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PLoS One
2013 Jan 01;86:e66113. doi: 10.1371/journal.pone.0066113.
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Intracellular and extracellular pH and Ca are bound to control mitosis in the early sea urchin embryo via ERK and MPF activities.
Ciapa B
,
Philippe L
.
Abstract
Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Cai) and pH (pHi) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na(+)/H(+) exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Cai and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pHi and Cai determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life.
Figure 1. ERK and CDK activities control Cai and pHi after fertilization.A. Time courses of Cai changes after fertilization in eggs treated or not (a) 10 mins after sperm addition (time zero) with 2 µM U0126 (b) or 20 µM Roscovitine (c). One typical recording is shown for each condition. Cai transients occur at time of pronuclear migration (black arrows) and at mitosis (grey arrow). Inset images taken 80 mins after fertilization show normal division in control eggs (a), mitotic alterations with U0126 (b) and absence of mitosis with Roscovitine (c). B. Time course of pHi changes after fertilization of control eggs. C. Relative changes in pHi (a) and Cai (b) levels in eggs treated or not (control) with 2 µM U0126 or 20 µM Roscovitine. The mean levels of pHi recorded from 60 and 65 mins following sperm addition are expressed relative to that of unfertilized egg (arbitrarily taken as 1). Mean levels of Cai recorded during 5 mins at time of the mitotic peak (grey arrow and line in Fig. A), i.e. between 35–40 mins in control eggs, 30–35 mins in U0126 and 25–30 mins in Roscovitine treated eggs, are expressed as the percentage of the fertilization Cai peak arbitrarily taken as 100. The total number of eggs monitored is indicated for each condition (brackets). Values (mean +/− sem) are significantly higher than that of control eggs (**, student test, p<0.01) or not significantly different (ns).
Figure 2. Changes in Cai and pHi during mitosis depend on external Ca++ and Na+.Eggs were transferred in the different media after fertilization (arrow). Two examples representative of n measurements (number in brackets) are shown in each condition. Variations in pHi (a), evolution of Cai (b) and images of eggs taken 70 min after fertilization (insets in a). A. Transfer in 0Na (1), Am (2) or Ac (3). B. Effect of additional absence of external Ca after transfer in 0Na0Ca (1), Am0Ca (2) or Ac0Ca (3). C. Mean levels of pHi (a) and Cai (b) changes recorded from 60–65 mins after sperm addition in Figs. A and B. pHi changes (a) are expressed relative to that of unfertilized egg (arbitrarily taken as 1) while Cai changes (b) are expressed as the percentage of the fertilization Cai peak arbitrarily taken as 100 (zero is the unfertilized level). Values (means +/− sem) obtained in the absence of external Ca are significantly different (student test, p<0.01, two black stars) or not (ns, black letters) from control. Values obtained in the absence of Ca are significantly different (p<0.01, two grey stars) or not (ns, grey letters) from those obtained in the presence of Ca. Number of eggs is indicated for each condition (brackets).
Figure 4. P-ERK activity controls mitosis and survival of fertilized eggs.A. Images taken 3 hours after fertilization of eggs transferred or not (control) 25 mins after fertilization in Ac containing or not 2 µM U0126. B. The experiment shown in A was performed three times and the data obtained pooled. Eggs were also transferred in 0Na or Am. % of divided eggs (a), embryos (b) or fragmented eggs (c) were determined 3 hours after fertilization (filled histograms) except for divided control eggs that were counted 80 mins after sperm addition (hatched histograms). 42–78 total embryos were counted in each condition. Values (means +/− sem) obtained in the presence of U0126 (grey histogram) are significantly different (student test, p<0.01, two stars) or not (ns) to those obtained without this drug (black histogram).
Figure 5. Cai and pHi depend on the presence of external HCO3–.A. Observation 80 mins after fertilization. Eggs develop in ASW (Control) or are transferred 20 mins after fertilization into ASW deprived of Ca (0Ca), HCO3
− (0HCO3) or of Na+ and Ca++ (0Na0Ca). B. Time course after fertilization of pHi (a) and Cai (b) in ASW, 0Na and 0Na0Ca in the presence or not of HCO3
−. Examples of results obtained in absence of HCO3-, i.e. in 0HCO3 (1), 0Na0HCO3 (2) and 0Na0Ca0HC03 (3), are depicted in different grey colours and representative of n determinations (grey number in brackets). In each condition, one typical control experiment performed with the same population of eggs in the presence of HCO3
−, i.e. in ASW (1), 0Na (2) and 0Na0Ca (3) is shown in black curve and representative of n determinations (black number in brackets).
Figure 6. Na/Ca inhibitors reduce inhibition of mitosis induced in the absence of external Na.Eggs were let to develop in ASW (Control) or transferred 20 min after fertilization in 0Na containing or not (0Na) the different inhibitors, BHC, SN6 or KB. Observation 80 min after fertilization (A) and compiled assessment of results (means +/− sem) from 3 experiments that gave similar results (B). Total number of eggs counted in each condition is indicated in brackets in B.
Amirand,
Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis.
2000, Pubmed
Amirand,
Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis.
2000,
Pubmed
Arion,
M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.
1989,
Pubmed
,
Echinobase
Berridge,
Inositol trisphosphate and calcium signalling mechanisms.
2009,
Pubmed
Carballeira,
Influence of salinity on fertilization and larval development toxicity tests with two species of sea urchin.
2011,
Pubmed
,
Echinobase
Casey,
Sensors and regulators of intracellular pH.
2010,
Pubmed
Chiri,
Evidence for MAP kinase activation during mitotic division.
