Precise transmittance of genetic information to the daughter cell during mitosis is dependent on the proper alignment of sister chromatids as well as bipolar mitotic spindle formation. The mitotic spindle is nucleated from the major microtubule-organizing centers, centrosomes, at opposite ends of the cell (Luders and Stearns 2007). The centrosome is comprised of two centrioles which detach from each other, in a process referred to as disengagement, at the exit from mitosis concurrently with the segregation of sister chromatids. These processes are both dependent on the action of the protease, separase (Uhlmann, Wernic et al. 2000;Tsou and Stearns 2006). While it is known that separase cleaves the protein complex cohesin to release sister chromatids, it is unknown which protein is responsible for the attachment of centrioles. The objective of this proposal is to determine which protein is cleaved by separase to release centrioles. I propose that in addition to being responsible for the physical linkage between sister chromatids, the cohesin complex is also required for centriole cohesion. It has been difficult to study the many functions of the cohesin protein complex during the cell cycle through traditional inactivation techniques such as RNA interference (RNAi), due to the interdependence of cell cycle events, the presence of feedback regulation, and the relatively fast timescale of mitotic processes. The objective of experiments proposed in the first aim is to develop a new method for cohesin inactivation specifically during mitosis by chemically induced activation of an exogenously expressed protease targeting cohesin. In the second aim, this method will be applied to determine if cleavage of cohesin at the centrosome is sufficient for centriole disengagement. The inducible protease will be specifically targeted to the centrosome in order to discriminate if centriole disengagement is due solely to cohesin cleavage at the centrosome and not a secondary effect of sister chromatid segregation. Development of this technique is of general importance since it can be applied to the inactivation of a broad range of proteins and will thus impact future studies on mitotic regulators. If the cohesin protein complex is responsible for centriole cohesion, it will reveal the coordinated control of centrosome dynamics and the chromosome cycle. Defects in these processes result in cells with either extra or fewer chromosomes. This state, referred to as aneuploidy, is associated with the malignant transformation of cancer cells (Griffin 1996). Many aneuploid cancer cells exhibit centrosome amplification;therefore, it is essential to understand the mechanism governing centriole cohesion and disengagement (Carter, Eklund et al. 2006;Patel and Gordon 2009).
When cells divide, chromosomes, which contain our genes, are segregated into two daughter cells. Aberrant segregation of our genetic material during cell division results in an abnormal number of chromosomes, which is a hallmark of some cancers. Results from this proposal will identify one mechanism in the cell to prevent the formation of multipolar spindles and thus the prevention of chromosome missegregation.
