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International Journal of Molecular... May 2021The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism....
The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes' parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain "imprinting" of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent's age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual's gametes with those in chromosomes inherited from different individuals' gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual's reaction norm.
Topics: Chromosomes, Human; Fertilization in Vitro; Humans; Metaphase; Telomere; Telomere Homeostasis; Triploidy; Zygote
PubMed: 34070406
DOI: 10.3390/ijms22115579 -
Chromosome Research : An International... Sep 2016We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues...
We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.
Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Cell Line; Chromosomal Proteins, Non-Histone; Chromosomes, Human; Cysteine; DNA-Binding Proteins; Dithiothreitol; Electrophoresis, Gel, Two-Dimensional; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Karyotype; Mass Spectrometry; Metaphase; Micromanipulation; Multiprotein Complexes; Notophthalmus viridescens; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Octamer Transcription Factors; Oxidation-Reduction; Phosphines; RNA-Binding Proteins; Cohesins
PubMed: 27145786
DOI: 10.1007/s10577-016-9528-6 -
The Journal of Biological Chemistry Aug 2016The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell...
The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.
Topics: Anaphase; CDC2 Protein Kinase; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Cyclin-Dependent Kinases; HeLa Cells; Humans; Kinetochores; Metaphase; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 27325694
DOI: 10.1074/jbc.M115.712745 -
Current Biology : CB Feb 2016Spindle positioning is essential for tissue morphogenesis and homeostasis. The signaling network synchronizing spindle placement with mitotic progression relies on...
Spindle positioning is essential for tissue morphogenesis and homeostasis. The signaling network synchronizing spindle placement with mitotic progression relies on timely recruitment at the cell cortex of NuMA:LGN:Gαi complexes, in which NuMA acts as a receptor for the microtubule motor Dynein. To study the implication of Aurora-A in spindle orientation, we developed protocols for the partial inhibition of its activity. Under these conditions, in metaphase NuMA and Dynein accumulate abnormally at the spindle poles and do not reach the cortex, while the cortical distribution of LGN remains unperturbed. FRAP experiments revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle pole-localized pools of NuMA. We show that Aurora-A phosphorylates directly the C terminus of NuMA on three Ser residues, of which Ser1969 determines the dynamic behavior and the spindle orientation functions of NuMA. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif. Our study demonstrates that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in transformed and non-transformed cells in culture. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules or for LGN but rather determines the mobility of the protein at the spindle poles. The finding that NuMA can associate concomitantly with LGN and microtubules suggests that its microtubule-binding activity contributes to anchor Dynein-loaded microtubule +TIPs at cortical sites with LGN.
Topics: Antigens, Nuclear; Aurora Kinase A; Cell Cycle; Cell Cycle Proteins; Dyneins; HeLa Cells; Humans; Metaphase; Nuclear Matrix-Associated Proteins; Phosphorylation; Protein Binding; Spindle Apparatus
PubMed: 26832443
DOI: 10.1016/j.cub.2015.12.051 -
Scientific Reports Sep 2017Mitotic spindles, which consist of microtubules (MTs) and associated proteins, play critical roles in controlling cell division and maintaining tissue homeostasis. The...
Mitotic spindles, which consist of microtubules (MTs) and associated proteins, play critical roles in controlling cell division and maintaining tissue homeostasis. The orientation of the mitotic spindle is closely related with the duration of mitosis. However, the molecular mechanism in regulating the orientation of the mitotic spindles is largely undefined. In this study, we found that Palladin is a novel MT-associated protein and regulator of spindle orientation, which maintains proper spindle orientation by stabilizing astral MTs. Palladin depletion distorted spindle orientation, prolonged the metaphase, and impaired proliferation of HeLa cells. Results showed that Palladin depletion-induced spindle misorientation and astral MT instability could be rescued by constitutively active AKT1 or dominant negative GSK3β. Our findings revealed that Palladin regulates spindle orientation and mitotic progression mainly through the AKT1-GSK3β pathway.
Topics: Cytoskeletal Proteins; Glycogen Synthase Kinase 3 beta; HeLa Cells; Humans; Metaphase; Microtubule-Associated Proteins; Microtubules; Phosphoproteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Spindle Apparatus
PubMed: 28924223
DOI: 10.1038/s41598-017-12051-w -
Molecular Biology of the Cell Nov 2016Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are...
Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.
Topics: Animals; Cell Cycle; Cell Membrane; Clathrin; Cytokinesis; Endosomes; Imaging, Three-Dimensional; Metaphase; Microscopy; Microscopy, Fluorescence; Mitosis; Zebrafish
PubMed: 27535432
DOI: 10.1091/mbc.E16-03-0164 -
Frontiers in Endocrinology 2021This retrospective study assessed the effect of the co-administration of clomiphene citrate (CC) and letrozole in mild ovarian stimulation, compared to conventional... (Comparative Study)
Comparative Study
This retrospective study assessed the effect of the co-administration of clomiphene citrate (CC) and letrozole in mild ovarian stimulation, compared to conventional regimens, among Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) Group 4 patients. There were 114 POSEIDON Group 4 patients undergoing fertilization treatments with 216 stimulation cycles recruited from a Taiwan's reproductive center during 2016-2020. Main outcomes were the numbers, quality of retrieved oocytes and embryo development. Pregnancy outcomes were assessed after embryo transfers. Per stimulation cycle, patients receiving mild stimulation with a combination of CC and letrozole (study group) versus those with COS (control group) had lower numbers of pre-ovulatory follicles (2.00 ± 1.23 vs. 2.37 ± 1.23, =0.0066) and oocytes retrieved (1.83 ± 1.17 vs. 2.37 ± 1.23, =0.0017), and lower follicular output rate (58.6% vs. 68.38%, =0.0093) and mature oocyte output rate (44.29% vs. 52.88%, =0.0386) but a higher top-quality metaphase II oocyte ratio (66.7% vs. 54.59%, =0.0444) and a similar fertilization rate (91.67% vs. 89.04%, =0.4660). With adjustment for significant between-group baseline differences using multivariable logistic generalized estimating equation model analyses, there was no statistical difference in oocytes retrieved and embryo development between the study and control groups, and insignificant increases in successful pregnancies in the study group were found compared to the control group (i.e., odds ratios [95% CIs]: 1.13 [0.55, 232] and 1.50 [0.65, 3.49] for ongoing pregnancy and live birth, respectively). For POSEIDON Group 4 patients, cotreatment of CC and letrozole in mild stimulation may increase the high-quality oocyte ratio and yield comparable fertilization rate and pregnancy outcomes.
Topics: Adult; Clomiphene; Drug Therapy, Combination; Embryo Transfer; Female; Fertility Agents, Female; Fertilization in Vitro; Humans; Letrozole; Logistic Models; Metaphase; Middle Aged; Multivariate Analysis; Oocyte Retrieval; Oocytes; Ovulation Induction; Pregnancy; Pregnancy Rate; Retrospective Studies
PubMed: 35095758
DOI: 10.3389/fendo.2021.780392 -
Cell Cycle (Georgetown, Tex.) Jun 2021ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to...
ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase-anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.
Topics: Adenosine Triphosphate; Anaphase; Aurora Kinase B; Chromosome Segregation; Energy Metabolism; Female; HeLa Cells; Humans; Interphase; Kinetochores; Metaphase; Microscopy; NAD; Signal Transduction; Spindle Apparatus; Time-Lapse Imaging; Uterine Cervical Neoplasms
PubMed: 34048314
DOI: 10.1080/15384101.2021.1930679 -
ELife Jun 2022The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have...
The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the α2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22's functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest that the motor domain α2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of α2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.
Topics: Anaphase; Chromosome Segregation; DNA-Binding Proteins; Kinesins; Metaphase; Mitosis; Mutation; Nuclear Proteins; Spindle Apparatus
PubMed: 35730929
DOI: 10.7554/eLife.78653 -
Nature Communications May 2020To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of...
To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in 20-40% of unperturbed yeast cells, DNA synthesis continues during anaphase, late in mitosis. High cyclin-Cdk activity inhibits DNA synthesis in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA synthesis to finish at subtelomeric and some difficult-to-replicate regions. DNA synthesis during late mitosis correlates with elevated mutation rates at subtelomeric regions, including copy number variation. Thus, yeast cells temporally overlap DNA synthesis and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.
Topics: Anaphase; Cell Nucleus; Chromatin; Chromosome Segregation; Chromosomes, Fungal; Cyclin-Dependent Kinases; DNA Replication; DNA, Fungal; Genes, Fungal; Metaphase; Mitosis; Mutation Rate; Saccharomyces cerevisiae Proteins; Saccharomycetales; Telomere
PubMed: 32385287
DOI: 10.1038/s41467-020-16100-3