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上结构来执行其不同的细胞功能：径向细胞质阵列，短的，高度平行的轴突阵列，纺锤体阵列或平铺长的轴突阵列。微管细胞骨架是许多单元操作的复杂功能，细胞骨架调节因子的个体行为：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运输的简单途径。α和β微管具有多种亚型，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，微管调节因子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，如帕金森病、阿尔茨海默病和肌萎缩性侧索硬化症，这并不奇怪。

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. We are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes to investigate their mechanism of action.We have recently reported in collaboration with Martin Tanner's laboratory (University of British Columbia) the identification of the first small molecule inhibitors for the polyglutamylase TTLL7 (Liu et al., 2013) and we are actively working towards identifying new inhibitor classes as well as increasing the affinity of our current inhibitors. In addition, we have made significant progress on deciphering the biophysical mechanism of action of tubulin tyrosine ligase. My laboratory determined the first structure of tubulin tyrosine ligase, the enzyme responsible for the post-translational addition of a tyrosine to the C-terminus of alpha-tubulin as part of the tubulin detyrosination/tyrosination cycle (Szyk et al., 2011). The C-terminal tyrosine in alpha-tubulin serves as an ON/OFF signal for the recruitment of microtubule dynamics regulators. Tubulin tyrosine ligase loss is associated with aggressive tumor progression and metastasis in several cancers, including neuroblastomas, breast and prostate cancers. Moreover, tubulin tyrosine ligase is homologous to enzymes that are part of the larger family of tubulin modifying enzymes, the tubulin tyrosine ligase-like family that includes polyglutamylases and polyglycylases that are important in microtubule motor regulation and have been recently involved in neurodegeneration. Our crystal structure of tubulin tyrosine ligase reveals how the tubulin tyrosine ligase scaffold supported the expansion of the repertory of tubulin post-translational modification enzymes that recognize either the alpha- or beta-tubulin tail and resolves a long-standing puzzle as to why tubulin tyrosine ligase modifies only monomeric tubulin and not tubulin that is already incorporated into microtubules. We also discovered that tubulin tyrosine ligase uses a novel strategy to prevent tubulin incorporation into microtubules by capping the tubulin dimer on an interface that would otherwise be involved in microtubule lattice interactions (Szyk et al., 2011). Recently, we found that tubulin tyrosine ligase competes for tubulin binding with stathmin (Szyk et al., 2013). These findings open the interesting possibility that changes in microtubule dynamics and morphology observed in cancer cells with low tubulin tyrosine ligase levels (a hallmark of drug resistant tumors) may be due not only to down-regulation of tubulin tyrosination, but also an increase in the polymerization competent tubulin pool. Recent work from my group also shed light on the structure and mechanism of action of tubulin acetyltransferase, an unusual enzyme that acetylates a site that is situated inside the microtubule lumen (Kormendi et al., 2012). We are now actively investigating how this enzyme gains access to the luminal acetylation site and the effects of acetylation on microtubule dynamics. Lastly, we are now using TTL and other tubulin modification enzymes 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.