Timing of calcium and protein synthesis requirements for the first mitotic cell cycle in fertilised Xenopus eggs

1999 ◽  
Vol 112 (22) ◽  
pp. 3975-3984
Author(s):  
C. Beckhelling ◽  
C. Penny ◽  
S. Clyde ◽  
C. Ford
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Calcium Concentration ◽  
Mitotic Cell ◽  
Sensitive Period ◽  
Free Calcium ◽  
Calcium Buffer ◽  
M Phase ◽  
Egg Extracts ◽  
Histone Kinase

Mitosis is governed by the activity of the M-phase promoting factor (MPF). In some systems, particularly early embryos, transient increases in calcium concentration have been shown to be necessary for mitosis and regulate its timing. By microinjection of the calcium buffer, dibromoBAPTA, into fertilised Xenopus eggs, we have assessed whether calcium events are required to initiate MPF activation and inactivation. Since initial experiments showed that this buffer inhibited protein synthesis, we measured when mitosis and cleavage became independent of translation. We found that, after a period of protein synthesis essential for cleavage, there was a phase during which continued translation affected the timing of cleavage, but was not essential for its occurrence. Measurement of MPF activity in single embryos injected with calcium buffer at different times in the first cell cycle, showed that there were two sensitive periods. The first period of sensitivity blocked MPF activation and coincided with the time at which cleavage became completely independent of protein synthesis. The second sensitive period occurred just before histone kinase activity peaked, and was necessary for kinase inactivation. Preventing inactivation in this way arrested egg extracts in mitosis. These results support the view that transient increases in free calcium concentration contribute to mitotic progression by first triggering MPF activation and subsequently, with elevated MPF activity, inducing its inactivation.

scholarly journals Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts

1995 ◽  
Vol 108 (11) ◽  
pp. 3557-3568 ◽  
Author(s):  
H.D. Lindsay ◽  
M.J. Whitaker ◽  
C.C. Ford
Keyword(s):  
Mitotic Cell ◽  
Free Calcium ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Cell Cycles ◽  
Calcium Buffer ◽  
Egg Extracts ◽  
Calcium Buffers ◽  
Xenopus Egg Extracts ◽  
Activation Phase

Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translation modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500 nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2A) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.

scholarly journals Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle.

1990 ◽  
Vol 110 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
D L Gard ◽  
S Hafezi ◽  
T Zhang ◽  
S J Doxsey
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Spindle Assembly ◽  
Centrosome Duplication ◽  
Microtubule Organizing Centers ◽  
Xenopus Embryos ◽  
M Phase ◽  
Pericentriolar Material ◽  
Histone Kinase

Cycloheximide (500 micrograms/ml) rapidly arrests cleavage, spindle assembly, and cycles of an M-phase-specific histone kinase in early Xenopus blastulae. 2 h after cycloheximide addition, most cells contained two microtubule asters radiating from perinuclear microtubule organizing centers (MTOCs). In contrast, blastomeres treated with cycloheximide for longer periods (3-6 h) contained numerous microtubule asters and MTOCs. Immunofluorescence with an anticentrosome serum and EM demonstrated that the MTOCs in cycloheximide-treated cells were typical centrosomes, containing centrioles and pericentriolar material. We conclude that centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. In addition, these observations suggest that Xenopus embryos contain sufficient material to assemble 1,000-2,000 centrosomes in the absence of normal protein synthesis.

scholarly journals Role of CDK1 in Translational Regulation in the M-Phase

2020 ◽  
Author(s):  
Jaroslav Kalous ◽  
Denisa Jansova ◽  
Andrej Susor
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Translational Regulation ◽  
Gene Expression Regulation ◽  
Translational Initiation ◽  
Initiation Factors ◽  
Cellular Events ◽  
M Phase ◽  
Mitosis And Meiosis

Cyclin dependent kinase 1 (CDK1) has been primarily identified as a key cell cycle regulator in both mitosis and meiosis. Recently, an extramitotic function of CDK1 emerged when evidence was found that CDK1 is involved in many cellular events that are essential for cell proliferation and survival. In this review we summarize the involvement of active CDK1 in the initiation and elongation steps of protein synthesis in eukaryotes. During its activation CDK1 influences the initiation of protein synthesis, promotes the activity of specific translational initiation factors and affects the functioning of a subset of elongation factors. Our review provides insights into gene expression regulation during the transcriptionally silent cell cycle/M-phase and describes quantitative and qualitative translational changes based on the extramitotic role of the cell cycle master regulator CDK1, to optimize temporal synthesis of proteins to sustain division-related processes: mitosis and cytokinesis.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

