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The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Centromeres are chromatin structures specialized in sister chromatid cohesion, kinetochore assembly, and microtubule attachment during chromosome segregation. The regional centromere of vertebrates consists of long regions of highly repetitive sequences occupied by the Histone H3 variant CENP-A, and which are flanked by pericentromeres. The three-dimensional organization of centromeric chromatin is paramount for its functionality and its ability to withstand spindle forces. Alongside CENP-A, key contributors to the folding of this structure include components of the Constitutive Centromere-Associated Network (CCAN), the protein CENP-B, and condensin and cohesin complexes. Despite its importance, the intricate architecture of the regional centromere of vertebrates remains largely unknown. Recent advancements in long-read sequencing, super-resolution and cryo-electron microscopy, and chromosome conformation capture techniques have significantly improved our understanding of this structure at various levels, from the linear arrangement of centromeric sequences and their epigenetic landscape to their higher-order compaction. In this review, we discuss the latest insights on centromere organization and place them in the context of recent findings describing a bipartite higher-order organization of the centromere., (© 2024. The Author(s).)

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation., (© 2023 Wu et al.)

Aneuploidy-the karyotype state in which the number of chromosomes deviates from a multiple of the haploid chromosome set-is common in cancer, where it is thought to facilitate tumor initiation and progression. However, it is poorly tolerated in healthy cells: during development and tissue homeostasis, aneuploid cells are efficiently cleared from the population. It is still largely unknown how cancer cells become, and adapt to being, aneuploid. P53, the gatekeeper of the genome, has been proposed to guard against aneuploidy. Aneuploidy in cancer genomes strongly correlates with mutations in TP53, and p53 is thought to prevent the propagation of aneuploid cells. Whether p53 also participates in preventing the mistakes in cell division that lead to aneuploidy is still under debate. In this review, we summarize the current understanding of the role of p53 in protecting cells from aneuploidy, and we explore the consequences of functional p53 loss for the propagation of aneuploidy in cancer., (© 2023. The Author(s).)

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation., (© 2023. Springer Nature Limited.)

Background & Aims: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation., Methods: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues., Results: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases., Conclusions: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 ( PKP2 ). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeasts are largely homologous, their ability to track depolymerizing microtubules, however, is carried out by the nonhomologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found among a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer could have played a role in the evolutionary history of Dam1-C. Here, we present an expanded analysis of Dam1-C evolution, using additional genome as well as transcriptome sequences and recently published 3D structures. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intracomplex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no horizontal gene transfer was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-versus-profile searches. Homology among 8 out of the 10 Dam1-C subunits suggests that the complex largely evolved from a single multimerizing subunit that diversified into a hetero-octameric core via stepwise subunit duplication and subfunctionalization of the subunits before the origin of the last eukaryotic common ancestor., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Aneuploidy is a hallmark of cancer and a major cause of miscarriages in humans. It is caused by chromosome segregation errors during cell divisions. Evidence is mounting that the probability of specific chromosomes undergoing a segregation error is non-random. In other words, some chromosomes have a higher chance of contributing to aneuploid karyotypes than others. This could have important implications for the origins of recurrent aneuploidy patterns in cancer and developing embryos. Here, we review recent progress in understanding the prevalence and causes of non-random chromosome segregation errors in mammalian mitosis and meiosis. We evaluate its potential impact on cancer and human reproduction and discuss possible research avenues.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity., (© 2022. The Author(s).)

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis 1-5 . Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer 6,7 -but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing 8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes 9 and aneuploidies in early development 10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development., (© 2022. The Author(s).)

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease., (© 2021. The Author(s).)

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes., (© 2021. The Author(s).)

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq-a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness., (© 2021. The Author(s).)

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.

