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。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。我们正在积极地从生物物理量上纯化几种微管蛋白修饰酶以研究它们的作用机制。此外，我们在破译微管蛋白酪氨酸连接酶的生物物理作用机制方面也取得了重大进展。微管蛋白酪氨酸连接酶在α微管蛋白上增加一个C末端的酪氨酸，这是大多数真核细胞中存在的酪氨酸化/去酪氨酸化循环的一部分。这种C端酪氨酸作为微管相互作用蛋白募集的开/关开关。微管蛋白酪氨酸连接酶是发育和细胞功能所必需的，它的抑制导致形成富含微管蛋白的触须，穿透内皮层，促进循环中的肿瘤细胞在转移过程中重新附着，从而与神经母细胞瘤、乳腺癌和前列腺癌患者的预后不良有关。我们现在正在使用这种酶在体外对微管进行修饰，以研究引入的微管蛋白修饰对微管动力学以及马达和微管结合蛋白招募的影响。

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. 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. We are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes to investigate their mechanism of action. In addition, we have made significant progress on deciphering the biophysical mechanism of action of tubulin tyrosine ligase. Tubulin tyrosine ligase adds a C-terminal Tyr to alpha−tubulin as part of a tyrosination/detyrosination cycle present in most eukaryotic cells. This C-terminal tyrosine acts as an ON/OFF switch for the recruitment of microtubule interacting proteins. Tubulin tyrosine ligase is essential for development and cellular function and its suppression leads to formation of tubulin rich tentacles that penetrate endothelial layers to facilitate the reattachment of circulating tumor cells during metastasis, thus correlating with poor prognosis in neuroblastoma, breast and prostate cancer patients. We are now using this enzyme to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment of motors and microtubule association proteins.