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When Chlamydomonas reinhardtii cells mate, a zygotic maturation program is activated, part of which leads to destruction of chloroplast DNA (cpDNA) from the mating type minus (mt-) parent, and, therefore, to uniparental inheritance of mating type plus (mt+) cpDNA. A long-standing model that explains the selective destruction of mt(-) cpDNA in zygotes invokes a methylation-restriction system. We tested this model by using the potent methylation inhibitor 5-aza-2'-deoxycytidine (5adc) to hypomethylate parental cpDNA and found that the pattern of cpDNA inheritance is altered by 5adc in a manner that is consistent with the model. Surprisingly, however, hypomethylated mt+ cpDNA is not destroyed in zygotes as the methylation-restriction model predicts it should be. Destruction of mt- cpDNA is also unaffected when the parental mt+ cpDNA is hypomethylated. Instead, loss of methylation affects the relative rates of replication of residual mt- cpDNA and mt+ cpDNA in germinating zygotes. The mode of action for 5adc on cpDNA replication in germinating zygotes may be via hypomethylation of mt+ cpDNA, but is also consistent with its action as a DNA-damaging agent. Interestingly, 5adc causes reduced cpDNA replication only in germinating zygotes, not in vegetatively grown cells, indicating that cpDNA replication is qualitatively different in these two stages of the life cycle. Our results demonstrate that methylation is not necessary for protection of the mt+ cpDNA in early zygotes and uncover a novel stage of the Chlamydomonas life cycle when replication of cpDNA is highly susceptible to perturbation. Our data support a model in which differential cpDNA replication in germinating zygotes is used as a mechanism to selectively amplify intact and properly methylated cpDNA molecules.

A key pathway that controls both cell division and differentiation in animal cells is mediated by the retinoblastoma (RB) family of tumor suppressors, which gate the passage of cells from G(1) to S and through S phase. The role(s) of the RB pathway in plants are not yet clearly defined, nor has there been any evidence for its presence in unicellular organisms. Here we have identified an RB homolog encoded by the mat3 gene in Chlamydomonas reinhardtii, a unicellular green alga in the land plant lineage. Chlamydomonas cells normally grow to many times their original size during a prolonged G(1) and then undergo multiple alternating rounds of S phase and mitosis to produce daughter cells of uniform size. mat3 mutants produce small daughter cells and show defects in two size-dependent cell cycle controls: They initiate the cell cycle at a below-normal size, and they undergo extra rounds of S phase/mitosis. Unlike mammalian RB mutants, mat3 mutants do not have a shortened G(1), do not enter S phase prematurely, and can exit the cell cycle and differentiate normally, indicating that the RB pathway in Chlamydomonas has a different role than in animals.

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.

The temperature-sensitive gametogenesis-defective mutant, gam-1 is sex-limited, expressed only in mating type minus (mt-), and can sexually agglutinate but not fuse at the restrictive temperature (35 degrees C) with gametes of wild type (wt) mt+. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gam-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35 degrees C, each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gam-1 flagella. The two mutant phenotypes displayed by gam-1 have been denoted gam-1-I and gam-1-II. An agglutination reaction involving gam-1-I cells does not result in activation of the wt mt+ mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-1-II cells, but because the gam-1 mt- mating sturcture still fails to activate, cell fusion is precluded. We conclude that the gam-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins; we also domonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane.

