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Current Biology : CB Mar 2024The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona...
The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins. CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression, interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358). We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined. Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.
Topics: Humans; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Kinetochores; Microtubules; Mad2 Proteins; Mitosis; Dyneins; Spindle Apparatus; HeLa Cells
PubMed: 38354735
DOI: 10.1016/j.cub.2024.01.042 -
Scientific Reports Mar 2021Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new...
Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.
Topics: Animals; Apoptosis; Burkitt Lymphoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; DNA; DNA Repair Enzymes; Deoxyguanine Nucleotides; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, B-Cell; Mice; Phosphoric Monoester Hydrolases; Pyrimidines; Xenograft Model Antitumor Assays
PubMed: 33737576
DOI: 10.1038/s41598-021-85613-8 -
Current Biology : CB Jun 2024Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents...
Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.
Topics: Saccharomyces cerevisiae Proteins; Kinetochores; Saccharomyces cerevisiae; Protein Serine-Threonine Kinases; Microtubules; Cell Cycle Proteins; Microtubule-Associated Proteins; Nuclear Proteins
PubMed: 38776902
DOI: 10.1016/j.cub.2024.03.062 -
Genes & Development Jul 2020During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the...
During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division.
Topics: Animals; Cell Cycle Checkpoints; Chromatin; Chromosomes; Enhancer Elements, Genetic; Genome; Histone Code; Histones; Humans; Mitosis; Promoter Regions, Genetic; Protein Binding; Protein Processing, Post-Translational; Time Factors; Transcriptional Activation
PubMed: 32499403
DOI: 10.1101/gad.335794.119 -
The Journal of Cell Biology Dec 2021The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the...
The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.
Topics: Animals; Cell Line, Tumor; Centrosome; Cytoplasmic Dyneins; Dynactin Complex; Evolution, Molecular; Golgi Apparatus; Humans; Interphase; Kinetochores; Metaphase; Microtubule-Associated Proteins; Mitosis; Mutant Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Nuclear Envelope; Phosphorylation; Protein Binding; Protein Subunits; Rats; Zebrafish
PubMed: 34709360
DOI: 10.1083/jcb.202005184 -
Current Biology : CB Oct 2023During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation....
During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation. The signaling activity of these kinetochores and the likelihood of chromosome missegregation depend on the amount of SAC signaling proteins each kinetochore recruits. Therefore, factors that control SAC protein recruitment must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins due to the concerted action of many kinases and phosphatases is a significant determinant of the SAC protein recruitment to signaling kinetochores. Whether the abundance of SAC proteins also influences the recruitment and signaling activity of human kinetochores has not been studied. Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits its own recruitment and that of BubR1 and restricts the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher recruitment of SAC proteins, producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins, including Bub1. The inverse correlation may allow the dividing cell to prevent a large number of signaling kinetochores in early prophase from generating an overly large signal while enabling the last unaligned kinetochore in late prometaphase to signal at the maximum strength.
Topics: Humans; Cell Cycle Proteins; M Phase Cell Cycle Checkpoints; Signal Transduction; Kinetochores; Mitosis; Spindle Apparatus
PubMed: 37738972
DOI: 10.1016/j.cub.2023.08.074 -
Molecular Systems Biology Aug 2020The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human...
The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.
Topics: Cell Nucleolus; Chromosomes, Human; HEK293 Cells; Humans; Ki-67 Antigen; Microscopy, Confocal; Mitosis; Nuclear Proteins; Phenotype; Proteomics; Single-Cell Analysis
PubMed: 32744794
DOI: 10.15252/msb.20209469 -
BMC Psychiatry Mar 2022The World Health Organization (WHO) proposed COVID-19 vaccination as an emergent and important method to end the COVID-19 pandemic. Since China started vaccination...
Subjective health status: an easily available, independent, robust and significant predictive factor at the prometaphase of vaccination programs for the vaccination behavior of Chinese adults.
BACKGROUND
The World Health Organization (WHO) proposed COVID-19 vaccination as an emergent and important method to end the COVID-19 pandemic. Since China started vaccination programs in December 2020, vaccination has spread to provinces and municipalities nationwide. Previous research has focused on people's vaccination willingness and its influencing factors but has not examined vaccination behavior. We examine the effectiveness of psychosocial factors in predicting vaccination behavior.
METHODS
A cross-sectional online survey was performed among Chinese adults on 8 May and 4 June 2021. The statistical analysis of the data included univariate analysis, receiver operator characteristics (ROC) analysis and ordinal multiclassification logistic regression model analysis.
RESULTS
Of the 1300 respondents, 761 (58.5%) were vaccinated. Univariate analysis showed that a high education level and good subjective health status were protective factors for vaccination behavior, while suffering from chronic diseases was a risk factor. ROC analysis showed that subjective health status (AUC = 0.625, 95% CI: 0.594-0.656, P < 0.001) was the best predictor of vaccination behavior. Logistic regression analysis with subjective health status as a dependent variable indicated that older age, female sex, depression, neurasthenia, obsession, hypochondriasis and chronic disease were significant risk factors, while positive coping tendencies were a significant protective factor.
CONCLUSION
Our study found a simple and effective marker, subjective health status, that can predict vaccination behavior. This finding can guide future epidemic prevention work.
Topics: Adult; COVID-19; COVID-19 Vaccines; China; Cross-Sectional Studies; Diagnostic Self Evaluation; Female; Humans; Pandemics; Prometaphase; Vaccination
PubMed: 35287644
DOI: 10.1186/s12888-022-03830-5 -
Current Biology : CB Mar 2022Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via...
Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.
Topics: Chromosome Segregation; Humans; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35108523
DOI: 10.1016/j.cub.2022.01.013 -
Molecular Biology Reports Jun 2022This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides.
BACKGROUND
This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides.
METHODS AND RESULTS
Chicken cathelicidins were used as anticancer agent against the breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116). In addition, the mechanism of action of the interaction of cationic peptides with breast cancer cell line MCF-7 was also investigated. An in vivo investigation was also achieved to evaluate the role of chicken cathelicidin in Ehrlich ascites cell (EAC) suppression as a tumor model after subcutaneous implantation in mice. It was found during the study that exposure of cell lines to 40 µg/ml of chicken cathelicidin for 72 h reduced cell lines growth rate by 90-95%. These peptides demonstrated down-regulation of (cyclin A1 and cyclin D genes) of MCF-7 cells. The study showed that two- and three-fold expression of both of caspase-3 and - 7 genes in untreated MCF-7 cells compared to treated MCF-7 cells with chicken cathelicidin peptides. Our data showed that chicken (CATH-1) enhance releasing of TNFα, INF-γ and upregulation of granzyme K in treated mice groups, in parallel, the tumor size and volume was reduced in the treated EAC-bearing groups. Tumor of mice groups treated with chicken cathelicidin displayed high area of necrosis compared to untreated EAC-bearing mice. Based on histological analysis and immunohistochemical staining revealed that the tumor section in Ehrlich solid tumor exhibited a strong Bcl2 expression in untreated control compared to mice treated with 10 & 20 µg of cathelicidin. Interestingly, low expression of Bcl2 were observed in mice taken 40 µg/mL of CATH-1.
CONCLUSIONS
This study drive intention in treatment of cancer through the efficacy of anticancer efficacy of chicken cathelicidin peptides.
Topics: Animals; Antineoplastic Agents; Cathelicidins; Cell Line, Tumor; Chickens; Humans; MCF-7 Cells; Mice; Neoplasms; Proto-Oncogene Proteins c-bcl-2
PubMed: 35449320
DOI: 10.1007/s11033-022-07267-7