Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

Molecular convergence by differential domain acquisition is a hallmark of chromosomal passenger complex evolution

The chromosomal passenger complex (CPC) is a heterotetrameric regulator of eukaryotic cell division, consisting of an Aurora-type kinase and a scaffold built of INCENP, Borealin and Survivin. While most CPC components are conserved across eukaryotes, orthologs of the chromatin reader Survivin have previously only been found in animals and fungi, raising the question of how its essential role is carried out in other eukaryotes. By characterizing proteins that bind to the Arabidopsis Borealin ortholog, we identified BOREALIN RELATED INTERACTOR 1 and 2 (BORI1 and BORI2) as redundant Survivin-like proteins in the context of the CPC in plants. Loss of BORI function is lethal and a reduced expression of BORIs causes severe developmental defects. Similar to Survivin, we find that the BORIs bind to phosphorylated histone H3, relevant for correct CPC association with chromatin. However, this interaction is not mediated by a BIR domain as in previously recognized Survivin orthologs, but by an FHA domain, a widely conserved phosphate-binding module. We propose that the unifying criterion of Survivin-type proteins is a helix that facilitates complex formation with the other two scaffold components, and that the addition of a phosphate-binding domain, necessary for concentration at the inner centromere, evolved in parallel in different eukaryotic groups. Using sensitive similarity searches, we indeed find conservation of this helical domain between animals and plants, and identify the missing CPC component in most eukaryotic supergroups. Interestingly, we also detect Survivin orthologs without a defined phosphate-binding domain, possibly reflecting the situation in the last eukaryotic common ancestor.

The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier ID to be provided when available, PXD000xxx.

The Abscission Checkpoint: A Guardian of Chromosomal Stability

The abscission checkpoint contributes to the fidelity of chromosome segregation by delaying completion of cytokinesis (abscission) when there is chromatin lagging in the intercellular bridge between dividing cells. Although additional triggers of an abscission checkpoint-delay have been described, including nuclear pore defects, replication stress or high intercellular bridge tension, this review will focus only on chromatin bridges. In the presence of such abnormal chromosomal tethers in mammalian cells, the abscission checkpoint requires proper localization and optimal kinase activity of the Chromosomal Passenger Complex (CPC)-catalytic subunit Aurora B at the midbody and culminates in the inhibition of Endosomal Sorting Complex Required for Transport-III (ESCRT-III) components at the abscission site to delay the final cut. Furthermore, cells with an active checkpoint stabilize the narrow cytoplasmic canal that connects the two daughter cells until the chromatin bridges are resolved. Unsuccessful resolution of chromatin bridges in checkpoint-deficient cells or in cells with unstable intercellular canals can lead to chromatin bridge breakage or tetraploidization by regression of the cleavage furrow. In turn, these outcomes can lead to accumulation of DNA damage, chromothripsis, generation of hypermutation clusters and chromosomal instability, which are associated with cancer formation or progression. Recently, many important questions regarding the mechanisms of the abscission checkpoint have been investigated, such as how the presence of chromatin bridges is signaled to the CPC, how Aurora B localization and kinase activity is regulated in late midbodies, the signaling pathways by which Aurora B implements the abscission delay, and how the actin cytoskeleton is remodeled to stabilize intercellular canals with DNA bridges. Here, we review recent progress toward understanding the mechanisms of the abscission checkpoint and its role in guarding genome integrity at the chromosome level, and consider its potential implications for cancer therapy.

Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This is an essential step towards the accuracy of cell division and therefore must be precisely regulated in both mitosis and meiosis. One of the main centromeric regulatory signaling pathways is the Haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. In mitosis, Haspin kinase phosphorylates H3 at threonine 3 (H3T3ph), the essential histone mark that recruits the CPC whose catalytic component is Aurora B kinase. To date, no data has yet been presented about the action of the centromeric Haspin-H3T3ph-CPC pathway in mammalian male meiosis. We have analyzed the consequences of Haspin chemical inhibition in cultured spermatocytes using LDN-192960. Our in vitro studies suggest that Haspin kinase activity is required for proper chromosome congression during both meiotic divisions and for the recruitment of phosphorylated Aurora B at meiotic centromeres. These results have been confirmed by the characterization of the meiotic phenotype of the genetic mouse model Haspin-/-, which displays similar defects. In addition, our work demonstrates that the absence of H3T3ph histone mark does not alter SGO2 localization to meiotic centromeres. These results add new and relevant information regarding the regulation of centromere function during meiosis.

Distinct roles of haspin in stem cell division and male gametogenesis

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Potential Role of S-Palmitoylation in Cancer Stem Cells of Lung Adenocarcinoma

S-palmitoylation, catalyzed by a family of 23 zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases localized on the cell membrane. However, stemness genes modulated by ZDHHCs in lung adenocarcinoma (LUAD) remain to be defined. Previously, we have constructed a network of cancer stem cell genes, including INCENP, based on mRNA stemness indices (mRNAsi) of LUAD. INCENP has the function of a chromosomal passenger complex locating to centromeres, which is performed by the conserved region of its N-terminal domain. INCENP protein with a deletion of the first non-conserved 26 amino acid sequence failed to target centromeres. However, the exact function of the deleted sequence has not been elucidated. To identify novel cancer stem cell-relevant palmitoylated proteins and responsible ZDHHC enzymes in LUAD, we analyzed multi-omics data obtained from the database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA). ZDHHC5 is distinguished from the ZDHHC family for being up-regulated in mRNA and protein levels and associated with malignant prognosis. ZDHHC5 was positively associated with INCENP, and the correlation score increased with LUAD stages. CSS-Palm results showed Cys15 was the S-palmitoylation site of INCENP. Interestingly, Cys15 locates in the 1–26 aa sequence of INCENP, and is a conserved site across species. As INCENP is a nuclear protein, we predicted that the nuclear localization signal of ZDHHC5 was specific to the importin αβ pathway, and the result of immunofluorescence proves that ZDHHC5 is located in the nucleoplasm, in addition to the plasma membrane. Therefore, our study indicates the S-palmitoylation of INCENP mediated by ZDHHC5 as a potential mechanism of S-palmitoylation to modulate CSCs in LUAD.

Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPC’s intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated ‘kinetochore proximal centromere’ pool of CPC.

Multiple pools of PP2A regulate spindle assembly, kinetochore attachments, and cohesion in Drosophila oocytes

Meiosis in female oocytes lacks centrosomes, the microtubule-organizing center. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, we examined the role of Protein Phosphatase 2A (PP2A) in oocyte meiosis. We found that both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which is encoded by two partially redundant paralogs, wdb and wrd, is also required for maintaining sister chromatid cohesion, establishing end-on microtubule attachments, and the metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332, and kinetochore protein SPC105R. While BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. We propose at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle.