Understanding the effects of chronic exposure to pollutants over generations is of primary importance for the protection of humans and the environment; however, to date, knowledge on the molecular mechanisms underlying multigenerational adverse effects is scarce. We employed a systems biology approach to analyze effects of chronic exposure to gamma radiation at molecular, tissue and individual levels in the nematode Caenorhabditis elegans. Our data show a decrease of 23% in the number of offspring on the first generation F0 and more than 40% in subsequent generations F1, F2 and F3. To unveil the impact on the germline, an in-depth analysis of reproductive processes involved in gametes formation was performed for all four generations. We measured a decrease in the number of mitotic germ cells accompanied by increased cell-cycle arrest in the distal part of the gonad. Further impact on the germline was manifested by decreased sperm quantity and quality. In order to obtain insight in the molecular mechanisms leading to decreased fecundity, gene expression was investigated via whole genome RNA sequencing. The transcriptomic analysis revealed modulation of transcription factors, as well as genes involved in stress response, unfolded protein response, lipid metabolism and reproduction. Furthermore, a drastic increase in the number of differentially expressed genes involved in defense response was measured in the last two generations, suggesting a cumulative stress effect of ionizing radiation exposure. Transcription factor binding site enrichment analysis and the use of transgenic strain identified daf-16/FOXO as a master regulator of genes differentially expressed in response to radiation. The presented data provide new knowledge with respect to the molecular mechanisms involved in reproductive toxic effects and accumulated stress resulting from multigenerational exposure to ionizing radiation.
X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34cdc2 but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1. X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34cdc2 but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1. In Xenopus laevis, oocytes are naturally arrested at the G2/M transition of meiosis I. Upon release of progesterone by the follicle cells surrounding the oocytes, maturation occurs and the cell cycle resumes. Translation of the proto-oncogene c-Mos (a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown. kinase kinase), is an essential preliminary event (2Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar, 3Kanki J.P. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5794-5798Crossref PubMed Scopus (71) Google Scholar) in turning on the MAPK cascade, which eventually results in almost simultaneous activation of MAPK and maturation-promoting factor (MPF), a heterodimer composed of p34cdc2 kinase and cyclin B (4Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Dorée M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (375) Google Scholar, 5Gautier J. Maller J.L. EMBO J. 1991; 10: 177-182Crossref PubMed Scopus (155) Google Scholar). Eggs eventually arrest again at metaphase of second meiosis with high MPF and cytostatic factor (in which c-Mos participates (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar)) activities until fertilization. Conversion of inactive pre-MPF complexes, stored in oocytes, into active MPF requires dephosphorylation of residues Thr14 and Tyr15 on p34cdc2 kinase (7Strausfeld U. Labbé J.C. Fesquet D. Cavadore J.C. Picard A. Sadhu K. Russell P. Dorée M. Nature. 1991; 351: 242-245Crossref PubMed Scopus (443) Google Scholar). Dephosphorylation of these amino acids is crucial for MPF activation, and this process is highly regulated by an activating phosphatase, Cdc25 (for review see Ref. 8Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), and two inhibitory kinases, Myt1 (9Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar) and Wee1 (10Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (268) Google Scholar).Although oocyte maturation has been extensively studied over the past 10 years as a model to understand the mechanisms involved in reentry into the cell cycle, the signal transduction pathway between progesterone binding to its receptor and the activation of the MAPK cascade and MPF is ill understood. Protein kinase A (PKA) appears to be a crucial player in these events, because activation of progesterone receptor is followed by a sudden drop in cAMP concentration (11Speaker M.C. Butcher F.R. Nature. 1977; 267: 848-849Crossref PubMed Scopus (105) Google Scholar) and likely a subsequent inactivation of protein kinase A. Indeed, microinjection of the PKA catalytic subunit prevents progesterone-induced maturation in oocytes, whereas the expression of the PKA regulatory subunit is sufficient to induce maturation (12Maller J. Krebs E. J. Biol. Chem. 1977; 252: 1712-1718Abstract Full Text PDF PubMed Google Scholar). Matten et al. (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar) reported that PKA negatively regulates maturation by controlling both c-Mos translation and Cdc25 activation. How PKA acts on c-Mos de novo translation is, however, unclear, and we have demonstrated that in conditions in oocytes in which activation of MAPK is allowed, PKA does not inhibit the c-Mos translation (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar), raising the possibility that PKA could act on a single target in the negative regulation of maturation.A number of studies demonstrate the importance of the MAPK cascade in MPF activation (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar, 15Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 9: 2131-2138Crossref Scopus (189) Google Scholar) during Xenopus oocyte maturation. Indeed, microinjection of recombinant c-Mos is itself sufficient to induce oocyte maturation in the absence of hormonal treatment (16Yew N. Mellini M.L. Vande Woude G.F. Nature. 