Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera

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.     

Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild- type and mutant strains

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.     

Isolation and Genetic Analysis of Mutant Strains of CHLAMYDOMONAS REINHARDI Defective in Gametic Differentiation

Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-l, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or mt+ gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt- gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that imp-1 gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.     

Monoclonal antibodies directed against the sexual binding site of Chlamydomonas eugametos gametes

Monoclonal antibodies were raised against the mt- sexual agglutinin of Chlamydomonas eugametos gametes. Those that blocked the agglutination site were selected. They were divided into two classes dependent upon whether they gave a weak (class A) or clear positive (class B) reaction with mt- flagellar membranes in an ELISA and an indirect immunofluorescence test using glutaraldehyde-fixed mt- gametes. Class A antibodies were shown to be specific for the agglutinin in an extract of mt- gametes, based on results from immunoblotting, immunoprecipitation, affinity chromatography, and the absence of a reaction with nonagglutinable cells. Surprisingly, class A mAbs also recognized two mt+ glycoproteins, one of which is the mt+ agglutinin. Class B antibodies were shown to bind to several glycoproteins in both mt- and mt+ gametes, including the mt- agglutinin. Fab fragments from class A mAbs blocked the sexual agglutination process, but those from class B did not, even though the parent antibody did. We conclude that the class A epitope lies in or close to the agglutination site of the mt- agglutinin, whereas the class B epitope lies elsewhere on the molecule. We also conclude that the mt- agglutinin is the only component on the mt- flagellar surface directly involved in agglutination. Class A mAbs were found to elicit several reactions displayed by the mt+ agglutinin. They bound to the mt- agglutinin on gamete flagella and induced most of the reactions typical of sexual agglutination, with the exception of flagellar tip activation. None of these reactions was induced by Fab fragments. High concentrations of class A mAbs completely repressed the sexual competence of live mt- gametes, but low concentrations stimulated cell fusion.     

A monoclonal antibody that blocks adhesion of Chlamydomonas mt+ gametes

During the mating reaction (fertilization) in the biflagellated alga, Chlamydomonas reinhardtii, mt+ and mt- gametes adhere to each other via their flagella and subsequently fuse to form quadriflagellated zygotes. In the studies reported here, we describe a monoclonal antibody directed against an mt+ flagellar surface molecule. The antibody blocks the adhesiveness of mt+ gametes, isolated mt+ flagella, and detergent extracts thereof. It has no effect on mt- gametes. Cyanogen bromide- activated Sepharose beads derivatized with the antibody bind only mt+ gametes; mt- gametes and mt+ and mt- vegetative cells are unreactive with the derivatized beads. The interaction of mt+ gametes with the beads is dynamic and cells continuously bind, detach, and rebind to the beads. Surprisingly, antibody-derivatized beads that have been incubated with mt+ gametes acquire the ability to bind mt- gametes. Moreover, extraction of the preincubated beads with detergents releases active mt+ adhesion molecules. The evidence suggests that binding of the antibody to the flagellar surface adhesion molecules causes their release from the flagellar surface, possibly mimicking the normal mechanism of flagellar de-adhesion.     

Membrane differentiations at sites specialized for cell fusion

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.     

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     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

Mucilage secretion regulated by sex pheromones in Closterium peracerosum-strigosum-littorale complex

When mating-type plus (mt+) and minus (mt-) cells of the Closterium peracerosum-strigosum-littorale complex were mixed in nitrogen-depleted mating medium, secretion of mucilage containing uronic acid from cells was markedly activated and the mucilage accumulated around the cells. Substances with the ability to stimulate mucilage secretion from mt+ and mt- cells were detected in media in which mt- and mt+ cells had been separately cultured, respectively. We designated the active substances secreted from mt+ and mt- cells mucilage secretion-stimulating pheromone (MS-SP)-plus and MS-SP-minus, respectively. Activity of MS-SP-plus and MS-SP-minus decreased to 20% level by incubation at 80 degrees C for 10 min. Light was indispensable for the secretion of mucilage. The secretion of MS-SP-plus and MS-SP-minus decreased with dark treatment. MS-SP-plus eluted at around 95 k from a gel filtration column, and reacted with antibodies against two subunits of protoplast-release-inducing protein (PR-IP), which induces protoplast release from mt- cells. MS-SP-minus eluted at around 20 k from a gel filtration column, and reacted with an antibody against the PR-IP inducer, which induces the secretion of PR-IP from mt+ cells. In addition, purified PR-IP and PR-IP inducer stimulated mucilage secretion from mt- and mt+ cells, respectively. These results strongly suggested that MS-SP-plus and MS-SP-minus were the same molecules as the PR-IP and the PR-IP inducer, respectively.     

