Chromosome segregation fidelity in mitosis: Consequences of a crippled CPC

Abstract

The chromosomal passenger complex (CPC), plays an important role in faithful chromosome segregation during ­mitosis, as it mediates destabilization of incorrect attachments between chromosomes and microtubules of the mitotic spindle (error correction). During prometaphase and metaphase, the CPC localizes to the inner centromere, a specialized chromatin region that lies at the intersection of the inter-kinetochore and inter-sister chromatid axes. This localization of the CPC is tightly controlled by two kinases, Haspin and Bub1. However, the importance of CPC inner centromere localization for chromosome bi-orientation and error-free segregation has become subject to debate. Disruption of CPC centromere localization does not severely compromise chromosome segregation fidelity in budding yeast. In addition, artificially re-locating Aurora B away from the inner centromere and in closer proximity of the kinetochore in human cells, does not preclude the stabilization of bi-oriented KT-MT interactions. In Xenopus laevis egg extracts, however, cytosolic CPC activity does not fully support chromosome bi-orientation, suggesting that in this model system proper CPC function does require its chromosomal localization. This means that the relationship between CPC localization and function has remained incompletely understood and it was therefore studied in this thesis. To investigate the consequences of altered CPC localization for Aurora B function, we first focused on setting up a system for efficient generation of knockout and knockin cell lines. Genome editing by CRISPR/Cas9 is a powerful method to study protein behavior, however its ease of use is limited by difficulties in delivery of the system due to its large size. We describe the use of baculovirus for the delivery of CRISPR/Cas9 machinery, resulting in the knockout of target genes, in a range of human cell lines. Baculoviral delivery of CRISPR/Cas9 components in combination with an HDR template can also be utilized to introduce point mutations or tag endogenous proteins. Tagging of Haspin allowed us the studied the localization of this protein during mitosis and cytokinesis. We subsequently explored the contribution of Haspin and Bub1 to CPC function. We found that Haspin and Bub1 appeared to be redundant for CPC function during normal mitotic progression. Compared to Aurora B kinase inhibition, loss of both Haspin and Bub1 activity induced only mild segregation errors, despite dramatic mis-localization of Aurora B. This indicates that without centromeric concentration of the CPC residual Aurora B activity is present which limits the severity of chromosome segregation errors. Chromosome mis-segregation during mitosis leads to aneuploidy, a hallmark of cancer cells. We therefore investigated if mutations in the CPC could underlie CIN in tumors. We took the CPC subunit Borealin as an example and characterized the consequences of tumor-derived somatic mutations in the CDCA8 gene (encoding Borealin) on Borealin protein function and chromosome segregation fidelity. We found that a subset of mutation indeed perturbed Borealin function. However, these CDCA8 mutations were annotated as being heterozygous in tumors, and chromosome segregation was not perturbed in heterozygous knockin cell lines harboring these mutations at the endogenous locus. This renders it unlikely that somatic mutations in Borealin underlie chromosomal instability in cancer.