Control of microtubule length by polymerases and depolymerases

描述(申请人提供)：细胞生物学中的一个基本问题是细胞器的大小如何控制，但人们对此知之甚少。例如，有丝分裂纺锤体和轴丝的长度在同一类型的细胞之间只有几个百分点的差异。此外，正确的大小和形态对于细胞分裂的有丝分裂纺锤体和运动的轴丝是必不可少的。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。在没有分子统治者的情况下 模板微管长度，人们认为长度控制是微管聚合和解聚之间微妙平衡的结果。这是如何实现的还不得而知。在我们之前的工作中，我们证明了马达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(MAP)一起，可以在长度和动力学之间提供反馈，从而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，结合数学建模，了解另外两种蛋白质-酵母运动蛋白Kip2和脊椎动物聚合酶XMAP215的酵母同源蛋白Stu2-与Kip3一起调节酵母微管的长度。我们设计了一种新的纯化天然萌芽酵母微管蛋白的方案，这使得我们能够使用酵母作为我们的模型系统，这具有明显的优势，因为微管蛋白亚型的数量很少，并且在脊椎动物中没有潜在的令人混淆的翻译后修饰，特别是在大脑中发现的微管蛋白。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)检验Kip3与Kip2和Stu2共同控制微管长度的精确度。这些研究将为有丝分裂纺锤体的组装和功能提供重要的洞察力，并建立适用于其他生物医学相关细胞器系统的长度调节原则，如轴丝、微绒毛、立体纤毛和丝足。

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.