The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere

Kinase and Phosphatase Cross-Talk at the Kinetochore

Multiple kinases and phosphatases act on the kinetochore to control chromosome segregation: Aurora B, Mps1, Bub1, Plk1, Cdk1, PP1, and PP2A-B56, have all been shown to regulate both kinetochore-microtubule attachments and the spindle assembly checkpoint. Given that so many kinases and phosphatases converge onto two key mitotic processes, it is perhaps not surprising to learn that they are, quite literally, entangled in cross-talk. Inhibition of any one of these enzymes produces secondary effects on all the others, which results in a complicated picture that is very difficult to interpret. This review aims to clarify this picture by first collating the direct effects of each enzyme into one overarching schematic of regulation at the Knl1/Mis12/Ndc80 (KMN) network (a major signaling hub at the outer kinetochore). This schematic will then be used to discuss the implications of the cross-talk that connects these enzymes; both in terms of why it may be needed to produce the right type of kinetochore signals and why it nevertheless complicates our interpretations about which enzymes control what processes. Finally, some general experimental approaches will be discussed that could help to characterize kinetochore signaling by dissociating the direct from indirect effect of kinase or phosphatase inhibition in vivo. Together, this review should provide a framework to help understand how a network of kinases and phosphatases cooperate to regulate two key mitotic processes

Chromosome segregation fidelity in mitosis: Consequences of a crippled CPC

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

Chromosome segregation fidelity in mitosis: Consequences of a crippled CPC

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

Cancer Stem Cells, EMT, and Developmental Pathway Activation in Pancreatic Tumors

www.mdpi.com/journal/cancer

The Ins and Outs of Aurora B Inner Centromere Localization

Error-free chromosome segregation is essential for the maintenance of genomic integrity during cell division. Aurora B, the enzymatic subunit of the Chromosomal Passenger Complex (CPC), plays a crucial role in this process. In early mitosis Aurora B localizes predominantly to the inner centromere, a specialized region of chromatin that lies at the crossroads between the inter-kinetochore and inter-sister chromatid axes. Two evolutionarily conserved histone kinases, Haspin and Bub1, control the positioning of the CPC at the inner centromere and this location is thought to be crucial for the CPC to function. However, recent studies sketch a subtler picture, in which not all functions of the CPC require strict confinement to the inner centromere. In this review we discuss the molecular pathways that direct Aurora B to the inner centromere and deliberate if and why this specific localization is important for Aurora B function