Cell Cycle Control of the Cytoskeleton

描述（由申请人提供）：微管长度的精确控制是所有基于微管的细胞过程的基础，包括有丝分裂、纺锤体定向、轴突导向和囊泡运输3-6。这项建议的目标是确定微管长度控制的机制，并表征这些机制如何有助于有丝分裂的保真度。在以往供资期间工作的基础上，我们将从两个层面处理这一问题。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，驱动蛋白8马达（芽殖酵母中的Kip 3 5，7 -9）的分子机制。酵母驱动蛋白8，Kip 3向前走到微管正端，在那里它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中心问题。Kip 3结合MT+端和调节MT动力学的机制将使用大量生物化学、结构研究、单分子成像、体内成像和遗传分析的组合来确定。基于我们最近的发现，Kip 3具有滑动反平行微管的能力，我们将定义Kip 3如何有助于纺锤体组装和后期纺锤体伸长。这些实验将建立在新的体外系统上，包括在微图案化的细胞上重建纺锤体延伸样反应的能力。 玻璃基板第二系列实验将采用体外微生物进化作为无偏方法来定义最佳MT长度控制和纺锤体功能如何从基因组加倍的不稳定效应中出现2，11。我们先前表明，芽殖酵母中的四倍体导致纺锤体几何形状的深刻扭曲，这反过来又导致染色体错误分离增加>200倍11。基因组加倍或四倍体在生物体进化和肿瘤发生过程中很常见12-15。

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.