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 mating type plus and mating type minus gametes activates a flagellar protein-tyrosine kinase during fertilization in Chlamydomonas

When Chlamydomonas gametes of opposite mating type are mixed together, flagellar adhesion through sex-specific adhesion molecules triggers a transient elevation of intracellular cAMP, leading to gamete activation in preparation for cell-cell fusion and zygote formation. Here, we have identified a protein-tyrosine kinase (PTK) activity that is stimulated by flagellar adhesion. We determined that the protein-tyrosine kinase inhibitor genistein inhibited fertilization, and that fertilization was rescued by dibutyryl cAMP, indicating that the genistein-sensitive step was upstream of the increase in cAMP. Incubation with ATP of flagella isolated from non-adhering and adhering gametes followed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies showed that adhesion activated a flagellar PTK that phosphorylated a 105-kDa flagellar protein. Assays using an exogenous protein-tyrosine kinase substrate confirmed that the activated PTK could be detected only in flagella isolated from adhering gametes. Our results indicate that stimulation of the PTK is a very early event during fertilization. Activation of the PTK was blocked when gametes underwent flagellar adhesion in the presence of the protein kinase inhibitor staurosporine, but not in the presence of the cyclic nucleotide-dependent protein kinase inhibitor, H8, which (unlike staurosporine) does not block the increases in cAMP. In addition, incubation of gametes of a single mating type in dibutyryl cAMP failed to activate the PTK. Finally, flagella adhesion between plus and minus fla10-1 gametes, which have a temperature-sensitive lesion in the microtubule motor protein kinesin-II, failed to activate the PTK at elevated temperatures. Our results show that kinesin-II is essential for coupling flagellar adhesion to activation of a flagellar PTK and cAMP generation during fertilization in Chlamydomonas.     

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.     

Cyclic AMP enhances the sexual agglutinability of Chlamydomonas flagella

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.     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Identification of the carboxy terminus as important for the isoform- specific subcellular targeting of glucose transporter proteins

Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd2+. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40 degrees C, first described by Zhang et al. (1991), is insensitive to Cd2+ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40 degrees C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type flagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of flagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different flagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell- body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.     

Calmodulin Sensitivity of the Flagellar Membrane Adenylate Cyclase and Signaling of Motile Responses by cAMP in Gametes of Chlamydomonas reinhardtii

Cyclic AMP (cAMP) has been shown to be a primary signal of the agglutination-induced mating events of flagellar tip activation, cell wall loss, and mating structure activation in the unicellular alga Chlamydomonas reinhardtii (Pasquale and Goodenough, Cell Biol. 105 (1987), 2279–2293). The flagellar membrane adenylate cyclase of Chlamydomonas is here shown to be inhibited in vitro by EGTA, La³⁺, and trifluoperazine, and to be stimulated in the presence of calcium by incubation with exogenous calmodulin. Also, the motility of detergent-extracted models of Chlamydomonas is shown to be enhanced by cAMP. These observations suggest the hypothesis that the twitching motility characteristic of agglutinating Chlamydomonas gametes may be signaled by cAMP produced locally within the flagella by a calmodulin-sensitive adenylate cyclase.     

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 in Chlamydomonas: a system for the study of specific cell adhesion. II. A radioactive flagella-binding assay for quantitation of adhesion

To measure the flagellar adhesiveness of Chlamydomonas gametes in a more quantitative manner than agglutination assays permit, a binding assay was developed which measured the binding of radioactive flagella of one mating type to unlabeled gametes of the opposite mating type. With the appropriate assay conditions, the number of [3H] flagella specifically bound was shown to be proportional to the number of cells in the incubation mixture and, therefore, to the number of binding sites that were present. The assay was used to study the effects of trypsin treatment on the loss and development of flagellar binding sites. It was shown that after trypsin treatment at least 9 h were required for the return of a full complement of binding sites to the flagellar surface; moreover, the results indicated that these sites reappeared on existing, extended flagella.     

