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EMBO Reports Jun 2001The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned... (Review)
Review
The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned sister chromatid pairs split and separate. During metaphase, cohesion between sister chromatids is maintained by the chromosomal multi-subunit cohesin complex. Here, I review recent findings as to how anaphase is initiated by proteolytic cleavage of the Scc1 subunit of cohesin. Scc1 is cleaved by a site-specific protease that is conserved in all eukaryotes, and is now called 'separase'. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms seem to impose regulation on Scc1 cleavage, acting on both the activity of separase and the susceptibility of the substrate.
Topics: Anaphase; Animals; Binding Sites; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosomes; Fungal Proteins; Metaphase; Nuclear Proteins; Phosphoproteins; Saccharomyces cerevisiae Proteins; Cohesins
PubMed: 11415980
DOI: 10.1093/embo-reports/kve113 -
International Journal of Molecular... Jan 2021The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and... (Randomized Controlled Trial)
Randomized Controlled Trial
The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration.
Topics: Cell Survival; Chromosomes, Human; Cryopreservation; Female; Humans; In Vitro Oocyte Maturation Techniques; Metaphase; Oocytes; Spindle Apparatus; Time Factors; Vitrification
PubMed: 33498768
DOI: 10.3390/ijms22031125 -
Journal of Cell Science Jul 2021Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been...
Proper control of division orientation and symmetry, largely determined by spindle positioning, is essential to development and homeostasis. Spindle positioning has been extensively studied in cells dividing in two-dimensional (2D) environments and in epithelial tissues, where proteins such as NuMA (also known as NUMA1) orient division along the interphase long axis of the cell. However, little is known about how cells control spindle positioning in three-dimensional (3D) environments, such as early mammalian embryos and a variety of adult tissues. Here, we use mouse embryonic stem cells (ESCs), which grow in 3D colonies, as a model to investigate division in 3D. We observe that, at the periphery of 3D colonies, ESCs display high spindle mobility and divide asymmetrically. Our data suggest that enhanced spindle movements are due to unequal distribution of the cell-cell junction protein E-cadherin between future daughter cells. Interestingly, when cells progress towards differentiation, division becomes more symmetric, with more elongated shapes in metaphase and enhanced cortical NuMA recruitment in anaphase. Altogether, this study suggests that in 3D contexts, the geometry of the cell and its contacts with neighbors control division orientation and symmetry. This article has an associated First Person interview with the first author of the paper.
Topics: Anaphase; Animals; Intercellular Junctions; Metaphase; Mice; Mitosis; Spindle Apparatus; Stem Cells
PubMed: 34323278
DOI: 10.1242/jcs.255018 -
Science (New York, N.Y.) Nov 2013Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied...
Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type-specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
Topics: Biopolymers; Cell Cycle; Chromatin; Chromosomes, Human, Pair 21; HeLa Cells; Humans; Metaphase; Mitosis; Models, Chemical
PubMed: 24200812
DOI: 10.1126/science.1236083 -
Molecular Biology of the Cell Apr 2021Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase...
Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.
Topics: Chromatids; Chromosome Segregation; HeLa Cells; Humans; Image Processing, Computer-Assisted; Kinetochores; Metaphase; Microscopy, Fluorescence; Microtubules; Mitosis; Models, Theoretical; Spindle Apparatus
PubMed: 33085580
DOI: 10.1091/mbc.E20-09-0585 -
Cellular and Molecular Life Sciences :... Aug 2022Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation,...
Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.
Topics: Animals; Histones; Male; Mammals; Meiosis; Metaphase; Mice; Phosphorylation; Semen; Spermatocytes
PubMed: 35930080
DOI: 10.1007/s00018-022-04507-8 -
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 -
The Journal of Cell Biology Nov 1995We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase...
We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.