1998,
Pubmed
,
Echinobase
Davies,
Specificity and mechanism of action of some commonly used protein kinase inhibitors.
2000,
Pubmed
Edgecombe,
A cyclin-abundance cycle-independent p34cdc2 tyrosine phosphorylation cycle in early sea urchin embryos.
1991,
Pubmed
,
Echinobase
Epel,
Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes.
1978,
Pubmed
,
Echinobase
Frémin,
Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.
2009,
Pubmed
Grainger,
Intracellular pH controls protein synthesis rate in the sea urchine egg and early embryo.
1979,
Pubmed
,
Echinobase
Grynkiewicz,
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
1985,
Pubmed
Haccard,
Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase.
1993,
Pubmed
Hamaguchi,
Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs.
2001,
Pubmed
,
Echinobase
Hara,
Start of the embryonic cell cycle is dually locked in unfertilized starfish eggs.
2009,
Pubmed
,
Echinobase
Harrison,
Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos.
2002,
Pubmed
,
Echinobase
Huang,
Differentiation impairs low pH-induced Ca2+ signaling and ERK phosphorylation in granule precursor tumour cells.
2009,
Pubmed
Iwamoto,
Forefront of Na+/Ca2+ exchanger studies: molecular pharmacology of Na+/Ca2+ exchange inhibitors.
2004,
Pubmed
Johnson,
Intracellular pH of sea urchin eggs measured by the dimethyloxazolidinedione (DMO) method.
1981,
Pubmed
,
Echinobase
Kishimoto,
Cell-cycle control during meiotic maturation.
2003,
Pubmed
Kleyman,
Amiloride and its analogs as tools in the study of ion transport.
1988,
Pubmed
Kumano,
Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.
2001,
Pubmed
,
Echinobase
Liu,
The physiology of bicarbonate transporters in mammalian reproduction.
2012,
Pubmed
Lopo,
The rise and fall of intracellular pH of sea urchin eggs after fertilisation.
1977,
Pubmed
,
Echinobase
Machaca,
Ca2+ signaling differentiation during oocyte maturation.
2007,
Pubmed
Malo,
Physiological role and regulation of the Na+/H+ exchanger.
2006,
Pubmed
Martin,
Early development and molecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO2-driven acidification.
2011,
Pubmed
,
Echinobase
McCarty,
Manipulating tumor acidification as a cancer treatment strategy.
2010,
Pubmed
Mukai,
Effects of extracellular acidic-alkaline stresses on trigeminal ganglion neurons in the mouse embryo in vivo.
2011,
Pubmed
Murakami,
Analysis of the early embryonic cell cycles of Xenopus; regulation of cell cycle length by Xe-wee1 and Mos.
1998,
Pubmed
Patel,
Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells.
1999,
Pubmed
Payan,
Mechanisms regulating intracellular pH in sea urchin eggs.
1983,
Pubmed
,
Echinobase
Philipova,
Inhibiting MAP kinase activity prevents calcium transients and mitosis entry in early sea urchin embryos.
2005,
Pubmed
,
Echinobase
Place,
Effects of seawater acidification on cell cycle control mechanisms in Strongylocentrotus purpuratus embryos.
2012,
Pubmed
,
Echinobase
Putney,
Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition.
2003,
Pubmed
Rees,
Protein synthesis increases after fertilization of sea urchin eggs in the absence of an increase in intracellular pH.
1995,
Pubmed
,
Echinobase
Roderick,
Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival.
2008,
Pubmed
Ruknudin,
The regulation of the Na/Ca exchanger and plasmalemmal Ca2+ ATPase by other proteins.
2007,
Pubmed
Santo-Domingo,
The plasma membrane Na+/Ca2+ exchange inhibitor KB-R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter.
2007,
Pubmed
Sato,
Bepridil, an antiarrhythmic drug, opens mitochondrial KATP channels, blocks sarcolemmal KATP channels, and confers cardioprotection.
2006,
Pubmed
Schreiber,
Ca2+ signaling, intracellular pH and cell volume in cell proliferation.
2005,
Pubmed
Shaul,
The MEK/ERK cascade: from signaling specificity to diverse functions.
2007,
Pubmed
Soliman,
Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression.
2009,
Pubmed
Stumpp,
Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification.
2012,
Pubmed
,
Echinobase
Sunda,
Eutrophication induced CO₂-acidification of subsurface coastal waters: interactive effects of temperature, salinity, and atmospheric PCO₂.
2012,
Pubmed
Suprynowicz,
Fluctuation of the Ca-sequestering activity of permeabilized sea urchin embryos during the cell cycle.
1985,
Pubmed
,
Echinobase
Toyoshima,
How Ca2+-ATPase pumps ions across the sarcoplasmic reticulum membrane.
2009,
Pubmed
Tseng,
Matriptase activation, an early cellular response to acidosis.
2010,
Pubmed
Uhlén,
Biochemistry of calcium oscillations.
2010,
Pubmed
Vaughan-Jones,
Intracellular pH regulation in heart.
2009,
Pubmed
Whitaker,
Calcium microdomains and cell cycle control.
2006,
Pubmed
Wilding,
Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.
1996,
Pubmed
,
Echinobase
Wu,
Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.
2008,
Pubmed
Yue,
Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts.
2004,
Pubmed
Zhang,
Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca2+ -dependent pathway but without completion of S phase.
2006,
Pubmed
,
Echinobase
Zhang,
A MAPK pathway is involved in the control of mitosis after fertilization of the sea urchin egg.
2005,
Pubmed
,
Echinobase
Zorec,
Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy.
1993,
Pubmed
