Cell Cycle Control of the Cytoskeleton

描述（由申请人提供）：精确控制微管长度是所有细胞微管过程的基础，包括有丝分裂、纺锤体定向、轴突引导和囊泡运输3-6。本建议的目标是定义微管长度控制的机制，并描述这些机制如何有助于有丝分裂的保真度。在之前资助期工作的基础上，我们将从两个层面解决这个问题。首先，我们将定义真核生物中微管长度、纺锤体大小和纺锤体几何形状的主要调节因子，即运动蛋白8马达（出芽酵母中的Kip3 5,7-9）的分子机制。酵母驱动蛋白8，Kip3在微管+端以高亲和力结合并促进微管分解8-10。由于大多数微管动力学调节和长度控制发生在微管正端，微管相关蛋白（MAPs）和马达如何与正端相互作用是该领域的核心问题。Kip3结合MT +端并调控MT动力学的机制将通过整体生物化学、结构研究、单分子成像、体内成像和遗传分析相结合来确定。基于我们最近的发现，Kip3具有将反平行微管滑动分开的能力，我们将定义Kip3如何有助于主轴组装和后期主轴伸长。这些实验将建立在新的体外系统上，包括在微图案上重建纺锤体拉长样反应的能力

DESCRIPTION (provided by applicant): Accurate control of microtubule length is fundamental to all cellular microtubule -based processes including mitosis, spindle orientation, axon guidance, and vesicular transport 3-6. The goal of this proposal is to define the mechanisms underlying microtubule length control and to characterize how these mechanisms contribute to the fidelity of mitosis. Building on work in prior funding periods, we will approach this problem a two levels. First, we will define the molecular mechanism of a major regulator of microtubule length, spindle size, and spindle geometry in eukaryotes, the kinesin 8 motor (Kip3 in budding yeast 5,7-9). The yeast kinesin 8, Kip3 walks processively to the microtubule plus end where it binds with high affinity and promotes microtubule disassembly 8-10. Because most microtubule dynamics regulation and length control occurs at the microtubule plus end, how microtubule-associated proteins (MAPs) and motors interact with the plus end is a central question in the field. The mechanism by which Kip3 binds the MT plus end and regulates MT dynamics will be determined using a combination of bulk biochemistry, structural studies, single molecule imaging, in vivo imaging and genetic analysis. Building on our recent finding that Kip3 has the ability to slide antiparallel microtubules apart, we will define how Kip3 contributes to spindle assembly and anaphase spindle elongation. These experiments will build on novel in vitro systems, including the ability to reconstitute a spindle elongation-like reaction on micropatterned glass substrates. The second line of experiments will employ in vitro microbial evolution as an unbiased approach to define how optimal MT length control and spindle function can emerge from the destabilizing effects of a genome doubling 2,11. We previously showed that tetraploidy in budding yeast causes a profound distortion in spindle geometry, which in turn results in a >200-fold increase in chromosome missegregation11. Genome doubling or tetraploidy is common during organismal evolution and during tumorigenesis 12-15.