Single-molecule interrogation of microtubule dynamics mechanisms

微管（MT）细胞骨架对真核细胞是必不可少的：微管是所需的动态聚合物 染色体分离和细胞内组织，是抗癌的直接靶点 化疗药物如紫杉醇和紫堇生物碱。MT的动态特性是其性能的核心。 功能，它们来自单个αβ-微管蛋白亚基的生化特性，以及它们如何在细胞中发挥作用。 在MT晶格中相互作用。MT动力学的定量机制一直难以理解 因为MT末端是一个复杂的生物化学环境，其中单个微管蛋白采用不同的 构象，并可以有不同数量的邻居接触，因为它一直不可能， 直接测量个体的相互作用。目的是定量研究 纵向和横向相互作用，核苷酸状态，MT末端构型，以及晶格诱导 构象变化的MT组装和转换，我们最近表明，酵母之间的相互作用， αβ-微管蛋白和MT末端可以在单分子水平上观察到，并在高时间内定量。 使用干涉散射（iSCAT）显微镜的分辨率。酵母和人类的比较研究 微管蛋白被提出来建立微管动力学的一般机制。Aim 1将使用iSCAT来 测量和定量人和酵母αβ-微管蛋白与稳定微管末端的相互作用 种子，以及这些相互作用如何取决于核苷酸状态。Aim 2将使用iSCAT和其他技术， 测量干扰αβ-微管蛋白构象变化倾向的突变如何影响生物化学 与微管末端的相互作用以及微管生长和收缩动力学。目标3将 使用iSCAT和其他技术来测量不同剂量的αβ-微管蛋白突变体与其正末端 “阻断”影响微管的伸长和突变。结果将用于构建生化 微管动力学模型，将加深对灾难的理解。

The microtubule (MT) cytoskeleton is essential to eukaryotic cells: microtubules are dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the biochemical properties of individual αβ-tubulin subunits and how they interact within the MT lattice. The quantitative mechanisms of MT dynamics have been difficult to understand because the MT end is a complex biochemical environment where individual tubulins adopt different conformations and can have different numbers of neighbor contacts, and because it has not been possible to measure individual interactions directly. With the goal of quantitatively examining the contributions of longitudinal and lateral interactions, nucleotide state, MT end configurations, and lattice-induced conformational changes to MT assembly and switching, we recently showed that interactions between yeast αβ-tubulin and the MT end can be observed at the single-molecule level and quantified with high temporal resolution using interferometric scattering (iSCAT) microscopy. Comparative studies of yeast and human tubulin are proposed to establish general mechanisms of microtubule dynamics. Aim 1 will use iSCAT to measure and quantify the interactions of human and yeast αβ-tubulin with the end of a stable microtubule seed, and how these interactions depend on nucleotide state. Aim 2 will use iSCAT and other techniques to measure how a mutation that perturbs the αβ-tubulin propensity for conformational change affects biochemical interactions with the microtubule end and microtubule growth and shrinking kinetics more generally. Aim 3 will use iSCAT and other techniques to measure how different doses of an αβ-tubulin mutant with its plus-end “blocked” affect microtubule elongation and catastrophe. The results will be used to construct a biochemical model for microtubule dynamics that will deepen the understanding of catastrophe.