1992; 355: 649-652Crossref PubMed Scopus (202) Google Scholar, 17Sagata N. Daar I. Oskarsson M. Showalter S.D. Vande Woude G.F. Science. 1989; 245: 643-646Crossref PubMed Scopus (249) Google Scholar), whereas CL100 (MAPK phosphatase-1) inhibits progesterone-induced maturation (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Important work in Saccharomyces cerevisiaetoward the understanding of the control of the MAPK cascade has been undertaken in the last few years. The response to the mating pheromone signal is mediated by the direct interaction of the Gβ subunit of G protein heterotrimers to Ste20, a serine threonine kinase (19Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-194Crossref PubMed Scopus (183) Google Scholar). Activation of Ste20 results in activation of the MAPK kinase kinase Ste11, which allows the MAPK cascade to be turned on and results in MAPK (Kss1 and Fus3) activation (reviewed in Ref. 20Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 162-167Crossref Scopus (188) Google Scholar). InXenopus oocytes, Gotoh et al. (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) showed that microinjection of constitutively active Ste11 is able to efficiently induce the maturation process. Moreover, addition of the N-terminal regulatory domain of Ste11 to Xenopus extracts prevents a constitutive form of Ste20 from activating MAPK (21Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).p21-activated kinases (PAKs) are Ste20 homologues. Understanding of their functional importance in signal transduction and in regulation of the actin cytoskeleton is rapidly growing (see Refs. 22Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 23Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Manser E.H.Y. Huang T.H. Loo X.Q. Chen J.M. Dong T. Leung L. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). We have cloned Xenopus PAKs and recently demonstrated that a member of this family is involved in regulation of the oocyte maturation process (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). Indeed, we showed that microinjection of a constitutive truncated version of X-PAK1 (X-PAK1-Cter) into oocytes completely blocks both insulin and progesterone mediated maturation, and conversely, the dominant negative version of the kinase facilitated this process. In this present report, we focus on how X-PAK1 negatively regulates maturation. Our results demonstrate that X-PAK1-Cter prevents c-Mos synthesis and MAPK activation in progesterone-treated oocytes. Even if MAPK is activated by injection of recombinant c-Mos, no H1 kinase activity can be detected. All the enzymes involved in regulation of p34cdc2 kinase are affected by X-PAK1-Cter expression in progesterone-treated oocytes. We show that in the presence of X-PAK1 kinase activity, pre-MPF complexes can efficiently form but activation of pre-MPF fails. Furthermore, in the presence of active MPF, X-PAK1 kinase activity prevents the amplification loop from being switched on. We have analyzed a possible link of transduction pathways used by PKA and X-PAK in controlling maturation and show that X-PAK1-Cter is still capable of inhibiting maturation induced by the regulatory domain of PKA. Finally, using cell-free Xenopus egg cycling extracts, which mimic the alternation of S and M phases of the first embryonic cycles, we can reproduce the inhibition of entry into mitosis with the catalytic domain of X-PAK1. Thus the involvement of XenopusPAK in G2/M transition is not restricted to oocyte meiotic maturation and can be extended to the first embryonic cell cycles.DISCUSSIONOocyte maturation is a highly regulated process, and a complete understanding of the many intertwining regulations involved is far from being a reality. GVBD is always associated with high cyclin B-p34cdc2 activity, which results from the activation of stockpiled complexes, occurring by a well described amplification loop. Although experimental conditions have been described in which GVBD can occur in the absence of MAPK activity (34Rime H. Haccard O. Ozon R. Dev. Biol. 1992; 151: 105-110Crossref PubMed Scopus (43) Google Scholar), it is widely accepted that the MAPK cascade participates in normal maturation in the activation of MPF. Understanding the event required for activation of the first cyclin B-p34cdc2 complex, which in turn will start the amplification loop, is crucial to explain the maturation process, and in this regard identification of new regulatory proteins will certainly help to reconstitute the sequence of activation of the different pathways.In this report we investigate how constitutive Xenopusp21-activated kinase negatively controls the oocyte maturation process. Our results indicate that X-PAK1 mimics the effects previously reported for the