Ectopic expression of a Chlamydomonas mt+-specific homeodomain protein in mt- gametes initiates zygote development without gamete fusion.

The molecular mechanisms that activate expression of zygote genes after fertilization are obscure. In animals, receptor-ligand interactions during sperm-egg membrane fusion as well as delivery of putative regulatory molecules by the sperm into the egg cytoplasm are proposed to activate zygote development and subsequent transcription of zygote genes. The mechanisms of activation of zygote development in higher plants also are mysterious, in part because of the difficulty of isolating female gametes of higher plants. In the unicellular, biflagellated green alga Chlamydomonas, the early steps in zygote development are much more accessible to investigation. Within minutes after mating type plus (mt+) and mating type minus (mt-) gametes fuse, expression of several zygote-specific transcripts is induced independently of protein synthesis. Here, we show that ectopic expression in mt- gametes of an mt+ gamete-specific, homeodomain protein, GSP1, induces a zygote-like phenotype and activates expression of zygote genes. One of the genes, zsp2, expressed in these "haploid zygotes" encodes a zygote cell surface adhesion molecule that promotes formation of multicellular aggregates. In total, expression of six out of seven zygote genes examined was induced by ectopic expression of GSP1. Our experiments show that in addition to contributing their genomes to the zygote cytoplasm, gametes also deliver proteins that can activate gene transcription.

Signal transduction during fertilization in the unicellular green alga, Chlamydomonas.

Sexual reproduction in the green alga, Chlamydomonas, is regulated by environmental conditions and by cell-cell interactions. After gametogenesis, flagellar adhesion between gametes triggers gamete activation, leading to cell fusion and zygote formation. Recent studies have identified new molecular events that underlie signal transduction during Chlamydomonas fertilization, including expression of a sex-determining protein, phosphorylation of a homeodomain protein, activity of a kinesin II and regulated translocation of an aurora/Ip11-like protein kinase from the cell body to the flagella.

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas.

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.

Flagellar adhesion between mt(+) and mt(-) Chlamydomonas gametes regulates phosphorylation of the mt(+)-specific homeodomain protein GSP1.

During fertilization in Chlamydomonas, flagellar adhesion between mt(+) and mt(-) gametes induces a cAMP-dependent signal transduction pathway that prepares the gametes for cell fusion and zygote formation. Previously, our laboratory identified a homeodomain protein (GSP1) whose expression was restricted to the cell bodies of mt(+) gametes and whose transcript level was up-regulated during flagellar adhesion. In this report, we describe a new form of GSP1 that appears early during gamete interactions. Immunoblot analysis showed that in addition to the 120-kDa form of GSP1 normally present in mt(+) gametes, a 122-kDa form was detected when the cells were mixed with mt(-) gametes. The more slowly migrating form of GSP1 was detectable within minutes after gametes were mixed together, and its appearance did not require new protein synthesis. Thus, the 122-kDa form represents a post-translational modification of the pre-existing 120-kDa form of GSP1. Moreover, conversion to the 122-kDa form did not require cell fusion. Although the 120-kDa form was expressed 10 h after vegetative cells were transferred to gametic induction medium, the 122-kDa form was detected only after mt(+) gametes were induced to undergo the sexual signaling that accompanies fertilization. Incubation of mt(+) gametes with dibutyryl cAMP led to the appearance of the 122-kDa form of GSP1, and the cyclic nucleotide-dependent protein kinase inhibitor H-8 inhibited the adhesion-induced conversion. Incubation of GSP1 immunoprecipitated from signaling mt(+) gametes with alkaline phosphatase showed that the conversion was due to phosphorylation. The results indicate that flagellar adhesion induces a rapid, cAMP-dependent phosphorylation of the homeodomain protein GSP1 early during fertilization in Chlamydomonas.

A gamete-specific, sex-limited homeodomain protein in Chlamydomonas.

