-
Molecular Biology of the Cell Nov 2021The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important...
The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19's localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.
Topics: Cell Cycle; Cell Nucleus; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 34495712
DOI: 10.1091/mbc.E21-05-0279 -
Yakugaku Zasshi : Journal of the... 2022Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process...
Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process with high accuracy and precision, various motor proteins work in a concerted manner. Especially in the metaphase, mitotic chromosomes are delivered by the motor protein of centromere-associated protein E (CENP-E) to the cell equatorial plane (metaphase plate) along mitotic spindles. However, the critical functional failure of CENP-E can activate the spindle assembly checkpoint through the misalignment of chromosomes at the metaphase plate. In this symposium review, the reversibly photoswitchable CENP-E inhibitor PCEI-HU (5) is reported. Compound 5 exhibited almost quantitative trans-cis photoisomerization of the arylazopyrazole photoswitch by illuminating light at 365 nm and 510 nm. Depending on the photoisomerization, CENP-E activity was regulated not only in vitro but also in cells. We successfully established a novel technique using 5 to dynamically photocontrol the CENP-E-dependent chromosome movement and mitotic progression in a living cell.
Topics: Cell Division; Metaphase; Spindle Apparatus
PubMed: 35491157
DOI: 10.1248/yakushi.21-00203-5 -
Scientific Reports Sep 2023The aim of our study was to evaluate the feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation after...
Feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation and childbearing desire after fertility-impairing treatment.
The aim of our study was to evaluate the feasibility and efficiency of delayed ovarian stimulation and metaphase II oocyte banking for fertility preservation after fertility-impairing treatment regardless of the initial disease. We conducted a cohort study based on population of women < 40 years of age with diminished ovarian reserve caused by fertility-impairing treatment (n = 129). Three groups of women were compared according to the type of initial disease: hematological malignancies, solid tumors, and benign diseases. The primary endpoint was the number of metaphase II oocytes collected per woman. We studied the cumulative live-birth rate per cycle with fertilized metaphase II oocyte, for women who wanted to conceive. We studied 245 delayed controlled ovarian stimulation cycles in 129 women: 201 for fertility preservation and 44 for in vitro fertilization and fresh embryo transfers. The number of metaphase II oocytes collected per woman after banking was similar in the three groups, with a mean of 10.7 ± 4.6, 12.3 ± 9.1, and 10.1 ± 7.6 metaphase II oocytes (p = 0.46), respectively. In the subgroup of women who wanted to conceive, the cumulative live birth rate per woman was 38%, with 8 live births for these 21 women. After fertility-impairing treatment, practitioners should discuss a fertility preservation procedure for banking metaphase II oocytes.
Topics: Female; Humans; Fertility Preservation; Metaphase; Cohort Studies; Feasibility Studies; Oocytes; Ovulation Induction
PubMed: 37730827
DOI: 10.1038/s41598-023-42583-3 -
British Journal of Haematology Sep 2009Karyotypic analysis provides useful diagnostic information in many haematological malignancies. However, standard metaphase cytogenetics has technical limitations that... (Review)
Review
Karyotypic analysis provides useful diagnostic information in many haematological malignancies. However, standard metaphase cytogenetics has technical limitations that result in the underestimation of the degree of chromosomal changes. Array-based technologies can be used for karyotyping and can supplant some of the shortcomings of metaphase cytogenetics, and include single nucleotide polymorphism arrays (SNP-A) and comparative genomic hybridization arrays (CGH-A). Array-based cytogenetic tools do not rely on cell division, have superb resolution for unbalanced lesions and allow for the detection of copy number-neutral loss of heterozygosity, a type of lesion not seen with metaphase cytogenetics. Moreover, genomic array analysis is automated and results can be objectively and systematically analysed using biostatistical algorithms. As a potential advantage over genomic approaches, metaphase cytogenetics can detect balanced chromosomal defects and resolves clonal mosaicism. Initial studies performed in various haematological malignancies indicate the potential of SNP-A-based karyotyping as a useful clinical cytogenetic detection tool. The current effort is aimed at developing rational diagnostic algorithms for the detection of somatic defects and the establishment of clinical correlations for novel SNP-A-detected chromosomal defects, including acquired somatic uniparental disomy. SNP-A can complement metaphase karyotyping and will probably play an important role in clinical cytogenetic diagnostics.