The increase in intracellular pH associated with Xenopus egg activation is a Ca(2+)-dependent wave

1992 ◽  
Vol 101 (1) ◽  
pp. 55-67 ◽  
Author(s):  
N. Grandin ◽  
M. Charbonneau
Keyword(s):  
Cell Cycle ◽  
Intracellular Ph ◽  
Egg Activation ◽  
Free Calcium ◽  
Slowing Down ◽  
M Phase ◽  
Xenopus Egg ◽  
General Slowing ◽  
The Impact ◽  
Kinetics Of

In Xenopus eggs, the transient increase in intracellular free calcium ([Ca2+]i), or Ca2+ transient, which occurs 1–3 min after egg activation, is likely to be partly responsible for the release of the cell cycle blockade. In the present study, we have used microinjection of BAPTA or EGTA, two potent chelators of Ca2+, to buffer [Ca2+]i at various steps during Xenopus egg activation and evaluate the impact on some of the associated events. Microinjection of either one of the Ca2+ chelators into unactivated eggs prevented egg activation without, however, lowering [Ca2+]i, suggesting that only physiological [Ca2+]i changes, but not [Ca2+]i levels, were affected by the Ca2+ buffer. When BAPTA was microinjected around the time of occurrence of the Ca2+ transient, the egg activation-associated increase in intracellular pH (pHi) was clearly delayed. That delay was not due to a general slowing down of the cell cycle, since under the same conditions of microinjection of BAPTA the kinetics of MPF (a universal M-phase promoting factor) inactivation were unaffected. These results represent the first indication that the Ca2+ transient participates in determining the time of initiation of the pHi increase during Xenopus egg activation. The present results also demonstrate that the egg activation-associated pHi changes (a slight, transient decrease in pHi followed by a permanent increase in pHi) proceed as a wave propagating from the site of triggering of egg activation. Experiments of local microinjection of BAPTA support the view that the pH wave is a consequence of the Ca2+ wave, which it follows closely.

Regulatory effect of thromboxane A2 on proliferation of vascular smooth muscle cells from rats

1992 ◽  
Vol 263 (5) ◽  
pp. H1331-H1338 ◽  
Author(s):  
T. Nagata ◽  
Y. Uehara ◽  
A. Numabe ◽  
T. Ishimitsu ◽  
N. Hirawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cell Cycle Progression ◽  
Thromboxane A2 ◽  
Cycle Progression ◽  
M Phase ◽  
Rapid Transition

We investigated the regulatory effects of the vasoconstrictor thromboxane A2 on the proliferation of vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats using 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2. STA2 dose dependently increased incorporation of [3H]thymidine into DNA in randomly cycling VSMC and significantly shortened the doubling time. Cell cycle analysis revealed that the increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, STA2 enhanced protein synthesis in VSMC during the G2/M phase, whereas the protein synthesis was unaffected in the G0/G1 period. In fact, STA2 prompted the cells in G2/M phase to synthesize actin, a major cytoskeleton protein. Conversely, inhibition of protein synthesis by puromycin retarded the transition from S to G2/M. In addition, depolymerization of the actin molecules by cytochalasin D offset the quick progression to the G2/M phase by STA2. These data indicate that thromboxane A2 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. This enhanced progression is attributable partly to a rapid buildup of the cytoskeleton proteins during the G2/M period.

scholarly journals Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins

2009 ◽  
Vol 29 (18) ◽  
pp. 4891-4905 ◽  
Author(s):  
Santhi Pondugula ◽  
Daniel W. Neef ◽  
Warren P. Voth ◽  
Russell P. Darst ◽  
Archana Dhasarathy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Dependent Manner ◽  
Phosphate Homeostasis ◽  
Periodic Cell ◽  
M Phase ◽  
Cycle Dependent ◽  
Nutrient Homeostasis

ABSTRACT Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G2/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G1. These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

scholarly journals Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly

2008 ◽  
Vol 181 (2) ◽  
pp. 241-254 ◽  
Author(s):  
Michael J. Emanuele ◽  
Weijie Lan ◽  
Miri Jwa ◽  
Stephanie A. Miller ◽  
Clarence S.M. Chan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Culture Cells ◽  
Kinetochore Assembly ◽  
M Phase ◽  
Egg Extracts ◽  
Cycle Dependent

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle–dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

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