Centromeres License the Mitotic Condensation of Yeast Chromosome Arms.

During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation.

Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores.

Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.

Cohesin Removal Reprograms Gene Expression upon Mitotic Entry.

As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.

Aurora B-dependent Ndc80 degradation regulates kinetochore composition in meiosis.

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

Low tension recruits the yeast Aurora B protein Ipl1 to centromeres in metaphase.

Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.

PBAF chromatin remodeler complexes that mediate meiotic transitions in mouse.

Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Proteomic and Phosphoproteomic Reprogramming in Epithelial Ovarian Cancer Metastasis.

Epithelial ovarian cancer (EOC) is a high-risk cancer presenting with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. We used cellular models of spheroid formation and readherence to investigate cellular signaling dynamics in each step toward EOC metastasis. In our system, adherent cells model primary tumors, spheroid formation represents the initiation of metastatic spread, and readherent spheroid cells represent secondary tumors. Proteomic and phosphoproteomic analyses show that spheroid cells are hypoxic and show markers for cell cycle arrest. Aurora kinase B abundance and downstream substrate phosphorylation are significantly reduced in spheroids and readherent cells, explaining their cell cycle arrest phenotype. The proteome of readherent cells is most similar to spheroids, yet greater changes in the phosphoproteome show that spheroid cells stimulate Rho-associated kinase 1 (ROCK1)-mediated signaling, which controls cytoskeletal organization. In spheroids, we found significant phosphorylation of ROCK1 substrates that were reduced in both adherent and readherent cells. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of readherence and altered spheroid density. The data suggest ROCK1 inhibition increases EOC metastatic potential. We identified novel pathways controlled by Aurora kinase B and ROCK1 as major drivers of metastatic behavior in EOC cells. Our data show that phosphoproteomic reprogramming precedes proteomic changes that characterize spheroid readherence in EOC metastasis.

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 find 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 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, likely reflecting the situation in the last eukaryotic common ancestor.

The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation.

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.

Revisiting Aurora Kinase B: A promising therapeutic target for cancer therapy.

Cancer continues to be a major health concern globally, although the advent of targeted therapy has revolutionized treatment options. Aurora Kinase B is a serine-threonine kinase that has been explored as an oncology therapeutic target for more than two decades. Aurora Kinase B inhibitors show promising biological results in in-vitro and in-vivo experiments. However, there are no inhibitors approved yet for clinical use, primarily because of the side effects associated with Aurora B inhibitors. Several studies demonstrate that Aurora B inhibitors show excellent synergy with various chemotherapeutic agents, radiation therapy, and targeted therapies. This makes it an excellent choice as an adjuvant therapy to first-line therapies, which greatly improves the therapeutic window and side effect profile. Recent studies indicate the role of Aurora B in some deadly cancers with limited therapeutic options, like triple-negative breast cancer and glioblastoma. Herein, we review the latest developments in Aurora Kinase B targeted research, with emphasis on its potential as an adjuvant therapy and its role in some of the most difficult-to-treat cancers.

An analysis of AURKB's prognostic and immunological roles across various cancers.

Aurora kinase B (AURKB), an essential regulator in the process of mitosis, has been revealed through various studies to have a significant role in cancer development and progression. However, the specific mechanisms remain poorly understood. This study, therefore, seeks to elucidate the multifaceted role of AURKB in diverse cancer types. This study utilized bioinformatics techniques to examine the transcript, protein, promoter methylation and mutation levels of AURKB. The study further analysed associations between AURKB and factors such as prognosis, pathological stage, biological function, immune infiltration, tumour mutational burden (TMB) and microsatellite instability (MSI). In addition, immunohistochemical staining data of 50 cases of renal clear cell carcinoma and its adjacent normal tissues were collected to verify the difference in protein expression of AURKB in the two tissues. The results show that AURKB is highly expressed in most cancers, and the protein level of AURKB and the methylation level of its promoter vary among cancer types. Survival analysis showed that AURKB was associated with overall survival in 12 cancer types and progression-free survival in 11 cancer types. Elevated levels of AURKB were detected in the advanced stages of 10 different cancers. AURKB has a potential impact on cancer progression through its effects on cell cycle regulation as well as inflammatory and immune-related pathways. We observed a strong association between AURKB and immune cell infiltration, immunomodulatory factors, TMB and MSI. Importantly, we confirmed that the AURKB protein is highly expressed in kidney renal clear cell carcinoma (KIRC). Our study reveals that AURKB may be a potential biomarker for pan-cancer and KIRC.

microRNA-486-5p Regulates DNA Damage Inhibition and Cisplatin Resistance in Lung Adenocarcinoma by Targeting AURKB.

Lung adenocarcinoma (LUAD) severely affects human health, and cisplatin (DDP) resistance is the main obstacle in LUAD treatment, the mechanism of which is unknown. Bioinformatics methods were utilized to predict expression and related pathways of AURKB in LUAD tissues, as well as the upstream regulated microRNAs. qRT-PCR assayed expression of AURKB and microRNA-486-5p. RIP and dual-luciferase experiments verified the binding and interaction between the two genes. CCK-8 was used to detect cell proliferation ability and IC50 values. Flow cytometry was utilized to assess the cell cycle. Comet assay and western blot tested DNA damage and γ-H2AX protein expression, respectively. In LUAD, AURKB was upregulated, but microRNA-486-5p was downregulated. The targeted relationship between the two was confirmed by RIP and dual-luciferase experiments. Cell experiments showed that AURKB knock-down inhibited cell proliferation, reduced IC50 values, induced cell cycle arrest, and caused DNA damage. The rescue experiment presented that high expression of microRNA-486-5p could weaken the impact of AURKB overexpression on LUAD cell behavior and DDP resistance. microRNA-486-5p regulated DNA damage to inhibit DDP resistance in LUAD by targeting AURKB, implying that microRNA-486-5p/AURKB axis may be a possible therapeutic target for DDP resistance in LUAD patients.