catalytic subunit of protein kinase A by preventing c-Mos accumulation as well as MAPK and MPF activation (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar, 14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar). We show, in a context in which MAPK activation is activated by injection of c-Mos, that accumulation of the endogenous proto-oncogene does occur, in the presence of X-PAK1. Thus X-PAK1 does not interfere with the amplification loop in which active MAPK controls c-Mos synthesis (42Matten W.T. Copeland T.D. Ahn N.G. Vande Woude G.F. Dev. Biol. 1996; 179: 485-492Crossref PubMed Scopus (88) Google Scholar) but rather prevents the initial MAPK activation. Even on c-Mos microinjection, no H1 kinase activity is detected, indicating that X-PAK1 also affects MPF activation. We used recombinant cyclin B, and active MPF, to demonstrate that formation of new pre-MPF complexes is not affected but that they are not activated in the presence of active X-PAK1.Indeed, we show that the regulatory kinases and Cdc25 phosphatase identified in MPF amplification during the maturation process remain in their initial state. A well accepted concept is that activation of the first MPF molecules is the result of activation and/or inactivation of Cdc25 and Myt1, respectively, by an upstream regulating element different from MPF itself. Indeed, in SDS-PAGE, Cdc25 displays a complex phosphorylation pattern and can also be phosphorylated and activated by kinases other than MPF itself, Plx-1 (36Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar), and possibly Raf-1(43).Raf-1 may be a Cdc25-activating kinase (43Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar), and it also acts as a MAPK kinase kinase (44Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar) whose involvement in regulation of maturation is subject to controversy. Using a dominant negative mutant approach it has been reported (45Muslin A.J. Mac Nicol A. Williams L.T. Mol. Cell. Biol. 1993; 13: 4197-4202Crossref PubMed Scopus (98) Google Scholar) that Raf-1 activation is required for progesterone-induced maturation, but this result was challenged (46Fabian J.R. Morrison D.K. Daar I. J. Cell Biol. 1993; 122: 645-652Crossref PubMed Scopus (119) Google Scholar). It was recently shown that autophosphorylation of Raf-1 on serine 621 (47Mischak H. Seitz T. Janosch P.K. Enlitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar) is a way to down-regulate its activity, and this same site can also be phosphorylated by PKA. We discussed in a previous paper a possible transient activation of Raf-1 in Xenopus oocytes, upon down-regulation of PKA, during normal maturation induced by progesterone (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar). Because Raf-1 mobility shift observed during maturation by progesterone is usually equated to the kinase activation, we verified that Raf-1 is indeed prevented from shifting upon progesterone treatment of oocytes injected with X-PAK1-Cter (data not shown). As X-PAK1 belongs to the well known family of PAK proteins that were first isolated as upstream regulators of MAPK cascades, involvement of X-PAK in Raf-1 kinase regulation is an interesting hypothesis.We show that X-PAK1-Cter negatively targets the same events in inhibition of maturation as PKA/C and moreover that X-PAK1-Cter prevents maturation induced by the regulatory subunit of protein kinase A, but we have been unable to demonstrate any direct activation of endogenous X-PAK1 or two other closely related family members X-PAK2 and X-PAK3 by PKA/C. Both pathways could also be linked in a different manner; for example, X-PAK could interfere with the ability of PKA to form a tetramer (for example by phosphorylation of PKA/R) and consequently inhibit its inactivation. Indeed it has previously been demonstrated that PKA/RII can be phosphorylated in vitro by different kinases, and recently Keryer et al. (41Keryer G. Yassenko M. Labbé J.C. Castro A. Lohmann S.M. Evain-Brion D. Taskén K. J. Biol. Chem. 1998; 273: 34594-34602Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) reported that MPF can also phosphorylate it. We verified that neither PKA/R nor PKA/C are substrates of X-PAK1-Cter. At the moment we cannot rule out other possible cross-links in the pathways used by the two kinases; however, another possible explanation of our results would be that both pathways are parallel and that active X-PAK and PKA target an identical substrate that allows the MPF amplification loop to be turned on.Both PKA and X-PAK kinases are also capable of inhibiting the G2/M transition during the first mitotic cycle, at which point cyclin B-p34cdc2 is still regulated by phosphorylation on Tyr15, but control by MAPK activity is absent. We show that X-PAK1-Cter does not prevent cyclin A-p34cdc2 activation in cell-free egg cycling extracts, and in fact the kinase reaches an abnormally high level of activity compared with a control cycling extract supplemented with MalBP fusion protein. Nevertheless, under these conditions, high cyclin A-p34cdc2activity does not allow cyclin B-p34cdc2 activation, and as a consequence the cyclin degradation pathway is not turned on. We show that cyclin B-p34cdc2 complexes are indeed formed, but no H1 activity is associated with them, and the p34cdc2 subunit displays a slow electrophoretic mobility indicative of phosphorylation on Tyr15. We conclude that X-PAK1-Cter activity is very specific because it does not interfere with the activity of cyclin A-p34cdc2 complexes, which are not subject to a regulation by phosphorylation on Tyr15 (30Abrieu A. Brassac T. Galas S. Fisher D. Labbé J.C. Dorée M. J. Cell Sci. 1998; 111: 1751-1757Crossref PubMed Google Scholar, 48Clarke P.R. Leiss D. Pagano M. Karsenti E. EMBO J. 1992; 11: 1751-1761Crossref PubMed Scopus (61) Google Scholar), but it does interfere specifically with the activation of cyclin B-p34cdc2 complexes by preventing dephosphorylation of Tyr15. In 1994 Griecoet al. (49Grieco D. Avvedimento E. Gottesman M.