scholarly journals Phosphatase PP2A and microtubule pulling forces disassemble centrosomes during mitotic exit

2017 ◽  
Author(s):  
Stephen J. Enos ◽  
Martin Dressler ◽  
Beatriz Ferreira Gomes ◽  
Anthony A. Hyman ◽  
Jeffrey B. Woodruff
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mitotic Exit ◽  
Scaffold Proteins ◽  
Regulatory Subunit ◽  
Pulling Forces ◽  
Dividing Cells ◽  
Pericentriolar Material ◽  
Embryonic Death

ABSTRACTCentrosomes are major microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ~25% of PCM persisted from one cell cycle into the next, resulting in cytokinetic furrow ingression errors, excessive centrosome accumulation, and embryonic death. Purified pp2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and catastrophic rupture of the PCM scaffold.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Transcription of the histone H5 gene is not S-phase regulated

1986 ◽  
Vol 6 (2) ◽  
pp. 601-606
Author(s):  
S Dalton ◽  
J R Coleman ◽  
J R Wells
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Northern Blotting ◽  
S Phase ◽  
Erythroid Cells ◽  
Dividing Cells ◽  
Steady State Levels ◽  
Avian Erythroblastosis Virus ◽  
Histone H5

Levels of the tissue-specific linker histone H5 are elevated in mature erythroid cells as compared with levels in dividing cells of the same lineage. We examined levels of H5 mRNA in relation to the cell cycle in early erythroid cells transformed by avian erythroblastosis virus to determine whether the gene for this unusual histone is S-phase regulated. Northern blotting analyses revealed that during the cell cycle steady-state levels of H5 mRNA remained relatively constant in contrast to levels of the major core and H1 mRNAs which increased approximately 15-fold during S phase. In vitro pulse-labeling experiments involving nuclei isolated from synchronized cells at various stages of the cell cycle revealed that transcription of the H5 gene was not initiated at any particular stage of the cell cycle but was constitutive. In contrast, transcription of the H2A gene(s) initiated in early S phase, was present throughout the DNA replicative phase, and was essentially absent in G1 and G2 phases.

Stage specificity in the mesenchyme requirement of rodent lung epithelium in vitro: a matter of growth control?

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 183-206
Author(s):  
Kirstie A. Lawson
Keyword(s):  
Cell Cycle ◽  
Bronchial Epithelium ◽  
Branching Morphogenesis ◽  
S Phase ◽  
Growth Fraction ◽  
Early Event ◽  
Rat Lung ◽  
Lung Epithelium ◽  
Dividing Cells

Epithelia from lung rudiments in which secondary bronchial buds are already established (14th and 13th gestational day for rat and mouse respectively) are able to undergo branching morphogenesis and cytodifferentiation in submandibular mesenchyme in vitro, whereas lung epithelium from one day younger foetuses rarely gives a morphogenetic response to submandibular mesenchyme and usually differentiates into primary (non-budding) bronchial epithelium. The failure of 13-day rat lung epithelium to respond to submandibular mesenchyme can be prevented by peeling off the submandibular mesenchyme from the lung epithelium after 2½ days culture and replacing the same mesenchyme, or renewing it with fresh salivary mesenchyme ex vivo. Changes in the epithelial contour are visible by 10 h and buds form within 24 h; this is followed by branching morphogenesis in more than 66% of the samples. The number of cells in S-phase in the epithelium is doubled within 3 to 5 h after the operation and the number of mitotic cells (colchicine block) is increased during an 11 to 19 h period after the operation. Substituting stomach mesenchyme for submandibular mesenchyme after the operation failed to elicit morphogenesis or an increase in the number of S-phase cells in the epithelium. The proportion of epithelial cells in S-phase in unoperated recombinates does not differ from the proportion in the primary bronchial epithelium (non-budding) of homotypic lung recombinates, whereas the proportion of S-phase cells in operated recombinates approaches that found in the buds of homotypic lung recombinates. The distribution of S-phase cells in visibly responding recombinates 15 to 17 h after operation shows the same heterogeneity as in homotypic lung recombinates, newly formed buds having twice as many cells labelled with [3H]thymidine as the non-budding area. Cell cycle parameters of intact rat lung growing in vitro were estimated using the labelled mitoses method. Primary bronchial epithelium and bronchial buds both had a total cell cycle time of about 13 h and an S-phase of about 10 h. The growth fraction was 0·54 in the primary bronchus and 0·95 in the buds. It is suggested that, also in the recombinates, differences in the proportion of S-phase cells at any one time in morphogenetically active and inactive areas of the epithelium are due to differences in the growth fraction. It is concluded that an early event in the morphogenetic response of lung epithelium to submandibular mesenchyme after removing and restoring the mesenchyme is an increase in the size of the population of dividing cells and it is suggested that a high proportion of dividing cells in an epithelial population is a prerequisite for further interaction of epithelium and mesenchyme leading to branching morphogenesis.

