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Chromosoma Sep 2023Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a...
Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.
PubMed: 37728741
DOI: 10.1007/s00412-023-00810-w -
The Journal of Cell Biology Nov 2023UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion...
UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest.
Topics: Apoptosis; Cell Line, Tumor; Epigenesis, Genetic; G2 Phase Cell Cycle Checkpoints; Mitosis; Humans; Ubiquitin-Protein Ligases; Kinesins; Ubiquitination; DNA Damage; Spindle Apparatus; Chromosomes
PubMed: 37728657
DOI: 10.1083/jcb.202210093 -
The EMBO Journal Oct 2023Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the...
Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.
Topics: Humans; M Phase Cell Cycle Checkpoints; Kinetochores; Protein Serine-Threonine Kinases; Cell Cycle Proteins; Chromosome Segregation; Phosphorylation; Microtubules; Mitosis; HeLa Cells
PubMed: 37712330
DOI: 10.15252/embj.2022112630 -
Ecotoxicology (London, England) Sep 2023Cytogenetic studies of Pinus sylvestris seed progeny have been carried out for the first time in the Murmansk region of Russia. Seeds were collected in the territory of...
Cytogenetic studies of Pinus sylvestris seed progeny have been carried out for the first time in the Murmansk region of Russia. Seeds were collected in the territory of 6 district forestries: Zelenoborsky, Kovdorsky, Kandalakshsky, Zasheikovsky, Umbsky and Nickelsky, which were at different distances from large copper-nickel plants. Analysis revealed higher S, Cu, and Ni content in Pinus sylvestris tree seeds from the Zasheikovsky and Nickelsky district forestries. Seeds from the Zelenoborsky district forestry had a higher Cu content (13.6 ± 0.5 mg/kg) compared to other areas. It was found that the frequency of mitotic pathologies in all areas of the study exceeded the level of spontaneous mutation in 5%. The most frequent aberrant cells were registered in the root meristem of seedlings from the Zasheikovsky district forestry, and their proportion averaged 9.4 ± 1.3% of the total number of cells studied at the metaphase and ana-telophase of mitosis stages. In Pinus sylvestris seedlings, micronuclei were noted in the cells at the interphase stage, often varying on average from 0.2 ± 0.1% in plants from the Kandalakshsky district forestry to 0.9 ± 0.3% from the Zasheikovsky district forestry. The data obtained testify to the colossal impact of heavy metals on the living organism cell genetic apparatus. The negative effect from industries, as sources of air pollutants, extends over tens of kilometers. Therefore, regularly monitoring the cytogenetic parameters of bioindicators such as Pinus sylvestris is necessary.
PubMed: 37676552
DOI: 10.1007/s10646-023-02696-x -
Cytoskeleton (Hoboken, N.J.) 2023Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across...
Accurate placement of the cleavage furrow is crucial for successful cell division. Recent advancements have revealed that diverse mechanisms have evolved across different branches of the phylogenetic tree. Here, we employed Dictyostelium cells to validate previous models. We observed that during metaphase and early anaphase, mitotic spindles exhibited random rotary movements which ceased when the spindle elongated by approximately 7 μm. At this point, astral microtubules reached the polar cell cortex and fixed the spindle axis, causing cells to elongate by extending polar pseudopods and divide along the spindle axis. Therefore, the position of the furrow is determined when the spindle orientation is fixed. The distal ends of astral microtubules stimulate the extension of pseudopods at the polar cortex. One signal for pseudopod extension may be phosphatidylinositol trisphosphate in the cell membrane, but there appears to be another unknown signal. At the onset of polar pseudopod extension, cortical flow began from both poles toward the equator. We suggest that polar stimulation by astral microtubules determines the furrow position, induces polar pseudopod extension and cortical flow, and accumulates the elements necessary for the construction of the contractile ring.
Topics: Dictyostelium; Phylogeny; Cytokinesis; Microtubules; Spindle Apparatus; Anaphase
PubMed: 37650534
DOI: 10.1002/cm.21784 -
Chromosoma Nov 2023Nucleolin is a multifunctional RNA-binding protein that resides predominantly not only in the nucleolus, but also in multiple other subcellular pools in the cytoplasm in...
Nucleolin is a multifunctional RNA-binding protein that resides predominantly not only in the nucleolus, but also in multiple other subcellular pools in the cytoplasm in mammalian cells, and is best known for its roles in ribosome biogenesis, RNA stability, and translation. During early mitosis, nucleolin is required for equatorial mitotic chromosome alignment prior to metaphase. Using high resolution fluorescence imaging, we reveal that nucleolin is required for multiple centrosome-associated functions at the G2-prophase boundary. Nucleolin depletion led to dissociation of the centrosomes from the G2 nuclear envelope, a delay in the onset of nuclear envelope breakdown, reduced inter-centrosome separation, and longer metaphase spindles. Our results reveal novel roles for nucleolin in early mammalian mitosis, establishing multiple important functions for nucleolin during mammalian cell division.
Topics: Animals; Nucleolin; Spindle Apparatus; Centrosome; Mitosis; Vertebrates; Mammals
PubMed: 37615728
DOI: 10.1007/s00412-023-00808-4 -
Research in Pharmaceutical Sciences 2023Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By...
Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.
BACKGROUND AND PURPOSE
Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death.
EXPERIMENTAL APPROACH
Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively.
FINDINGS/RESULTS
CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells.
CONCLUSION AND IMPLICATIONS
Our data suggested the proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.
PubMed: 37614620
DOI: 10.4103/1735-5362.378083 -
Chromosome Research : An International... Aug 2023Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural...
Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.
Topics: Humans; Induced Pluripotent Stem Cells; Aphidicolin; Caffeine; Chromosomes, Human, Pair 12; In Situ Hybridization, Fluorescence; Intracellular Signaling Peptides and Proteins
PubMed: 37597021
DOI: 10.1007/s10577-023-09729-5 -
Archives of Microbiology Aug 2023Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata...
Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.
Topics: Armillaria; Cluster Analysis; Gene Expression Profiling; Mycelium
PubMed: 37594611
DOI: 10.1007/s00203-023-03621-w -
BioRxiv : the Preprint Server For... Aug 2023The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles...
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected by manipulating mechanical properties of motors.
PubMed: 37577480
DOI: 10.1101/2023.08.03.551815