Aneuploidy originating during meiosis in oocytes is the major cause of reduced fertility, implantation failure and miscarriage in women beyond their mid-thirties. Loss of chromosome cohesion, and defective microtubule dynamics and spindle assembly are, in turn, the major contributors to the error-prone nature of chromosome segregation in the oocytes of older women. However, the underlying molecular defects are not well understood. Altered function of MPS1 and AURKC have been shown to induce multipolar spindle phenotypes in murine oocytes and cancer cells, however, their role in reproductive aging associated oocyte aneuploidy is not known. Although age-related gamete and embryonic aneuploidy has been studied in female rodents, the horse may be a more appropriate animal model. Similar to women, aged mares suffer from reduced fertility and an increased incidence of oocyte aneuploidy. Moreover, mares show a long interval (decades) to reproductive senescence and, unlike rodents but similar to women, horse oocytes assemble the meiotic spindle in a slow and unstable manner, independent of microtubule organizing centers. In this study we found that oocytes from aged mares have lower expression of mRNA for Mps1, Spc25 and AurkC than oocytes from young mares while gene expression for other meiosis regulators did not differ. To assess the ability of horse oocytes to correctly form a bipolar spindle, in vitro matured MII oocytes were allowed to re-form their spindle after nocodazole-induced microtubule depolymerization. To investigate the importance of MPS1 and AURKC function in spindle (re)assembly, various concentrations of a MPS1 inhibitor (MPS1i, Compound 5) or an AURK inhibitor (AURKi, ZM447439) were included after nocodazole washout. MII oocytes from aged mares showed a higher incidence of spindle abnormalities after exposure to MPS1i. In contrast, Aurora kinase inhibition severely impaired microtubule organization and spindle formation in all oocytes, irrespective of mare age. In conclusion, gene expression for the kinases Mps1, Spc25 , and AurkC is reduced in oocytes from aged mares. Moreover, spindle (re)assembly in aged mares' oocytes is more unstable when Mps1 is inhibited. Overall, this suggests that compromised Mps1 activity predisposes to meiotic spindle instability in aged mare oocytes. This spindle instability could predispose to chromosome segregation errors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rizzo, Stout, Cristarella, Quartuccio, Kops and De Ruijter-Villani.)

Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus ∼9-12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes., Competing Interests: Declaration of Interests A patent application has been filed by Stichting Oncode Institute for the technology disclosed in this publication., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The kinetochore is at the heart of chromosome segregation in mitosis and meiosis. Rather than a static linker complex for chromatin and spindle microtubules, it is highly dynamic in composition, size, and shape. While known for decades that it can expand and grow a fibrous meshwork known as the corona, it was until recently unclear what constitutes this 'crown' and what its relevance is for kinetochore function. Here, we highlight recent discoveries in fibrous corona biology, and place them in the context of the processes that orchestrate high-fidelity chromosome segregation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The tremendous diversity in eukaryotic life forms can ultimately be traced back to evolutionary modifications at the level of molecular networks. Deep understanding of these modifications will not only explain cellular diversity, but will also uncover different ways to execute similar processes and expose the evolutionary 'rules' that shape the molecular networks. Here, we review the evolutionary dynamics of the spindle assembly checkpoint (SAC), a signaling network that guards fidelity of chromosome segregation. We illustrate how the interpretation of divergent SAC systems in eukaryotic species is facilitated by combining detailed molecular knowledge of the SAC and extensive comparative genome analyses. Ultimately, expanding this to other core cellular systems and experimentally interrogating such systems in organisms from all major lineages may start outlining the routes to and eventual manifestation of the cellular diversity of eukaryotic life., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (Apc Min/+ ), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.

The monopolin complex is a multifunctional molecular crosslinker, which in S. pombe binds and organises mitotic kinetochores to prevent aberrant kinetochore-microtubule interactions. In the budding yeast S. cerevisiae, whose kinetochores bind a single microtubule, the monopolin complex crosslinks and mono-orients sister kinetochores in meiosis I, enabling the biorientation and segregation of homologs. Here, we show that both the monopolin complex subunit Csm1 and its binding site on the kinetochore protein Dsn1 are broadly distributed throughout eukaryotes, suggesting a conserved role in kinetochore organisation and function. We find that budding yeast Csm1 binds two conserved motifs in Dsn1, one (termed Box 1) representing the ancestral, widely conserved monopolin binding motif and a second (termed Box 2-3) with a likely role in enforcing specificity of sister kinetochore crosslinking. We find that Box 1 and Box 2-3 bind the same conserved hydrophobic cavity on Csm1, suggesting competition or handoff between these motifs. Using structure-based mutants, we also find that both Box 1 and Box 2-3 are critical for monopolin function in meiosis. We identify two conserved serine residues in Box 2-3 that are phosphorylated in meiosis and whose mutation to aspartate stabilises Csm1-Dsn1 binding, suggesting that regulated phosphorylation of these residues may play a role in sister kinetochore crosslinking specificity. Overall, our results reveal the monopolin complex as a broadly conserved kinetochore organiser in eukaryotes, which budding yeast have co-opted to mediate sister kinetochore crosslinking through the addition of a second, regulatable monopolin binding interface.

