Control of microtubule length by polymerases and depolymerases

摘要/摘要 细胞生物学中一个基本但知之甚少的问题是细胞器的大小是如何控制的。这个 例如，有丝分裂纺锤体和轴丝的长度在细胞之间的差异只有几个百分点。 同样的类型。此外，正确的大小和形态对于细胞的有丝分裂纺锤体是必不可少的。 用于运动的分裂和轴丝。细胞通过严格控制细胞器的长度来调节细胞器的大小。 它们的组成微管。在没有模板微管长度的分子尺子的情况下，它是 认为长度控制是由于聚合和解聚之间的微妙平衡 微管。这是如何实现的还不得而知。 好了！基于我们以前的工作，我们证明了运动Kinesin-8 Kip3是一个长度依赖的 微管解聚酶，我们假设马达蛋白与其他微管- 相关蛋白质(MAP)，可以提供长度和动态之间的反馈，从而严格调节 微管的长度。 好了！这项资助的总体目标是使用单分子技术，以及数学 建模，以了解另外两种蛋白质-酵母Kinesin Kip2和酵母同源物 脊椎动物聚合酶XMAP215，Stu2-与Kip3一起调节酵母微管的长度。我们 我设计了一种新的天然发芽酵母微管蛋白的纯化方案，这使得我们可以使用酵母 作为我们的模型系统，这具有明显的优势，因为微管蛋白异构体的数量很少， 在脊椎动物，特别是大脑中，没有发现潜在的令人困惑的翻译后修饰， 微管蛋白。 好了！我们的具体目标是(1)表征Stu2对酵母微管生长的促进作用，(Ii) 确定Kip2如何促进微管组装，以及(Iii)检查Kip3在 与Kip2和Stu2结合，控制微管长度。这些研究将提供重要的洞察力 有丝分裂纺锤体的组装和功能，并建立长度调节原则，这将是 适用于其他与生物医学相关的细胞器系统，如轴丝、微绒毛、立体纤毛和 丝状伪足。

Summary/Abstract 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 the 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 will be applicable to other biomedically relevant organellar systems such axonemes, microvilli, stereocilia and filopodia.