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Cells Apr 2020Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome...
Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.
Topics: Cell Culture Techniques; Cell Cycle; Cell Nucleus; Centrosome; Golgi Apparatus; Humans; Microtubules; Mitosis; Nuclear Envelope; Retinal Pigment Epithelium
PubMed: 32344866
DOI: 10.3390/cells9051069 -
Science Advances Apr 2020During mitotic prophase, cohesins are removed from chromosome arms by Wapl to ensure faithful sister chromatid separation. However, during female meiosis I, the...
During mitotic prophase, cohesins are removed from chromosome arms by Wapl to ensure faithful sister chromatid separation. However, during female meiosis I, the resolution of chiasmata requires the proteolytic cleavage of cohesin subunit Rec8 along chromosome arms by Separase to separate homologs, and thus the role of Wapl remained unknown. Here, we report that Wapl functions as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Depletion of Wapl accelerates meiotic progression, inactivates SAC, and causes meiotic defects such as aberrant spindle/chromosome structure and incorrect kinetochore-microtubule (K-MT) attachment, consequently leading to aneuploid eggs. Notably, we identify Bub3 as a binding partner of Wapl by immunoprecipitation and mass spectrometry analysis. We further determine that Wapl controls the SAC activity by maintaining Bub3 protein level and document that exogenous Bub3 restores the normal meiosis in Wapl-depleted oocytes. Together, our findings uncover unique, noncanonical roles for Wapl in mediating control of the SAC in female meiosis I.
Topics: Aneuploidy; Animals; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Female; M Phase Cell Cycle Checkpoints; Meiosis; Mice; Models, Biological; Oocytes; Poly-ADP-Ribose Binding Proteins; Proteins
PubMed: 32284991
DOI: 10.1126/sciadv.aax3969 -
Life Science Alliance Mar 2020In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays...
In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditional knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.
Topics: Acetylation; Acetyltransferases; Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Chromosome Segregation; Chromosome Structures; Gametogenesis; Male; Meiosis; Mice; Mice, Inbred C57BL; Nuclear Proteins; Sex Chromosomes; Spermatocytes; Synaptonemal Complex; Cohesins
PubMed: 32051254
DOI: 10.26508/lsa.201900564 -
Molecular Cell Apr 2020As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that...
As cells enter mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA polymerase II (Pol II) in mitosis. Here, we demonstrate that chromatin-bound cohesin is necessary to retain elongating Pol II at centromeres. We find that WAPL-mediated removal of cohesin from chromosome arms during prophase is required for the dissociation of Pol II and nascent transcripts, and failure of this process dramatically alters mitotic gene expression. Removal of cohesin/Pol II from chromosome arms in prophase is important for accurate chromosome segregation and normal activation of gene expression in G1. We propose that prophase cohesin removal is a key step in reprogramming gene expression as cells transition from G2 through mitosis to G1.
Topics: Anaphase; Animals; Aurora Kinase B; Cell Cycle; Cell Cycle Proteins; Cell Line; Centromere; Chromosomal Proteins, Non-Histone; Chromosome Segregation; G1 Phase; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation; Humans; Metaphase; Mitosis; Prophase; RNA Polymerase II; Transcription, Genetic; Xenopus laevis; Cohesins
PubMed: 32035037
DOI: 10.1016/j.molcel.2020.01.023 -
The Plant Cell Apr 2020Meiosis consists of two highly conserved nuclear divisions, which allow eukaryotes to maintain their chromosome number through sexual reproduction. The successful...
Meiosis consists of two highly conserved nuclear divisions, which allow eukaryotes to maintain their chromosome number through sexual reproduction. The successful completion of meiosis depends on homologous chromosome pairing. Centromere interactions during early meiotic prophase I facilitate homologous chromosome pairing, but the underlying mechanism is unclear. Here, we performed chromatin immunoprecipitation-mass spectrometry analysis of maize () anthers during early meiotic prophase I using anti-centromeric histone H3 (CENH3) antibodies and determined that the cohesin subunit Structural Maintenance of Chromosome3 (SMC3) interacts with CENH3 during this period. SMC3 is enriched at centromeres and along chromosome arms in threads from leptotene to pachytene and might promote interactions between homologous centromeres. We observed dysfunctional SMC3 assembly in meiotic-specific maize mutants with defective centromere pairing. In meiocytes, centromere pairing defects were observed during early meiotic prophase I, SMC3 was weakly associated with centromeres, and SMC3 did not localize to the chromosome arms. In wild-type mitosis, SMC3 is associated with chromatin and is enriched at centromeres from prophase to anaphase. CRISPR-Cas9-induced mutants showed premature loss of sister chromatid cohesion and mis-segregation of chromosomes in mitotic spreads. Our findings suggest that in addition to sister chromatid cohesion, ZmSMC3 participates in meiotic centromere pairing.
Topics: Cell Cycle Proteins; Centromere; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Chromosomes, Plant; Meiosis; Meiotic Prophase I; Mitosis; Mutation; Phenotype; Plant Proteins; Protein Binding; Protein Subunits; Recombination, Genetic; Spindle Apparatus; Zea mays; Cohesins
PubMed: 31996400
DOI: 10.1105/tpc.19.00834 -
Reproductive Biology and Endocrinology... Dec 2019Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by...
BACKGROUND
Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis.
METHODS
Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique.
RESULTS
MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage.
CONCLUSIONS
We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.