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9896-9900Crossref PubMed Scopus (51) Google Scholar) showed that PKA activity oscillates during the first embryonic cycles in Xenopus extracts, being high at interphase and falling at the onset of mitosis. They demonstrated that sustained PKA activity inhibits cycle progression in a dose-dependent manner and that extracts eventually arrest in interphase with a high level of inactive pre-MPF complexes. In this context, X-PAK1–1Cter and PKA probably act independently of a control over MAPK activity, because MAPK is inactive in the cycling extract, and so this cannot be compared with the G2/M transition block described previously (50Abrieu A. Fisher D. Simon M.N. Dorée M. Picard A. EMBO J. 1997; 21: 6407-6413Crossref Scopus (72) Google Scholar), in which the proto-oncogene c-Mos is added to cycling extracts. The work of Grieco et al. (49Grieco D. Avvedimento E. Gottesman M.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9896-9900Crossref PubMed Scopus (51) Google Scholar) indicates that PKA could stimulate an okadaic acid-sensitive serine/threonine phosphatase pathway that dephosphorylates Cdc25.Plx kinase is another Cdc25 positive regulatory kinase (36Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar) recently shown (30Abrieu A. Brassac T. Galas S. Fisher D. Labbé J.C. Dorée M. J. Cell Sci. 1998; 111: 1751-1757Crossref PubMed Google Scholar) to be required for the G2/M transition during the first mitotic cell cycle in Xenopus eggs. Cyclin B-p34cdc2 enhances Plx activation, which triggers Cdc25 and MPF activation in a positive feedback loop. Adding blocking anti-Plx antibodies to an egg cycling extract suppressed entry into mitosis, although a strong cyclin A-p34cdc2 activity is detected, as we observed ourselves in the X-PAK1-Cter block in cycling extracts. Moreover, pre-MPF complexes also remain inactive and phosphorylated on Tyr15, if Plx is blocked with these antibodies. We did not investigate the status of Plx in cycling extracts incubated with X-PAK1-Cter, but we show that during the maturation process Plx activation is prevented by active Xenopus p21-activated kinase.In summary, our data demonstrate that PKA and X-PAK pathways target the same components in their inhibition of G2/M progression during meiosis as well as in the first mitotic cycle inXenopus and that their transduction pathways are likely to be connected at the point where the MPF amplification loop is turned on. We are currently in the process of isolating physiological substrates of X-PAK active kinase in Xenopus extracts to get new insights into factors regulating the G2/M transition. In Xenopus laevis, oocytes are naturally arrested at the G2/M transition of meiosis I. Upon release of progesterone by the follicle cells surrounding the oocytes, maturation occurs and the cell cycle resumes. Translation of the proto-oncogene c-Mos (a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown. kinase kinase), is an essential preliminary event (2Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar, 3Kanki J.P. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5794-5798Crossref PubMed Scopus (71) Google Scholar) in turning on the MAPK cascade, which eventually results in almost simultaneous activation of MAPK and maturation-promoting factor (MPF), a heterodimer composed of p34cdc2 kinase and cyclin B (4Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Dorée M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (375) Google Scholar, 5Gautier J. Maller J.L. EMBO J. 1991; 10: 177-182Crossref PubMed Scopus (155) Google Scholar). Eggs eventually arrest again at metaphase of second meiosis with high MPF and cytostatic factor (in which c-Mos participates (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar)) activities until fertilization. Conversion of inactive pre-MPF complexes, stored in oocytes, into active MPF requires dephosphorylation of residues Thr14 and Tyr15 on p34cdc2 kinase (7Strausfeld U. Labbé J.C. Fesquet D. Cavadore J.C. Picard A. Sadhu K. Russell P. Dorée M. Nature. 1991; 351: 242-245Crossref PubMed Scopus (443) Google Scholar). Dephosphorylation of these amino acids is crucial for MPF activation, and this process is highly regulated by an activating phosphatase, Cdc25 (for review see Ref. 8Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), and two inhibitory kinases, Myt1 (9Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar) and Wee1 (10Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (268) Google Scholar). Although oocyte maturation has been extensively studied over the past 10 years as a model to understand the mechanisms involved in reentry into the cell cycle, the signal transduction pathway between progesterone binding to its receptor and the activation of the MAPK cascade and MPF is ill understood. Protein kinase A (PKA) appears to be a crucial player in these events, because activation of progesterone receptor is followed by a sudden drop in cAMP concentration (11Speaker M.C. Butcher F.R. Nature. 