CHARACTERIZATION OF MOB1 DYNAMICS IN LIVING CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maintenance of chromosomal ploidy during cell division requires a precise coordination of chromosome segregation and cytoplasmic partitioning (cytokinesis) such that the contractile ring does not assemble before the onset of anaphase. Progression of dividing cells into anaphase into cytokinesis is dependent on the spindle assembly checkpoint, which monitors chromosome attachment to the spindle and regulates the onset of sister chromatid segregation and CDK1 inactivation (mitotic exit). In yeast, the mitotic checkpoint also regulates a signaling cascade termed the Septation Initiation Network (SIN) that regulates the initiation of cytokinesis. Few functional homologues of the SIN have been identified in higher eukaryotes, but a terminal component named Mob1 has been identified in all eukaryotes. Expression of GFP chimeras of the four human Mob1s reveal that Mob1 localizes to the spindle poles and kinetochores up until anaphase onset, and the central spindle and midbody during cytokinesis. Additionally, we have identified a novel interaction between Mob1A and LATS2 kinase, which may serve as the functional homolog of the yeast Dbf2/Sid2 kinase in mammalian cells. Using these preliminary studies as a foundation, this application seeks to evaluate the roles that Mob1 plays in coordinating mitosis and cytokinesis in mammalian cells. The lines of experimentation proposed in this application will characterize the localization dynamics of the four human Mob1 family members using co-localization studies and live cell imaging. Further, the role of Mob1 in the coordination of mitosis and cytokinesis will be assessed by specifically disrupting Mob1/Lats2 interactions in vivo by siRNA knockdown of Mob1 or by expression of Lats2-binding mutants of Mob1. These efforts should ultimately lead to a clearer understanding of how mitosis and cytokinesis is coordinated, and confirm whether the strategies employed by unicellular fungi to regulate septation are conserved in higher eukaryotes.