scholarly journals The Roles of Cyclin A2, B1, and B2 in Early and Late Mitotic Events

2010 ◽  
Vol 21 (18) ◽  
pp. 3149-3161 ◽  
Author(s):  
Delquin Gong ◽  
James E. Ferrell
Keyword(s):  
Chromatin Condensation ◽  
Time Lapse ◽  
Cyclin B1 ◽  
S Phase ◽  
Epifluorescence Microscopy ◽  
Nuclear Accumulation ◽  
H3 Phosphorylation ◽  
Resistant Form ◽  
Cyclin A2 ◽  
Histone H3 Phosphorylation

Here we have used siRNAs and time-lapse epifluorescence microscopy to examine the roles of various candidate mitotic cyclins in chromatin condensation in HeLa cells. Knocking down cyclin A2 resulted in a substantial (∼7 h) delay in chromatin condensation and histone H3 phosphorylation, and expressing an siRNA-resistant form of cyclin A2 partially rescued chromatin condensation. There was no detectable delay in DNA replication in the cyclin A2 knockdowns, arguing that the delay in chromatin condensation is not secondary to a delay in S-phase completion. Cyclin A2 is required for the activation and nuclear accumulation of cyclin B1-Cdk1, raising the possibility that cyclin B1-Cdk1 mediates the effects of cyclin A2. Consistent with this possibility, we found that chromatin condensation was tightly associated temporally with the redistribution of cyclin B1 to the nucleus. Moreover, a constitutively nuclear cyclin B1 rescued chromatin condensation in cyclin A2 knockdown cells. On the other hand, knocking down cyclin B1 delayed chromatin condensation by only about one hour. Our working hypothesis is that active, nuclear cyclin B1-Cdk1 normally cooperates with cyclin A2 to bring about early mitotic events. Because cyclin A2 is present only during the early stages of mitosis, we asked whether cyclin B knockdown might have more dramatic defects on late mitotic events. Consistent with this possibility, we found that cyclin B1- and cyclin B1/B2-knockdown cells had difficulty in maintaining a mitotic arrest in the presence of nocodazole. Taken together, these data suggest that cyclin A2 helps initiate mitosis, in part through its effects on cyclin B1, and that cyclins B1 and B2 are particularly critical for the maintenance of the mitotic state.

scholarly journals Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1–Cdk1 at the centrosome

2005 ◽  
Vol 171 (1) ◽  
pp. 35-45 ◽  
Author(s):  
Arne Lindqvist ◽  
Helena Källström ◽  
Andreas Lundgren ◽  
Emad Barsoum ◽  
Christina Karlsson Rosenthal
Keyword(s):  
Chromatin Condensation ◽  
Three Dimensional ◽  
Time Lapse ◽  
Cyclin B1 ◽  
Hek293 Cells ◽  
Unique Role ◽  
Mitotic Entry ◽  
Mitotic Cyclin ◽  
Interfering Rna ◽  
G2 Delay

Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.

scholarly journals Cyclin A2/Cdk1 is Essential for the in vivo S Phase Entry by Phosphorylating Top2a

2019 ◽  
Author(s):  
Miaomiao Jin ◽  
Ruikun Hu ◽  
Baijie Xu ◽  
Weilai Huang ◽  
Hong Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
S Phase ◽  
Downstream Target ◽  
Early Embryonic Lethality ◽  
M Phase ◽  
Phase Progression ◽  
Cyclin A2 ◽  
Phase Entry

AbstractCyclin-dependent kinase 1 (CDK1) plays essential roles in cell cycle regulation. However, due to the early embryonic lethality of mouse Cdk1 mutants, the in vivo role of CDK1 in regulating cell cycle and embryonic development remains unclear. Here, by generating zebrafish cdk1 mutants using CRISPR/Cas9 system, we show that cdk1−/− embryos exhibit severe microphthalmia accompanied with multiple defects in polarized cell division, S phase entry and M phase progression, cell apoptosis and cell differentiation, but not in interkinetic nuclear migration (IKNM). By informatics analysis, we identified Top2a as a potential downstream target, and Cyclin A2 and Cyclin B1 as partners of Cdk1 in cell cycle. Depletion of either Cyclin A2 or Top2a leads to decreased S phase entry and increased DNA damage response in zebrafish retinal cells, and depletion of Cyclin B1 leads to M phase arrest. Immunoprecipitation shows that Cdk1 and Cyclin A2 physically interact in vivo. Moreover, phosphorylation of Top2a on Serine 1213 (S1213) site is almost absent in either cdk1 or ccna2 mutants, but in not ccnb1 mutants. Furthermore, overexpression of TOP2AS1213, the phosphomimetic form of human TOP2A, rescues S phase entry and microphthalmia defects in cdk1−/− and ccna2−/− embryos. Taken together, our data suggests that Cdk1 interacts with Cyclin A2 to regulate S phase entry through phosphorylating Top2a, and with Cyclin B1 to regulate M phase progression in vivo.

NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 79-79
Author(s):  
Florian C. Bassermann ◽  
Christine von Klitzing ◽  
Silvia Kluempen ◽  
Ren-Yuan Bai ◽  
Tao Ouyang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
S Phase ◽  
Cyclin B ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Checkpoint Control ◽  
Mitotic Entry ◽  
F Box Protein ◽  
Constitutively Active

Abstract Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity. We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation. We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.

H3 phosphorylation: dual role in mitosis and interphaseThis paper is one of a selection of papers published in this Special Issue entitled 30th Annual International Asilomar Chromatin and Chromosomes Conference and has undergone the Journal’s usual peer review process.

2009 ◽  
Vol 87 (5) ◽  
pp. 695-709 ◽  
Author(s):  
Beatriz Pérez-Cadahía ◽  
Bojan Drobic ◽  
James R. Davie
Keyword(s):  
Gene Transcription ◽  
Transcriptional Activation ◽  
Peer Review Process ◽  
Chromatin Condensation ◽  
Post Translational Modifications ◽  
Cellular Events ◽  
H3 Phosphorylation ◽  
Functional Consequences ◽  
Genomic Regions ◽  
Histone H3 Phosphorylation

Chromatin condensation and subsequent decondensation are processes required for proper execution of various cellular events. During mitosis, chromatin compaction is at its highest, whereas relaxation of chromatin is necessary for DNA replication, repair, recombination, and gene transcription. Since histone proteins are directly complexed with DNA in the form of a nucleosome, great emphasis is put on deciphering histone post-translational modifications that control the chromatin condensation state. Histone H3 phosphorylation is a mark present in mitosis, where chromatin condensation is necessary, and in transcriptional activation of genes, when chromatin needs to be decondensed. There are four characterized phospho residues within the H3 N-terminal tail during mitosis: Thr3, Ser10, Thr11, and Ser28. Interestingly, H3 phosphorylated at Ser10, Thr11, and Ser28 has been observed on genomic regions of transcriptionally active genes. Therefore, H3 phosphorylation is involved in processes requiring opposing chromatin states. The level of H3 phosphorylation is mediated by opposing actions of specific kinases and phosphatases during mitosis and gene transcription. The cellular contexts under which specific residues on H3 are phosphorylated in mitosis and interphase are known to some extent. However, the functional consequences of H3 phosphorylation are still unclear.

scholarly journals Cyclin A2 Regulates Nuclear-Envelope Breakdown and the Nuclear Accumulation of Cyclin B1

2007 ◽  
Vol 17 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Delquin Gong ◽  
Joseph R. Pomerening ◽  
Jason W. Myers ◽  
Christer Gustavsson ◽  
Joshua T. Jones ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Nuclear Envelope Breakdown ◽  
Cyclin A2

scholarly journals Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis

1999 ◽  
Vol 146 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Christina Karlsson ◽  
Stephanie Katich ◽  
Anja Hagting ◽  
Ingrid Hoffmann ◽  
Jonathon Pines
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Time Lapse ◽  
Cyclin B1 ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
G2 Phase ◽  
Dna Synthesis Inhibitors

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.

scholarly journals Mitotic Histone H3 Phosphorylation by Vaccinia-Related Kinase 1 in Mammalian Cells

2007 ◽  
Vol 27 (24) ◽  
pp. 8533-8546 ◽  
Author(s):  
Tae-Hong Kang ◽  
Do-Young Park ◽  
Yoon Ha Choi ◽  
Kyung-Jin Kim ◽  
Ho Sup Yoon ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Chromatin Condensation ◽  
Histone H3 ◽  
Cycle Phase ◽  
Aurora B ◽  
H3 Phosphorylation ◽  
Histone H3 Phosphorylation

ABSTRACT Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.

Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation

1998 ◽  
Vol 111 (23) ◽  
pp. 3497-3506 ◽  
Author(s):  
A. Van Hooser ◽  
D.W. Goodrich ◽  
C.D. Allis ◽  
B.R. Brinkley ◽  
M.A. Mancini
Keyword(s):  
Cell Cycle Progression ◽  
Histone H3 ◽  
Chromosome Morphology ◽  
Chromosome Condensation ◽  
S Phase ◽  
Condensed State ◽  
Cycle Progression ◽  
H3 Phosphorylation ◽  
Excess Substrate ◽  
Histone H3 Phosphorylation

The temporal and spatial patterns of histone H3 phosphorylation implicate a specific role for this modification in mammalian chromosome condensation. Cells arrest in late G2 when H3 phosphorylation is competitively inhibited by microinjecting excess substrate at mid-S-phase, suggesting a requirement for activity of the kinase that phosphorylates H3 during the initiation of chromosome condensation and entry into mitosis. Basal levels of phosphorylated H3 increase primarily in late-replicating/early-condensing heterochromatin both during G2 and when premature chromosome condensation is induced. The prematurely condensed state induced by okadaic acid treatment during S-phase culminates with H3 phosphorylation throughout the chromatin, but in an absence of mitotic chromosome morphology, indicating that the phosphorylation of H3 is not sufficient for complete condensation. Mild hypotonic treatment of cells arrested in mitosis results in the dephosphorylation of H3 without a cytological loss of chromosome compaction. Hypotonic-treated cells, however, complete mitosis only when H3 is phosphorylated. These observations suggest that H3 phosphorylation is required for cell cycle progression and specifically for the changes in chromatin structure incurred during chromosome condensation.

Abstract 32: The Role of Cyclin A2 in Adult Human Cardiomyocyte Plasticity

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Amaresh Ranjan ◽  
Sangeetha Vadakke Madathil ◽  
Hina Chaudhry
Keyword(s):  
Cardiac Regeneration ◽  
Time Lapse ◽  
Heart Tissue ◽  
Functional Restoration ◽  
Epifluorescence Microscopy ◽  
Cardiac Marker ◽  
Human Cardiomyocytes ◽  
Adult Human ◽  
Cyclin A2

The adult mammalian heart is known to have a very low abundance of progenitor cells which can take part in active cycling and regeneration after damage. Cardiomyocytes exit the cell cycle soon after birth coincident with the silencing of cyclin A2 (CCNA2). In our previous studies, we demonstrated that viral delivery of Ccna2 induces cardiac regeneration in infarcted hearts of small and large animal models. However, the molecular mechanism whereby Ccna2 induces cardiac regeneration and increase in cardiac function deserves further study. To explore further, we isolated adult mouse cardiomyocytes and induced Ccna2 expression by using adenovirus transfection and cultured them for 3 weeks. Co-expression of the mature cardiac marker troponin Tc with the immature cardiac marker non-muscle myosin IIB was observed. Additionally, expression of epithelial to mesenchymal transition markers (vimentin and FSP1) was observed. Also, decreased expression of mature cardiac markers α-MHC , ckmt2 and cTnT was noted. To study the factors responsible for human cardiomyocyte plasticity and cell division, we have optimized a novel method for culturing adult human cardiomyocytes in our laboratory. We cultured cardiomyocytes isolated from heart tissue obtained from a 55 yr old male patient. After transfection with CCNA2 adenovirus made for human use (cTnT promoter driving human CCNA2 cDNA), they were co-transfected with two more adenoviruses (1) cTnT-GFP to label cardiomyocytes (green) and (2) CMV-α-actinin-m-Cherry to label the sarcomere (red). Time lapse live epifluorescence microscopy was carried out for 70 hrs and time lapse movies were prepared (please refer the youtube link to see a representative time lapse movie https://youtu.be/OBrJGCq7YCA ). Movies were analyzed to calculate the cytokinesis index in samples transfected with (Test) and without (Null) CCNA2 adenoviruses. We observed a significantly higher cytokinesis index in CCNA2 samples versus Null. We are further investigating the role of cyclin A2 in dedifferentiation of adult human cardiomyocytes to generate immature or progenitor cardiac cells and their contractile status, which could be utilized for regeneration and functional restoration of damaged adult heart tissue.

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