The role of calcium in the Chlamydomonas reinhardtii mating reaction.

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.

Improved media for testing the mating reaction and genetic complementation of Ustilago hordei

The sexual cycle of Ustilago hordei, which results in the formation of teliospores, requires growth on its barley host for completion. However, the early steps of mating, including conjugation and the formation of dikaryotic mycelium, can occur on artificial media. The addition of activated charcoal to a variety of media enhanced the stability and intensity of the mating reaction as measured by mycelium formation. The incubation time at which the strongest mating reaction occurred was also reduced. The dikaryotic nature of the mycelia that resulted from mating on charcoal-containing media was confirmed by fluorescence microscopy. Complementation assays using minimal medium containing activated charcoal demonstrated allelism of mutations in auxotrophic sporidial strains of opposite mating type. The ease and reliability of this mating test allow for rapid identification of the mating type of unknown isolates and progeny of crosses, as well as providing a dependable procedure for performing complementation tests. Key words: barley, covered smut, Hordeum vulgare, mating type.

Monoclonal antibody against a conjugation-specific nuclear antigen in Paramecium caudatum

To understand molecular mechanisms controlling the sexual reproduction process (conjugation) in Paramecium caudatum, we have tried to detect conjugation-specific antigens with monoclonal antibodies. We obtained a monoclonal antibody (CSN-1) against an antigen that appears in the nuclei only during conjugation. This nuclear antigen began to appear both in micro- and macronuclei at micronuclear stage II or III early in the conjugation process (4 h after the mating reaction at 25°C). In the macronucleus, the nuclear antigen persisted until the stage of macronuclear fragmentation (about 35 h) and then disappeared before degeneration of the macronuclear fragments. In the micronucleus, the antigen existed until the crescent stage (stage V) of the first meiotic division (8h). The antigen in the micronucleus disappeared after the crescent stage but reappeared again in the eight nuclei produced by the third postzygotic division (25 h). Then it persisted only in the four macronuclear anlagen differentiated from the eight nuclei (about 30 h). When exconjugant cells had undergone two successive postconjugational cell divisions and thus possessed only one new macronucleus as in the vegetative cells, the antigen disappeared completely from the new macronucleus in most cells. These cells without the antigen began to appear about 50 h after the mating reaction. As the antigen is specific to conjugation and localized in nuclei, it may play some important role(s) in the conjugation process.

A monoclonal antibody that blocks adhesion of Chlamydomonas mt+ gametes.

During the mating reaction (fertilization) in the biflagellated alga, Chlamydomonas reinhardtii, mt+ and mt- gametes adhere to each other via their flagella and subsequently fuse to form quadriflagellated zygotes. In the studies reported here, we describe a monoclonal antibody directed against an mt+ flagellar surface molecule. The antibody blocks the adhesiveness of mt+ gametes, isolated mt+ flagella, and detergent extracts thereof. It has no effect on mt- gametes. Cyanogen bromide-activated Sepharose beads derivatized with the antibody bind only mt+ gametes; mt- gametes and mt+ and mt- vegetative cells are unreactive with the derivatized beads. The interaction of mt+ gametes with the beads is dynamic and cells continuously bind, detach, and rebind to the beads. Surprisingly, antibody-derivatized beads that have been incubated with mt+ gametes acquire the ability to bind mt- gametes. Moreover, extraction of the preincubated beads with detergents releases active mt+ adhesion molecules. The evidence suggests that binding of the antibody to the flagellar surface adhesion molecules causes their release from the flagellar surface, possibly mimicking the normal mechanism of flagellar de-adhesion.