Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

Overexpression of CENPU promotes cancer growth and metastasis and is associated with poor survival in patients with nasopharyngeal carcinoma.

Background: Centromere protein U (CENPU) is key for mitosis in the carcinogenesis of cancers. However, the roles of CENPU have not been inspected in nasopharyngeal carcinoma (NPC). Thus, we aimed to explore the functions and mechanisms of CENPU in NPC. Methods: Expression of CENPU was evaluated by real-time quantitative polymerase chain reaction, western blotting and immunohistochemistry. The biological functions of CENPU were evaluated in vitro and in vivo. Gene chip analysis, ingenuity pathway analysis, and coimmunoprecipitation experiments were used to explore the mechanisms of CENPU. Results: CENPU was highly expressed in NPC. High expression of CENPU was associated with advanced tumor, node and metastasis (TNM) stage and poor overall survival. Cox regression analysis demonstrated that CENPU expression was an independent prognostic factor in NPC. Knockdown of CENPU inhibited proliferation and migration in vitro and in vivo. Knockdown of CENPU upregulated dual specificity phosphatase 6 (DUSP6) expression. The expression of CNEPU was inversely correlated with the expression of DUSP6 in NPC tissues. Mechanistic studies confirmed that CENPU increased the activation of the ERK1/2 and p38 signaling pathways by suppressing the expression of DUSP6. Conclusions: CENPU acts as an oncogene in NPC by interacting with DUSP6, and may represent a promising prognostic biomarker for patients with NPC.

An unusual duo: Immunodeficiency disorder and scleroderma.

A 45-year-old woman on treatment for HIV infection with highly active antiretroviral therapy for the past 10 years presented to us with a history of Raynaud's phenomenon and hyperpigmentation of the skin for 2 years. She was diagnosed to have pulmonary arterial hypertension 8 months ago. On examination, she had salt-and-pepper pigmentation and sclerodactyly. Her biochemical workup was normal. She had positive antinuclear antibody by indirect immunofluorescence method. Skin biopsy was consistent with systemic sclerosis. HIV has its own musculoskeletal manifestations. The paradox of autoimmunity in the background of immunodeficiency was intriguing. Treating autoimmunity in the presence of immunodeficiency was challenging. The attribution and differentiation of pulmonary hypertension were difficult. There has been a homology identified between human immunodeficiency virus 1 (HIV 1) and centromere B protein (CENP B). This case is reported because of the unusual occurrence of systemic sclerosis in an HIV patient.

Mck1-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in Saccharomyces cerevisiae.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to non-centromeric regions contributes to chromosomal instability (CIN) in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers is associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box (SCF) ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and CIN under normal physiological conditions. Mck1-mediated phosphorylation of SCF-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4 and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus site (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, CIN, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the CIN phenotype of GAL-cse4-3A strain, suggesting that the CIN phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and CIN. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

JRK binds satellite III DNA and is necessary for the heat shock response.

JRK is a DNA-binding protein of the pogo superfamily of transposons, which includes the well-known centromere binding protein B (CENP-B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates ß-catenin-TCF activity but little is known of its cellular functions. Based on its homology to CENP-B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK-GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti-JRK antiserum we show that endogenous JRK co-localises with a subset of centromeres in non-stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat-shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines.

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

High expression levels of centromere protein O participates in cell proliferation of human ovarian cancer.

Ovarian cancer is a common malignant tumor in women, with a high mortality rate ranking first among gynecological tumors. Currently, there is insufficient understanding of the causes, pathogenesis, recurrence and metastasis of ovarian cancer, and early diagnosis and treatment still face great challenges. The sensitivity and specificity of existing ovarian cancer screening methods are still unsatisfactory. Centromere protein O (CENP-O) is a recently discovered structural centromere protein that is involved in cell death and is essential for spindle assembly, chromosome separation, and checkpoint signaling during mitosis. The abnormal high expression of CENP-O was detected in various tumors such as bladder cancer and gastric cancer, and it participates in the regulation of tumor cell proliferation. In this study, we detect the expression abundance of CENP-O mRNA in different ovarian cancer cells ( ES-2, A2780, Caov-3, OVCAR-3 and SK-OV-3). The biological function changes of cell proliferation and apoptosis were detected and the role of CENP-O in ovarian cancer cell proliferation and apoptosis was explored by knocking down the expression of CENP-O gene. The results showed that CENP-O gene was significantly expressed in 5 types of ovarian cancer cell lines. After knocking down the CENP-O gene, the proliferation and cloning ability of ovarian cancer cells decreased, and the apoptosis increased. This study indicates that CENP-O has the potential to be a molecular therapeutic target, and downregulating the expression of CENP-O gene can break the unlimited proliferation ability of cancer cells and promote their apoptosis, providing a foundation and new ideas for subsequent molecular mechanism research and targeted therapy.