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

Six cDNA clones have been identified that are complementary to transcripts present in young zygotes of Chlamydomonas reinhardtii but absent from vegetative and gametic cells. Five early transcripts are synthesized within 5 to 10 min of fertilization; the sixth, late, transcript is not synthesized until 90 min following fertilization. Synthesis of both classes requires cell fusion between gametes. Cycloheximide fails to inhibit early mRNA synthesis, indicating that transcription factors must preexist in the gametes and be activated by cytoplasmic confluence. By contrast, cycloheximide blocks synthesis of the late transcript, suggesting that an early protein product(s) is required for expression of the late gene. Restriction fragment length polymorphism analysis of inter- and intraspecific genetic crosses demonstrates that one of the early genes is very tightly linked to the mating-type locus.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Membrane adhesions between the flagella of mating-type "plus" and "minus" gametes of Chlamydomonas reinhardi are shown to stimulate a rapid change in the ultrastructure of the flagellar tips, designated as flagellar tip activation (FTA). A dense substance, termed fibrous tip material (FTM), accumulates between the flagellar membrane and the nine single A microtubules of the tip. The A microtubules then elongate, growing into the distal region of the tip, increasing tip length by 30%. This study describes FTA kinetics during normal and mutant matings, presents experiments designed to probe its role in the mating reaction, and offers the following conclusions: (a) FTA is elicited by agents that cross-link flagellar membrane components (including natural sexual agglutinins, antiflagellar antisera, and concanavalin A) but not by flagellar adherence to polylysine-coated films. (b) FTA is reversed by flagellar disadhesion. (c) Gametes can undergo repeated cycles of FTA during successive rounds of adhesion/disadhesion. (d) FTA, flagellar tipping, and sexual signaling are simultaneously blocked by colchicine and by vinblastine, suggesting that tubulinlike molecules, perhaps exposed at the membrane surface, are involved in all three responses. (e) FTA is not blocked by short exposure to chymotrypsin, by cytochalasins B and D, nor by concanavalin A, even though all block cell fusion; the response is therefore autonomous and experimentally dissociable from later stages in the mating reaction. (f) Under no experimental conditions is mating-structure activation observed to occur unless FTA also occurs. This study concludes that FTA is a necessary event in the sexual signaling sequence, and presents a testable working model for its mechanism.

The substructure of the outer dynein arm has been analyzed in quick-frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function.

Tetrahymena and Chlamydomonas cells treated with high (0.25-0.5%) concentrations of the detergent Nonidet P-40 in appropriate buffers retain the shape of the intact cells but are devoid of any ciliary activity unless supplied with MgATP. ATP causes them to swim actively, with beat parameters and swimming patterns indistinguishable from those of intact cells. Both types of detergent-extracted cells are completely devoid of ciliary membranes. The Tetrahymena preparations also lack all cellular membranes, whereas cellular membranes remain intact in the Chlamydomonas preparations. Experiments demonstrating the effects of ATP, ADP, vanadate, erythro-9-[3-2-(hydroxynonyl)]-adenine, and Ca++ are described to illustrate the use of these detergent-extracted cells in research on ciliary motility.

The mutant strain bald-2 is unique among "flagellaless" strains of Chlamydomonas reinhardtii isolated to date, in that it possesses a mutant basal body: it is only capable of forming a ring of nine singlet microtubules, 180 nm in diameter, instead of the usual triplet basal body which is 225 nm in diameter. This singlet basal body lacks structural stability and the ability to associate with striated fiber material but retains two critical properties of basal bodies, namely, information specifying the length to which it should elongate and the ability to induce, albeit rarely, a flagellar transition region, a short, singlet-containing axoneme, and a specialized tunnel in the cell wall through which flagella normally emerge. The mutation seems to be specific for B- and C-microtubule synthesis or assembly since all other cytoplasmic sets of microtubules appear normal in numbers, orientation, and stability.

The microtubule system associated with the Chlamydomonas reinhardi flagellar apparatus is shown to differ from previous descriptions; two of the four flagellar "roots" possess only two microtubules and are associated with a finely striated fiber. In gametic cells this fiber underlies the gametic mating structure and makes contact with it. Functional interpretations are offered.

Experimental manipulations of gametes of Chlamydomonas reinhardi and ultrastructural observation were used to examine the composition of the microfilaments in the fertilization tubule, their probable mode of formation, and their interaction with intracellular signals. Decoration with myosin subfragment-1 was used to demonstrate that the microfilaments in the fertilization tubule were actin filaments having uniform polarity: Myosin subfragment-1 arrowheads pointed away from the membrane at the tip of the process. Filaments were attached to the cone-shaped "doublet zone" at the base of the process by their pointed ends. Discrete attachment sites for filaments on the surface of the doublet zone were seen in stereo view. To test whether actin polymerization might accompany elongation of the fertilization tubule, mating gametes were exposed to cytochalasin D in an attempt to block actin polymerization. Treatment of mating type "plus" gametes with cytochalasin D prior to and during mating inhibited the appearance of actin filaments in fertilization tubules, suppressed fertilization tubule outgrowth, and lowered mating efficiency from 90 to 15%. The role of signals generated by flagellar adhesion in maintaining the structural integrity of the microfilament-doublet zone complex was examined by correlating flagellar disadhesion with the kinetics of breakdown of the complex. In zygotes, where flagellar disadhesion occurred after cell fusion, the complex disassembled within 3 h after mating. In gametes that had been agglutinated by isolated mating type "minus" flagella, microfilaments and fertilization tubules progressively disassembled over a 3-h time course following flagellar disadhesion. Disassembly of microfilaments was inhibited by maintaining flagellar agglutination, suggesting that signals generated by flagellar adhesion were necessary to maintain microfilaments intact.