During fertilization in Chlamydomonas, gametes of opposite mating types interact with each other through sex-specific adhesion molecules on their flagellar surfaces. Flagellar adhesion brings the cell bodies of the gametes into close contact and initiates a signal transduction pathway in preparation for cell-cell fusion. We have identified a cDNA, gsp1, whose transcript levels are upregulated during flagellar adhesion. The GSP1 polypeptide is a novel, gamete-specific homeodomain protein, the first to be identified in an alga. Its homeodomain shows significant identity with several higher plant homeodomain proteins. Although encoded by a single copy gene present in cells of both mating types, immunoblot analysis showed that GSP1 was expressed in mating type (mt)+ gametes, but was not detectable in mt- gametes or in vegetative cells of either mating type. Moreover, GSP1 appeared late during gametogenesis, suggesting that it may function during adhesion with mt- gametes or after zygote formation. GSP1 is expressed in imp11, mt- mutant gametes, which have a lesion in the mid gene involved in sex determination and exhibit many phenotypic characteristics of mt+ gametes. Thus, gsp1 is negatively regulated by mid and is the first molecule to be identified in Chlamydomonas that shows sex-limited expression.

Microvilli and cell-cell fusion during fertilization.

A number of fundamental biological processes, ranging from fertilization and embryonic development to viral infections, depend upon a complex interplay between cells that results in the fusion of their plasma membranes. Surprisingly, the cellular and molecular mechanisms underlying cell-cell fusion remain largely unknown. Here, the authors discuss evidence suggesting that microvilli play a central role in fusion of many cells and present features of these actin-filled, cell-surface protrusions that might make them particularly well suited as cell-fusion organelles.

The Chlamydomonas mating type plus fertilization tubule, a prototypic cell fusion organelle: isolation, characterization, and in vitro adhesion to mating type minus gametes.

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this approximately 3-microm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt-) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt- gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2-3 x 10(6) fertilization tubules/microg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt- gametes between the two flagella, the site of the mt- mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt- gametes.

SksC, a fertilization-related protein kinase in Chlamydomonas, is expressed throughout the cell cycle and gametogenesis, and a phosphorylated form is present in both flagella and cell bodies.

Fertilization in the biflagellated eukaryote, Chlamydomonas, is initiated by flagellar adhesion between gametes of opposite mating types. An early event in the signal transduction pathway induced by these cell-cell interactions is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48 kDa flagellar protein. Molecular cloning and characterization indicated that the 48 kDa substrate, termed SksC, itself is a novel protein kinase. Here, we have determined that its transcript levels were unchanged during prolonged flagellar adhesion. Moreover, resynthesis of new flagellar proteins following deflagellation was not accompanied by increases in transcript levels of SksC, suggesting that expression of this soluble protein kinase might not be restricted to flagella. Immunoblot analysis indicated that expression of SksC was ubiquitous: this soluble protein was found in both flagella and cell bodies and was expressed throughout the cell cycle and gametogenesis. Immunoprecipitation experiments indicated that SksC was phosphorylated in both flagella and cell bodies. Thus, in addition to its potential role in fertilization, this novel protein kinase may play a role in other signaling events in Chlamydomonas.

Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation.

Molecular cloning of a protein kinase whose phosphorylation is regulated by genetic adhesion during Chlamydomonas fertilization.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine beta-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.

Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes.

Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.

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.

Differential regulation of adenylylcyclases in vegetative and gametic flagella of Chlamydomonas.

To learn more about the mechanism of regulation of cAMP during fertilization in Chlamydomonas, we have begun to study the flagellar adenylylcyclase. Recently (Zhang, Y., Ross, E. M., and Snell, W. J. (1991) J. Biol. Chem. 266, 22954-22959) we reported that the adenylylcyclase in gametic flagella is inhibited by ATP and activated by pretreatment at 45 degrees C or by incubation with the protein kinase inhibitor staurosporine. Here we present evidence that this novel regulatory mechanism is unique to gametes and may be required for sexual signaling between mt+ and mt- gametes during fertilization. The vegetative form of the enzyme, which has a specific activity 3-5-fold less than the gametic form, was neither inhibited by ATP nor activated at 45 degrees C. 5'-Adenylylimidodiphosphate, staurosporine, and Mn2+, which activated the gametic enzyme, had no effect on the vegetative adenylylcyclase. In addition the gametic enzyme was inhibited by low (0.1-1 microM) Ca2+ concentrations, whereas the vegetative form was unaffected by 10 microM Ca2+. During gametogenesis acquisition of the ability to undergo sexual signaling was coincident with the appearance of the gametic flagellar adenylylcyclase. Our results suggest that gametogenesis is accompanied by appearance of a new adenylylcyclase that may play a central role in signal transduction during fertilization in Chlamydomonas.