Mating Structure Differences Demonstrated by Freeze-Fracture Analysis of Fusion-Defective Chlamydomonas Gametes

While the mating structure of unmated mating type minus ( mt ^-) gametes of Chlamydomonas reinhardtii has few intramembrane particles (IMPs), activation results in movement of IMPs to its center. Analysis of freeze-fractured replicas of wild type (wt) mt ^- and 3 mt ^- fusion-defective mutants, gam -1, gam -10 and gam -11, before and after activation with wt ⁺ flagella, provides a basis for suggesting that some of the IMPs in mt ^- mating structures, particularly a subset of particles that partitions to the E face, may be fusion-controlling molecules. Unmated gametes of gam -10 show a full range of images, from particle-free to fully activated, with both the P and E face of the mating structure revealing approximately twice as many IMPs as those observed on wt . Unactivated gametes of gam -1 and gam -11 appear identical to wt ^-. After activation, the mating structures of all of these gametes appear to have approximately the same number of IMPs. If the sizes of particles for these mutants are compared to wild type at the restrictive temperature, all 3 mutants have significantly smaller IMPs on the E face; before mating, in the plasma membrane and after mating, in the mating structure. At 34° C, the gam -1-II mating structure appears to be missing most of the particles from 15.5 to 16.5 nm in diameter, while all gametes with the ability to fuse have an equivalent percentage of their mating structure particles in this size range. The possibility that an IMP in this size range represents a protein that may be responsible for gamete fusion is discussed.     

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.     

Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.     

Filipin labeling in Chlamydomonas gametes exposes site-specific lipid specializations.

A thin section study of mating Chlamydomonas cell wall-less CW 15 mating type plus (mt+) and mating type minus (mt-) gametes utilized filipin. The results show extensive labeling of mt+ and mt- plasma membranes. No labeling was seen on the mating structure membranes of activated mt+ or mt- gametes. These results indicate that differences exist between the plasma membrane and the mating structure membrane of gametes. If filipin is specific for the 3-beta-OH sterol, ergosterol and/or other Chlamydomonas sterols, then these results imply that the fusing mating structure membranes may be altered or reduced in sterol content. Such lipid specializations may increase local membrane fluidity and thereby facilitate the site-specific cell fusion associated with mating Chlamydomonas gametes.     

Sexual agglutinin of mating-type minus gametes in Chlamydomonas reinhardii : I. Loss and recovery of agglutinability of gametes treated with EDTA

The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin.     

Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling

Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase.     

Mating Type Linked Mutations Which Disrupt the Uniparental Transmission of Chloroplast Genes in Chlamydomonas

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.     

The Unusual Sexual Preferences of a Chlamydomonas Mutant May Provide Insight into Mating-Type Evolution

Chlamydomonas monoica undergoes intraclonal mating-type differentiation (homothallism). Although the species differs in this regard from the more commonly studied heterothallic C. reinhardtii, cell-cell interactions and progression of the sexual cycle are similar for many homothallic and heterothallic species of the genus. Regulation of chloroplast gene transmission by the nuclear mating-type alleles (mt+ and mt-) is another common denominator for Chlamydomonas species studied thus far. We have previously reported the use of chloroplast inheritance patterns to identify mutants of C. monoica that have lost the potential to function as the mt+ mating-type. A similar screening procedure led to the isolation of an unusual mutant, mtl-3 whose phenotype is less readily explained. Chloroplast gene transmission patterns in crosses involving mtl-3 suggest that the mtl-3 strain mates preferentially as mt+. However, normal mating efficiencies and high zygospore viability are observed in clonal culture, indicating the unbiased production of functional opposite mating-types. By construction of appropriately marked strains we have been able to show that mtl-3 mt- gametes prefer the mt+ gametes of their own strain. A model is presented which invokes unequal crossing over between highly homologous flagellar agglutinin genes to account for the unusual properties of the mtl-3 strain and for the evolution of mating barriers within the genus.     

The iso1 gene of Chlamydomonas is involved in sex determination.

Sexual differentiation in the heterothallic alga Chlamydomonas reinhardtii is controlled by two mating-type loci, mt+ and mt-, which behave as a pair of alleles but contain different DNA sequences. A mutation in the mt minus-linked imp11 gene has been shown previously to convert a minus gamete into a pseudo-plus gamete that expresses all the plus gametic traits except the few encoded by the mt+ locus. Here we describe the iso1 mutation which is unlinked to the mt- locus but is expressed only in minus gametes (sex-limited expression). A population of minus gametes carrying the iso1 mutation behaves as a mixture of minus and pseudo-plus gametes: the gametes isoagglutinate but they do not fuse to form zygotes. Further analysis reveals that individual gametes express either plus or minus traits: a given cell displays one type of agglutinin (flagellar glycoprotein used for sexual adhesion) and one type of mating structure. The iso1 mutation identifies a gene unlinked to the mating-type locus that is involved in sex determination and the repression of plus-specific genes.     

Kinesin-II is required for flagellar sensory transduction during fertilization in Chlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt-) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt- flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32 degrees C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32 degrees C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32 degrees C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32 degrees C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.