Background: Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies., Methods: Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR., Results: MMC was more effective in eliminating peritoneal metastasis-derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis-derived organoids to MMC, as the IC50 decreased 2·6-12·4-fold to well below concentrations commonly attained in clinical practice. Live-cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC-induced cell cycle arrest, but caused increased replication stress and accelerated cell death., Conclusion: Peritoneal metastasis-derived organoids can be used to evaluate existing HIPEC regimens on an individual-patient level and for development of more effective treatment strategies. Surgical relevance Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient-derived organoids can accurately predict clinical therapeutic response in vitro. A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient-derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery-HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC. Peritoneal metastasis-derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent-tailored HIPEC regimens., (© 2019 The Authors. BJS published by John Wiley & Sons Ltd on behalf of BJS Society Ltd.)

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro . Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice. See related commentary by Hill and D'Andrea, p. 828 . This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

The emergence of eukaryotes from ancient prokaryotic lineages embodied a remarkable increase in cellular complexity. While prokaryotes operate simple systems to connect DNA to the segregation machinery during cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually similar, prokaryotic segregation systems and the eukaryotic kinetochore are not homologous. Here we investigate the origins of the kinetochore before the last eukaryotic common ancestor (LECA) using phylogenetic trees, sensitive profile-versus-profile homology detection, and structural comparisons of its protein components. We show that LECA's kinetochore proteins share deep evolutionary histories with proteins involved in a few prokaryotic systems and a multitude of eukaryotic processes, including ubiquitination, transcription, and flagellar and vesicular transport systems. We find that gene duplications played a major role in shaping the kinetochore; more than half of LECA's kinetochore proteins have other kinetochore proteins as closest homologs. Some of these have no detectable homology to any other eukaryotic protein, suggesting that they arose as kinetochore-specific folds before LECA. We propose that the primordial kinetochore evolved from proteins involved in various (pre)eukaryotic systems as well as evolutionarily novel folds, after which a subset duplicated to give rise to the complex kinetochore of LECA., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. SAC proteins are shed from kinetochores once stable attachments are achieved. Human kinetochores consist of hundreds of SAC protein recruitment modules and bind up to 20 microtubules, raising the question of how the SAC responds to intermediate attachment states. We show that one protein module ('RZZS-MAD1-MAD2') of the SAC is removed from kinetochores at low microtubule occupancy and remains absent at higher occupancies, while another module ('BUB1-BUBR1') is retained at substantial levels irrespective of attachment states. These behaviours reflect different silencing mechanisms: while BUB1 displacement is almost fully dependent on MPS1 inactivation, MAD1 (also known as MAD1L1) displacement is not. Artificially tuning the affinity of kinetochores for microtubules further shows that ∼50% occupancy is sufficient to shed MAD2 and silence the SAC. Kinetochores thus respond as a single unit to shut down SAC signalling at submaximal occupancy states, but retain one SAC module. This may ensure continued SAC silencing on kinetochores with fluctuating occupancy states while maintaining the ability for fast SAC re-activation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.

Comparative genomics has proven a fruitful approach to acquire many functional and evolutionary insights into core cellular processes. Here it is argued that in order to perform accurate and interesting comparative genomics, one first and foremost has to be able to recognize, postulate, and revise different evolutionary scenarios. After all, these studies lack a simple protocol, due to different proteins having different evolutionary dynamics and demanding different approaches. The authors here discuss this challenge from a practical (what are the observations?) and conceptual (how do these indicate a specific evolutionary scenario?) viewpoint, with the aim to guide investigators who want to analyze the evolution of their protein(s) of interest. By sharing how the authors draft, test, and update such a scenario and how it directs their investigations, the authors hope to illuminate how to execute molecular evolution studies and how to interpret them. Also see the video abstract here https://youtu.be/VCt3l2pbdbQ., (© The Authors. Bio Essays Published by WILEY Periodicals, Inc.)

Faithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to kinetochores to initiate SAC signaling and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here, we show that a helical fragment within the kinetochore-targeting N-terminal extension (NTE) module of MPS1 is required for interactions with kinetochores and forms intramolecular interactions with its adjacent tetratricopeptide repeat (TPR) domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, inefficient MPS1 delocalization upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MPS1-NDC80-C interactions., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Discovered in 1991 in a screen for genes involved in spindle pole body duplication, the monopolar spindle 1 (Mps1) kinase has since claimed a central role in processes that ensure error-free chromosome segregation. As a result, Mps1 kinase activity has become an attractive candidate for pharmaceutical companies in the search for compounds that target essential cellular processes to eliminate, for example, tumour cells or pathogens. Research in recent decades has offered many insights into the molecular function of Mps1 and its regulation. In this review, we integrate the latest knowledge regarding the regulation of Mps1 activity and its spatio-temporal distribution, highlight gaps in our understanding of these processes and propose future research avenues to address them., (© 2018 The Authors.)

