FORMATION, INTERACTION, & FUNCTION OF SPINDLE COMPONENTS

这项提议的主要目标是确定中心体如何 和动粒在有丝分裂过程中起作用并相互作用， 纺锤体，以及如何产生染色体分离的力量， 监管. 目标#1涉及同细胞相关性的应用 视频光显微镜（V-LM）和电子显微镜（EM）方法， 研究海洋中心粒/中心体复制的机制 海胆胚胎在体内，并在提取物中的Spisula solvesima卵母细胞 体外 目的#2是评估中心体微管（MT） 当细胞进入有丝分裂时，行为发生变化， 影响MT在体内的行为。 对于本研究，牛MT蛋白，或 没有荧光标记，将被注射到新的肺细胞（NLC） 然后是V-LM。 目标#3是量化aster 有丝分裂过程中的顶出力是染色体大小的函数，有丝分裂 阶段和距离星体中心，并评估这是否 力有助于双极附着和聚集。 为这些 研究表明，NLC中的染色体臂将从动粒中分离出来， 区域与激光微束和操纵校准的光学 镊子 目的#4是通过同池V-LM/EM方法确定 姐妹PtK（1）着丝粒处的MT数量与 聚集的染色体移动的方向， 这是区分国会假说的关键。 目标5 是生成动粒结构的高分辨率3D模型， 在MT采集之前和之后，通过EM层析成像方法。 等 需要重建以评估现有的动粒模型 结构和功能。 目的#6是确定向极染色体是否 速度受动粒纤维成熟度或 分裂。 这项研究还探讨了 染色体运动在有丝分裂阶段之间不同，将基于V-LM 诱导附着在纺锤体上的未附着染色体的分析 在后期。 最后，目标7是确定中期是否 纺锤体结构依赖于两极取向的染色体，如果 后期纺锤体伸长的产力机制是主动的 整个中期或仅在后期触发。 回答这些 问题中期PtK（1）细胞将在以下之一后进行V-LM 每条染色体上的动粒都被激光破坏了 消融术 这些目标将大大加强我们的概念， 有丝分裂是如何工作的--这是理解各种 出生缺陷和癌症，以及设计新的治疗策略 用于控制与疾病状态相关的细胞增殖。

The broad objectives of this proposal are to determine how centrosomes and kinetochores function and interact during mitosis to form the spindle, and how the forces for chromosome segregation are generated and regulated. Aim #1 involves the application of same-cell correlative video light microscopic (V-LM) and electron microscopic (EM) methods to investigate the mechanism of centriole/centrosome replication in sea urchin embryos in vivo, and in extracts of Spisula solidissima oocytes in vitro. Aim #2 is to evaluate how centrosomal microtubule (MT) behavior changes as the cell enters mitosis, and how exogenous tubulin affects MT behavior in vivo. For this study bovine MT protein, with or without a fluorescent label, will be injected into new lung cells (NLCs) and the cells followed by V-LM. Aim #3 is to quantitate the aster ejection force during mitosis as a function of chromosome size, mitotic stage and distance from the astral center, and to evaluate whether this force contributes to bipolar attachment and congression. For these studies chromosome arms in NLCs will be severed from the kinetochore region with a laser microbeam and manipulated by calibrated optical tweezers. Aim #4 is to determine, by same-cell V-LM/EM methods, the relationship between the number of MTs at sister PtK(1) kinetochores and the direction the congressing chromosome is moving--information that is critical for distinguishing between hypotheses of congression. Aim #5 is to generate high-resolution 3D models of kinetochore structure, prior to and after the acquisition of MTs, by EM tomographic methods. Such reconstructions are needed to evaluate existing models of kinetochore structure and function. Aim#6 is to determine if poleward chromosome velocity is regulated by kinetochore fiber maturity or the stage of mitosis. This study, which also addresses whether the mechanism for chromosome motion differs between mitotic stages, will be based on V-LM analyses of unattached chromosomes induced to attach to the spindle during anaphase. Finally, Aim #7 is to determine whether metaphase spindle structure is dependent on bipolar-oriented chromosomes, and if the force-producing mechanism for anaphase spindle elongation is active throughout metaphase or triggered only during anaphase. To answer these questions metaphase PtK(1) cells will be followed by V-LM after one of the kinetochores on every chromosome has been destroyed by laser ablation. Together these aims will significantly enhance our concept of how mitosis works--a requisite for understanding the etiology of various birth defects and cancers, and for designing new therapeutic strategies for the control of cell proliferation related to disease states.