lated Histone H3 found across all chromosomes. These results suggest that deficiency of UBASH3B has a dramatic effect on localization of Aurora B by mistargeting the active kinase at the chromosome. We next tested if the spindle-associated UBASH3B is required for the chromosomal localization of Aurora B. Strikingly, depolymerization of microtubules with micromolar doses of nocodazole abolished the chromosomal localization defects of Aurora B observed upon downregulation of UBASH3B and the centromere levels of Aurora B and P-T3-H3 were reduced only in the presence of intact microtubules. Thus, the microtubule-associated UBASH3B contributes to the histone marks pathways and provides a molecular link between the centromeric and the microtubule-associated fractions of Aurora B. UBASH3B is a limiting factor sufficient for Aurora B localization to microtubules Monoubiquitylation can serve as a signal mediating reversible recruitment of the proteins to specific compartments. In agreement with previous findings, regulation of Aurora B localization by ubiquitin is likely to be dynamic and to contribute to Aurora B function in faithful chromosome segregation. If this were the case, UBASH3B may regulate the balance of centromeric and spindle-associated Aurora B by actively recruiting Aurora B to microtubules. To test this assumption, we Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Dev Cell. Author manuscript; available in PMC 2017 April 21. Krupina et al. Page 7 overexpressed UBASH3B in cells arrested in prometaphase by STLC. In control cells, the majority of Aurora B localized to the centromeric regions of circularly arranged chromosomes. Strikingly, overexpression of the FLAG-WT-UBASH3B was sufficient to drive association of endogenous Aurora PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811302 B with microtubules, and both FLAG-UBASH3B and Aurora B were localized to the monopolar spindles in these cells. In contrast, FLAG-UBASH3B mutated in both UBA and eUBA domains failed to trigger Aurora B relocalization to microtubules despite its efficient localization to microtubules. The GFP-tagged form of Aurora B was also actively recruited to the spindle by UBASH3B and it could be visualized within the microtubule bundles in prometaphase-arrested cells. Furthermore, overexpression of UBASH3B prometaphase arrested cells increased association of Aurora B with stable microtubules. Since a drop in CDK1 activity at anaphase onset was reported to contribute to Aurora B microtubule association, we analyzed the levels of the activatory subunit of CDK1 kinase, Cyclin B. Importantly, the average levels of Cyclin B were not dramatically influenced by overexpression of FLAG-UBASH3B, and both low and high Cyclin B levels were observed in prometaphase cells with microtubule-associated Aurora B. This result suggests that overexpression of UBASH3B may target Aurora B to microtubules also in the presence of high CDK1 activity. To corroborate these findings, we analyzed localization of Aurora B in prometaphase-arrested cells, in which degradation of Cyclin B was inhibited by the proteasome inhibitor MG132. Indeed, overexpression 
of GFP-UBASH3B caused relocalization of Aurora B to microtubules. Next, we tested if other CPC components can be recruited to microtubules by UBASH3B. Indeed, Aurora Bphosphorylated INCENP colocalized with the microtubule-associated Aurora B upon UBASH3B overexpression in prometaphase in Oleandrin contrast to control cells in which Aurora B and INCENP were found at the centro
