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.
