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的，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突体的长度在相同类型的细胞之间仅相差几个百分点。此外，正确的大小和形态对功能至关重要-有丝分裂纺锤体用于细胞分裂，轴突用于运动。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。在没有分子标尺的情况下

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.