The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.

Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.

Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

Gametogenesis in Chlamydomonas reinhardtii has been studied in mating-type plus cells utilizing several different culture conditions, all of which are shown to depend on the depletion of nitrogen from the medium, and the fine structure of gametes prepared under these conditions has been compared by using thin sections of fixed materials. We document alterations in ribosome levels, in chromatin morphology, in starch levels, in the organization of chloroplast membranes, and in the appearance of nuclear envelope and endoplasmic reticulum membranes during gametogenesis. We also noted the acquisition of two new organelles: a mating structure (Friedman, L., A. L. Colwin, and L. H. Colwin. 1968. j. cell Sci. 3:115-128; goodenough, U. W., and R. L. Weiss. 1975. J. Cell Biol. 67:623-637), and Golgi-derived vesicles containing a homogeneous material. We chart the time course of these morphological changes during synchronous gametogenesis. We note that many of these changes may represent adjustments to nitrogen starvation rather than direct features of gametic differentiation, and we also document that cells can differentiate so that they survive conditions of nitrogen starvation for many weeks after they become gametes. We conclude that metabolic alterations, the acquisition of mating ability, and the preparation for long-term survival are all elicited in this organism by nitrogen withdrawal, and we discuss how the various structural alterations observed in this study may relate to these three interrelated avenues of cellular differentiation.

A structural and biochemical study is presented concerning the agglutination of gametic flagella, the initial step in the mating reaction of Chlamydomonas reinhardtii. An alteration in the distribution of the intramembranous particles revealed by freeze-fracturing of flagella membranes is shown to accompany gametic differentiation in both mating types. The isolation and electrophoretic analysis of flagellar membranes and mastigonemes are reported; no electrophoretic differences can be detected when the membrane or mastigoneme glycoproteins from vegative and gametic cells are compared, nor when glycoproteins from the two mating types are compared, and no novel polypeptides are present in gametic preparations. The membrane vesicles, after they are freed of mastigonemes by sedimentation through a discontinuous sucrose gradient, are extremely active as an isoagglutinin, indicating a direct involvement of the membrane in the mating reaction.

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

Freeze-fracture replicas reveal that five distinct types of intramembranous particle arrays coexist within a small sector of the C. reinhardtii cell flagellar membrane. Of these, three are newly described in this report. (a) Flagellar bracelets, which encircle the flagellar bases, appear to be intrinsically ordered strands of particles of unknown function. (b) Strut arrays, representing nine sites where the basal body attaches to the membrane, appear to serve a mechanical function. (c) Contractile vacuole arrays, which develop into circular plaques of particles, appear to serve as "membrane gates" through which water is discharged from the cell.

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.

Sexual adhesion between Chlamydomonas reinhardtii gametes elicits a rise in intracellular cAMP levels, and exogenous elevation of intracellular cAMP levels in gametes of a single mating type induces such mating responses as cell wall loss, flagellar tip activation, and mating structure activation (Pasquale, S. M., and U. W. Goodenough. 1987. J. Cell Biol. 105:2279-2292). Here evidence is presented that sexual adhesion mobilizes agglutinin to the flagellar surface, and that this mobilization can be induced by exogenous presentation of cAMP to gametes of a single mating type. It is proposed that Chlamydomonas adhesion entails a positive feedback system--initial contacts stimulate the presentation of additional agglutinin--and that this feedback is mediated by adhesion-induced cAMP generation.