Mos limits the number of meiotic divisions in urochordate eggs.

Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca(2+) oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.

Inactivation of MAPK in mature oocytes triggers progression into mitosis via a Ca2+ -dependent pathway but without completion of S phase.

Unfertilized sea urchin eggs that are arrested at G1 phase after completion of meiosis contain a highly phosphorylated mitogen-activated protein (MAP) kinase (MAPK), the ERK-like protein (ERK-LP). Several data including our previous results show that ERK-LP is inactivated after fertilization, which agrees with results obtained in other species including Xenopus, starfish and mammals. The question is to elucidate the function of a high MAPK activity in sea urchin eggs. We report here that dephosphorylation of ERK-LP with very low concentrations of two MEK inhibitors, PD98059 or U0126, triggers entry into mitosis. Under these conditions, recurrent oscillations of the phosphorylation of ERK-LP and of a tyrosine residue in Cdc2 occur, and the intracellular Ca2+ level (Ca2+ i) progressively and slowly increases. Nuclear envelope breakdown and all mitotic events initiated after dephosphorylation of ERK-LP are inhibited when changes in Ca2+ i are prevented; however, they are independent of the intracellular pH. These results suggest that inactivation of a MEK-ERK pathway, normally induced after fertilization of sea urchin eggs, triggers entry into mitosis by altering Ca2+ i but cannot trigger full DNA replication. We discuss the hypothesis that neither inactivation nor activation of a MEK-ERK pathway is required for S phase completion in sea urchin egg.

Jupiter, a new Drosophila protein associated with microtubules.

In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

A MAPK pathway is involved in the control of mitosis after fertilization of the sea urchin egg.

Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos. We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea urchin embryo.

Two anti-radial spoke monoclonal antibodies inhibit Chlamydomonas axonemal motility by different mechanisms.

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

Effects of methoxychlor, dieldrin and lindane on sea urchin fertilization and early development.

We have studied the effects of methoxychlor (MXC), dieldrin, and lindane on fertilization and early development of sea urchin egg. These organochlorine pesticides have often been found in polluted ground and water near agricultural sites, and have therefore been detected from time to time in the food chain and in drinking water. They have been reported to alter various reproduction functions in various animals including marine populations. We observed that the rate of fertilization decreased when the sperm was incubated with dieldrin or lindane. Treatment of eggs with each pesticide did not prevent fertilization, but increased the rate in polyspermy, delayed or blocked the first mitotic divisions, and altered early embryonic development. Moreover, all pesticides could alter several intracellular biochemical pathways that control first mitotic divisions and early development, including intracellular calcium homeostasis, MPF (mitosis promoting factor) activity and formation of the bipolar mitotic spindle. We found that lindane was the most potent of the three pesticides to alter all biochemical events. All these effects were observed at relatively high concentrations. However, bio-accumulation in sediments and aquatic organisms have been reported. Sea urchin eggs may then be in contact with very high concentrations of these pesticides in areas where these pesticides are not handled or stocked properly, and then develop into abnormal embryos.

Differential distribution of glutamylated tubulin isoforms along the sea urchin sperm axoneme.

Tubulin belongs to a highly conserved multigenic family, in which several gene products usually coexist in the same tissue or the same cell. Moreover, seven classes of post-translational modifications of these gene products lead to an amazing diversity of tubulin polypeptide chains, within the same cell type, whose physiological function remains elusive. Such diversity has been found in a very stable microtubular organelle, the sea urchin sperm flagellum, where some tubulin isoforms have been directly implicated in motility, whereas others may play a more structural role. In particular, polyglutamylated tubulin has been shown to be crucial for motility (Gagnon et al., 1996: J Cell Sci 109:1545 p). Here, we show with the GT335 antibody that polyglutamylated tubulin is distributed according to a decreasing gradient along the sea urchin sperm axoneme, since a semi-quantitative measurement of immunofluorescence intensity reveals that in its proximal half, the axoneme is sixfold more labeled than in its distal half. This gradient along the length of the axoneme is confirmed by immunogold labeling procedures which, in addition, demonstrate a uniform distribution of polyglutamylated tubulin among peripheral doublets and a lesser content in the central pair within a same section. Moreover, our data obtained with B3, an antibody that recognizes both mono- and poly-glutamylated tubulin, suggest that the number of glutamate residues in the lateral poly-glutamyl chain of tubulin varies along the whole length of the axoneme. These novel results coupled with those published earlier may be important to understand the role of polyglutamylation in flagellar motility.