1977; 267: 848-849Crossref PubMed Scopus (105) Google Scholar) and likely a subsequent inactivation of protein kinase A. Indeed, microinjection of the PKA catalytic subunit prevents progesterone-induced maturation in oocytes, whereas the expression of the PKA regulatory subunit is sufficient to induce maturation (12Maller J. Krebs E. J. Biol. Chem. 1977; 252: 1712-1718Abstract Full Text PDF PubMed Google Scholar). Matten et al. (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar) reported that PKA negatively regulates maturation by controlling both c-Mos translation and Cdc25 activation. How PKA acts on c-Mos de novo translation is, however, unclear, and we have demonstrated that in conditions in oocytes in which activation of MAPK is allowed, PKA does not inhibit the c-Mos translation (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar), raising the possibility that PKA could act on a single target in the negative regulation of maturation. A number of studies demonstrate the importance of the MAPK cascade in MPF activation (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar, 15Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 9: 2131-2138Crossref Scopus (189) Google Scholar) during Xenopus oocyte maturation. Indeed, microinjection of recombinant c-Mos is itself sufficient to induce oocyte maturation in the absence of hormonal treatment (16Yew N. Mellini M.L. Vande Woude G.F. Nature. 1992; 355: 649-652Crossref PubMed Scopus (202) Google Scholar, 17Sagata N. Daar I. Oskarsson M. Showalter S.D. Vande Woude G.F. Science. 1989; 245: 643-646Crossref PubMed Scopus (249) Google Scholar), whereas CL100 (MAPK phosphatase-1) inhibits progesterone-induced maturation (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Important work in Saccharomyces cerevisiaetoward the understanding of the control of the MAPK cascade has been undertaken in the last few years. The response to the mating pheromone signal is mediated by the direct interaction of the Gβ subunit of G protein heterotrimers to Ste20, a serine threonine kinase (19Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-194Crossref PubMed Scopus (183) Google Scholar). Activation of Ste20 results in activation of the MAPK kinase kinase Ste11, which allows the MAPK cascade to be turned on and results in MAPK (Kss1 and Fus3) activation (reviewed in Ref. 20Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 162-167Crossref Scopus (188) Google Scholar). InXenopus oocytes, Gotoh et al. (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) showed that microinjection of constitutively active Ste11 is able to efficiently induce the maturation process. Moreover, addition of the N-terminal regulatory domain of Ste11 to Xenopus extracts prevents a constitutive form of Ste20 from activating MAPK (21Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). p21-activated kinases (PAKs) are Ste20 homologues. 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Ca(2+) channel inactivation is a key element in controlling the level of Ca(2+) entry through voltage-gated Ca(2+) channels. Interaction between the pore-forming alpha(1) subunit and the auxiliary beta subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the beta(2a) subunit strongly reduces alpha(1A) (Ca(V)2.1) Ca(2+) channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the beta(2a) subunit. Inhibition of inactivation by beta(2a) also requires a link between MAS and another important molecular determinant, the beta interaction domain (BID). Our data suggest that mobility of the Ca(2+) channel I-II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the beta(2a) subunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.
The design of experimental protocols that use animal models to assess the impact of a stress on a population or to determine the life span expectancy impact can be time-consuming due to the need for direct observations of dead and living animals. These experiments are usually based on the detectable activity of animals such as food intake or mobility and can sometimes produce either under- or overestimated results. The tardigrade Hypsibius exemplaris is an emerging model for the evolutionary biology of the tardigrade phylum because of its convenient laboratory breeding and the recent introduction of new molecular tools. In this report, we describe the use of a new fluorescent dye that can specifically stain dead tardigrades. Furthermore, we also monitored the absence of a toxic side effect of the death-linked fluorescent dye on tardigrade populations. Finally, we conclude that tardigrade experiments that require survival counting of the Hypsibius exemplaris species can be greatly improved by using this technique in order to limit underestimation of alive animals.
Imiqualines are analogues of the immunomodulatory drug imiquimod. EAPB02303, the lead of the second-generation imiqualines, is characterized by significant anti-tumor effects with IC50s in the nanomolar range. We used
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