Microtubule reorganization between interphase and mitosis

 描述(申请人提供)：有丝分裂纺锤体的组装需要间期微管(MT)细胞骨架的快速溶解，包括动态和非动态微管的分解。虽然微管在间期和有丝分裂过程中的周转已经被很好地描述了，但我们对间期MT阵列如何被移除以便为两极纺锤体的组装让路知之甚少。间期和有丝分裂的MT阵列存在于接近稳定的状态，而间期MT阵列的溶解代表了这两种稳定状态之间的过渡。整个MT阵列如何在自组织状态之间转换尚未被探索，但对于理解MT阵列在不同模式的阵列之间移动的事件(例如有丝分裂和神经元突起形成)至关重要。无论MT阵列从G2向有丝分裂前期转变的机制是什么，它出人意料地没有受到紫杉醇的抑制，紫杉醇是一种用于治疗几种人类癌症的MT稳定剂。在这里，我们提出了计算机模拟和基于细胞的实验相结合的方法来定义间期MT阵列是如何在有丝分裂之前被移除的，并建立相对于活跃的CDK/细胞周期蛋白B水平的不同步骤何时发生。我们的实验利用了我们最近的发现，即我们可以通过用CDK1抑制剂(RO3306)将细胞阻断在细胞周期的G2中，然后在药物洗脱后30分钟内跟随有丝分裂进入，从而显著增加研究中的细胞数量。我们提出了3个目标来定义有丝分裂进入时MT的周转和溶解。在目标1中，我们将通过将对整个MT阵列的长度分布、MT数量和总聚合物的计算模拟与这些相同特征的实验测量相结合来确定动态MT重组的途径。在目标2中，我们定义了稳定的、非动态的MT何时解聚，以及它们是否形成了前期存在的MT束。在目标3中，我们通过MT标记实验将解聚定位于MT的正端和/或负端，并通过探索MT马达、MT解聚运动蛋白和MT切断蛋白在MT束溶解中的作用，来探索MT束解聚的机制。

 DESCRIPTION (provided by applicant): Mitotic spindle assembly requires the rapid dissolution of the interphase microtubule (MT) cytoskeleton, including disassembly of both dynamic and non-dynamic MTs. While the turnover of microtubules during interphase and mitosis has been well characterized, we know relatively little about how the interphase MT array is removed to make way for assembly of a bipolar spindle. Interphase and mitotic MT arrays exist at near steady states, while the dissolution of the interphase MT array represents a transition between these two steady states. How the entire MT array transitions between self-organized states has not been explored, yet is critical to understanding events where the MT array shifts between arrays of different patterns (e.g. mitosis, and neuronal process formation). Whatever the mechanism underlying MT array transition from G2 to prometaphase of mitosis, it is surprisingly uninhibited by Taxol, a MT stabilizing drug used to treat several human cancers. Here we propose a combination of computer simulation and cell-based experiments to define how the interphase MT array is removed just prior to mitosis and to establish when various steps occur relative to the level of active CDK/cyclin B. Our experiments take advantage of our recent discovery that we can significantly increase the number of cells for study by blocking cells in G2 of the cell cycle with a CDK1 inhibitor (RO3306) and then follow mitotic entry within 30 min of drug washout. We propose 3 aims to define MT turnover and dissolution at mitotic entry. In Aim 1 we will determine the pathway of dynamic MT reorganization by combining computational simulations of the entire MT array's length distribution, MT number and total polymer with experimental measures of these same features. In Aim 2 we define when stable, non-dynamic MTs are depolymerized and whether they form the MT bundles present at prophase. In Aim 3 we explore mechanisms underlying MT bundle disassembly, using both MT marking experiments to localize depolymerization to MT plus and/or minus ends, and by probing the roles of MT motors, MT depolymerizing kinesins and MT severing proteins in dissolution of MT bundles.