scholarly journals The spindle checkpoint, APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

2013 ◽  
Vol 201 (7) ◽  
pp. 1013-1026 ◽  
Author(s):  
Linda Clijsters ◽  
Janneke Ogink ◽  
Rob Wolthuis
Keyword(s):  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
S Phase ◽  
Animal Cells ◽  
Proliferating Cells ◽  
Cell Cycle Entry ◽  
Sister Chromatids ◽  
Mitotic Cyclin ◽  
Bona Fide ◽  
Subsequent Activation

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/CCdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/CCdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/CCdc20, and APC/CCdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.

scholarly journals Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

2002 ◽  
Vol 157 (7) ◽  
pp. 1125-1137 ◽  
Author(s):  
Anja Hagting ◽  
Nicole den Elzen ◽  
Hartmut C. Vodermaier ◽  
Irene C. Waizenegger ◽  
Jan-Michael Peters ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Mutant Form ◽  
Exact Time ◽  
Sister Chromatids ◽  
Sister Chromatid Separation

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

scholarly journals Cyclin-Specific Control of Ribosomal DNA Segregation

2008 ◽  
Vol 28 (17) ◽  
pp. 5328-5336 ◽  
Author(s):  
Matt Sullivan ◽  
Liam Holt ◽  
David O. Morgan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Ribosomal Dna ◽  
S Phase ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Early Anaphase ◽  
Mitotic Cyclin ◽  
Mitosis And Meiosis ◽  
Specific Control

ABSTRACT Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.

scholarly journals p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

1999 ◽  
Vol 19 (1) ◽  
pp. 205-215 ◽  
Author(s):  
Zoe A. Stewart ◽  
Steven D. Leach ◽  
Jennifer A. Pietenpol
Keyword(s):  
Cyclin E ◽  
Ectopic Expression ◽  
Cyclin B1 ◽  
S Phase ◽  
Nuclear Antigen ◽  
Microtubule Inhibitors ◽  
Cdk2 Activity ◽  
Integral Role ◽  
Subsequent Activation ◽  
Proliferating Cell

ABSTRACT During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21+/+ and p21−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G1-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21+/+ cells paralleled the onset of endoreduplication in HCT116 p21−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.

scholarly journals Expression of Mitotic Cyclin B1 is not Confined to Proliferating Cells in the Rat Testis1

1997 ◽  
Vol 57 (6) ◽  
pp. 1312-1319 ◽  
Author(s):  
Jörg Gromoll ◽  
Judith Wessels ◽  
Giesa Rosiepen ◽  
Martin H. Brinkworth ◽  
Gerhard F. Weinbauer
Keyword(s):  
Cyclin B1 ◽  
Proliferating Cells ◽  
Mitotic Cyclin

Cyclin A and Nek2A: APC/C–Cdc20 substrates invisible to the mitotic spindle checkpoint

2010 ◽  
Vol 38 (1) ◽  
pp. 72-77 ◽  
Author(s):  
Wouter van Zon ◽  
Rob M.F. Wolthuis
Keyword(s):  
Nuclear Envelope ◽  
Spindle Checkpoint ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Spindle Microtubules

Active cyclin B1–Cdk1 (cyclin-dependent kinase 1) keeps cells in mitosis, allowing time for spindle microtubules to capture the chromosomes and for incorrect chromosome-spindle attachments to be repaired. Meanwhile, securin, an inhibitor of separase, secures cohesion between sister chromatids, preventing anaphase onset. The spindle checkpoint is a signalling pathway emerging from improperly attached chromosomes that inhibits Cdc20, the mitotic activator of the APC/C (anaphase-promoting complex/cyclosome) ubiquitin ligase. Blocking Cdc20 stabilizes cyclin B1 and securin to delay mitotic exit and anaphase until all chromosomes reach bipolar spindle attachments. Cells entering mitosis in the absence of a functional spindle checkpoint degrade cyclin B1 and securin right after nuclear-envelope breakdown, in prometaphase. Interestingly, two APC/C substrates, cyclin A and Nek2A, are normally degraded at nuclear-envelope breakdown, even when the spindle checkpoint is active. This indicates that the APC/C is activated early in mitosis, whereas cyclin B1 and securin are protected as long as the spindle checkpoint inhibits Cdc20. Remarkably, destruction of cyclin A and Nek2A also depends on Cdc20. The paradox of Cdc20 being both active and inhibited in prometaphase could be explained if cyclin A and Nek2A are either exceptionally efficient Cdc20 substrates, or if they are equipped with ‘stealth’ mechanisms to effectively escape detection by the spindle checkpoint. In the present paper, we discuss recently emerging models for spindle-checkpoint-independent APC/C–Cdc20 activity, which might even have implications for cancer therapy.

