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     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

An antiserum against a glycoprotein functional in flagellar adhesion between Chlamydomonas eugametos gametes

Chlamydomonas eugametos gametes agglutinate sexually by their flagellar surfaces. The agglutination factor on mating type minus (mt^-) gametes is thought to be a glycoprotein named PAS-1.2. To test this idea, an antiserum was raised against purified PAS-1.2., which reacted with isolated PAS-1.2 (immunoprecipitation tests) and blocked the ability of isolated PAS-1.2 to induce sexual twitching in mt ⁺ gametes. When tested with living cells, the antiserum specifically agglutinated mt ^- gametes and induced a reaction resembling twitching. Mt ⁺ flagella were shown to bind the antiserum (indirect immunofluorescence) but much less than mt ^- gametes. Mt ^- gametes pretreated with Fab fragments of the antiserum were unable to reproduce sexually, while treated mt ⁺ gametes were unaffected. This effect presumably results from the ability of the serum to block mt ^- sexual agglutination, for mt ^- isoagglutinin was completely inactivated by the serum, while mt ⁺ isoagglutinin was unaffected. It is therefore argued that PAS-1.2 is the in vivo mt ^- agglutination factor. However it is shown that the antiserum was able to react in vitro not only with PAS-1.2 but with several other proteins in both mt ^- and mt ⁺ flagella.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid-Schiff - GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PBS phosphate buffer-saline - Fab univalent antibody fragment 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 (Z.W.O.)     

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     

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     

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     

Agglutination and glycosyltransferase activity of isolated gametic flagella from Chlamydomonas reinhardii

Isolated flagella from gametes of both mating types (mt⁺ and mt^-) of Chlamydomonas reinhardii were suspended in buffer containing 7% sucrose. After mixing instantaneous agglutination occurred, giving rise to clumps which seem to be stable for at least 24 h. Control experiments show that no aggregates are formed when gametic flagella of one mating type are mixed with flagella prepared from vegetative cells of the other mating type.This in vitro agglutination is inhibited by a number of salt solutions in the same concentration range in which the agglutination of live gametes is affected. Moreover the clumps of flagella tend to disaggregate completely when the salt solutions are added after agglutination has occurred, or by treatment with trypsin. These observations suggest that the in vitro agglutination of isolated gametic flagella indeed reflects their physiological role in the recognition step of the mating process, which appears to be possible without participation of live gametes.We have also investigated the activity of glycosyl transferases on isolated gametic flagella before and during the in vitro agglutination reaction. As there was no detectable increase in the activity of glycosyl transferases, our results do not favour the hypothesis that these enzymes are involved in the primary step of recognition between gametic flagella.Dedicated to Prof. Dr. Otto Kandler on the occasion of his 60th birthday     

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     

The relationship of flagellar length to sexual signalling in Chlamydomonas

The flagella of Chlamydomonas reinhardi are required for the initiation of mating between opposite mating type gametes. It has been suggested that flagellar length is a crucial factor in a cell's ability to transmit and receive the sexual signals necessary for fusion. Mating type + (mt⁺) cells of gam-5, a mutant which is characterized by variable length, paralyzed flagella, were mated with wild-type, mt⁻ cells. Activation of the mating structures of the gam-5 gametes, and therefore successful signalling, was demonstrated for cells with flagella as short as 1.5 μm (less than 1/6 normal length). Because this mutant displays aberrant axonemal structures, and because various mutants with other defects in axonemal structure are also able to mate, it seems likely that the flagellar membrane may provide the main conduit for gametic sexual signals.     

Two tunicamycin-sensitive components involved in agglutination and fusion ofChlamydomonas reinhardtii gametes

To find out glycoproteins involved in the mating reaction ofChlamydomonas reinhardtii, the effect of tunicamycin (TM), a potent inhibitor of glycosylation of proteins, was studied. TM, when present during gametogenesis, blocked the acquisition of agglutinability ofmt ⁺ cells. TM also inhibited the recovery of agglutinability ofmt ⁺ gamete after trypsin treatment. On the contrary, TM blocked neither the acquisition of agglutination during gametogenesis ofmt ^- cells nor the recovery of their agglutinability after trypsinization. It was found, however, that the TM-treatedmt ^- gametes can agglutinate but do not fuse with non treatedmt ⁺ gametes at all. When gametes of gam-1mt ^-, a conditional mutant strain for cell fusion, were induced at non permissive temperature of 35°C and then transferred to 25°C, the ability of cell fusion was acquired after about 5 h incubation. Presence of TM completely blocked this acquisition. Based on these evidence, we conclude that at least two TM-sensitive glycoproteins are included in the mating reaction. The first component is located on the flagellar surface ofmt ⁺ gamete and responsible for agglutination withmt ^- flagella. The second component occurs on the surface ofmt ^- gamete and plays a role in the fusion withmt ⁺ gamete.Abbreviations CHI cycloheximide - mt mating type - TM tunicamycin     