The Correlation of Centromere Protein Q with Diagnosis and Prognosis in Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) is one of the major types of liver cancer. Previous studies have shown that the centromere protein family is associated with malignant biological behaviors such as HCC proliferation. As a member of the centromere protein family, centromere protein Q (CENPQ) is closely associated with immunotherapy and immune cell infiltration in various tumors. However, the role and mechanism of CENPQ in HCC remain unclear. Methods: Multiple public databases and RT-qPCR were used to study the expression of CENPQ in HCC. Based on TCGA data, the correlation between CENPQ and clinicopathological characteristics and prognosis of HCC patients was analyzed, and its diagnostic value was evaluated. The potential biological functions of CENPQ in HCC were explored by functional enrichment analysis of differentially expressed genes. The distribution of tumor-infiltrating immune cell types was assessed using single-sample GSEA, and immune checkpoint gene expression was analyzed using Spearman correlation. Subsequently, loss-of-function experiments were performed to determine the function of CENPQ on the cell cycle and proliferation of HCC cells in vitro. Results: CENPQ was found highly expressed in HCC and correlated with weight, BMI, age, AFP, T stage, pathologic stage, histologic grade, and prothrombin time (all p < 0.05). ROC and Kaplan-Meier analyses indicated that CENPQ may be potentially used as a diagnostic marker for HCC (AUC = 0.881), and its upregulation is associated with decreased OS (p = 0.002), DSS (p < 0.001), and PFI (p = 0.002). Functional enrichment analysis revealed an association of CENPQ with biological processes such as immune cell infiltration, cell cycle, and hippo-merlin signaling deregulation in HCC. Furthermore, knockdown of CENPQ manifested in HCC cells with G0/1 phase cycle arrest and decreased proliferative capacity. Conclusion: CENPQ expression was higher in HCC tissues than in normal liver tissues. It was significantly associated with poor prognosis, immune cell infiltration, cell cycle, and proliferation. Therefore, CENPQ may become a promising prognostic biomarker for HCC patients.

Vertebrate centromere architecture: from chromatin threads to functional structures.

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.

Native and tagged CENP-A histones are functionally inequivalent.

BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

RNAi-mediated downregulation of AcCENH3 can induce in vivo haploids in onion (Allium cepa L.).

Haploid induction (HI) holds great promise in expediting the breeding process in onion, a biennial cross-pollinated crop. We used the CENH3-based genome elimination technique in producing a HI line in onion. Here, we downregulated AcCENH3 using the RNAi approach without complementation in five independent lines. Out of five events, only three could produce seeds upon selfing. The progenies showed poor seed set and segregation distortion, and we were unable to recover homozygous knockdown lines. The knockdown lines showed a decrease in accumulation of AcCENH3 transcript and protein in leaf tissue. The decrease in protein content in transgenic plants was correlated with poor seed set. When the heterozygous knockdown lines were crossed with wild-type plants, progenies showed HI by genome elimination of the parental chromosomes from AcCENH3 knockdown lines. The HI efficiency observed was between 0 and 4.63% in the three events, and it was the highest (4.63%) when E1 line was crossed with wildtype. Given the importance of doubled haploids in breeding programmes, the findings from our study are poised to significantly impact onion breeding.

Centromere sequence-independent but biased loading of subgenome-specific CENH3 variants in allopolyploid Arabidopsis suecica.

Centromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species Arabidopsis suecica. Synthetic A. thaliana x A. arenosa hybrids were generated and analyzed to mimic the early evolution of A. suecica. Our expression analyses indicated that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana, and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards the A. arenosa-type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization.

CENPF May Act as a Novel Marker and Highlight the Influence of Pericyte in Infantile Hemangioma.

Infantile hemangioma (IH), a benign microvascular tumor, is marked by early and extensive proliferation of immature hemangioma endothelial cells (Hem-ECs) that naturally regress through differentiation into fibroblasts or adipocytes. However, a challenge persists, as the unique biological behavior of IH remains elusive, despite its general sensitivity to propranolol treatment. Recent evidence suggests that abnormal volume proliferation in IH is primarily attributed to the accumulation of hemangioma pericytes (Hem-Pericytes), in addition to Hem-ECs. Centromere protein F (CENPF) is involved in regulating mitotic processes and has been associated with malignant tumor cell proliferation. It is a key player in maintaining genomic stability during cell division. Our findings revealed specific expression of CENPF in Hem-Pericytes, with a proliferation index (PI) approximately half that of Ki67 (3.28 vs 6.97%) during the proliferative phase of IH. This index decreased rapidly in the involuting phase (P < .05), suggesting that the contribution of pericytes to IH development was comparable to that of Hem-ECs. Tumor expansion and shrinkage may be due to the proliferation, reduction, and differentiation of Hem-Pericytes. In conclusion, we speculate CENPF as a novel marker for clinical pathological diagnosis and a potential therapeutic target, fostering advancements in drug development.

