Centrosomal and centrosome-independent mechanisms in mitotic spindle assembly

描述（由申请人提供）：本项目的总体目标是揭示确保有丝分裂期间染色体无错误分离的机制。虽然较高水平的分离错误是致命的，但相对罕见的单个染色体的错误分离是“染色体不稳定性”（CIN）的基础，这是癌症转化的标志。因此，确定CIN的确切原因并找到一种降低染色体错分离率的方法可能会导致选择性消除癌细胞的新策略。为了实现这一目标，我们正在使用复杂的成像技术，结合分子和细胞生物学技术来表征“有丝分裂纺锤体”，这是一种自组装的分子机器，可以实现染色体分离。在目前的资助期内，我们计划实现三个目标：1)通过基于超分辨率光学显微镜的新方法重建正常和CIN细胞中纺锤体的三维结构。对正常细胞和CIN细胞纺锤体组织的比较分析将揭示染色体丢失的结构基础。具体来说，我们将验证一些染色体与纺锤极缺乏直接连接的假设，这些染色体在CIN细胞中数量更多。2)描述着丝点（将染色体连接到纺锤体微管上的大分子组件）从最初的“横向”附着到最终的“端上”附着过渡期间的结构重组。尽管它们很重要，但由于技术限制，侧向附着物的超微结构尚未被描述。我们将利用一种新的方法，将多色光学显微镜与在同一着丝点上进行的高分辨率电子断层扫描直接相关。这种方法将允许我们测试相同的分子机制（Hec1）介导外侧和末端附着的假设，并揭示有丝分裂检查点满足的基础外板的重组。3)验证动力蛋白的轻度抑制（在正常细胞中是良性的）选择性抑制CIN细胞有丝分裂的假设。这一想法源于初步数据，即CIN细胞中大量染色体缺乏与纺锤极的直接连接，这些染色体依赖动力蛋白介导的运输进行分离。我们将系统地描述动力蛋白抑制剂对正常细胞和CIN细胞中细胞增殖、细胞死亡水平、染色体运动模式和纺锤体组装机制的影响。如果我们的假设被证明是正确的，部分抑制动力蛋白将成为一种强有力的治疗方法。

DESCRIPTION (provided by applicant): The overarching goal of this project is to reveal the mechanisms that ensure error-free segregation of chromosomes during mitosis. While higher levels of segregation errors are lethal, relatively infrequent mis- segregation of individual chromosomes underlies 'chromosomal instability' (CIN), a hallmark of cancer transformation. Therefore, identifying the exact cause(s) of CIN and finding a way to attenuate the rate chromosome missegregation will likely lead to novel strategies for selective elimination of cancer cells. Toward this goal we are employing sophisticated imaging in conjunction with molecular and cell-biology techniques to characterize the 'mitotic spindle', a self-assembling molecular machine that enacts chromosome segregation. In the current funding period we plan to achieve three objectives: 1) Reconstruct 3-D architecture of the spindle in normal and CIN cells via a novel approach based on super-resolution light microscopy. Comparative analyses of spindle organization in normal vs. CIN cells will reveal the structural foundation of chromosome loss. Specifically, we will test the hypothesis that some chromosomes lack direct connections to spindle pole and that these chromosomes are more numerous in CIN cells. 2) Characterize the structural reorganization of kinetochores (macromolecular assemblies that attach chromosomes to spindle microtubules) during the transition from the initial 'lateral' to the final 'end-on' attachments. In spite of their importance, the ultrastructure of lateral attachments has not been described due to technological limitations. We will capitalize on a novel approach that involves direct correlation of multi-color light-microscopy with high-resolution electron tomography conducted on the same kinetochore. This approach will allow us test the hypothesis that the same molecular machinery (Hec1) mediates both lateral and end-on attachments and also reveal reorganization of the outer plate that underlies satisfaction of the mitotic checkpoint. 3) Test the hypothesis that mild inhibition of dynein, benign in normal cells, selectively suppresses mitosis in CIN cells. This idea stems from the preliminary data that a significant number of chromosomes in CIN cells lack a direct attachment to the spindle poles and these chromosomes rely on dynein-mediated transport for segregation. We will systematically characterize the effects of dynein inhibitors on cell proliferation, levels of cell death, patternsof chromosome movement, and mechanisms of spindle assembly in normal vs. CIN cells. If our hypothesis is proven correct, partial inhibition of dynein will emerge as a powerful therapeutic approach.