Wieger L. Homan

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or α, α′-dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.

Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos

Previously, we have shown that the monomeric-sugar composition of cell-surface-associated glycoconjugates of two strains of Chlamydomonas eugametos, of different mating type, differs strikingly (Gerwig et al. 1984, Carbohydr. Res. 127, 245–251). Besides the common occurrence of various pentoses and hexoses, the glycoconjugates of one strain contain 4-O-methyl xylose, a 2-O-methyl pentose (probably 2-O-methyl arabinose) and 3-O-methyl galactose, whereas those of the other strain contain 6-O-methyl mannose and 3-O-methyl glucose. In order to investigate whether these differences are relevant to the mating process of this organism, the sugar composition of the sexual progeny of these strains was analyzed. The ability to produce 4-O-methyl xylose, 2-O-methyl pentose and 3-O-methyl galactose on the one hand, and the ability to produce 6-O-methyl mannose and 3-O-methyl glucose on the other hand, appear to be genetically linked. However, the ability to produce either set of O-methyl sugars was inherited independently of mating type. O-Methylated sugars do not occur in the cell wall of C. eugametos, or in the cell-free medium, but only in surface-membrane-associated glycoconjugates, extractable with salt or detergent solutions.

Isolation of monovalent sexual binding components from Chlamydomonas eugametos flagellar membranes

Several treatments were tested to extract the sexual binding site from membrane vesicles derived from the flagellar surface of Chlamydomonas eugametos. Extraction with detergents, chaotropic and hydrogen bond-disrupting agents, as well as sonication, was effective in reducing the isoagglutination activity of these membrane vesicles. Complementary with this reduction, a sex-specific biological activity related to isoagglutination, called “twitch activity” appeared in the extract. This was only observed with vesicles derived from minus mating type (mt-) gametes. After fractionation of the extract, one high-molecular weight glycoprotein fraction appeared to be responsible for this activity. When extracts were treated with cross-linking agents, a pelletable fraction was obtained with isoagglutinative activity. We conclude that the mt- factor, responsible for twitch activity, causes isoagglutination when it is rendered multivalent.

Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.

Transport of membrane receptors and the mechanics of sexual cell fusion in Chlamydomonas eugametos

During sexual reproduction in the heterothallic, biflagellate, green alga Chlamydomonas, gametes adhere together via their agglutinins, sex‐specific glycoproteins extrinsically bound to the flagellar membrane. Using an antibody specific for a C. eugametos agglutinin, we illustrate that agglutinins engaged in adhesion are transported to the flagellar tips. This tipping phenomenon, together with a particular orientation of the flagella, forms part of the mechanism by which gametes position themselves properly for fusion in pairs.

Cell-cell adhesion in conjugatingChlamydomonas gametes: a self-enhancing process

SummaryThe flagellar adhesiveness of gametes ofChlamydomonas eugametos increases during conjugation such that the cell-cell contacts are intensified. The rise in adhesiveness is due to an increase in agglutinin exposure which can be visualized by immunolabeling. The adhesiveness in the one cell is stimulated by the agglutinins of the adherent partner cell, and vice versa. Thus, sexual cell-cell adhesion is a self-enhancing process. In addition, it is shown that the gametes are able to activate potential partners at distance via agglutinin-rich vesicles which they shed into their environment.

Reconstitution of biological activity in isoagglutinins fromChlamydomonas eugametos

Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt- isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt- isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt+ gametes could be reactivated to gainmt- properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt- isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.

Characteristic differences in monosaccharide composition of glycoconjugates from opposite mating types of Chlamydomonas eugametos

Abstract The monosaccharide composition of the isoagglutinins derived from the flagellar membranes of the opposite mating types of Chlamydomonas eugametos (mt+ and mt−) differ strikingly. Besides the common occurrence of l -arabinose, l -rhamnose, d -xylose, d -mannose, d -galactose, l -galactose, d -glucose, and 2-acetamido-2-deoxy- d -glucose, the mt+ isoagglutinin carbohydrate chains contain gulose, 4-O-methyxylose, a 2-O-methylpentose, and 3-O-methyl- d -galactose, whereas those of the mt− isoagglutinin contain 6-O-methyl- d -mannose and 3-O-methyl- l -glucose. Monosaccharide constituents were analysed by g.l.c. and g.l.c.-m.s. of trimethylsilylated methyl glycosides and alditol acetates. For the assignment of the absolute configurations using g.l.c., trimethylsilylated (−)-2-butyl glycosides were analysed. In view of the absolute specificity of the agglutination process displayed by the isoagglutinins of the sexually different mating types, the carbohydrate differences might be significant for the sexual interaction.

Endogenous oscillator controls surface density of mating receptors in Chlamydomonas eugametos

SummaryWe describe a circadian rhythm in the surface density of receptors that play a dominant role in the mating process of the unicellular green alga Chlamydomonas eugametos.These receptors — called agglutinins — are large glycoproteins extrinsically bound to the membrane of gamete flagella. We found circadian fluctuations in their density. Since inhibition of protein synthesis affected the agglutinin density without a lag period at any time,we conclude that the density was dependent on de novo synthesis and that the fluctuations in density are caused by circadian oscillations in the rate of agglutinin synthesis. This phenomenon evidently underlies the pronounced endogenous rhythm in mating competence that we described previously (Demets et al. 1987). Finally, we speculate on the nature of the time keeping mechanism that is generating these rhythmic events.

Fusion-defective mutants ofChlamydomonas eugametos

SummaryMutant strains of the unicellular green algaChlamydomonas eugametos are described which are defective in sexual fusion. All mutants are mating type plus (mt+). They are unable to fuse because none of them is capable of protruding a mating structure through the cell wall, neither during sexual agglutination nor after adding dibutyryl-cAMP or compounds that raise the intracellular calcium level, treatments that are effective in wild type cells. Evidence is presented that these mutants lack the lytic enzyme activity which is normally involved in the local hydrolysis of the cell wall to allow the protrusion of the mating structure. Furthermore, a simple light microscopic method is presented to determine the presence of activated mating structures.