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PLoS Genetics Aug 2011In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the...
In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.
Topics: Animals; Cell Cycle Checkpoints; Chromosomes; Drosophila Proteins; Drosophila melanogaster; Female; Kinetochores; Male; Meiosis; Metaphase; Microtubules; Mutation, Missense; Oocytes; Prometaphase; Protein Binding; Spindle Apparatus; Tubulin
PubMed: 21852952
DOI: 10.1371/journal.pgen.1002209 -
Scientific Reports Oct 2019We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible...
We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.
Topics: Administration, Oral; Alcohol Oxidoreductases; Animals; Antineoplastic Agents, Phytogenic; Carrier Proteins; Cell Death; Cell Division; Cell Movement; Cell Proliferation; Cellular Senescence; Curcumin; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Glutathione Transferase; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Lactoylglutathione Lyase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MCF-7 Cells; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Peroxiredoxins; Prometaphase; Reactive Oxygen Species; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 31619723
DOI: 10.1038/s41598-019-51244-3 -
Heterochromatic threads connect oscillating chromosomes during prometaphase I in Drosophila oocytes.PLoS Genetics Jan 2009In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We...
In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.
Topics: Animals; Chromosome Segregation; Chromosomes; Drosophila melanogaster; Heterochromatin; Models, Biological; Models, Genetic; Oocytes; Oscillometry; Prometaphase; Spindle Apparatus
PubMed: 19165317
DOI: 10.1371/journal.pgen.1000348 -
Molecular Cell Aug 2007During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is...
During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; CDC2 Protein Kinase; Cdc20 Proteins; Cell Cycle Proteins; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Inhibitors; Fibroblasts; Glioblastoma; Humans; Immunoblotting; Immunoprecipitation; Lung; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Prometaphase; S-Phase Kinase-Associated Proteins; Ubiquitin; Ubiquitin-Protein Ligase Complexes; Ubiquitin-Protein Ligases
PubMed: 17679094
DOI: 10.1016/j.molcel.2007.06.013 -
Journal of Visualized Experiments : JoVE Oct 2016Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of...
Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.
Topics: Animals; Drosophila; Drosophila melanogaster; Meiosis; Metaphase; Oocytes; Prometaphase; Spindle Apparatus
PubMed: 27842371
DOI: 10.3791/54666 -
Methods in Molecular Biology (Clifton,... 2022The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate,...
The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.
Topics: Anaphase; HeLa Cells; Humans; Metaphase; Mitosis; Telophase
PubMed: 36045201
DOI: 10.1007/978-1-0716-2736-5_8 -
Prenatal Diagnosis Jul 1987A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures is described. The technique is based upon several key steps including: (1)...
A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures is described. The technique is based upon several key steps including: (1) reduced colcemid concentration, (2) reduced exposure to trypsin-EDTA, and (3) maintaining cells in single suspension by adjusting cell concentration appropriately. Chromosomes with banding resolution up to 800 bands per haploid set can be routinely produced. The described methodology is particularly useful in defining and establishing the clinical significance of subtle structural aberrations.
Topics: Amniotic Fluid; Cells, Cultured; Chromosomes, Human; Female; Humans; Metaphase; Pregnancy; Prenatal Diagnosis
PubMed: 3658911
DOI: 10.1002/pd.1970070602 -
The Journal of Cell Biology Sep 2002When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt...
When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP-alpha-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.
Topics: Animals; Biological Transport; Cell Line; Cells, Cultured; Cytoplasm; Cytoskeleton; Dynactin Complex; Dyneins; Enzyme Inhibitors; G2 Phase; Green Fluorescent Proteins; Kinesins; Luminescent Proteins; Meiosis; Microtubule-Associated Proteins; Microtubules; Myosin Type II; Prophase; Pyrimidines; Spindle Apparatus; Thiones; Tubulin; Xenopus Proteins
PubMed: 12235119
DOI: 10.1083/jcb.200204109 -
Biochimica Et Biophysica Acta Aug 2012Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G(2)/M cell cycle arrest (based on...
Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G(2)/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME(2)-induced cell cycle arrest. We found that 2ME(2) can selectively induce mitotic prometaphase arrest, but not G(2) phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12-24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME(2)-induced prometaphase arrest. The 2ME(2)-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME(2)-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME(2) causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.
Topics: 2-Methoxyestradiol; Antineoplastic Agents; Breast Neoplasms; CDC2 Protein Kinase; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus Shape; Cyclin B; Cyclin B1; Cyclin-Dependent Kinases; Estradiol; Female; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; Mad2 Proteins; Mitogen-Activated Protein Kinase 8; Prometaphase; RNA Interference; Repressor Proteins; Up-Regulation
PubMed: 22580043
DOI: 10.1016/j.bbamcr.2012.05.003 -
Cell Discovery May 2022Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show...
Noncoding RNAs are known to associate with mitotic chromosomes, but the identities and functions of chromosome-associated RNAs in mitosis remain elusive. Here, we show that rRNA species associate with condensed chromosomes during mitosis. In particular, pre-rRNAs such as 45S, 32S, and 30S are highly enriched on mitotic chromosomes. Immediately following nucleolus disassembly in mitotic prophase, rRNAs are released and associate with and coat each condensed chromosome at prometaphase. Using unbiased mass spectrometry analysis, we further demonstrate that chromosome-bound rRNAs are associated with Ki-67. Moreover, the FHA domain and the repeat region of Ki-67 recognize and anchor rRNAs to chromosomes. Finally, suppression of chromosome-bound rRNAs by RNA polymerase I inhibition or by using rRNA-binding-deficient Ki-67 mutants impair mitotic chromosome dispersion during prometaphase. Our study thus reveals an important role of rRNAs in preventing chromosome clustering during mitosis.
PubMed: 35637200
DOI: 10.1038/s41421-022-00400-7