Single-molecule interrogation of microtubule dynamics mechanisms

微管动力学机制的单分子研究

• 批准号: 10454249
• 负责人: Luke W Rice
• 金额: $ 38万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10454249
• 关键词: Address; Adopted; Affect; Behavior; Binding; Biochemical; Biochemistry; Cells; Chromosome Segregation; Comparative Study; Complex; Computer Models; Cytoskeleton; Data; Dose; Environment; Eukaryotic Cell; Evolution; Frequencies; Genetic Materials; Goals; Growth; Human; Individual; Kinetics; Knowledge; Label; Lateral; Measurement; Measures; Mediating; Methods; Microscopy; Microtubule Polymerization; Microtubules; Mind; Modeling; Molecular; Molecular Conformation; Mutation; Nucleotides; Paclitaxel; Pharmaceutical Preparations; Plus End of the Microtubule; Poison; Polymers; Property; Reagent; Recombinants; Seeds; Site; Specificity; Structure; Techniques; Testing; Tubulin; Tubulin Interaction; Variant; Vinca Alkaloids; Work; Yeasts; anti-cancer; beta Tubulin; biochemical model; comparative; experimental study; innovation; mutant; nanoGold; polymerization; reconstitution; segregation; single molecule; temporal measurement

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.

微管 (MT) 细胞骨架对于真核细胞至关重要：微管是所需的动态聚合物 负责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长春花生物碱。 MT 的动态特性对其至关重要 功能，它们源自各个 αβ-微管蛋白亚基的生化特性以及它们如何 MT 晶格内相互作用。机器翻译动力学的定量机制一直难以理解 因为MT端是一个复杂的生化环境，各个微管蛋白采用不同的 构象并且可以有不同数量的邻居接触，并且因为不可能 直接测量个体交互。为了定量检查的贡献的目标 纵向和横向相互作用、核苷酸状态、MT 末端构型和晶格诱导 MT 组装和转换的构象变化，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并用高时间定量 使用干涉散射 (iSCAT) 显微镜进行分辨率。酵母和人类的比较研究 微管蛋白被提议建立微管动力学的一般机制。目标 1 将使用 iSCAT 来 测量和量化人和酵母 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依赖于核苷酸状态。目标 2 将使用 iSCAT 和其他技术 测量扰乱 αβ-微管蛋白构象变化倾向的突变如何影响生化 与微管末端的相互作用以及更普遍的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-微管蛋白突变体及其正端 “阻塞”影响微管伸长和灾难。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。