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     

Agglutination factor in the cell body of Chlamydomonas eugametos

Chlamydomonas eugametos gametes agglutinate via the surfaces of their flagella. The mating-type minus (mt ^-) agglutination factor is a high-molecular-weight glycoprotein called PAS-1.2, present on the exterior surface of the flagellar membrane. During flagellar regeneration, mt ^- gametes were able to agglutinate as soon as the flagella protruded as short stumps. This was also observed when protein synthesis was blocked, indicating that gametes possess a pool of PAS-1.2. When the exterior surface of flagella-less gametes was extracted and the proteins were subjected to gel electrophoresis, large quantities of PAS-1.2 were detected. Using anti-PAS-1.2 serum, the presence of PAS-1.2-like material was visualized on the plasma membrane of mt ^- gamete cell bodies. By assaying the biological activity of extracts of the cell bodies and of isolated flagella, it was calculated that the plasma membrane of the cell bodies contains 25 times the activity present in the flagella and could, therefore, represent a large pool of mt ^- agglutination factor.     

Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )     

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.     

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     

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.     

Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes

A quantitative assay for cell wall release in Chlamydomonas has been used to study the timing of release of cell wall degrading enzyme (lysin) during adhesion. Lysin activity, which shows a broad pH range and requires divalent cations, is released as a pulse within 1–2 min after mixing of mt^? and mt⁺ gametes. Thereafter, there is no further lysin release. Gametes of both mating types release the activity during aggregation with isolated gametic flagella of the opposite mating type, although mt⁺ gametes appear to release more lysin activity than mt^? gametes. Electrophoretic analysis of cell wall proteins before and after lysin degradation indicate that the major wall proteins are unchanged after wall breakdown.     

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     

Transmission of mitochondrial DNA in crosses involving diploid gametes homozygous or heterozygous for the mating-type locus in Chlamydomonas

Summary The two interfertile algal species Chlamydomonas reinhardtii and C. smithii possess physically distinct mitochondrial (mit) genomes. Recently, use was made of this difference to demonstrate that sexual zygotes transmit the mit DNA from the mating-type minus (mt ^-, or paternal) parent exclusively. Diploid clones homozygous or heterozygous for the mt locus and carrying the mit genome of either of the two species were constructed by sexual crosses or artificially induced fusions. Haploid x diploid and diploid x diploid crosses were performed in order to analyze the role of both the mt locus and ploidy on the mode of transmission of mit DNA to the meiotic progeny. The inheritance of the mit DNA was determined by use of two molecular probes which hybridize to different regions of the organelle genomes. The mt ^u+/mt ^- gametes, which behave as mt ^- in the mating reaction, usually transmit their mit genome to the meiotic progeny, as do mt ^- or mt ^-/mt ^- gametes, regardless of the ploidy of the mt ⁺ gametes. In the cross mt ⁺ x mt ⁺/mt ^- however, 2 zygospore clones (out of 14) transmitted recombinant DNA molecules containing a large segment of the C. reinhardtii mit genome and a 1 kb fragment typical of C. smithii. It can thus be concluded that, contrary to what was observed earlier for chloroplast gene transmission: (1) mt ^- is dominant to mt ⁺with regard to mit DNA transmission, and (2) nuclear ploidy has little, if any, effect on mit DNA transmission.     

–SH and S–S involvement in Chlamydomonas mating

Reagents that block or cross-link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild-type (wt) gametes of mating type + (mt⁺) and mt^?, and a fusion-defective mt^? mutant, gam-11, were studied. Differential sensitivities of mt⁺ vs mt^? and of wt mt^? vs gam-11 mt^? were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt⁺ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt^? gametes. Wt mt^? is more sensitive than wt mt⁺ to inhibition by low concentrations of p-chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane-impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt^?. Gam-11 mt^?, a fusion-defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane-associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt^?. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam-11. The differential inhibition of pair and group formation during gam-11 × wt mt⁺ matings has suggested a possible mechanism for mating structure adhesion.