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Folia Biologica 2021The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100...
The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.
Topics: Actins; Animals; Dinoprost; Meiosis; Metaphase; Mice; Oocytes; Spindle Apparatus
PubMed: 35439854
DOI: No ID Found -
Oncotarget Apr 2017Multicellular spheroids are very attractive models in oncology because they mimic the 3D organization of the tumour cells with their microenvironment. We show here using...
Multicellular spheroids are very attractive models in oncology because they mimic the 3D organization of the tumour cells with their microenvironment. We show here using 3 different cell types (mammary TSA/pc, embryonic kidney Hek293 and cervical cancer HeLa), that when the cells are growing as spheroids the frequency of binucleated cells is augmented as occurs in some human tumours.We therefore describe mitosis in multicellular spheroids by following mitotic markers and by time-lapse experiments. Chromosomes alignment appears to be correct on the metaphasic plate and the passenger complex is well localized on centromere. Moreover aurora kinases are fully active and histone H3 is phosphorylated on Ser 10. Consequently, the mitotic spindle checkpoint is satisfied and, anaphase proceeds as illustrated by the transfer of survivin on the spindle and by the segregation of the two lots of chromosomes. However, the segregation plane is not well defined and oscillations of the dividing cells are observed. Finally, cytokinesis fails and the absence of separation of the two daughter cells gives rise to binucleated cells.Division orientation is specified during interphase and persists throughout mitosis. Our data indicate that the cancer cells, in multicellular spheroids, lose their ability to regulate their orientation, a feature commonly encountered in tumours.Moreover, multicellular spheroid expansion is still sensitive to mitotic drugs as pactlitaxel and aurora kinase inhibitors. The spheroids thus represent a highly relevant model for studying drug efficiency in tumours.
Topics: Aurora Kinase B; Cell Polarity; Chromosome Segregation; Cytokinesis; HEK293 Cells; HeLa Cells; Histones; Humans; Inhibitor of Apoptosis Proteins; Mitosis; Neoplasms; Phosphorylation; Protein Transport; Spheroids, Cellular; Spindle Apparatus; Survivin
PubMed: 28430635
DOI: 10.18632/oncotarget.15673 -
Journal of Cell Science Feb 2022The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous...
The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed as reflecting kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex in U2OS cells. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, although the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1 (also known as Ndc80) depletion suggests that other subpopulations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, although Aurora B kinase functions to destabilize kinetochore bound microtubules it might also stabilize certain slow turnover non-kinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement and segregation during mitosis.
Topics: Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Nuclear Proteins; Spindle Apparatus
PubMed: 34907446
DOI: 10.1242/jcs.258745 -
Journal of Ginseng Research May 2022Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet....
BACKGROUND
Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer.
MATERIALS AND METHODS
Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines.
RESULTS
Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation.
CONCLUSION
Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.
PubMed: 35600766
DOI: 10.1016/j.jgr.2021.11.004 -
Journal of Biomedical Optics Jun 2024Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond...
SIGNIFICANCE
Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye.
AIM
This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.
APPROACH
We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy.
RESULTS
We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization.
CONCLUSIONS
This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.
Topics: Animals; Mice; Oocytes; Humans; Female; Lasers; Spindle Apparatus; Microscopy, Confocal; Metaphase; Microscopy, Polarization
PubMed: 38812963
DOI: 10.1117/1.JBO.29.6.065002 -
FEBS Letters Apr 2019Early results showed the emanation of chromatin fibers from mitotic chromosomes and nuclei swollen with water. In contrast, under metaphase ionic conditions, it was...
Early results showed the emanation of chromatin fibers from mitotic chromosomes and nuclei swollen with water. In contrast, under metaphase ionic conditions, it was found that chromatin from mitotic chromosomes is planar and forms multilayered plates. Here, we show that in buffers containing interphase cation concentrations, the chromatin emanated from disrupted nuclei also has a planar morphology. Furthermore, the chromatin fragments produced by micrococcal nuclease digestion of nuclei form the typical beads-on-a-string fibers in the absence of cations, but they self-assemble into plate-like structures in buffers containing magnesium. The plates from interphase nuclei do not form the thick multilayered structures observed in metaphase chromosomes, suggesting that they are more exposed to the medium to facilitate DNA replication and gene expression.
Topics: Cell Nucleus; Chromatin; HeLa Cells; Humans; Interphase; Microscopy, Electron
PubMed: 30908620
DOI: 10.1002/1873-3468.13370 -
The EMBO Journal Aug 2020MIWI, a murine member of PIWI proteins mostly expressed during male meiosis, is crucial for piRNA biogenesis, post-transcriptional regulation, and spermiogenesis....
MIWI, a murine member of PIWI proteins mostly expressed during male meiosis, is crucial for piRNA biogenesis, post-transcriptional regulation, and spermiogenesis. However, its meiotic function remains unknown. Here, we report that MIWI deficiency alters meiotic kinetochore assembly, significantly increases chromosome misalignment at the meiosis metaphase I plate, and causes chromosome mis-segregation. Consequently, Miwi-deficient mice show elevated aneuploidy in metaphase II and spermatid death. Furthermore, in Miwi-null and Miwi slicer-deficient mutants, major and minor satellite RNAs from centromeric and pericentromeric satellite repeats accumulate in excess. Over-expression of satellite repeats in wild-type spermatocytes also causes elevated chromosome misalignment, whereas reduction of both strands of major or minor satellite RNAs results in lower frequencies of chromosome misalignment. We show that MIWI, guided by piRNA, cleaves major satellite RNAs, generating RNA fragments that may form substrates for subsequent Dicer cleavage. Furthermore, Dicer cleaves all satellite RNAs in conjunction with MIWI. These findings reveal a novel mechanism in which MIWI- and Dicer-mediated cleavage of the satellite RNAs prevents the over-expression of satellite RNAs, thus ensuring proper kinetochore assembly and faithful chromosome segregation during meiosis.
