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     

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     

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     

TheChlamydomonas plus and minus agglutinin: difference in sensitivity to dithiothreitol

The effect of disulfide-reducing agent dithiothreitol (DTT) on the plus and minus agglutinins ofChlamydomonas reinhardtii gametes was studied. Live gametes of mating-type plus (mt ⁺) lost their flagellar agglutinability by DTT treatment without any loss of cell motility and concurrently released into the medium agglutinin in an inactive form. DTT treated cells also lost completely their cell body-agglutinin. By contrast, the mating-type minus (mt ^-) gametes neither lost their agglutinability nor released agglutinin into the medium by DTT even at very high concentrations. In vitro experiments showed that plus agglutinin in solution is just as sensitive as that in vivo to DTT, whereas minus agglutinin is totally insensitive, and the sulfhydryl-oxidizing agent diamide restores the plus agglutinin activity immediately and completely. Isolated flagella from themt ⁺ gametes were also inactivated by DTT, but they retained the inactivated agglutinin on the surfaces. The results indicate that plus agglutinin, but not minus agglutinin, possesses disulfide bonds which are essential for the recognition/adhesion activity.Abbreviations mt ^+/- mating-type plus or minus - DTT dithiothreitol     

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     

Cell surface differentiation of Chlamydomonas during gametogenesis. I. Mating and concanavalin A agglutinability

Sexual agglutination of opposite gametes and agglutination of like gametes by concanavalin A (Con A) were studied in Chlamydomonas moewusii and C. reinhardtii for the possibility that the surface sites involved in these agglutinating phenomena may be the same. Our data show that the two agglutinating phenomena appeared at different times after the beginning of gametogenesis. Sucrose and mannose block agglutination of the gametes by Con A but do not affect mating ability. Trypsin eliminates mating ability, except in (?) gametes of C. moewusii, while Con A agglutinability remains. Monovalent Con A can selectively bind to Con A-binding sites to block agglutination of gametes by multivalent Con A while mating ability is unaffected. The data indicate that the mating agglutinin and the Con A-binding sites are two different flagellar surface agglutinins that occur coincidentally during gametic differentiation of both mating types of C. moewusii and C. reinhardtii. The function of the Con A-binding site on Chlamydomonas gametes is not known.     

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     

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     

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     

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).     

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 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.     

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.     

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.)     

Ultrastructure and properties of the sexual agglutinins of the biflagellate green alga Chlamydomonas moewusii

Summary The mt^– agglutinins of the interfertile species Chlamydomonas moewusii and Chlamydomonas eugametos are very similar fibrous molecules. The mt^– agglutinin of C. moewusii has the same Stokes radius (39 nm) and sedimentation coefficient (9.3 S) as its counterpart in C. eugametos; its length (336 nm) and its ultrastructure, including the position of four kinks are also the same as in C. eugametos. The sugar compositions of both agglutinins are very similar, and they react equally well with the monoclonal antibody Mab 66.3 raised against the mt^– agglutinin of C. eugametos. Finally, they are equally thermoresistant, with half-lives at 100 °C of 50 min (C. moewusii) and 57 min (C. eugametos). The mt⁺ agglutinins of both species are different. Both are fibrous molecules with a terminal head, but the fibrous part of the molecule in C. moewusii is shorter (210 nm compared to 276 nm). The mt⁺ agglutinin of C. moewusii is also significantly more sensitive to heating with a half-life of 6 min at 40 °C compared to the 20 min shown by the mt⁺ agglutinin of C. eugametos. Their sugar compositions are, however, very similar, and they react equally well with Mab 66.3. The mt⁺ agglutinin of C. moewusii is sensitive to denaturing reagents and proteolytic attack, whereas the mt^– agglutinin is highly resistant. It is proposed that the globular head of the mt⁺ agglutinin acts as its recognition domain and interacts with a carbohydrate ligand on the mt^– agglutinin.     

–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.     

Sexual reproduction and sex determination in green algae

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt⁺) and mating type minus (mt^?), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt⁺ and mt^? mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.     

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.     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

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