scholarly journals Diamond Nanoparticles Downregulate Expression of CycD and CycE in Glioma Cells

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1549 ◽  
Author(s):  
Marta Grodzik ◽  
Jaroslaw Szczepaniak ◽  
Barbara Strojny-Cieslak ◽  
Anna Hotowy ◽  
Mateusz Wierzbicki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Control Group ◽  
Ki 67 ◽  
Normal Cells ◽  
Diamond Nanoparticles ◽  
Dividing Cells

Our previous studies have shown that diamond nanoparticles (NDs) exhibited antiangiogenic and proapoptotic properties in vitro in glioblastoma multiforme (GBM) cells and in tumors in vivo. Moreover, NDs inhibited adhesion, leading to the suppression of migration and invasion of GBM. In the present study, we hypothesized that the NDs might also inhibit proliferation and cell cycle in glioma cells. Experiments were performed in vitro with the U87 and U118 lines of GBM cells, and for comparison, the Hs5 line of stromal cells (normal cells) after 24 h and 72 h of treatment. The analyses included cell morphology, cell death, viability, and cell cycle analysis, double timing assay, and gene expression (Rb, E2F1, CycA, CycB, CycD, CycE, PTEN, Ki-67). After 72 h of ND treatment, the expression level of Rb, CycD, and CycE in the U118 cells, and E2F1, CycD, and CycE in the U87 cells were significantly lower in comparison to those in the control group. We observed that decreased expression of cyclins inhibited the G1/S phase transition, arresting the cell cycle in the G0/G1 phase in glioma cells. The NDs did not affect the cell cycle as well as PTEN and Ki-67 expression in normal cells (Hs5), although it can be assumed that the NDs reduced proliferation and altered the cell cycle in fast dividing cells.

A New Abl Kinase Inhibitor (AMN107) Has In Vitro Activity on Chronic Myeloid Leukaemia (CML) Ph+ Cells Resistant to Imatinib.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2004-2004 ◽  
Author(s):  
Giovanni Martinelli ◽  
Alberto M. Martelli ◽  
Tiziana Grafone ◽  
Irina Mantovani ◽  
Alessandra Cappellini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Imatinib Resistance ◽  
First Line Therapy

Abstract Imatinib mesylate (Novartis Pharma), an inhibitor of the bcr/abl tyrosine kinase, has rapidly become the first-line therapy for CML. Imatinib has proved remarkably effective at reducing the number of leukaemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, in patients in advanced phases of the disease, the development of resistance to this drug is a frequent setback. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107 (Novartis Pharma), has been developed. We have tested AMN107 on human leukaemia cell lines and on blasts isolated from imatinib-resistant CML patients. After a 24 h incubation, AMN107 (10 nM) blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 had no affect on cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 500 nM. After 48 h incubation, AMN107 (10 nM) was capable of inducing a massive apoptosis of K562 cells whereas, once again, 200 nM imatinib was required to obtain the same effect. Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (50 nM) markedly down-regulated autophosphorylation of bcr/abl Tyr177 and Tyr412, whereas autophosphorylation of Thr735 was unaffected. In contrast, imatinib even if used at 200 nM, did not diminish phosphorylation of either bcr/abl Tyr177 or Tyr412. This finding seems particularly important because recent evidence has demonstrated that the signalling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr177 forms a high-affinity binding site for the SH2 domain of the adapter Grb2. The main effectors of Grb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2-Gab2 complex, which results in activation of phosphoinositide 3-kinase (PI3K)/Akt and Erk signalling networks. Consistently, we found by immunoprecipitation decreased levels of bcr/abl-associated Gab2, Grab2, and p85 regulatory subunit of PI3K in AMN107-treated cells. AMN107 treatment of K562 cells also caused a reduction of STAT5, cCBL, CRKL, and Akt phosphorylation levels, as well as Bcl-XL expression. AMN107 (5 μM for 24h) significantly increased the apoptosis rate of CML blasts isolated from patients resistant to imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.

