Mechanisms of molecular machines that regulate the neuronal cytoskeleton

微管是细胞形态发生、细胞分裂和细胞内运输所必需的聚合物。微管通过形成具有非常独特的几何形状的超结构来执行其不同的细胞角色：放射状细胞质阵列、短的、高度平行的轴丝阵列、纺锤体阵列或平铺的长轴突阵列。微管细胞骨架是许多单位操作的复杂功能，是细胞骨架调节因子的个体作用：成核、生长和收缩、断裂和运动。此外，微管本身不仅仅是细胞成分传递的幼稚道路。α微管蛋白和β微管蛋白有多种异构体，并受到高度多样化、丰富和进化保守的翻译后修饰的影响，这些修饰标志着微管亚群(Garnham和Roll-Mecak，2012)。鉴于微管在基本细胞过程中发挥的核心作用，微管调节器与许多人类疾病有关也就不足为奇了，这些疾病包括癌症、心血管疾病、真菌、细菌和病毒感染，以及帕金森氏症、阿尔茨海默氏症和肌萎缩侧索硬化症等神经退行性疾病。 我们的工作集中在微管调节的两个家族：微管切断酶和翻译后修饰微管蛋白的酶。我们的研究计划是高度跨学科的，整合了生物物理学、结构生物学、分子生物学和细胞生物学的技术和概念，以回答两个紧密交错的问题：当微管结构被这些调节器参与时，微管的结构是如何局部扰动的，以及这些调节器如何在细胞水平上影响微管的结构和动力学？微管动力学的扰动已经成为各种神经退行性疾病的共同主题，我们的工作对所有这些疾病的病因都有影响。 在过去的一年里，我们启动了几项旨在了解微管翻译后修饰功能的机制基础的研究，并继续我们对痉挛蛋白切断微管的机制的研究。 我的团队最近的工作集中在微管蛋白乙酰转移酶(TAT)的作用机制上，TAT是一种不寻常的酶，用于乙酰化位于微管腔中的α-微管蛋白Lys40。TAT是极性建立和定向迁移所必需的。我的团队确定了微管蛋白乙酰转移酶与底物的络合物的第一结构，揭示了它的催化机制。出乎意料的是，尽管Lys40在腔内的位置有限，但我们发现该酶有效地双向扫描微管并随机乙酰化，而不是偏爱末端。利用第一性原理模型和单分子扩散测量，我们证明了TAT的催化活性，而不是限制的管腔扩散，是乙酰化的速度限制。因此，由于其对微管的偏好而不是游离微管蛋白，以及其适度的催化速度，TAT可以作为微管寿命的慢时钟，并选择性地标记细胞中稳定的微管(Szyk等人，2014)。 去年，我的团队还负责开发新的方法来产生均一的乙酰化、谷氨酰化或酪氨酸化的微管和微管，用于体外分析(Vemu等人，2014年)。现在，差异修饰微管的产生使得对微管蛋白翻译后修饰对微管的动力学和机械性能的影响以及对发动机和微管相关蛋白的招募和行为的机械剖析成为可能。 最后，我们正在积极地纯化几种微管蛋白修饰酶，以研究它们的作用机制。我们目前还在使用这些酶制剂对微管进行体外修饰，以研究引入的微管蛋白修饰对微管动力学以及运动和微管切断酶spastin和katanin的招募和活性的影响。

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. Microtubules execute their diverse cellular roles by forming suprastructures with highly distinctive geometries: the radial cytoplasmic array, the short, highly parallel axonemal array, the spindle array or the tiled long axonal array. The microtubule cytoskeleton is a complex function of many unit operations, the individual actions of cytoskeletal regulators: nucleation, growth and shrinkage, severing and motor movement. Moreover, the microtubule itself is more than just a naive roadway for cellular components to transit along. Alpha and beta tubulins have multiple isoforms and are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules (Garnham and Roll-Mecak, 2012). Given the central role microtubules play in basic cellular processes, it is not surprising that microtubule regulators have been implicated in many human diseases, including cancers, cardiovascular disease, fungal, bacterial and viral infections, as well as neurodegenerative disorders such as Parkinson's, Alzheimer's and Amyotrophic lateral sclerosis. Our efforts concentrate on two families of microtubule regulators: microtubule severing enzymes and enzymes that post-translationally modify tubulin. Our research plan is highly interdisciplinary, integrating techniques and concepts from biophysics, structural, molecular and cell biology to answer two closely interdigitated questions: how is the structure of the microtubule locally perturbed when it is engaged by these regulators and how do these regulators affect microtubule architecture and dynamics at the cellular level? Perturbation of microtubule dynamics has emerged as a common theme in a variety of neurodegenerative diseases and our work has implications for the etiologies of all these disorders. In the last year we initiated several studies aimed at understanding the mechanistic underpinnings of the functions of microtubule post-translational modifications as well as continued our work on the mechanism of microtubule severing by spastin. Recent work from my group focused on the mechanism of action of the of tubulin acetyltransferase (TAT), an unusual enzyme that acetylates alpha-tubulin Lys40 situated in the microtubule lumen. TAT is required for polarity establishment and directional migration. My group determined the first structure of the tubulin acetyltransferase in complex with its substrate shedding light on its catalytic mechanism. Unexpectedly, despite the confined intraluminal location of Lys40, we found that the enzyme efficiently scans the microtubule bidirectionally and acetylates stochastically without a preference for ends. Using first-principles modeling and single-molecule diffusion measurements we demonstrated that TAT catalytic activity, not constrained luminal diffusion, is rate-limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes and selectively marks stable microtubules in cells (Szyk et al., 2014). My group has also been responsible in the last year for the development of novel methods for generating homogenous acetylated, glutamylated, or tyrosinated tubulin and microtubules for in vitro assays (Vemu et al., 2014). The generation of differentially modified microtubules now enables a mechanistic dissection of the effects of tubulin post-translational modifications on the dynamics and mechanical properties of microtubules as well as the recruitment and behavior of motors and microtubule-associated proteins. Lastly, we are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes to investigate their mechanism of action. We are currently also using these enzyme preparations to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment and activity of motors and microtubule severing enzymes spastin and katanin.