Topics: Comparative Genomic Hybridization; Cytogenetic Analysis; Hematologic Neoplasms; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Metaphase; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 19563474
DOI: 10.1111/j.1365-2141.2009.07757.x -
Fertility and Sterility Dec 2019To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects. (Observational Study)
Observational Study
OBJECTIVE
To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects.
DESIGN
Retrospective observational study.
SETTING
University medical center research laboratory and andrology clinic.
PATIENT(S)
Forty-eight patients with confirmed spermatogenic impairment (Johnsen scores 3-6) and 15 controls (Johnsen score 10).
INTERVENTION(S)
None.
MAIN OUTCOME MEASURE(S)
Quantitative assessment of immunofluorescent analyses of specific markers to determine meiotic entry, chromosome pairing, progression of DNA double-strand break repair, crossover formation, formation of meiotic metaphases, metaphase arrest, and spermatid formation, resulting in a novel classification of human meiotic arrest types.
RESULT(S)
Complete metaphase arrest was observed most frequently (27%), and the patients with the highest frequency of apoptotic metaphases also displayed a reduction in crossover number. Incomplete metaphase arrest was observed in 17% of the patients. Only four patients (8%) displayed a failure to complete meiotic chromosome pairing leading to pachytene arrest. Two new types of meiotic arrest were defined: premetaphase and postmetaphase arrest (15% and 13%, respectively).
CONCLUSION(S)
Meiotic arrest in men occurs most frequently at meiotic metaphase. This arrest can be incomplete, resulting in low numbers of spermatids, and often occurs in association with reduced crossover frequency. The phenotyping approach described here provides mechanistic insights to help identify candidate infertility genes and to assess genotype-phenotype correlations in individual cases.
Topics: Apoptosis; Azoospermia; Chromosome Pairing; DNA Breaks, Double-Stranded; Humans; Male; Metaphase; Pachytene Stage; Retrospective Studies; Spermatogenesis; Spermatozoa; Testis
PubMed: 31767154
DOI: 10.1016/j.fertnstert.2019.08.004 -
Developmental Cell Aug 2013Reporting in Developmental Cell, Schlaitz et al. (2013) show that endoplasmic reticulum (ER) membrane exclusion from the mitotic spindle is an active process requiring...
Reporting in Developmental Cell, Schlaitz et al. (2013) show that endoplasmic reticulum (ER) membrane exclusion from the mitotic spindle is an active process requiring REEP membrane proteins. REEP protein depletion results in ER membrane retention on the spindle and chromosomes, leading to defects in chromosome segregation and nuclear envelope assembly.
Topics: Chromatin; Endoplasmic Reticulum; Humans; Membrane Transport Proteins; Metaphase; Nuclear Envelope
PubMed: 23948250
DOI: 10.1016/j.devcel.2013.07.018 -
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 -
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 -
Cell Cycle (Georgetown, Tex.) Feb 2021DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle... (Review)
Review
DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle stalls due to failed ATP hydrolysis, the onset of anaphase is delayed, presumably to allow extra time for decatenation to be completed. Recent evidence revealed that, unlike the spindle assembly checkpoint, this TopoII checkpoint response requires Aurora B and Haspin kinases and is triggered by SUMOylation of the C-terminal domain of TopoII.
Topics: Animals; Aurora Kinase B; Cell Cycle Proteins; DNA Topoisomerases, Type II; Genes, cdc; Humans; Intracellular Signaling Peptides and Proteins; M Phase Cell Cycle Checkpoints; Metaphase; Mitosis; Protein Serine-Threonine Kinases
PubMed: 33459116
DOI: 10.1080/15384101.2021.1875671 -
STAR Protocols Dec 2022The syncytial Drosophila blastoderm embryo contains apical microvilli with filamentous actin that are remodeled during nuclear division cycles 10-13. Here, we describe a...
The syncytial Drosophila blastoderm embryo contains apical microvilli with filamentous actin that are remodeled during nuclear division cycles 10-13. Here, we describe a protocol for preparing whole embryo samples and capturing images of microvilli using confocal and super-resolution STED microscopy. This protocol enables visualization and quantification of lengths and numbers of microvilli oriented along the imaging plane. We provide information on identifying different nuclear division cycles and examples of quantification from the interphase and metaphase of cycle 12. For complete details on the use and execution of this protocol, please refer to Sherlekar et al. (2020).
Topics: Animals; Drosophila; Blastoderm; Microvilli; Interphase; Metaphase
PubMed: 36181681
DOI: 10.1016/j.xpro.2022.101736