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）：细胞生物学中的基本问题（但知之甚少）是如何控制细胞器的大小。例如，有丝分裂的纺锤体和轴突的长度在相同类型的细胞之间的变化略低于几％。此外，正确的大小和形态对于细胞分裂的功能有线纺锤来说至关重要。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尺寸。在没有分子统治者的情况下 模板微管长度，人们认为长度控制是由于微管的聚合和解聚之间的微妙平衡而产生的。如何实现这一目标。基于我们以前的工作，我们表明运动动力蛋白-8 KIP3是长度依赖性的微管解酯酶，我们假设运动蛋白与其他微管相关蛋白（MAP）结合使用，可以在长度和动力学之间提供紧密调节长度长度的小管的反馈。 该赠款的总体目的是使用单分子技术以及数学建模，以了解两种其他蛋白质 - 酵母驱动蛋白KIP2和脊椎动物聚合酶XMAP215的酵母同源物，stu2 t和Kip3如何调节酵母微管的长度。我们已经设计了一种新颖的纯化纯化方案，用于原生萌芽的酵母小管蛋白，这使我们能够使用酵母作为模型系统，由于微管蛋白同工型的数量少，并且缺乏潜在的混杂后翻译后修饰，因此具有明显的优势，脊椎动物，尤其是大脑，尤其是大脑，微管蛋白。 我们的具体目的是（1）表征通过Stu2，（ii）确定KIP2如何促进微管组装的TE加速酵母微管的生长，并且（iii）检查Kip3与Kip2和Stu2结合的精度控制微管长度。这些研究将为有丝分裂主轴的组装和功能提供重要的见解，并建立长度调节原理，这些原理适用于其他与生物医学相关的细胞器系统，例如轴突，微绒毛，立体尾膜和丝状。