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ELife Jul 2015Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric...
Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore-microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions.
Topics: Anaphase; Cell Size; Epithelial Cells; HeLa Cells; Humans; Metaphase; Spindle Apparatus
PubMed: 26188083
DOI: 10.7554/eLife.05124 -
Journal of Cell Science Jan 2021Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic...
Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin-kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which astrin-kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.
Topics: Alcian Blue; Cell Cycle Proteins; Chromosome Segregation; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Phenazines; Phenothiazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Resorcinols; Spindle Apparatus; Polo-Like Kinase 1
PubMed: 33288550
DOI: 10.1242/jcs.251025 -
Reproduction in Domestic Animals =... Oct 2022The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of...
Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes.
The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2',7'-dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10 M melatonin during in vitro maturation had no effect on these parameters.
Topics: Animals; Cryopreservation; Cryoprotective Agents; Dietary Supplements; Melatonin; Metaphase; Oocytes; Swine; Vitrification
PubMed: 35567517
DOI: 10.1111/rda.14158 -
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 -
International Review of Cell and... 2013During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This... (Review)
Review
During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This requires a complex process to ensure proper interactions between chromosomes and spindle microtubules. The kinetochore, the proteinaceous complex assembled at the centromere region on each chromosome, serves as the microtubule attachment site and powers chromosome movement in mitosis. Numerous proteins/protein complexes have been implicated in the connection between kinetochores and dynamic microtubules. Recent studies have advanced our understanding on the nature of the interface between kinetochores and microtubule plus ends in promoting and maintaining their stable attachment. These efforts have demonstrated the importance of this process to ensure accurate chromosome segregation, an issue which has great significance for understanding and controlling abnormal chromosome segregation (aneuploidy) in human genetic diseases and in cancer progression.
Topics: Chromosomes; Humans; Kinetochores; Metaphase; Microtubules; Models, Biological; Spindle Apparatus
PubMed: 23445812
DOI: 10.1016/B978-0-12-407697-6.00006-4 -
Experimental Cell Research Apr 2020The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found...
The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.
Topics: Animals; Blastomeres; Cell Division; Cell Fusion; Embryo, Mammalian; Female; Green Fluorescent Proteins; Lasers; Male; Metaphase; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pregnancy; Tetraploidy
PubMed: 32027865
DOI: 10.1016/j.yexcr.2020.111887 -
Journal of Assisted Reproduction and... Apr 2023Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we...
Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 10 or 0.5 × 10 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used.
Topics: Humans; Male; Animals; Cattle; Mice; Polar Bodies; Fertilization in Vitro; Metaphase; Cryopreservation; Semen; Oocytes; Blastocyst
PubMed: 36864182
DOI: 10.1007/s10815-023-02758-3 -
Physical Biology Jul 2021This perspective aims to identify the relationships between the structural and dynamic properties of chromosomes and the fundamental properties of soft-matter systems.... (Review)
Review
This perspective aims to identify the relationships between the structural and dynamic properties of chromosomes and the fundamental properties of soft-matter systems. Chromatin is condensed into metaphase chromosomes during mitosis. The resulting structures are elongated cylinders having micrometer-scale dimensions. Our previous studies, using transmission electron microscopy, atomic force microscopy, and cryo-electron tomography, suggested that metaphase chromosomes have a multilayered structure, in which each individual layer has the width corresponding to a mononucleosome sheet. The self-assembly of multilayer chromatin plates from small chromatin fragments suggests that metaphase chromosomes are self-organized hydrogels (in which a single DNA molecule crosslinks the whole structure) with an internal liquid-crystal order produced by the stacking of chromatin layers along the chromosome axis. This organization of chromatin was unexpected, but the spontaneous assembly of large structures has been studied in different soft-matter systems and, according to these studies, the self-organization of chromosomes could be justified by the interplay between weak interactions of repetitive nucleosome building blocks and thermal fluctuations. The low energy of interaction between relatively large building blocks also justifies the easy deformation and structural fluctuations of soft-matter structures and the changes of phase caused by diverse external factors. Consistent with these properties of soft matter, different experimental results show that metaphase chromosomes are easily deformable. Furthermore, at the end of mitosis, condensed chromosomes undergo a phase transition into a more fluid structure, which can be correlated to the decrease in the Mgconcentration and to the dissociation of condensins from chromosomes. Presumably, the unstacking of layers and chromatin fluctuations driven by thermal energy facilitate gene expression during interphase.
Topics: Chromatin; Chromosomes; Humans; Metaphase
PubMed: 34126606
DOI: 10.1088/1478-3975/ac0aff -
Cell Aug 2000Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces...
Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces exerted at kinetochores and on chromosome arms drive chromosome movements that culminate in their alignment on the metaphase plate. In this paper, we show that Xkid, a kinesin-like protein localized on chromosome arms, plays an essential role in metaphase chromosome alignment and in its maintenance. We propose that Xkid is responsible for the polar ejection forces acting on chromosome arms. Our results show that these forces are essential to ensure that kinetochores and chromosome arms align on a narrow equatorial plate during metaphase, a prerequisite for proper chromosome segregation.
Topics: Amino Acid Sequence; Animals; Chromosomes; Cloning, Molecular; DNA-Binding Proteins; Kinesins; Metaphase; Microscopy, Video; Microtubule Proteins; Molecular Sequence Data; Xenopus; Xenopus Proteins
PubMed: 10966105
DOI: 10.1016/s0092-8674(00)00048-9 -
The Journal of Cell Biology Mar 2010During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement....
During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.
Topics: Autoantigens; Biological Assay; Centromere; Centromere Protein A; Chromosomal Proteins, Non-Histone; Elasticity; HeLa Cells; Humans; Kinesins; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Periodicity; RNA, Small Interfering; Recombinant Fusion Proteins; Spindle Apparatus
PubMed: 20212316
DOI: 10.1083/jcb.200909005