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。微管通过形成具有高度独特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元操作的复杂功能，细胞骨架调节剂的单独作用：成核、生长和收缩、切断和运动。此外，微管本身不仅仅是细胞成分沿着运输的天然通道。α和β微管蛋白具有多种同种型，并且经历高度多样、丰富和进化上保守的翻译后修饰，其标记微管亚群（Garnham和Roll-Mecak，2012）。鉴于微管在基本细胞过程中发挥的核心作用，微管调节剂与许多人类疾病有关，包括癌症、心血管疾病、真菌、细菌和病毒感染以及神经退行性疾病如帕金森氏症、阿尔茨海默氏症和肌萎缩性侧索硬化症，这并不奇怪。 我们的努力集中在两个家庭的微管调节剂：微管切断酶和酶的微管蛋白后修饰。我们的研究计划是高度跨学科的，从生物物理学，结构，分子和细胞生物学的技术和概念相结合，回答两个密切相互交织的问题：微管的结构是如何局部扰动时，它是由这些监管机构和这些监管机构如何影响微管结构和动力学在细胞水平？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，我们发起了几项研究，旨在了解微管翻译后修饰功能的机制基础，并继续我们的工作微管切断痉挛的机制。 我的小组最近的工作集中在微管蛋白乙酰转移酶（达特）的作用机制上，TAT是一种不寻常的酶，它使位于微管腔中的α-微管蛋白Lys 40乙酰化。 极性建立和定向迁移需要达特。我的小组确定了微管蛋白乙酰转移酶与其底物复合物的第一个结构，揭示了其催化机制。出乎意料的是，尽管Lys 40的管腔内位置有限，我们发现该酶有效地双向扫描微管，并乙酰化stochemical没有偏好的结束。使用第一性原理建模和单分子扩散测量，我们证明了达特催化活性，而不是限制管腔扩散，是乙酰化的限速。 因此，由于其对微管的偏好超过游离微管蛋白及其适度的催化速率，达特可以用作微管寿命的慢时钟并选择性地标记细胞中的稳定微管（Szyk等人，2014）。 我的小组在去年还负责开发用于产生用于体外测定的同质乙酰化、谷氨酰化或酪氨酸化微管蛋白和微管的新方法（Vemu等人，2014）。 差异修饰的微管的产生现在使得微管蛋白翻译后修饰对微管的动力学和机械性质以及马达和微管相关蛋白的募集和行为的影响的机械解剖成为可能。 最后，我们正在积极致力于纯化几种微管蛋白修饰酶，使其达到均匀性和生物物理量，以研究其作用机制。 我们目前还使用这些酶制剂在体外修饰微管，以研究微管蛋白修饰对微管动力学和马达的招募和活性的影响，以及微管切断酶spastin和katanin。

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.