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii.

Adenylylcyclase activity in the flagella of gametes of Chlamydomonas reinhardtii was inhibited by prior incubation at or below 30 degrees C in the presence of ATP. This decrease did not occur in the absence of ATP, in the presence of the ATP analog 5'-adenylylimidodiphosphate (App(NH)p), or in the presence of ATP plus the protein kinase inhibitor staurosporine (2 microM). If ATP treatment was performed in the absence of an ATP-regenerating system, activity initially declined and subsequently recovered. Incubation of flagella at 45 degrees C in the absence of ATP or incubation at lower temperatures in the presence of either App(NH)p or staurosporine both increased adenylylcyclase activity (over 10-fold) and blocked subsequent ATP-dependent loss of activity at 30 degrees C. This heat-induced activation was prevented by the presence of ATP plus an ATP-regenerating system. Incubation of flagella with [gamma-32P]ATP followed by gel electrophoresis in sodium dodecyl sulfate indicated the presence of endogenous protein kinase and protein phosphatase activities. These data suggest that the flagellar adenylylcyclase in Chlamydomonas gametes is inhibited by phosphorylation and stimulated by dephosphorylation. This mechanism for regulating adenylylcyclase may underlie the rapid increase in cyclic AMP that is induced by flagellar adhesion during fertilization in Chlamydomonas.

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.

Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier.

Fertilization in Chlamydomonas reinhardtii is initiated when gametes of opposite mating types adhere to each other via adhesion molecules (agglutinins) on their flagella. Adhesion leads to loss of active agglutinins from the flagella and recruitment of new agglutinins from a pool associated with the cell body. We have been interested in determining the precise cellular location of the pool and learning more about the relationship between agglutinins in the two domains. In the studies reported here we describe methods for purification of mt+ cell body agglutinins by use of ammonium sulfate precipitation, chromatography (molecular sieve, ion exchange, and hydrophobic interaction), and sucrose gradient centrifugation. About 90% of the total agglutinins were associated with the cell body and the remainder were on the flagella. Cell body agglutinins were indistinguishable from mt+ flagellar agglutinins by SDS-PAGE, elution properties on a hydrophobic interaction column, and in sedimentation properties on sucrose gradients. The nonadhesiveness of cell bodies suggested that the cell body agglutinins would be intracellular, but our results are not consistent with this interpretation. We have demonstrated that brief trypsin treatment of deflagellated gametes destroyed all of the cell body agglutinins and, in addition, we showed that the cell body agglutinins were accessible to surface iodination. These results indicated that C. reinhardtii agglutinins have a novel cellular disposition: active agglutinins, representing approximately 10% of the total cellular agglutinins, are found only on the flagella, whereas the remaining 90% of these molecules are on the external surface of the cell body plasma membrane in a nonfunctional form. This segregation of cell adhesion molecules into distinct membrane domains before gametic interactions has been demonstrated in sperm of multicellular organisms and may be a common mechanism for sequestering these critical molecules until gametes are activated for fusion. In experiments in which surface-iodinated cell bodies were permitted to regenerate new flagella, we found that the agglutinins (as well as the 350,000 Mr, major flagellar membrane protein) on the newly regenerated flagella were iodinated. These results indicate that proteins destined for the flagella can reside on the external surface of the cell body plasma membrane and are recruited onto newly forming flagella as well as onto preexisting flagella during fertilization.