Molecular Dissection Of The Cellular Dynamics Underlying Chromosome Segregation In Mitosis

Mitosis is the most dramatic,as well as critical event in the cell cycle.Errors in the choreography of mitotic chromosome movements can lead to aneuploidy or genetic instability,fostering cell death or tumor development.Therefore it is essential to delineate how cellular plasticity and dynamics are achieved and orchestrated during mitotic progression.Combined with biophotonic and chemical biological approaches, here I attempted to delineate the molecular mechanisms underlying faithful chromosome segregation in mammalian cell division.Chromosome segregation in mitosis is orchestrated by dynamic interactions between spindle microtubules and kinetochores,which in turn are governed by protein kinase-and phosphatase-signaling cascades.Growing evidences indicate that Polo-like kinase 1 (PLK1) is activated during mitotic entry and plays an essential role in mitosis from start to finish.However,the molecular illustration of spatiotemporal dynamics of PLK1 in mitosis is unavailable.To illuminate of PLK1 activity in space and time,I have developed a fluorescence resonance energy transfer(FRET)-based optical sensor to report PLK kinase activity of kinetochore.Using this optical sensor,my study shows that kinetochore-centered PLK1 activity is high in prophase and prometaphase,and drops upon the achievement of chromosome bi-orientation in metaphase.Importantly,our analysis demonstrates that PLK1 kinase dynamics provides an accurate readout of interkinetochore tension.Using chemical modulators combined with real-time imaging the PLK1 sensor,my studies reveal that PLK1 kinase gradient at kinetochore is governed by Aurora B.Our findings suggest a novel regulatory mechanism by which Aurora B and PLK1 kinases cooperate to ensure accurate kinetochore-microtubule attachment and chromosome plasticity in mitosis.Our molecular delineation of PLK1 kinase function in mitosis led to the identification of a novel interaction between PLK1 and hCdc14A phosphoatase.Our biochemical characterization reveals that hCdc14A phosphatase activity latent in early mitosis via its intra-molecular association.PLK1 interacts with and phosphorylates hCdc14A,therefore stimulates hCdc14A's phosphatase activity by partially releasing its auto-inhibition. Importantly,overexpression of the phospho-mimicking mutant caused aberrant chromosome alignment with a prometaphase delay,suggesting the temporal regulation of hCdc14A activity by PLK1 is critical for orchestrating mitotic events.Accurate segregation of chromosomes,initiated by abrupt and irreversible dissolution of sister-chromatid cohesion at anaphase,is crucial for the faithful inheritance of parental genomes during cell division.The dissolution of sister-chromatid cohesion is catalyzed by separase after the destruction of securin by the anaphase-promoting complex. However,separase was localized to the mitotic centrosome,raising the question as how separase hydrolyzes sister-chromatid cohesion of centromere at the anaphase onset.Using a panel of separase antibodies,we found that separase protein is accumulated in mitosis and degraded at the end of telophase.Surprisingly,in our immunofluorescence microscopic analyses,separase was found to be associated with mitotic chromosomes from prophase to metaphase and dissociated from chromosomes in anaphase right after sister chromatids separation.Staining of isolated mitotic chromosomes from nocodazole-arrested cells revealed that separase is concentrated at the centromeric cohesion.To examine if any mitotic kinases are responsible for chromosomal localization of separase, we carried out RNAi-mediated knockdown and found the association of separase with mitotic chromosomes is a function of Aurora B.Consistent with the phenotype seen in the Aurora B-repressed cells,inhibition of Aurora B kinase by hesperadin prevents the association of separase with chromosomes.Our results suggested that Aurora B kinase activity helps coordinate the association of separase with chromosomes and the initiation of sister-chromatid separation.It has been postulated that accurate kinetochore-microtubule attachment and tension across the sister kinetochore initiates anaphase events.As the guardian spirit of centromeric cohesion,Sgol collaborates with PP2A to orchestrate the temporal order of sister chromatide separation at the anaphase onset.Temporal control of Sgo1 degradation allows a timely resolution of sister chromatid cohesion by separase.However,the mechanism regulating its displacement from kinetochore at metaphase-anaphase transition remains unclear.Our in vitro cleavage results suggested Sgo1 is a novel substrate of the Ca²⁺-dependcnt protease m-calpain.The physiological relevance of calpain-Sgo1 interaction was demonstrated by the liberation of Sgo1 from kinetochore upon elevation of intracellular calcium.Importantly,the Sgo1 dissociation from kinetochore in response to the intracellular calcium rise was blocked by calpain inhibitor E64d,demonstrating the role of calpain-mediated proteolysis in dissociation of Sgo1 from kinetochore.Our findings illustrate a novel regulatory mechanism by which spatiotemporal dynamics of calcium spike in mitosis cooperate with chromosome segregation machinery to ensure the chromosome plasticity in mitosis.In sum,my studies illustrate the spatiotemporal dynamics and plasticity of PLK1 activity in mitosis.In addition,my analyses reveal that Aurora B and PLK1 kinases cooperate to ensure accurate kinetochore-microtubule attachment in mitosis.Together with the discovery calpain-Sgo1 inter-relationship established,my studies shed new light on the mitotic orchestration underlying chromosome segregation.