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 aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

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 , -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.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate     

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.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

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.Abbreviations GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PAS periodic acid Schiff     

Endogenous oscillator controls surface density of mating receptors in Chlamydomonas eugametos

Summary We 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.     

Mating receptor complex in the flagellar membrane of Chlamydomonas eugametos gametes

Summary The protein composition of the flagellar membrane of C. eugametos mt ^–gametes was analyzed using SDS-polyacrylamide gel electrophoresis. The association of the proteins with the membrane was assessed by differential extraction and an assay for glycosylation. Particular attention was paid to integral membrane proteins that could be associated with the mt ^– agglutinin, the membrane-bound sexual receptor by which the mt ^– gamete binds to its mt ⁺ partner. This agglutinin is a peripheral membrane glycoprotein and must be bound to the flagellar surface by an integral membrane anchor protein that connects the agglutinin with the cell's interior. Immunoaffinity chromatography was performed using Mab 66.3, a monoclonal antibody specific for the mt ^– agglutinin, in order to isolate protein complexes consisting of agglutinin molecules and associated components. Only one integral membrane glycoprotein (M_r = 125 kDa) was isolated that has an association with the agglutinin. It did not bind Mab 66.3, but did bind the lectin wheat germ agglutinin. This was an expected property of the membrane anchor protein because previous research (Kooijman et al. 1989) has shown that cross-linking a WGA-binding glycoprotein by this lectin induces sexual responses that are similar to those induced by agglutinin-agglutinin interactions during mating. We conclude that the 125-kDa glycoprotein is the membrane anchor for the agglutinin.Abbreviations BSA Bovine serum albumin - CBB Coomassie Brilliant Blue - CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate - GTC guanidine thiocyanate - mt ^–/mt ⁺ mating type minus/plus - PAS periodic acid Schiff - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TBS TRIS-buffered saline - WGA wheat germ agglutinin     

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.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

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.Abbreviation mt ^+/- mating-type plus or minus     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas 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 fed³²PO 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 of³²PO 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 Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

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.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Gamete activity in mating strains of Chlamydomonas eugametos

The gamete activity of compatible mating strains of the isogamous, heterothallic species Chlamydomonas eugametos was investigated. Gamete activity was optimum within 4 h after flooding of agar slants and was maintained over a 24-h period. When male and female mating strains were mixed in proportions of 1:4, 2:3, 1:1, 3:2, and 4:1, the results based on zygote yield, indicated the strains exhibited different degrees of gamete activity. The male strain consistently showed less gamete activity than the female strain in a variety of culture conditions.     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Concanavalin A binding to Chlamydomonas eugametos flagellar proteins and its effect on sexual reproduction

The relative amounts of Concanavalin A (Con A) bound by gamete and vegetative flagella of both mating types (mt ⁺ and mt ^-) of Chlamydomonas eugametos were determined using ¹²⁵I-Con A. Con A agglutinated all cell types by cross-linking their flagella in a random manner. No correlation was found between the extent of Con A-binding and Con A-mediated isoagglutination. Con A inhibited the sexual interaction between gametes at various levels. In mt ⁺ gametes it blocked sexual agglutination, whereas in mt ^- gametes it prevented papillar fusion. By SDS-gel electrophoresis nine Con A-binding components were found to be present in flagella. However, it was not possible to allocate a role in sexual agglutination to any of these components since they were present in all cell types, including vegetative cells which are not able to sexually agglutinate.Abbreviations Con A concanavalin A - SDS sodium dodecyl sulphate - TB Tris buffer - PBS phosphate buffered saline - HRP horse radish peroxidase - SEM scanning electron microscope - PAS periodic acid Schiff     

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 ³²P-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 ³²P 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.Abbreviations Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PtdA phosphatidic acid - PtdIns phosphatidylinositol - PtdIns(4)P phosphatidylinositol 4-phosphate - PtdIns(4,5)P₂ phosphatidylinositol 4,5-bisphosphate To whom correspondence should be addressedWe thank Dr. P. van Haastert (Biochemistry, University of Groningen, The Netherlands) and his colleagues for introducing us to their Ins(1,4,5)P₃ assay, and Ben ten Brink (Molecular Cell Biology, University of Amsterdam, The Netherlands) for information about contractile vacuoles. We also thank Bas Nagelkerken, Marcel van der Vaart, Pieter van der Schoor, Gyuri Fenyvesi and Susan Kenter for their help.     

Flagellar tip activation in vis-à-vis pairs of Chlamydomonas eugametos

An alteration of the form and ultrastructure of the tips of the flagella of Chlamydomonas eugametos, occurring during sexual agglutination, is shown to be persistent in the mt ^- flagella of the resulting vis-à-vis pairs. It is argued that this phenomenon is related to the lack of motility of mt ^- flagella in vis-à-vis pairs of this species.     

Sex-specific glycoproteins in Chlamydomonas flagella an immunological study

To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Composition and properties of the sexual agglutinins of the flagellated green alga Chlamydomonas eugametos

Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt ⁺ agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·10⁶. The corresponding data for the mt ^- agglutinin were 38 nm, 9.7 S and 1.3·10⁶, respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.Abbreviations HP high performance - mt mating type - SDS sodium dodecyl sulfate