scholarly journals Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Pathway

2004 ◽  
Vol 24 (2) ◽  
pp. 514-526 ◽  
Author(s):  
Po-Yuan Ke ◽  
Zee-Fen Chang
Keyword(s):  
Cell Cycle ◽  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Mitotic Exit ◽  
Limiting Factor ◽  
Anaphase Promoting Complex ◽  
Thymidine Kinase 1 ◽  
Ubiquitin Proteasome ◽  
The Right

ABSTRACT The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G1 phase of the cell cycle in mammalian cells.

scholarly journals Regulation of Mitotic Exit by Cell Cycle Checkpoints: Lessons From Saccharomyces cerevisiae

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 195 ◽  
Author(s):  
Laura Matellán ◽  
Fernando Monje-Casas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Model Organism ◽  
Mitotic Exit ◽  
Cell Cycle Checkpoints ◽  
Dna Integrity ◽  
Intracellular Signals ◽  
Dividing Cells

In order to preserve genome integrity and their ploidy, cells must ensure that the duplicated genome has been faithfully replicated and evenly distributed before they complete their division by mitosis. To this end, cells have developed highly elaborated checkpoints that halt mitotic progression when problems in DNA integrity or chromosome segregation arise, providing them with time to fix these issues before advancing further into the cell cycle. Remarkably, exit from mitosis constitutes a key cell cycle transition that is targeted by the main mitotic checkpoints, despite these surveillance mechanisms being activated by specific intracellular signals and acting at different stages of cell division. Focusing primarily on research carried out using Saccharomyces cerevisiae as a model organism, the aim of this review is to provide a general overview of the molecular mechanisms by which the major cell cycle checkpoints control mitotic exit and to highlight the importance of the proper regulation of this process for the maintenance of genome stability during the distribution of the duplicated chromosomes between the dividing cells.

scholarly journals Dbf4p, an Essential S Phase-Promoting Factor, Is Targeted for Degradation by the Anaphase-Promoting Complex

2000 ◽  
Vol 20 (1) ◽  
pp. 242-248 ◽  
Author(s):  
Miguel Godinho Ferreira ◽  
Corrado Santocanale ◽  
Lucy S. Drury ◽  
John F. X. Diffley
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Rapid Degradation ◽  
N Terminus ◽  
Chromosome Separation ◽  
Apc Mutation ◽  
Redundant Role

ABSTRACT The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase. The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle. In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle. Dbf4p is absent from cells during G1and accumulates during the S and G2 phases. Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G1 phase. The rapid degradation of Dbf4p during G1 requires a functional anaphase-promoting complex (APC). Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p. We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire.

scholarly journals PP2A-Cdc55 phosphatase coordinates actomyosin ring contraction and septum formation during cytokinesis

2020 ◽  
Author(s):  
Yolanda Moyano-Rodríguez ◽  
Odena Vilalta-Castany ◽  
Magdalena Foltman ◽  
Alberto Sanchez-Diaz ◽  
Ethel Queralt
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Exit ◽  
In Vitro Assays ◽  
Ring Contraction ◽  
Mitotic Kinases ◽  
Septum Formation ◽  
Primary Septum ◽  
Complete Cell

SummaryEukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is regulated by exclusive elements of the process, and by some mitotic exit regulators. The mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate cytokinetic proteins in budding yeast, but very little is known about the phosphatases regulating cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylations. This prompted us to propose that PP2A-Cdc55 could also regulate cytokinesis by counteracting the mitotic kinases. Here, we demonstrate by in vivo and in vitro assays that PP2A-Cdc55 dephosphorylates the F-BAR protein Hof1 and the chitin synthase Chs2, two components of the Ingression Progression Complexes (IPC) involved in cytokinesis regulation. Primary septum formation and actomyosin ring contraction are impaired in absence of PP2A-Cdc55. Interestingly, the non-phosphorylable version of Chs2 rescue the asymmetric AMR contraction observed in absence of Cdc55, indicating that timely dephosphorylation of the IPC proteins by PP2A-Cdc55 is crucial for proper actomyosin ring contraction and septum formation. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge in the involvement of multiple phosphatases during cytokinesis.

Sign in / Sign up

Export Citation Format

Share Document