scholarly journals A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

1998 ◽  
Vol 46 (10) ◽  
pp. 1203-1209 ◽  
Author(s):  
Françoise Jaunin ◽  
Astrid E. Visser ◽  
Dusan Cmarko ◽  
Jacob A. Aten ◽  
Stanislav Fakan
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Salt Concentration ◽  
Chinese Hamster ◽  
S Phase ◽  
Tween 20 ◽  
Proliferating Cells ◽  
Specific Staining ◽  
Double Labeling ◽  
Chinese Hamster Cells

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

scholarly journals Phosphoinositide 3-Kinases p110α and p110β Regulate Cell Cycle Entry, Exhibiting Distinct Activation Kinetics in G1 Phase

2008 ◽  
Vol 28 (8) ◽  
pp. 2803-2814 ◽  
Author(s):  
Miriam Marqués ◽  
Amit Kumar ◽  
Isabel Cortés ◽  
Ana Gonzalez-García ◽  
Carmen Hernández ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinases ◽  
Control Cell ◽  
Growth Factor Receptor ◽  
Maximum Activity ◽  
S Phase ◽  
Selective Action ◽  
Cell Cycle Entry ◽  
Phosphoinositide 3 Kinase ◽  
Phase Entry

ABSTRACT Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G0/G1 transition) and again in late G1. The two ubiquitous PI3K isoforms (p110α and p110β) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110α was activated first in G0/G1, followed by a minor p110β activity peak. In late G1, p110α activation preceded that of p110β, which showed the maximum activity at this time. p110β activation required Ras activity, whereas p110α was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110α and -β activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110α in blocking early G1 events. We show that inhibition of either p110α or p110β reduced cell cycle entry. These results reveal that PI3Kα and -β present distinct activation requirements and kinetics in G1 phase, with a selective action of PI3Kα at the G0/G1 phase transition. Nevertheless, PI3Kα and -β both regulate S-phase entry.

Changes in p21Cip1 and p27Kip1 expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells

2000 ◽  
Vol 12 (9-10) ◽  
pp. 619-627 ◽  
Author(s):  
Waraporn Promwikorn ◽  
Shaun R Hawley ◽  
Stephen R Pennington
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
3T3 Cells ◽  
P27kip1 Expression ◽  
Cell Cycle Entry ◽  
Swiss 3T3

scholarly journals Structure of yellow lupine genes coding for mitotic cyclins.

1999 ◽  
Vol 46 (3) ◽  
pp. 759-769
Author(s):  
J Jeleńska ◽  
Z Zaborowska ◽  
A B Legocki
Keyword(s):  
Cell Cycle Progression ◽  
Cyclin B1 ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Cycle Progression ◽  
Coding Regions ◽  
Mitotic Cyclin ◽  
Plant Genes ◽  
Yellow Lupine ◽  
First Time

Cell cycle progression in eukaryotes is controlled by complexes of p34 protein kinases and cyclins. For the first time in plants, we have established the sequence of four yellow lupine mitotic cyclin B1 genes. Their coding regions and expression pattern were also characterised recently. Structure of all the four lupine genes is similar: they consist of nine exons and eight introns, analogously located, except Luplu;CycB1;3 lacking 7th intron. Analysis of 5'-regulatory sequences of two of them showed that both comprise M-specific activators (MSA), common to plant genes induced in late G2 and early M. Putative repressor binding sites CDE/CHR found in animal G2-specific promoters can also be detected in lupine genes. Controlling region of Luplu;CycB1;4 gene that is highly activated by IAA, contains up to 7 auxin response elements, while insensible to IAA Luplu;CycB1;4 gene have no such motifs. Further studies should be undertaken to determine precisely the functions of putative regulatory elements in the expression of lupine mitotic cyclins.

scholarly journals With Age Comes Maturity: Biochemical and Structural Transformation of a Human Centriole in the Making

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1429 ◽  
Author(s):  
Catherine Sullenberger ◽  
Alejandra Vasquez-Limeta ◽  
Dong Kong ◽  
Jadranka Loncarek
Keyword(s):  
S Phase ◽  
Human Cells ◽  
Cellular Structures ◽  
Age Difference ◽  
Proliferating Cells ◽  
Cell Cycles ◽  
Controlled Processes ◽  
The Third ◽  
Centriole Assembly ◽  
Lengthy Process

Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.

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