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     

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     

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 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     

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.)     

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     

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)     

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     

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     

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.     

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 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.     

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; )     

–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.     

Rearrangement of the flagellar apparatuses and eyespots of isogametes during the fertilization of the marine green alga,Monostroma nitidum (Ulvophyceae,Chlorophyta)

Behaviors of the flagellar apparatuses (flagella, basal bodies, microtubular roots, etc.), mating structures and eyespots of gametes during the fertilization of Monostroma nitidum were studied using field emission scanning electron microscopy and transmission electron microscopy. The biflagellate isogamete (mt⁺ and mt^?) mating structure has a position that is converse between mt⁺ and mt^? gametes relative to the flagellar beat plane and the eyespot. After the adhesion of mt⁺ and mt^? gametes, gamete fusion occurred between the two mating structures. The cell fusion plane expanded to the cell surface as circumscribed by 1s–2d roots in mt⁺ gamete and 1d–2s roots in the mt^? gamete. Two sets of flagellar apparatuses lay side by side in the planozygote and soon become mutually close. The no. 1 basal body of mt⁺ gamete and the no. 2 basal body of mt^? gamete rotated in a counterclockwise direction, as viewed from the cell anterior. Then, the no. 2 basal body of mt⁺ gamete and the no. 1 basal body of mt^? gamete slid into a face to face position. Finally, four flagella and basal bodies exhibited a cruciate arrangement. The basal bodies of the opposing pair (no. 1 and no. 2) were offset in a counterclockwise orientation by the basal body diameter. The 1s and 2d roots of the mt⁺ gamete lay nearly parallel to the 1d and 2s roots of the mt^? gamete, respectively, at the cell fusion plane. Because of the asymmetric localization of the mating structure, association, and subsequent rearrangement of basal bodies and microtubular roots, two eyespots lay on the same side of the planozygote. After the settlement of the planozygote, the flagellar apparatus started to disintegrate in the zygote cytoplasm.     

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.     

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

Flagellar adhesion and deadhesion in chlamydomonas gametes: Effects of tunicamycin and observations on flagellar tip morphology

The aggregation-dependent loss of flagellar adhesiveness in Chlamydomonas reinhardi has been correlated with changes in flagellar tip morphology during adhesion and deadhesion. As aggregating mt^? and impotent (able to adhere, but not fuse) mt⁺ gametes begin to disaggregate in the presence of the protein synthesis inhibitor cycloheximide, there is a concomitant change in flagellar tip morphology from the activated bulbous form to the nonactivated tapered shape. The requirement of protein-synthetic activity for the maintenance of flagellar adhesiveness during aggregation may be due in part to turnover of proteins involved in formation or stabilization of activated flagellar tips. Incubation of aggregating gametes with tunicamycin indicates that, like protein synthesis inhibitors, this inhibitor of glycosylation also causes adhering gametes to deadhere. The results suggest that protein glycosylation may be essential for maintenance of adhesiveness during aggregation.