Investigating late mitotic events using Xenopus egg extracts

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DESCRIPTION (provided by applicant): Distribution of a complete set of chromosomes to each daughter cell during mitosis is essential for organism viability, and defects in this process are thought to contribute to cancer. The irreversible and most important steps occur during anaphase. At anaphase A duplicated sister chromosomes separate and move to opposite poles of the microtubule-based mitotic spindle, while anaphase B is defined as spindle pole separation, which occurs as a microtubule structure called the central spindle forms between the segregating chromosomes that also contributes to cytokinesis (pinching of the cell into two). It is unknown how the central spindle assembles and what role it plays in anaphase chromosome separation. The experiments proposed are to elucidate anaphase events using Xenopus egg extracts, which reconstitute the cell cycle in vitro, and are open to biochemical manipulation and high resolution microscopy. Components of the central spindle will be examined that are implicated in late mitotic events, but whose roles and interactions are not understood. Many of these components have overlapping functions in other aspects of mitosis including cytokinesis. An advantage of the Xenopus egg extract system is that cytokinesis does not occur, allowing for the specific study of central spindle components involvement at anaphase. The specific aims are to examine three potential players of the central spindle whose role in the events surrounding anaphase are not clearly defined. Chemical inhibitors will be used in conjunction with immunofluorscence analysis to examine the role of the mitotic kinase Aurora B in anaphase events. Reagents will be developed to disrupt the microtubule bundling protein PRC1 and measure its effects on central spindle formation and anaphase progression. Experiments will be performed to identify and define the role of Golgi-derived membrane in central spindle assembly and organization. These experiments will reveal the molecular pathways that drive anaphase and may be useful developing novel targets for chemotherapeutic agents.