Hydrodynamic Analysis and Protein Interactions of the Chromosomal Passenger Complex

Abstract

The chromosomal passenger complex (CPC) is a conserved protein complex regulating cell division. Although the kinase subunit, Aurora B (AURKB), self-activates through autophosphorylation, it remains unclear how this happens and more broadly how phosphorylation regulates CPC function. There are also outstanding questions about CPC structure, protein-protein interactions, and transport along microtubules. In Chapter 1, I analyzed CPC hydrodynamic behavior to discover novel interacting partners with inactive CPC. Phosphorylated CPC in clarified mitotic and interphase Xenopus laevis egg extracts had a lower sedimentation coefficient on sucrose density gradients than unphosphorylated CPC. Therefore, phosphorylated CPC either is lighter or is in a more extended conformation. The addition of AURKB inhibitors prevented phosphorylation-induced sedimentation change, emphasizing the role of AURKB activity on CPC structure and function. I tagged the CPC subunit CDCA8 with GFP-3xFLAG in HeLa cells using CRISPR/Cas9. CPC diffusion speed, measured by fluorescence correlation spectroscopy, underwent an AURKB kinase activity-dependent increase upon phosphorylation, indicating a decrease in complex size. This would not be possible if phosphorylated CPC adopted a more extended conformation, which in conjunction with prior density sedimentation data implies that it must decrease in mass. The nucleophosmin/nucleoplasmin family of proteins were identified to be candidates for this mass decrease using immunoprecipitation-mass spectrometry in both X. laevis extract and HeLa cell lysate. As known oligomeric chaperones, binding and unbinding of these proteins may be large enough to cause observed changes in CPC sedimentation and diffusion. Nucleophosmin/nucleoplasmin may inhibit CPC activity or prevent complex aggregation as a means of regulation. In Chapter 2, I discussed tools developed to study CPC structure and function, including attempted targeting of the CPC to artificial lipid bilayers and purification of several recombinant CPC subunits and subunit domains. I also enumerated several efforts to isolate the CPC from biological sources. In Chapter 3, I identified two additional potential CPC interacting partners, myosin II and the kinesin KIF20AE, through sucrose density gradient analysis and conduct preliminary examinations of the effects of these interactions on cell division.