Topics: Anaphase; Animals; Cell Cycle; Cell Line; Kidney Tubules, Proximal; Kinetochores; Metaphase; Microtubules; Photochemistry; Spindle Apparatus; Swine; Temperature; Time Factors; Tubulin
PubMed: 7593192
DOI: 10.1083/jcb.131.3.721 -
Cytometry Mar 1994Chromosome painting is a term used to describe the direct visualisation using in situ hybridisation of specific chromosomes in metaphase spreads and in interphase... (Review)
Review
Chromosome painting is a term used to describe the direct visualisation using in situ hybridisation of specific chromosomes in metaphase spreads and in interphase nuclei. Chromosome painting, coupled with fluorescence in situ hybridisation (FISH), is now used routinely to enhance the identification of chromosomal rearrangements, the assignment of breakpoints, and the determination of the origin of extra chromosomal material. Amplification of small numbers of flow-sorted chromosomes by the polymerase chain reaction allows labelled chromosome paints to be generated in a matter of days. These technologies have enabled the development of reverse chromosome painting, in which the paint is produced from sorted aberrant chromosomes and hybridised back onto normal metaphase spreads to identify directly the composition of the aberrant chromosome. Reverse chromosome painting is able to identify not only the chromosomal origin of marker chromosomes but also the regions and breakpoints involved. In some cases, such as interstitial translocations and complex marker chromosomes, the combination of conventional (forward) chromosome painting and reverse chromosome painting combine to provide a definitive analysis of the rearrangement. With the availability of chromosome paints and painting kits from a variety of commercial sources, multicolour chromosome painting has now become a routine method of analysis in the clinical cytogenetic laboratory.
Topics: Cell Nucleus; Chromosome Banding; Chromosomes, Human; Humans; In Situ Hybridization, Fluorescence; Interphase; Karyotyping; Metaphase
PubMed: 8082483
DOI: 10.1002/cyto.990180103 -
Annals of Medicine Dec 2022Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological...
Combining metaphase cytogenetics with single nucleotide polymorphism arrays can improve the diagnostic yield and identify prognosis more precisely in myelodysplastic syndromes.
BACKGROUND
Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological aberrations, and a propensity for progression to acute myeloid leukaemia. Diagnosis and disease prognostic stratification are much based on genomic abnormalities. The traditional metaphase cytogenetics analysis (MC) can detect about 40-60% aberrations. Single-nucleotide polymorphism arrays (SNP-A) karyotyping can detect copy number variations with a higher resolution and has a unique advantage in detection of copy number neutral loss of heterozygosity (CN-LOH). Combining these two methods may improve the diagnostic efficiency and accuracy for MDS.
METHODS
We retrospectively analysed the data of 110 MDS patients diagnosed from January 2012 to December 2019 to compare the detection yield of chromosomal abnormalities by MC with by SNP-A, and the relationship between chromosomal abnormalities and prognosis.
RESULTS
Our results showed that SNP-A improved the detection yield of chromosomal aberrations compared with MC (74.5 vs. 55.5%, < .001). In addition, the positive yield could be further improved by combining MC with SNP-A to 77.3%, compared with MC alone. Univariate analysis showed that age >65 years, bone marrow blasts ≥5%, with acquired CN-LOH, new aberrations detected by SNP-A, TGA value > the median (81.435 Mb), higher risk by IPSS-R-MC, higher risk by IPSS-R-SNP-A all had poorer prognosis. More critically, multivariable analysis showed that age >65 years and higher risk by IPSS-R-SNP-A were independent predictors of inferior OS in MDS patients.
CONCLUSION
The combination of MC and SNP-A based karyotyping can further improve the diagnostic yield and provide more precise prognostic stratification in MDS patients. However, SNP-A may not completely replace MC because of its inability to detect balanced translocation and to detect different clones. From a practical point of view, we recommend the concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients on diagnosis and prognosis stratification.KEY MESSAGESSNP-A based karyotyping can further improve the MDS diagnostic yield and provide more precise prognostic stratification in MDS patients.Acquired CN-LOH is a characteristic chromosomal aberration of MDS, which should be integrated to the diagnostic project of MDS.The concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients can be recommended.
Topics: Aged; Humans; Chromosome Aberrations; Cytogenetic Analysis; DNA Copy Number Variations; Leukemia, Myeloid, Acute; Metaphase; Myelodysplastic Syndromes; Polymorphism, Single Nucleotide; Prognosis; Retrospective Studies
PubMed: 36148999
DOI: 10.1080/07853890.2022.2125173