Molecular regulation of abscission

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OVERVIEW: Successful cell division and the maintenance of a stable genome requires a coordinated spatio-temporal regulation of diverse cellular processes termed the cell cycle. Cell cycle progression relies on the alternate expression and degradation of distinct cyclin-kinase (CDK) complexes. The final stage of the cell cycle is the process of abscission at the end of cytokinesis when the two new cells physically separate. This process is regulated by the abscission checkpoint. Key to this checkpoint is the activity of the kinase Aurora B that blocks abscission. How Aurora B activity is overcome to drive abscission is not known. However, we have discovered a novel mitosis-specific Cyclin-CDK complex, Cyclin L-CDK11p58 whose activity is required for timely abscission in the presence of Aurora B activity. CDK11p58 does not switch off Aurora B activity, suggesting that the abscission checkpoint is overcome rather than terminated to promote mitotic progression. HYPOTHESIS: CDK11 activity is regulated by the completion of prior cell cycle events to promote timely assembly of the abscission machinery by opposing the inhibitory activity of Aurora B kinase. SPECIFIC AIMS: AIM1 Determine how CDK11p58 promotes abscission Our data suggests CDK11p58 and a PP2A phosphatase-B56 epsilon operate as positive effectors of abscission site assembly whereas Aurora B is a negative effector of the pathway. We will determine: A) How PP2A phosphatase-B56 epsilon and CDK11 oppose Aurora B activity to regulate ESCRT-III function in abscission using biochemical and imaging assays. B) Identify CDK11p58 substrates involved in abscission using biochemical assays. AIM2 Determine how CDK11p58 is regulated The abscission check point is sensitive to chromatin persisting in the cytokinetic machinery, DNA replication stress, nuclear envelope reassembly tension between the dividing cells. How these processes transmit signals to the abscission machinery is unknown. We will determine: A) Cyclin L-CDK11p58 interactors during the abscission process to determine how it is targeted to the abscission machinery. B) Which prior cell cycle pathways regulate CDK11p58 activity using biochemical and microscopy based assays. SUMMARY: In higher eukaryotes, the final stage of cell division, abscission, can only proceed once each copy of the genome has been segregated to opposite poles of the dividing mother cell and encased in a functional nuclear envelope. Our studies will identify and characterize new pathways that ensure that the final step in cell division occurs at the correct time thereby maintaining viable cells with a stable genome.