Control of microtubule length by polymerases and depolymerases

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，有丝分裂纺锤体和轴丝的长度在同一类型的细胞之间的差异只有百分之几。此外，正确的大小和形态是必不可少的功能有丝分裂纺锤体细胞分裂和轴丝运动。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， 作为微管长度的模板，长度控制被认为是微管聚合和解聚之间的微妙平衡的结果。这是如何实现的尚不清楚。基于我们以前的工作，我们表明，运动驱动蛋白-8 Kip 3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（MAPs），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，结合数学建模，以了解两种额外的蛋白质-酵母驱动蛋白Kip 2和脊椎动物聚合酶XMAP 215的酵母同系物Stu 2-与Kip 3一起调节酵母微管的长度。我们已经设计了一种新的天然芽殖酵母微管蛋白的纯化方案，这使我们能够采用酵母作为我们的模型系统，这具有明显的优势，由于微管蛋白同种型的数量少，并在脊椎动物，特别是脑，微管蛋白中发现的潜在混淆的翻译后修饰的情况下。 我们的具体目标是（1）表征Stu 2对酵母微管生长的加速作用，（ii）确定Kip 2如何促进微管组装，以及（iii）检查Kip 3与Kip 2和Stu 2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的细胞器系统，如轴丝，微绒毛，静纤毛和丝状伪足的长度调节原则。

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.