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Journal of Cell Science Jun 2023The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to...
The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.
Topics: Kinesins; Saccharomyces cerevisiae; Cell Cycle; Anaphase; Metaphase
PubMed: 37305999
DOI: 10.1242/jcs.260621 -
Comparative Cytogenetics 2023To have an insight into the karyotype variation of eight Cucurbitaceae crops including Linnaeus, 1753, Linnaeus, 1753, (Thunberg, 1794) Matsumura et Nakai, 1916,...
To have an insight into the karyotype variation of eight Cucurbitaceae crops including Linnaeus, 1753, Linnaeus, 1753, (Thunberg, 1794) Matsumura et Nakai, 1916, (Thunberg, 1784) Cogniaux, 1881, Linnaeus, 1753, (Linnaeus, 1753) Roemer, 1846, Lagenariasicerariavar.hispida (Thunberg, 1783) Hara, 1948 and Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CV, CV, M and Stebbins' category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of M vs. CV. The karyological relationships among these species revealed by PCoA based on , 2, TCL, M, CV and CV was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in , , , and , terminal GC-rich heterochromatin in . DAPI counterstaining after FISH revealed pericentromeric DAPI heterochromatin in . rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In , individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.
PubMed: 37305810
DOI: 10.3897/compcytogen.v17.i1.99236 -
Journal of Nanobiotechnology Jun 2023Due to its complicated pathophysiology, propensity for metastasis, and poor prognosis, colon cancer is challenging to treat and must be managed with a combination of...
Due to its complicated pathophysiology, propensity for metastasis, and poor prognosis, colon cancer is challenging to treat and must be managed with a combination of therapy. Using rolling circle transcription (RCT), this work created a nanosponge therapeutic medication system (AS1411@antimiR-21@Dox). Using the AS1411 aptamer, this approach accomplished targeted delivery to cancer cells. Furthermore, analysis of cell viability, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) content, and mitochondrial membrane potential (MMP) levels revealed that functional nucleic acid nanosponge drug (FND) can kill cancer cells. Moreover, transcriptomics uncovered a putative mechanism for the FND anti-tumor effect. These pathways, which included mitotic metaphase and anaphase as well as the SMAC-mediated dissociation of the IAP: caspase complexes, were principally linked to the cell cycle and cell death. In conclusion, by triggering cell cycle arrest and apoptosis, the nano-synergistic therapeutic system allowed for the intelligent and effective targeted administration of RNA and chemotherapeutic medicines for colon cancer treatment. The system allowed for payload efficiency while being customizable, targeted, reliable, stable, and affordable.
Topics: Humans; Doxorubicin; Drug Delivery Systems; Nucleic Acids; Colonic Neoplasms; Drug Carriers; Aptamers, Nucleotide; Oligodeoxyribonucleotides; Nanoparticles; Cell Line, Tumor
PubMed: 37280622
DOI: 10.1186/s12951-023-01941-z -
Pharmaceuticals (Basel, Switzerland) Jan 2023Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of...
Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of immunotherapy and targeted therapies. Thus, it is important to identify new drug candidates for melanoma. The design of hybrid molecules, with different pharmacophore fragments combined in the same scaffold, is an interesting strategy for obtaining new multi-target and more effective anticancer drugs. We designed nine hybrid compounds bearing piperine and chlorogenic acid pharmacophoric groups and evaluated their antitumoral potential on melanoma cells with distinct mutational profiles SK-MEL-147, CHL-1 and WM1366. We identified the compound named PQM-277 () to be the most cytotoxic one, inhibiting mitosis progression and promoting an accumulation of cells in pro-metaphase and metaphase by altering the expression of genes that govern G2/M transition and mitosis onset. Compound downregulated , , , , , and , and upregulated . Molecular docking showed that could interact with the CUL1-RBX1 complex, which activity is necessary to trigger molecular events essential for FOXM1 transactivation and, in turn, G2/M gene expression. In addition, compound effectively induced apoptosis by increasing ratio. Our findings demonstrate that is an important antitumor candidate prototype and support further investigations to evaluate its potential for melanoma treatment, especially for refractory cases to BRAF/MEK inhibitors.
PubMed: 37259298
DOI: 10.3390/ph16020145 -
Journal of Obstetrics and Gynaecology :... Dec 2023FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the...
FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the clinicopathological and prognostic significance of mRNA expression in gynecological cancers. We conducted a bioinformatics analysis of mRNA expression using Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), xiantao, The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier (KM) plotter databases. expression was elevated in breast, cervical, endometrial, and ovarian cancers when compared with normal tissue. Expression was positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade and TP53 mutation, and endometrial cancer serous subtype. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer patients. -correlated genes were involved in ligand-receptor interactions, and chromosomal, cell cycle, and DNA replication processes in breast, cervical, endometrial and ovarian cancers. Top hub genes primarily included cell cycle-related proteins in breast cancer, mucins and acetylgalactosaminyl transferases in cervical cancer, kinesin family members in endometrial cancer, and synovial sarcoma X and the cancer/testis antigen in ovarian cancer. mRNA expression was positively related to Th2 cell infiltration, but negatively associated with neutrophil and Th17 cell infiltration in breast, cervical, endometrial, and ovarian cancers. expression may be considered a potential biomarker reflecting carcinogenesis, histogenesis, aggressive behaviour, and prognosis in gynecological cancers.Impact statement FAM64A is located in cell nucleolar and nucleoplasmic regions, and during mitosis it putatively controls metaphase-to-anaphase transition. FAM64A appears to regulate different physiological processes, including apoptosis, tumorigenesis, neural differentiation, stress responses, and the cell cycle. expression was up-regulated in breast, cervical, endometrial, and ovarian cancers, and positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade, and TP53 mutation, and a serous subtype in endometrial cancer. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer. -correlated genes were involved in ligand-receptor interactions, chromosomal, cell cycle, and DNA replication processes, while mRNA expression was positively related to Th2 cell infiltration but negatively correlated with neutrophil and Th17 cell infiltration in four gynecological cancers. In the future, abnormal mRNA expression may serve as a biomarker of carcinogenesis, histogenesis, aggressiveness, and prognosis in gynecological malignancies.
Topics: Female; Humans; Male; Breast Neoplasms; Carcinogenesis; Carcinoma, Ductal; Computational Biology; Endometrial Neoplasms; Gene Expression Regulation, Neoplastic; Ligands; Ovarian Neoplasms; Prognosis; RNA, Messenger
PubMed: 37227120
DOI: 10.1080/01443615.2023.2216280 -
BioRxiv : the Preprint Server For... May 2023The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including...
The biorientation of sister chromatids on the mitotic spindle, essential for accurate sister chromatid segregation, relies on critical centromere components including cohesin, the centromere-specific H3 variant CENP-A, and centromeric DNA. Centromeric DNA is highly variable between chromosomes yet must accomplish a similar function. Moreover, how the 50 nm cohesin ring, proposed to encircle sister chromatids, accommodates inter-sister centromeric distances of hundreds of nanometers on the metaphase spindle is a conundrum. Insight into the 3D organization of centromere components would help resolve how centromeres function on the mitotic spindle. We used ChIP-seq and super-resolution microscopy to examine the geometry of essential centromeric components on human chromosomes. ChIP-seq demonstrates that cohesin subunits are depleted in α-satellite arrays where CENP-A nucleosomes and kinetochores assemble. Cohesin is instead enriched at pericentromeric DNA. Structured illumination microscopy of sister centromeres is consistent, revealing a non-overlapping pattern of CENP-A and cohesin. We used single particle averaging of hundreds of mitotic sister chromatids to develop an average centromere model. CENP-A clusters on sister chromatids, connected by α-satellite, are separated by ~562 nm with a perpendicular intervening ~190 nM wide axis of cohesin. Two differently sized α-satellite arrays on chromosome 7 display similar inter-sister CENP-A cluster distance, demonstrating different sized arrays can achieve a common spacing. Our data suggest a working model for a common core configuration of essential centromeric components that includes CENP-A nucleosomes at the outer edge of extensible α-satellite DNA and pericentromeric cohesion. This configuration helps reconcile how centromeres function and serves as a foundation for future studies of additional components required for centromere function.
PubMed: 37214893
DOI: 10.1101/2023.05.10.539634 -
Current Protocols May 2023The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The...
The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.
Topics: Animals; Humans; Karyotyping; Flow Cytometry; Cytogenetics; DNA; Chromosomes, Mammalian; Mammals
PubMed: 37200525
DOI: 10.1002/cpz1.785 -
The Journal of Cell Biology Jun 2023Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes...
Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant CENP-A. CENP-A nucleosome assembly is uncoupled from replication and occurs in G1, but how cells control this timing is incompletely understood. The formation of CENP-A nucleosomes in vertebrates requires CENP-C and the Mis18 complex which recruit the CENP-A chaperone HJURP to centromeres. Using a cell-free system for centromere assembly in X. laevis egg extracts, we discover two activities that inhibit CENP-A assembly in metaphase. HJURP phosphorylation prevents the interaction between HJURP and CENP-C in metaphase, blocking the delivery of soluble CENP-A to centromeres. Non-phosphorylatable mutants of HJURP constitutively bind CENP-C in metaphase but are not sufficient for new CENP-A assembly. We find that the M18BP1.S subunit of the Mis18 complex also binds to CENP-C to competitively inhibit HJURP's access to centromeres. Removal of these two inhibitory activities causes CENP-A assembly in metaphase.
Topics: Animals; Autoantigens; Carrier Proteins; Centromere; Centromere Protein A; Metaphase; Nucleosomes; Phosphorylation; Xenopus laevis; Xenopus Proteins; DNA-Binding Proteins
PubMed: 37141119
DOI: 10.1083/jcb.202110124 -
ELife Apr 2023During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size....
During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.
Topics: Animals; Xenopus laevis; Cell Nucleus; Chromosomes; Cytoplasm; Spindle Apparatus; Cell Size; Mitosis
PubMed: 37096661
DOI: 10.7554/eLife.84360 -
Biology Open May 2023In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies...
In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.
Topics: Animals; Zebrafish; Actins; Chromosomes; Mitosis; Actin Cytoskeleton
PubMed: 37071022
DOI: 10.1242/bio.059783