Inhibition of Aurora B kinase (AURKB) enhances the effectiveness of 5-fluorouracil chemotherapy against colorectal cancer cells.

BACKGROUND: 5-Fluorouracil (5-FU) remains a core component of systemic therapy for colorectal cancer (CRC). However, response rates remain low, and development of therapy resistance is a primary issue. Combinatorial strategies employing a second agent to augment the therapeutic effect of chemotherapy is predicted to reduce the incidence of treatment resistance and increase the durability of response to therapy. METHODS: Here, we employed quantitative proteomics approaches to identify novel druggable proteins and molecular pathways that are deregulated in response to 5-FU, which might serve as targets to improve sensitivity to chemotherapy. Drug combinations were evaluated using 2D and 3D CRC cell line models and an ex vivo culture model of a patient-derived tumour. RESULTS: Quantitative proteomics identified upregulation of the mitosis-associated protein Aurora B (AURKB), within a network of upregulated proteins, in response to a 24 h 5-FU treatment. In CRC cell lines, AURKB inhibition with the dihydrogen phosphate prodrug AZD1152, markedly improved the potency of 5-FU in 2D and 3D in vitro CRC models. Sequential treatment with 5-FU then AZD1152 also enhanced the response of a patient-derived CRC cells to 5-FU in ex vivo cultures. CONCLUSIONS: AURKB inhibition may be a rational approach to augment the effectiveness of 5-FU chemotherapy in CRC.

Haspin participates in AURKB recruitment to centromeres and contributes to chromosome congression in male mouse meiosis.

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. 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. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.

MKLP2 functions in early mitosis to ensure proper chromosome congression.

Mitotic kinesin-like protein 2 (MKLP2; also known as KIF20A) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small-molecule inhibitors of MKLP2 (MKLP2i) suggest that it also has a function earlier in mitosis, prior to anaphase. In this study, we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 facilitates error correction, as inhibited cells have a significant increase in unstable, syntelic kinetochore-microtubule attachments. We find that the aberrant attachments are accompanied by elevated Aurora kinase (A and B) activity and phosphorylation of the downstream target HEC1 (also known as NDC80) at Ser55. Finally, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability. This article has an associated First Person interview with the first author of the paper.

The noncanonical function of borealin, a component of chromosome passenger complex, promotes glycolysis via stabilization of survivin in squamous cell carcinoma cells.

The chromosome passenger complex (CPC) is a kinase complex formed by Aurora B, borealin, survivin and inner centromere protein (INCENP). The CPC is active during mitosis and contributes to proper chromosome segregation via the phosphorylation of various substrates. Overexpression of each CPC component has been reported in most cancers. However, its significance remains unclear, as only survivin is known to confer chemoresistance. This study showed that the overexpression of borealin, a CPC component, stabilized survivin protein depending on its interaction with survivin. Unexpectedly, the accumulation of survivin by borealin overexpression did not affect the well-characterized functions of survivin, such as chemoresistance and cell proliferation. Interestingly, the overexpression of borealin promoted lactate production but not the overexpression of the deletion mutant that lacks the ability to bind to survivin. Consistent with these findings, the expression levels of glycolysis-related genes were enhanced in borealin-overexpressing cancer cells. Meanwhile, the overexpression of survivin alone did not promote lactate production. Overall, the accumulation of the borealin-survivin complex promoted glycolysis in squamous cell carcinoma cells. This mechanism may contribute to cancer progression via excessive lactate production.

A functional genetic screen identifies aurora kinase b as an essential regulator of Sox9-positive mouse embryonic lung progenitor cells.

Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.

Aurora kinase B inhibits aurora kinase A to control maternal mRNA translation in mouse oocytes.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates mRNA polyadenylation. One message is aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout mouse oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function crucial to generating euploid eggs.

The Molecular Mechanism of Aurora-B Regulating Kinetochore-Microtubule Attachment in Mitosis and Oocyte Meiosis.

BACKGROUND: Aurora kinase B (Aurora-B), a member of the chromosomal passenger complex, is involved in correcting kinetochore-microtubule (KT-MT) attachment errors and regulating sister chromatid condensation and cytoplasmic division during mitosis. SUMMARY: However, few reviews have discussed its mechanism in oocyte meiosis and the differences between its role in mitosis and meiosis. Therefore, in this review, we summarize the localization, recruitment, activation, and functions of Aurora-B in mitosis and oocyte meiosis. The accurate regulation of Aurora-B is essential for ensuring accurate chromosomal segregation and correct KT-MT attachments. Aurora-B regulates the stability of KT-MT attachments by competing with cyclin-dependent kinase 1 to control the phosphorylation of the SILK and RVSF motifs on kinetochore scaffold 1 and by competing with protein phosphatase 1 to influence the phosphorylation of NDC80 which is the substrate of Aurora-B. In addition, Aurora-B regulates the spindle assembly checkpoint by promoting the recruitment and activation of mitotic arrest deficient 2. KEY MESSAGES: This review provides a theoretical foundation for elucidating the mechanism of cell division and understanding oocyte chromosomal aneuploidy.