Transmembrane signaling in cilia and flagella

Summary Ciliary and flagellar membranes are dynamic. Ciliary and flagellar membranes have diverged widely during evolution and perform many specialized functions. Transmembrane signaling is an important component of the function of ciliary and flagellar surfaces in general. In this review, I discuss some of the functions performed by ciliary and flagellar surfaces and I present three different ciliary and flagellar signaling systems associated with rather different dynamic events performed by ciliary and flagellar surfaces. Two of these are associated withChlamydomonas flagella and one is associated with vertebrate olfactory cilia. Calcium regulation of protein phosphorylation appears to be important in regulating glycoprotein movements in theChlamydomonas flagellar membrane. Changes in levels of cAMP and cAMP-dependent protein phosphorylation are clearly central to the signaling associated with mating events in gametic flagella ofChlamydomonas, although calcium clearly has an important, if poorly understood, role to play. There is no known role for G proteins in flagellar membrane events inChlamydomonas. In contrast, mammalian olfactory cilia possess an odorant activated, G protein regulated adenylate cyclase and conductance channels that are directly gated by cyclic nucleotides. A second class of odorants that do not affect adenylate cyclase activity appear to act through G protein activated phospholipase C and changes in IP₃ second messenger levels. These examples demonstrate the diversity in the signaling pathways associated with ciliary and flagellar membranes.Abbreviations CaPK-2 calcium-dependent protein kinase - db-cAMP dibutyryl cAMP - Fab fragment antigen binding - IgE immunoglobulin E - IP₃ myo-inositol trisphosphate - IP₄ myo-inositol tetrakisphosphate - OBP odorant binding protein - PIP₂ phosphoinositol bisphosphate - TFP trifluoperazine - WGA wheat germ agglutinin     

Transient increase in calcium efflux accompanies fertilization in Chlamydomonas

Mating in Chlamydomonas is a complex process initiated by contact of gametic flagellar surfaces, resulting in transmission of a signal from the flagella to the cell bodies. This signal triggers later events of cell wall loss, mating structure activation, and cell-cell fusion. Little is known about the nature of the signal or the role of Ca in these events. It was found that extracellular Ca is not necessary for successful mating in Chlamydomonas. However, cells will take up Ca from the medium in a linear manner for many hours and will accumulate micromolar concentrations, presumably by sequestering Ca within intracellular storage sites. If gametic cells of one mating type (preloaded with 45Ca) are mated with gametes of the opposite mating type (preloaded with unlabeled calcium), there is a rapid, transient increase in calcium efflux rate (20 times that of the control) that lasts approximately 6 min. This effect is not associated with cell-cell fusion, since the same observation is made if (+) gametes preloaded with 45-Ca are agglutinated by isolated flagella from (-) gametes preloaded with unlabeled Ca. Other experiments have shown that the increased efflux rate is not a simple consequence of cell wall release. Ca efflux in unmated gametes is greatly reduced in deflagellated cells, suggesting that much of the Ca movement is associated with the flagellar membrane. Although signaling itself may involve Ca fluxes across the flagellar membrane, it is also possible that a consequence of signaling is release of Ca from intracellular storage sites (perhaps functional equivalents of the sarcoplasmic reticulum). The observed transient increase in Ca efflux rate may reflect a transient increase in the cytoplasmic free-Ca concentration. This increase in cytoplasmic Ca may regulate the later events in mating (such as cell wall release and mating structure activation).     

Sex-specific glycoproteins in Chlamydomonas flagella an immunological study

To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

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.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

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.     

Booklist no. 52 June 1984

When plus and minus mating type gametes of Chlamydomonas eugametos were mixed, a rapid transient increase in the amount of cAMP was observed with a maximum at 20 s after the start of the sexual agglutination reaction. The transient increase only occurred within the cells and was also exhibited when cell suspensions of single mating type were presented with isolated flagella of the other mating type. Cyclic AMP-dependent protein kinase and cyclic AMP-phosphodiesterase activities were found in cell homogenates. Since the rise in cAMP concentration preceded all known morphological and physiological changes in the cells that prepare them for fusion, it might be a primary response, induced by sexual agglutination.     

Intracellular cAMP involvement in the synchronized activity of noradrenaline in response to evoked release of the transmitter quanta in the frog synapses

An analogue of cyclic AMP (db-cAMP) penetrating into the frog neuromuscular junction's cell, as well as the adenylyl cyclase activator forskolin, and inhibitor of nucleotide-depending phosphodiesterase isobutilmethylxantine alter the kinetics of the quanta secretion resulting in synchronizing of the process of the transmitter release. Following a db-cAMP preliminary action, no such synchronizing of the transmitter release occurred. Action of noradrenaline on the time course of the secretion seems to be realised through activation of presynaptic beta-adrenoreceptors, augmentation of the adenylyl cyclase activity, and the rise of the intracellular cAMP.     

Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas

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.