Ethanol stimulates phospholipid turnover and inositol 1,4,5-trisphosphate production in Chlamydomonas eugametos gametes.

Alcohols induce mating-structure activation in Chlamydomonas eugametos gametes. From the effect of ethanol on the (32)P-labelling of polyphosphoinositides, we conclude that the synthesis of these lipids is stimulated. Biologically inactive concentrations of ethanol (<6%) had no effect on synthesis, but 6-8% ethanol stimulated synthesis for upto 60 min. The (32)P incorporated into polyphosphoinositides and phosphatidic acid during ethanol treatment was readily chased out when 1 mM unlabelled Na3PO4 was added. Using a binding assay for inositol 1,4,5-trisphosphate, we show that the production of this phospholipid constituent is dramatically increased after ethanol treatment. This effect, coupled to a rise in intracellular calcium concentration, could explain gamete activation. The significance of these results in explaining other ethanol-induced phenomena in algae is discussed.

Polyphosphoinositol lipids in Chlamydomonas eugametos gametes.

In Chlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed (32)PO 4 (-) , the label was rapidly incorporated into PtdInsP and PtdInsP2 and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of (32)PO 4 (-) was chased with PO 4 (-) , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca(2+) is discussed.

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos.

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.

Wheat germ agglutinin induces mating reactions in Chlamydomonas eugametos by cross-linking agglutinin-associated glycoproteins in the flagellar membrane.

Species-specific binding between the flagellar surfaces of mating types plus and minus (mt+ and mt-) gametes of Chlamydomonas eugametos is mediated by mating type-specific agglutinins. Their interaction triggers several mating responses that are necessary for cell fusion, such as flagellar twitching, flagellar tip activation, redistribution of agglutinin molecules to the flagellar tip (tipping), and mating structure activation. Earlier, we reported that a monoclonal antibody (mAb 66.3) can induce mating reactions by cross-linking the agglutinins (Homan, W. L., A. Musgrave, H. de Nobel, R. Wagter, A. H. J. Kolk, D. de Wit, and H. van den Ende. 1988. J. Cell Biol. 107:177-189). Here we report that the lectin wheat germ agglutinin (WGA), which does not bind to the agglutinins, can also invoke all these mating reactions. We show, by immunofluorescence studies using anti-WGA and an agglutinin-specific monoclonal antibody (mAb 66.3), that WGA induces the redistribution of agglutinin to the flagellar tips of mt- gametes. Vice versa, when agglutinin tipping is induced by mAb 66.3, the WGA-binding glycoproteins are also tipped. Under the same conditions, the major flagellar glycoproteins are not redistributed, indicating that membrane transport is limited to a few components. We conclude that each agglutinin is associated with a WGA-binding glycoprotein. When cells lacking agglutinin or cells possessing inactive agglutinins are treated with WGA, mating responses are again elicited. The data suggest that clustering of agglutinin-containing complexes results in the production of intracellular signals, such as cAMP, and the coupling of the complex to a force generating system. In nature, the complexes are clustered via the agglutinins, but artificially they can be clustered by lectins or antibodies directed against other proteins in the complex.

Light Affects Flagellar Agglutinability in Chlamydomonas eugametos by Modification of the Agglutinin Molecules.

The effect of light on the sexual competence of a light-sensitive mating type minus strain (mt(-)) of Chlamydomonas eugametos obtained by crossing a light-sensitive mating type plus strain (mt(+)) with a light-insensitive mt(-) strain is described. As previously demonstrated for the mt(+) parent, this study of one of the mt(-) offspring shows that (a) a light-sensitive mechanism affects flagellar agglutinability in a rapid process that does not require protein synthesis; (b) only the activity of the flagellar agglutinins (glycoproteins responsible for agglutination) is susceptible to light while agglutinins on the cell body surface are not affected by light. We further demonstrate that (a) membrane vesicles naturally released from nonagglutinable dark gametes remain inactive. Extracts of these vesicles also remain inactive even though they contain agglutinin-like components; (b) inactive mt(-) agglutinin is present in extracts of flagella from nonagglutinable dark gametes by comparison of its chromatographic, electrophoretic, and immunogenic properties with those of active agglutinin. When purified of all other flagellar proteins, it remains inactive; (c) a monoclonal antibody directed against the sexual agglutination site of the mt(-) agglutintin discriminates between active and inactive agglutinins when present in a native state on the flagellar surface, but is unable to discriminate between them when they are denatured in sodium dodecyl sulfate-electrophoresis gels and blotted onto nitrocellulose. Taken collectively these observations suggest that light activation involves the chemical modification of the agglutinins in situ on the flagellar surface.

Light dependence of sexual agglutinability in Chlamydomonas eugametos.

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt (+) and mt (-) strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.

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.

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion.

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt(+) or mt(-)). It is shown that before mt(+) and mt(-) gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt(-) cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt(+) gametes constituting the outer surface of the clumps with the mt(-) gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt(+) flagellar tips then deactivate and swimming is resumed. The original mt(-) flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt(-) gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregation.

The cell wall of Chlamydomonas eugametos. Immunological aspects.

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L.

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 x 10(5) daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with beta-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.