Cyclin/CDK and Kinesin-5 Control of Spindle Assembly

本计画探讨细胞周期机制调控有丝分裂纺锤体的组装。 纺锤体的组装始于中心体的复制，中心体是细胞的主要微管组织中心（MTOC）。 在中心体复制后，中心体必须分离，以便在有丝分裂时组装成双极纺锤体。 中心体复制和纺锤体组装与其他细胞周期事件相协调，以确保染色体在有丝分裂时正确分离。 众所周知，细胞周期蛋白依赖性激酶（Cdk）复合物调节纺锤体组装的多个方面，包括中心体分离，尽管在分子水平上Cdk复合物如何触发这一步骤仍不清楚。 在酵母和后生动物中，驱动蛋白-5马达的活动对于中心体分离也是必不可少的。 芽殖酵母中心体的分离，称为纺锤体极体（SPB），需要两个驱动蛋白-5马达中至少一个的活性：Kip 1或Cin 8。 Kip 1和Cin 8作为多聚体（+）末端导向的微管马达发挥作用，并且也具有微管捆绑活性。Cin 8和Kip 1电机和/或捆绑活动可能会直接分离重复的SPB，从而组装一个短主轴。 Haase实验室的最新发现表明，Cin 8和Kip 1是芽殖酵母中Cdks的直接靶点，这表明Cdks可能通过调节Cin 8和Kip 1的活性来促进SPB分离。 在这个项目中的实验将确定驱动蛋白-5马达是如何调节细胞周期蛋白/Cdk介导的磷酸化。驱动蛋白-5马达的马达和微管捆绑活动如何促进SPB分离也将被检查。这个项目将产生新的见解调节有丝分裂纺锤体组装，并最终，细胞分裂的基本过程。 关于高等真核生物中心体分离的调控知之甚少，所以在实验上易处理的芽殖酵母中的工作应该能阐明这一重要问题。该项目还将对教育和培训产生重大影响。 该项目的一个主要目标是培养新一代的年轻研究人员，包括研究生和本科生，在多学科的方法来解决生物学中的基本问题。

This project addresses the mechanisms by which the cell cycle machinery regulates assembly of the mitotic spindle. Spindle assembly begins with the duplication of centrosomes, the major microtubule organizing center (MTOC) of the cell. Following centrosome duplication, the centrosomes must separate in order to assemble a bipolar spindle at mitosis. Centrosome duplication and spindle assembly are coordinated with other cell cycle events to insure that chromosomes are segregated properly at mitosis. It is well established that cyclin dependent kinase (Cdk) complexes regulate multiple aspects of spindle assembly, including centrosome separation, although it remains unclear at the molecular level how Cdk complexes trigger this step. In yeast and metazoans, the activities of kinesin-5 motors are also essential for centrosome separation. Separation of the budding yeast centrosome, called a spindle pole body (SPB), requires the activity of at least one of two kinesin-5 motors: Kip1 or Cin8. Kip1 and Cin8 function as multimeric (+) end-directed microtubule motors, and also have microtubule bundling activities. Cin8 and Kip1 motor and/or bundling activities are likely to direct the separation of duplicated SPBs, thus assembling a short spindle. Recent discoveries in the Haase lab indicate that Cin8 and Kip1 are direct targets of Cdks in budding yeast, suggesting that Cdks may promote SPB separation by regulating the activities of Cin8 and Kip1. Experiments in this project will determine how kinesin-5 motors are regulated by cyclin/Cdk mediated phosphorylation. How the motor and microtubule bundling activities of kinesin-5 motors contribute to SPB separation also will be examined.This project will yield new insights into the fundamental processes regulating mitotic spindle assembly, and ultimately, cell division. Little is known about the regulation of centrosome separation in higher eukaryotes, so work in the experimentally tractable budding yeast should shed light on this important problem. The project also will have substantial impact on education and training. A major goal of the project is to train new generations of young researchers, including both graduate and undergraduate students, in multi-disciplinary approaches to fundamental questions in biology.