研究微管蛋白方面具有成熟的专业知识 和蛋白质生物化学。我们在过去十年中的进步加深了我们对是如何- 微管蛋白CTT调节MT网络，以及更广泛地说，微管蛋白异质性如何影响细胞和 发育过程。该项目以我们的专业知识为基础，扩展了当前的微管模型 编写代码，并对MT功能的机制提供更广泛的见解。我们的目标包括1)定义CTTS如何指导 MT末端结构对MT动力学的调节2)决定-微管蛋白与不同氨基酸的共混 ACID序列和翻译后修饰导致MT网络水平上的复杂行为 在细胞中，3)确定CTTS如何促进运动蛋白沿MTS的方向性，以及4)建立一个新的 微管蛋白在缓冲细胞内阳离子浓度中的作用。我们的协同方法特别适合于 关于微管蛋白结构和功能高级知识，在广泛的背景下将是重要的，提供 对微管网络如何调节和响应微管蛋白亚单位水平变化的新见解， 以及这些如何影响不同的蜂窝环境。

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.