Topics: Aneuploidy; Animals; Argonaute Proteins; Chromosome Segregation; Chromosomes, Mammalian; DEAD-box RNA Helicases; Kinetochores; Meiosis; Mice; Mice, Transgenic; RNA Stability; RNA, Satellite; Ribonuclease III
PubMed: 32677148
DOI: 10.15252/embj.2019103614 -
Philosophical Transactions of the Royal... Dec 1998Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin-dependent kinases (CDKs) and their... (Review)
Review
Progress through the division cycle of present day eukaryotic cells is controlled by a complex network consisting of (i) cyclin-dependent kinases (CDKs) and their associated cyclins, (ii) kinases and phosphatases that regulate CDK activity, and (iii) stoichiometric inhibitors that sequester cyclin-CDK dimers. Presumably regulation of cell division in the earliest ancestors of eukaryotes was a considerably simpler affair. Nasmyth (1995) recently proposed a mechanism for control of a putative, primordial, eukaryotic cell cycle, based on antagonistic interactions between a cyclin-CDK and the anaphase promoting complex (APC) that labels the cyclin subunit for proteolysis. We recast this idea in mathematical form and show that the model exhibits hysteretic behaviour between alternative steady states: a Gl-like state (APC on, CDK activity low, DNA unreplicated and replication complexes assembled) and an S/M-like state (APC off, CDK activity high, DNA replicated and replication complexes disassembled). In our model, the transition from G1 to S/M ('Start') is driven by cell growth, and the reverse transition ('Finish') is driven by completion of DNA synthesis and proper alignment of chromosomes on the metaphase plate. This simple and effective mechanism for coupling growth and division and for accurately copying and partitioning a genome consisting of numerous chromosomes, each with multiple origins of replication, could represent the core of the eukaryotic cell cycle. Furthermore, we show how other controls could be added to this core and speculate on the reasons why stoichiometric inhibitors and CDK inhibitory phosphorylation might have been appended to the primitive alternation between cyclin accumulation and degradation.
Topics: Animals; Biological Evolution; Cell Cycle; Cyclin-Dependent Kinases; Cyclins; DNA Replication; Eukaryotic Cells; Models, Biological; Phosphorylation
PubMed: 10098216
DOI: 10.1098/rstb.1998.0352 -
Seminars in Cell & Developmental Biology Sep 2021The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to... (Review)
Review
The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to the spindle have occurred, the chromosomes have aligned at the metaphase plate and the spindle assembly checkpoint (a surveillance mechanism that halts cells form progressing in the cell cycle in case of spindle - microtubule attachment errors) has been satisfied, mitotic exit will occur. Mitotic exit has the purpose of completing the separation of the genomic material but also to rebuild the cellular structures necessary for the new cell cycle. This stage of mitosis received little attention until a decade ago, therefore our knowledge is much patchier than the molecular details we now have for the early stages of mitosis. However, it is emerging that mitotic exit is not just the simple reverse of mitotic entry and it is highly regulated in space and time. In this review I will discuss the main advances in the field that provided us with a better understanding on the key role of protein phosphorylation/de-phosphorylation in this transition together with the concept of their spatial regulation. As this field is much younger, I will highlight general consensus, contrasting views together with the outstanding questions awaiting for answers.
Topics: Chromosome Segregation; Humans; Mitosis
PubMed: 33810980
DOI: 10.1016/j.semcdb.2021.03.010 -
MBio Jul 2019Precise kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres, identified as scaffolding sites for kinetochore assembly,...
Precise kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres, identified as scaffolding sites for kinetochore assembly, are among the most rapidly evolving chromosomal loci in terms of the DNA sequence and length and organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics is well studied in plant-pathogenic fungi. Here, we sought to understand the process of chromosome segregation in the rice blast fungus High-resolution imaging of green fluorescent protein (GFP)-tagged inner kinetochore proteins CenpA and CenpC revealed unusual albeit transient declustering of centromeres just before anaphase separation of chromosomes in Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an apparent metaphase plate Using CenpA chromatin immunoprecipitation followed by deep sequencing, all seven centromeres in were found to be regional, spanning 57-kb to 109-kb transcriptionally poor regions. Highly AT-rich and heavily methylated DNA sequences were the only common defining features of all the centromeres in rice blast. Lack of centromere-specific DNA sequence motifs or repetitive elements suggests an epigenetic specification of centromere function in PacBio genome assemblies and synteny analyses facilitated comparison of the centromeric/pericentromeric regions in distinct isolates of rice blast and wheat blast and in Overall, this study revealed unusual centromere dynamics and precisely identified the centromere loci in the top model fungal pathogens that belong to and cause severe losses in the global production of food crops and turf grasses. is an important fungal pathogen that causes a loss of 10% to 30% of the annual rice crop due to the devastating blast disease. In most organisms, kinetochores are clustered together or arranged at the metaphase plate to facilitate synchronized anaphase separation of sister chromatids in mitosis. In this study, we showed that the initially clustered kinetochores separate and position randomly prior to anaphase in Centromeres in occupy large genomic regions and form on AT-rich DNA without any common sequence motifs. Overall, this study identified atypical kinetochore dynamics and mapped functional centromeres in to define the roles of centromeric and pericentric boundaries in kinetochore assembly on epigenetically specified centromere loci. This study should pave the way for further understanding of the contribution of heterochromatin in genome stability and virulence of the blast fungus and its related species of high economic importance.
Topics: Centromere; Edible Grain; Kinetochores; Magnaporthe
PubMed: 31363034
DOI: 10.1128/mBio.01581-19