Karyotypic Reshuffling in the Genus Rhipidomys (Rodentia: Cricetidae: Sigmodontinae) Revealed by Zoo-FISH.

INTRODUCTION: Rhipidomys is the second most specious and the most widespread genus of the tribe Thomasomyini. Chromosomal data have been an important tool in the taxonomy of the group that presents low variability of diploid number (2n) and highly variable fundamental numbers (FNs). Despite such diversity, the genus has been studied mainly by classical and banding cytogenetic techniques. METHODS: This study performed a comparative study between R. emiliae (2n = 44, FN = 52), R. macrurus (2n = 44, FN = 49), R. nitela (2n = 50, FN = 71), and R. mastacalis (2n = 44, FN = 72) using chromosome painting probes of two Oryzomyini species. RESULTS: Our analysis revealed pericentric inversion as the main rearrangement involved in the karyotype evolution of the group, although tandem fusions/fissions were also detected. In addition, we detected eight syntenic associations exclusive of the genus Rhipidomys, and three syntenic associations shared between species of the tribe Thomasomyini and Oryzomyini. CONCLUSION: Comparative cytogenetic analysis by ZOO-FISH on genus Rhipidomys supports a pattern of chromosomal rearrangement already suggested by comparative G-banding. However, the results suggest that karyotype variability in the genus could also involve the occurrence of an evolutionary new centromere.

The Aurora B-controlled PP1/RepoMan complex determines the spatial and temporal distribution of mitotic H2B S6 phosphorylation.

The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.

Cell Cycle-Related Centromere Protein F Deficiency Suppresses Ovarian Cancer Cell Growth by Inducing Ferroptosis.

OBJECTIVES: This study aimed to investigate the involvement of the cell cycle-related protein centromere protein F (CENPF) in the development of ovarian cancer (OC) and explored its relationship with ferroptosis. DESIGN: The databases were analysed to identify differential expression of cell cycle-related proteins between individuals with OC and normal individuals. Immunohistochemistry and statistical analysis were conducted on ovarian tissues obtained from 40 patients with epithelial OC and 20 normal individuals. In vitro experiments were performed using SKOV3 and HEY epithelial OC cell lines. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mRNA microarray dataset, consisting of GSE14001, GSE54388, GSE40595, and GSE14407, was downloaded from the Gene Expression Omnibus (GEO) database to investigate the genes associated with cell cycle regulation in OC cells. CENPF was selected as the subject of study through differential analysis.Assessed the expression of CENPF in both OC patients and normal ovarian tissues using immunohistochemistry. Lentivirus infection was employed to downregulate CENPF expression, and subsequent experiments including Cell Counting Kit-8 assay, cell cycle analysis, transwell assay, and wound-healing assay were conducted to investigate the effects of CENPF on proliferation, invasion, migration, and cell cycle regulation in OC cells. The reactive oxygen species (ROS) and the malondialdehyde (MDA) assays were performed to assess the involvement of CENPF in cellular redox reactions. Western blot analysis was conducted to examine the expression levels of ferroptosis-related proteins (GPX4, SLC7A11, DMT1, and protein 53 [p53]). RESULTS: By querying and integrating cell cycle-related genes from the GEO database, in silico analyses using The Cancer Genome Atlas database combined with immunohistochemical studies, we discovered that CENPF is upregulated in OC tissues and is related to survival. Downregulation of CENPF inhibited biological function of OC cells, increased intracellular ROS and MDA levels, and downregulated the GPX4 protein and the SLC7A11/xCT protein, but upregulated the DMT1 protein and the tumour p53 expression to induce ferroptosis. LIMITATIONS: This study did not investigate ferroptosis-related studies following CENPF overexpression, and the findings have not been validated in animal studies. CONCLUSIONS: Our findings demonstrated that the deficiency of CENPF played a crucial anti-oncogenic role in the progression of OC through the mechanism of ferroptosis.

Vertebrate centromeres in mitosis are functionally bipartite structures stabilized by cohesin.

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.

A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres.

The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces.

A communication hub for phosphoregulation of kinetochore-microtubule attachment.

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

The holocentricity in the dioecious nutmeg (Myristica fragrans) is not based on major satellite repeats.

Holocentric species are characterized by the presence of centromeres throughout the length of the chromosomes. We confirmed the holocentricity of the dioecious, small chromosome-size species Myristica fragrans based on the chromosome-wide distribution of the centromere-specific protein KNL1, α-tubulin fibers, and the cell cycle-dependent histone H3 serine 28 phosphorylation (H3S28ph) mark. Each holocentromere is likely composed of, on average, ten centromere units, but none of the identified and in situ hybridized high-copy satellite repeats is centromere-specific. No sex-specific major repeats are present in the high-copy repeat composition of male or female plants, or a significant difference in genome size was detected. Therefore, it is unlikely that M. fragrans possesses heteromorphic sex chromosomes.