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids., (© 2018 Elsevier Inc. All rights reserved.)

Through exome sequencing, we identified six individuals with biallelic loss-of-function mutations in TRIP13. All six developed Wilms tumor. Constitutional mosaic aneuploidies, microcephaly, developmental delay and seizures, which are features of mosaic variegated aneuploidy (MVA) syndrome, were more variably present. Through functional studies, we show that TRIP13-mutant patient cells have no detectable TRIP13 and have substantial impairment of the spindle assembly checkpoint (SAC), leading to a high rate of chromosome missegregation. Accurate segregation, as well as SAC proficiency, is rescued by restoring TRIP13 function. Individuals with biallelic TRIP13 or BUB1B mutations have a high risk of embryonal tumors, and here we show that their cells display severe SAC impairment. MVA due to biallelic CEP57 mutations, or of unknown cause, is not associated with embryonal tumors and cells from these individuals show minimal SAC deficiency. These data provide insights into the complex relationships between aneuploidy and carcinogenesis.

Faithful chromosome segregation relies on kinetochores, the large protein complexes that connect chromatin to spindle microtubules. Although human and yeast kinetochores are largely homologous, they track microtubules with the unrelated protein complexes Ska (Ska-C, human) and Dam1 (Dam1-C, yeast). We here uncovered that Ska-C and Dam1-C are both widespread among eukaryotes, but in an exceptionally inverse manner, supporting their functional analogy. Within the complexes, all Ska-C and various Dam1-C subunits are ancient paralogs, showing that gene duplication shaped these complexes. We examined various evolutionary scenarios to explain the nearly mutually exclusive patterns of Ska-C and Dam1-C in present-day species. We propose that Ska-C was present in the last eukaryotic common ancestor, that subsequently Dam1-C displaced Ska-C in an early fungus and was horizontally transferred to diverse non-fungal lineages, displacing Ska-C in these lineages too., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The cell cycle culminates in mitosis with the purpose of dividing the cell's DNA content equally over two daughter cells. Error-free segregation relies on correct connections between chromosomes and spindle microtubules. Kinetochores are complex multi-protein assemblies that mediate these connections and are the platforms for attachment-error-correction and spindle assembly checkpoint signaling. Proper kinetochore function is therefore key in preventing aneuploidization. Mutations in genes encoding kinetochore proteins are associated with several severe developmental disorders associated with microcephaly, and kinetochore defects contribute to chromosomal instability in certain cancers. This chapter gives an overview of the processes necessary for faithful chromosome segregation and how kinetochore malfunction causes various human pathologies.

Human cancers harbor great numbers of genomic alterations. One of the most common alterations is aneuploidy, an imbalance at the chromosome level. Some aneuploid cancer cell populations show varying chromosome copy number alterations over time, a phenotype known as 'chromosomal instability' (CIN). Chromosome segregation errors in mitosis are the most common cause for CIN in vitro, and these are also thought to underlie the aneuploidies seen in clinical cancer samples. However, CIN and aneuploidy are different traits and they are likely to have distinct impacts on tumor evolution and clinical tumor behavior. In this opinion article, we discuss these differences and describe scenarios in which distinguishing them can be clinically relevant., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Equal mitotic chromosome segregation is critical for genome integrity and is monitored by the spindle assembly checkpoint (SAC). We have previously shown that the consensus phosphorylation motif of the essential SAC kinase Monopolar spindle 1 (Mps1) is very similar to that of Polo-like kinase 1 (Plk1). This prompted us to ask whether human Plk1 cooperates with Mps1 in SAC signaling. Here, we demonstrate that Plk1 promotes checkpoint signaling at kinetochores through the phosphorylation of at least two Mps1 substrates, including KNL-1 and Mps1 itself. As a result, Plk1 activity enhances Mps1 catalytic activity as well as the recruitment of the SAC components Mad1:C-Mad2 and Bub3:BubR1 to kinetochores. We conclude that Plk1 strengthens the robustness of SAC establishment at the onset of mitosis and supports SAC maintenance during prolonged mitotic arrest., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface., (Copyright © 2015 Elsevier Inc. All rights reserved.)