Control of microtubule length by polymerases and depolymerases

通过聚合酶和解聚酶控制微管长度

• 批准号: 8842141
• 负责人: Jonathon Howard
• 金额: $ 40.51万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8842141
• 关键词: Acceleration; Aneuploidy; Biochemical; Biological Assay; Biological Models; Biophysics; Brain; Cell division; Cells; Cellular biology; Chromosome Segregation; Chromosomes; Computer Simulation; Congenital Abnormality; Daughter; Energy-Generating Resources; Ensure; Equilibrium; Feedback; Filament; Filopodia; G1/S Transition; Genes; Genetic; Grant; Growth; Guanosine Triphosphate; Health; Homologous Gene; Hydrolysis; Inherited; Kinesin; Lead; Length; Measurement; Measures; Methods; Microtubule Depolymerization; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic spindle; Molecular; Molecular and Cellular Biology; Morphology; Mothers; Motor; Neurons; Organelles; Phase; Polymerase; Polymers; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Regulation; Role; Saccharomycetales; Scheme; Stereocilium; System; Techniques; Testing; Tissues; Tubulin; Work; Yeasts; base; cancer cell; cell motility; cellular microvillus; genetic analysis; human disease; insight; mathematical model; novel; polymerization; segregation; single molecule; yeast protein

DESCRIPTION (provided by applicant): A fundamental, but poorly understood, problem in cell biology is how the sizes of organelles are controlled. The lengths of mitotic spindles and axonemes, for example, vary by as little as a few per cent between cells of the same type. Furthermore, the correct size and morphology are essential for function-mitotic spindles for cell division and axonemes for motility. Cells regulate the sizes of these organelles by tightly controlling the lengths of their constituent microtubules. In the absence of a molecular ruler that templates microtubule length, it is thought that length control results from a delicate balance between polymerization and depolymerization of the microtubules. How this is achieved is not known. Based on our previous work in which we showed that the motor kinesin-8 Kip3 is a length-dependent microtubule depolymerase, we hypothesize that motor proteins, in conjunction with other microtubule-associated proteins (MAPs), can provide feedback between length and dynamics that tightly regulates the lengths of microtubules. The general aim of this grant is to use single-molecule techniques, together with mathematical modeling, to understand how two additional proteins-the yeast kinesin Kip2 and the yeast homolog of the vertebrate polymerase XMAP215, Stu2-together with Kip3, regulate the lengths of yeast microtubules. We have devised a novel purification scheme for native budding-yeast tubulin and this allows us to employ yeast as our model system, which has distinct advantages due to the small number of tubulin isoforms and the absence of potentially confounding post-translational modifications found in vertebrate, and in particular brain, tubulin. Our specific aims are to (1) characterize te acceleration of growth of yeast microtubules by Stu2, (ii) determine how Kip2 promotes microtubule assembly, and (iii) examine the precision with which Kip3, in combination with Kip2 and Stu2, controls microtubule lengths. These studies will provide important insight into the assembly and function of the mitotic spindle and establish principles of length regulation that wil be applicable to other biomedically relevant organellar systems such axonemes, microvilli, stereocilia and filopodia.

描述（由申请人提供）：细胞生物学中一个基本但知之甚少的问题是如何控制细胞器的大小。例如，同一类型细胞之间有丝分裂纺锤体和轴丝的长度差异只有百分之几。此外，正确的尺寸和形态对于细胞分裂的有丝分裂纺锤体和运动的轴丝至关重要。细胞通过严格控制其组成微管的长度来调节这些细胞器的大小。在没有分子标尺的情况下 模板微管长度，据认为长度控制是微管聚合和解聚之间微妙平衡的结果。这是如何实现的尚不清楚。基于我们之前的工作，我们表明运动驱动蛋白 8 Kip3 是一种长度依赖性微管解聚酶，我们假设运动蛋白与其他微管相关蛋白 (MAP) 结合，可以提供长度和动力学之间的反馈，严格调节微管的长度。 这项资助的总体目标是使用单分子技术和数学模型来了解另外两种蛋白质——酵母驱动蛋白 Kip2 和脊椎动物聚合酶 XMAP215 的酵母同源物 Stu2——与 Kip3 一起如何调节酵母微管。我们为天然出芽酵母微管蛋白设计了一种新颖的纯化方案，这使我们能够采用酵母作为我们的模型系统，由于微管蛋白亚型数量较少且不存在脊椎动物中发现的潜在混淆的翻译后修饰，因此该系统具有明显的优势，特别是大脑，微管蛋白。 我们的具体目标是 (1) 表征 Stu2 对酵母微管生长的加速作用，(ii) 确定 Kip2 如何促进微管组装，以及 (iii) 检查 Kip3 与 Kip2 和 Stu2 结合控制微管长度的精度。这些研究将为有丝分裂纺锤体的组装和功能提供重要的见解，并建立适用于其他生物医学相关细胞器系统（如轴丝、微绒毛、静纤毛和丝状伪足）的长度调节原理。