Topics: Animals; Cell Cycle Proteins; DNA-Binding Proteins; Estradiol; Female; Fetus; Gene Expression Regulation, Developmental; Granulosa Cells; Meiotic Prophase I; Mice, Inbred C57BL; Oocytes; Organ Culture Techniques; Ovary; Pachytene Stage; Pregnancy; Progesterone; RNA-Binding Proteins
PubMed: 31791345
DOI: 10.1186/s12958-019-0548-x -
Turkish Journal of Biology = Turk... 2019is used to treat cancer patients in alternative healthcare systems. However, its cytotoxic and genotoxic effects have not been reported. Therefore, themethanol extract...
is used to treat cancer patients in alternative healthcare systems. However, its cytotoxic and genotoxic effects have not been reported. Therefore, themethanol extract of flower, the ethyl acetate fraction, and the pure compound patuletin were evaluatedusing the test.The methanol extract and fraction contained ~3% and ~36% patuletin, respectively, with ~98% purity. The methanol extract caused inhibition of root growth displaying an IC value of ~500 µg/mL, while the fraction and patuletin were more potent by ~2 and ~5 times, respectively. The root tips demonstrated a decline in prophase, metaphase, anaphase, and telophase stages with concomitant decrease in percent mitotic index in the methanol extract (~5.64), fraction, and patuletin (~4) as compared to the control (~7.61). However, in only methanol extract-treated root tips, an increase in metaphase stage was noted. In addition, the methanol extract predominantly induced c-type, misaligned, and multipolar chromosomal abnormalities while the fraction and patuletin displayed fragments and sticky chromosomes. The fraction and patuletin also produced micronuclei (~2%) In conclusion, flower methanol extract and ethyl acetate fraction are cytotoxicand genotoxic, which most likely could be due to the patuletin. Further preclinical and clinical studies are required to justify its clinical use.
PubMed: 31772498
DOI: 10.3906/biy-1906-7 -
Nucleic Acids Research Dec 2019We have explored the meiotic roles of cohesin modulators Pds5 and Rad61/Wapl, in relation to one another, and to meiotic kleisin Rec8, for homolog pairing, all...
We have explored the meiotic roles of cohesin modulators Pds5 and Rad61/Wapl, in relation to one another, and to meiotic kleisin Rec8, for homolog pairing, all physically definable steps of recombination, prophase axis length and S-phase progression, in budding yeast. We show that Pds5 promotes early steps of recombination and thus homolog pairing, and also modulates axis length, with both effects independent of a sister chromatid. [Pds5+Rec8] promotes double-strand break formation, maintains homolog bias for crossover formation and promotes S-phase progression. Oppositely, the unique role of Rad61/Wapl is to promote non-crossover recombination by releasing [Pds5+Rec8]. For this effect, Rad61/Wapl probably acts to maintain homolog bias by preventing channeling into sister interactions. Mysteriously, each analyzed molecule has one role that involves neither of the other two. Overall, the presented findings suggest that Pds5's role in maintenance of sister chromatid cohesion during the mitotic prophase-analogous stage of G2/M is repurposed during meiosis prophase to promote interactions between homologs.
Topics: Cell Cycle Proteins; Cells, Cultured; Chromosomal Proteins, Non-Histone; Chromosome Pairing; Chromosome Segregation; Chromosomes, Fungal; Meiosis; Organisms, Genetically Modified; Protein Binding; Recombination, Genetic; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sister Chromatid Exchange
PubMed: 31617566
DOI: 10.1093/nar/gkz903 -
Development (Cambridge, England) Nov 2019The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter...
The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 () is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele ( ) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.
Topics: Alleles; Animals; Apoptosis; CRISPR-Cas Systems; Cell Differentiation; Cell Lineage; Cell Proliferation; Cluster Analysis; Crosses, Genetic; Cyclin-Dependent Kinase 2; Germ Cells; Heterozygote; Homeostasis; Male; Mass Spectrometry; Meiosis; Mice; Mutagenesis, Site-Directed; Phenotype; Phosphorylation; RNA, Small Cytoplasmic; Seminiferous Tubules; Spermatogenesis; Spermatogonia; Testis; Transcriptome
PubMed: 31582414
DOI: 10.1242/dev.180273 -
FEBS Letters Oct 2019The primary function of cyclin-dependent kinases (CDKs) in complex with their activating cyclin partners is to promote mitotic division in somatic cells. This canonical... (Review)
Review
The primary function of cyclin-dependent kinases (CDKs) in complex with their activating cyclin partners is to promote mitotic division in somatic cells. This canonical cell cycle-associated activity is also crucial for fertility as it allows the proliferation and differentiation of stem cells within the reproductive organs to generate meiotically competent cells. Intriguingly, several CDKs exhibit meiosis-specific functions and are essential for the completion of the two reductional meiotic divisions required to generate haploid gametes. These meiosis-specific functions are mediated by both known CDK/cyclin complexes and meiosis-specific CDK-regulators and are important for a variety of processes during meiotic prophase. The majority of meiotic defects observed upon deletion of these proteins occur during the extended prophase I of the first meiotic division. Importantly a lack of redundancy is seen within the meiotic arrest phenotypes described for many of these proteins, suggesting intricate layers of cell cycle control are required for normal meiotic progression. Using the process of male germ cell development (spermatogenesis) as a reference, this review seeks to highlight the diverse roles of selected CDKs their activators, and their regulators during gametogenesis.
Topics: Animals; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Cyclin-Dependent Kinases; Cyclins; Gene Expression Regulation; Haploidy; Male; Meiosis; Mice; Nuclear Proteins; Recombination, Genetic; Signal Transduction; Spermatogenesis; Spermatozoa; Stem Cells
PubMed: 31566717
DOI: 10.1002/1873-3468.13627