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

项目概要 细胞生物学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) 该网络使用一组看似简单的蛋白质构建模块——-微管蛋白异二聚体。多年来， 细胞骨架领域重点关注 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，现在很明显，-微管蛋白的构建模块并不那么简单。 MT 表面不是一个统一的轨道，而是由遗传产生的分子多样化的景观。 以及 α-微管蛋白之间的翻译后差异。 “微管蛋白代码”模型假定改变 α-微管蛋白本质上无序的羧基末端尾部 (CTT) 结构域在 MT 处创建分子代码 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。 这个 结构 提案特点 和功能，以及 一种多系统、多尺度的方法来理解如何 这些如何导致 MT 网络功能的变化 CTT 影响 微管蛋白 在 细胞。 我的实验室有 使用整合遗传模型、活细胞成像的方法在研究微管蛋白方面建立了专业知识 和蛋白质生物化学。我们过去五年的进步加深了我们对 - 微管蛋白 CTT 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。拟议的项目将利用我们的专业知识来扩展当前的模型 微管蛋白代码，并为 MT 功能机制提供更广泛的见解。我们未来五年的目标 包括 1) 定义 CTT 如何指导 MT 末端的结构以调节 MT 动态，2) 确定如何混合 具有不同氨基酸序列和翻译后修饰的α-微管蛋白产生复杂的 细胞中 MT 网络水平的行为，3) 定义 CTT 如何促进驱动蛋白运动的方向性 沿着 MT，4) 确定微管蛋白在缓冲细胞内阳离子浓度方面的新作用。我们的 协同方法特别适合增进对微管蛋白结构和功能的了解，这将是 在广泛的背景下都很重要，为微管网络如何调节和提供新的见解 对微管蛋白亚基水平的变化做出反应，以及这些变化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 and function, and a multi-system, multi-scale approach to understanding how how these lead to changes in the function of MT networks CTTs impact tubulin 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 five years has furthered our understanding of how - tubulin CTTs regulate MT networks, and, more broadly, how tubulin heterogeneity impacts cellular and developmental processes. The proposed project will build upon our expertise to expand the current model of the tubulin code and give broader insights into mechanisms of MT function. Our goals for the next five years 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.