Molecular Dissection of the "Pacman-Flux" Machinery Used to Move Chromosomes

描述(申请人提供)：染色体朝向有丝分裂纺锤体的相反两极的能力是细胞正常分裂的基础，这一过程中的缺陷被认为是肿瘤发生的第一步。极向染色体的运动是通过“Pacman流”机制进行的：染色体诱导附着的微管正端解聚，称为“Pacman”，而极向微管被卷绕到纺锤体极，“由微管负端的解聚刺激”。这项建议中概述的研究的目标是阐明刺激和控制基于“吃豆子通量”的染色体运动的分子机制。这项工作的中心假设是，染色体的运动性由一系列机械上不同的蛋白质控制，这些蛋白质使用不同的靶向和作用机制来控制微管末端的聚合状态。目的1)阐明刺激和控制微管负端解聚和极向流速度的分子途径。本研究旨在评估微管切断蛋白、多个Kinesin-13s和Kinesin-13磷酸化的通量相关功能。目的2)阐明染色体诱导微管+末端解聚的分子途径。为此，研究评价了CLIP-170/190、微管切断蛋白和多个Kinesin-13s的Pacman相关功能。果蝇是这些研究中使用的主要实验系统。用于显示纺锤体和染色体动态的活细胞技术已经在果蝇S2细胞和胚胎中得到优化，因此这些细胞提供了一个理想的环境，在其中研究蛋白质功能的操纵如何影响“Pacman-Flux”。有丝分裂的活细胞显微镜研究将得到生化和分子方法的补充，以深入了解特定类别的蛋白质是否以及如何驱动染色体分离。在人类细胞中进行了其他研究，以确定我们提出的途径的某些方面是否在进化上是保守的。染色体分离缺陷会导致人类疾病，如出生缺陷和癌症。了解这一过程是如何正常发生的，应该为这些疾病的分子病因学提供洞察力，并为它们的治疗提出治疗策略。

DESCRIPTION (provided by applicant): The ability of chromosomes to move towards opposite poles of the mitotic spindle is fundamental for proper cell division and defects in this process are believed to be an initial step in tumorigenesis. Poleward chromosome motility occurs by a "Pacman-flux" mechanism: Chromosomes induce the depolymerization of attached microtubule plus-ends, termed "Pacman", while being reeled into spindle poles by poleward tubulin flux" stimulated by depolymerization of microtubule minus-ends. The goal of the studies outlined in this proposal is to elucidate the molecular machinery that stimulates and controls "Pacman-flux"-based chromosome motility. The central hypothesis of this work is that chromosome motility is controlled by a mechanistically diverse array of proteins which employ distinct targeting and mechanisms-of-action to control the polymerization state of microtubule ends. There are two specific aims: Aim 1) Elucidate the molecular pathway that stimulates and controls the velocity of microtubule minus-end depolymerization and poleward flux. Studies in this aim evaluate the flux-related functions of microtubule severing proteins, multiple kinesin-13s and kinesin-13 phosphorylation. Aim 2) Elucidate the molecular pathway by which chromosomes induce the depolymerization of microtubule plus-ends. Studies in this aim evaluate the Pacman-related functions of CLIP-170/190, microtubule severing proteins, and multiple kinesin-13s. The fruit fly Drosophila melanogaster is the primary experimental system used in these studies. Live cell techniques for visualizing spindle and chromosome dynamics have been optimized in Drosophila S2 cells and embryos and thus these cells provide an ideal context within which to study how the manipulation of protein function impacts "Pacman-Flux". Live cell microscopic studies of mitosis will be complemented by biochemical and molecular approaches to provide an in-depth understanding of whether and how specific classes of proteins drive chromosome segregation. Additional studies are performed in human cells to determine whether aspects of our proposed pathways are evolutionary conserved. Defects in chromosome segregation lead to human maladies such as birth defects and cancer. An understanding of how this process occurs normally should provide insights into the molecular etiology of these diseases and suggest therapeutic strategies for their treatment.