Genetic control of plasma membrane adhesion and fusion in Chlamydomonas gametes.

The final stages of gamete interaction in Chlamydomonas reinhardtii occur between the specialized plasma membrane organelles (mating structures) of opposite mating-type cells. A number of mutants affecting these interactions have been obtained in several laboratories; all have been defined as fusion-defective. This paper demonstrates that there are at least two genetically definable stages of mating-structure interaction: (1) the recognition/adhesion of mating structures, and (2) gamete fusion. The possibility that these two functions, the adhesion and subsequent fusion of mating structures, may be mating-type specific is raised, since of the seven independently isolated mutants analysed, all mutants defective in fusion are of mating-type (-), and only mating type (+) mutants show defects in adhesion.

Specific contact between mating structure membranes observed in conditional fusion-defective Chlamydomonas mutants.

Mating interactions between opposite mating type gametes in Chlamydomonas reinhardi can be conditionally manipulated by the use of mutants which control different stages in this process. Two independently isolated, sex-limited mutants (expressed only in mating type (-) (mt-] gam-10 and gam-11, have been shown to display a temperature-sensitive defect in mating. Gametes of these mutants can agglutinate, signal, and participate in a specific contact between their activated mating structures and the mating structures of wild-type mt+ gametes but are unable to fuse at the restrictive temperature. Studies of these mutants have resulted in a clear demonstration of this mating type-specific interaction between activated mating structures in this organism. This contact is trypsin-sensitive and therefore appears to involve protein. Both this interaction, and the defect in mating structure fusion, can be further analysed using these mutants.

Mutational disruption of the 9 + 2 structure of the axoneme of Chlamydomonas flagella.

A mutant of Chlamydomonas, gam-5, which has short but variable-length, paralysed flagella has been characterized using electron microscopy and genetics. The flagellar axoneme shows varying degrees of disorganization of its 9 + 2 structure, and the structures that attach to the microtubule pairs may be wholly or partly missing or, if present, abnormal. Flagellar length is inversely related to temperature, and gametes of this mutant are generally incapable of mating at higher temperatures. This pleiotropic mutation shows Mendelian (2:2) segregation and is unlinked to the mating-type locus. It is suggested that gam-5 leads to the production of a thermal-sensitive flagellar component, which results in increased assembly and/or maintenance difficulties as temperature is increased.

Membrane-membrane and membrane-ligand interactions in Chlamydomonas mating.

Our investigations of the mating reaction of Chlamydomonas revealed a surprisingly intricate series of interrelated events. Adhering sites are moved to the flagellar tips in a fashion highly reminiscent of the capping of surface ligands over the centriolar regions of lymphocytes (28). Tipping is prevented by the gam-1 mutation and by agents that interact with tubulin; the molecular mechanism(s) for the inhibition effects are currently being sought. Tip locking appears to be accompanied by the accumulation of a dense material beneath the tip membrane, a postulated alteration of axonemal structure, and an immobilization of component(s) involved in surface motility. Two mating signals are then transduced to the locked-in cells who respond by shedding cell walls, activating mating structures, and fusing together. Signal transmission and/or reception is sensitive to such agents as trypsin, chymotrypsin, and cold temperature. Once zygotic cell fusion has occurred, tip unlocking and a reversal of the tip activation response appear to occur in parallel. Since all of these events can occur within 30 sec, the mating reaction serves as an experimental paradigm for studying rapid cellular responses to specific membrane-membrane interactions.

Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.

The temperature-sensitive gametogenesis-defective mutant, gam-1 is sex-limited, expressed only in mating type minus (mt-), and can sexually agglutinate but not fuse at the restrictive temperature (35 degrees C) with gametes of wild type (wt) mt+. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gam-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35 degrees C, each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gam-1 flagella. The two mutant phenotypes displayed by gam-1 have been denoted gam-1-I and gam-1-II. An agglutination reaction involving gam-1-I cells does not result in activation of the wt mt+ mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-1-II cells, but because the gam-1 mt- mating sturcture still fails to activate, cell fusion is precluded. We conclude that the gam-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.

A selection procedure for obtaining conditional gametogenic mutants using a photosynthetically incompetent strain of Chlamydomonas reinhardi.

A new selection procedure has beed developed for the isolation of temperature sensitive gametogenic mutants in Chlamydomonas reinhardi. This technique makes use of pet-10-1, a mutant strain incapable of photosynthetic electron transport, and the redox dye, methyl viologen. Two mutants found by this method are discussed. The first, gam-4, does not agglutinate at 35 degrees, whereas the second, gam-5, is capable of sexual agglutination, but not of zygote formation at the restrictive temperature. Both mutants appear to be inherited in a Mendelian fashion and are expressed in mating type (+) and mating type (-).

Selection for conditional gametogenesis in Chlamydomonas reinhardi.

A new technique is described for selection of temperature-sensitive mutants affecting gametogenesis in Chlamydomonas reinhardi. The first mutant found by this technique is characterized. Cells exhibiting the gam-1 phenotype are capable of sexual agglutination, but cannot form zygotes at the restrictive temperature. The mutation, however, has been expressed only in gametes of mating type (-). Cells of mating type (+) which carry this gene are able to engage in normal zygote formation. Temperature shift experiments and antibiotic studies have indicated that this gene is expressed within 6 hr after the onset of gametogenesis in liquid culture, and its product has a lifetime of about 4 hr at 35 degrees.