Bipolar spindle assembly is required for the correct segregation of duplicated chromosomes during cell division. While spindle assembly has been studied for many years, the principles and mechanisms governing it remain unclear. The general goal of the project is to elucidate the principles that underlie spindle assembly and function, as well as to identify and study the roles of individual proteins. In the long term, this approach will allow the reconstitution of spindle assembly from purified components. Since uncontrolled cell division is at the heart of the cancer problem, a molecular understanding of spindle assembly and function could lead to new approaches for cancer therapy. To study spindle assembly mechanisms we have developed an in vitro system utilizing DNA linked to beads as artificial chromosomes, which function physiologically in cytoplasmic extracts prepared from Xenopus eggs. In mitotic extracts, DNA beads induce the formation of bipolar spindles in the absence of centrosomes and kinetochores. This assay has revealed that spindle assembly around chromatin proceeds in two phases. First microtubule nucleation and growth is induced around chromatin, then the stabilized microtubules are reorganized into a bipolar spindle by microtubule-based motors.
The specific aims of the proposal address the molecular mechanisms behind these two steps. The general strategies used take advantage of the open nature of extracts, which allows specific inactivation of individual components; while the use of DNA beads facilitates magnetic isolation of chromatin proteins for functional and biochemical analyses.
The aims are: (1) To examine the role of different microtubule-based motors in the bipolar organization of microtubules by specific inactivation through immunodepletion, or addition of specific antibodies or dominant negative constructs. (2) To explore the mechanism of specific microtubule growth around chromatin by gamma-tubulin inactivation and reconstitution experiments. (3) To examine the requirements for functionally active DNA beads by correlating chromatin assembly with the ability of beads to induce spindle assembly. (4) To identify chromatin factors important for microtubule stabilization through biochemical analysis of proteins and enzymatic activities bound to DNA beads. (5) To reconstitute aspects of anaphase chromosome segregation using beads coupled with centromeric DNA in order to develop an assay for kinetochore function and to identify DNA sequences and proteins required.
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