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The non-motor protein RHAMM locates TPX2 to coordinate spindle assembly and balance motor forces needed to segregate chromosomes and complete cell division

University of British Columbia

Cell division requires the assembly and organization of a microtubule-based mitotic spindle. Microtubule assembly at multiple sites is dependent on Aurora kinase A activity, which is promoted through a complex with TPX2 (targeting protein for XKlp2). Subsequent organization of these microtubules and progression into anaphase requires balance between forces orchestrated by antagonistic motor complexes. My studies show that the non-motor protein RHAMM (receptor for hyaluronan mediated motility) integrates structural and biochemical pathways to ensure the fidelity of cell division. Silencing RHAMM in HeLa cells delayed the kinetics of spindle assembly. I located RHAMM to centrosomes and non-centrosome sites for microtubule nucleation and found it necessary for TPX2 localization and Aurora A activity at kinetochores. The RHAMM-TPX2 complex requires a conserved leucine zipper motif in RHAMM and a domain that includes the nuclear localization signal in TPX2. These findings indicate RHAMM is needed for spatially-regulated activation of Aurora A by TPX2, which coordinates spindle assembly. I monitored mouse embryonic fibroblasts deficient for RHAMM through division and identified defects progressing through the spindle checkpoint. In RHAMM-silenced HeLa cells, I identified sustained activation of the checkpoint with unfocused spindles and unattached kinetochores, implicating unbalanced motor activities mediated by kinesins. In metaphase-delayed cells, the abundance or location of checkpoint proteins was not altered. Moreover, aberrant spindle orientation could not account for each delayed division. In RHAMM-silenced cells, I found that the reciprocal immunoprecipitation of Eg5-TPX2, an inhibitory complex, was reduced and that the concurrent inhibition of Eg5-generated force recovered division kinetics. I also observed a prolonged metaphase delay in a proportion of RHAMM-silenced cells, which resolved through cohesion fatigue. Together, my findings indicate that RHAMM-mediated attenuation of Eg5-dependent outward forces is needed to align chromosomes and progress through division. Lastly, I identified defects in spindle structure and function in redundant models for RHAMM over-expression. Collectively, my studies demonstrate that RHAMM coordinates Aurora A signaling and balances motor forces that are needed for cell division. These findings provide novel insights into processes that are essential for mammalian cell division and the maintenance of genome stability.

Text

2017-01-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/60235

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/