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).     

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.     

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.     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.     

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.     

Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii

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.     

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.     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.     

The role of calcium in the Chlamydomonas reinhardtii mating reaction

The mating reaction of Chlamydomonas reinhardtii entails a rapid series of cell-cell interactions leading to cell fusion. We have demonstrated (Pasquale, S. M., and U. Goodenough. 1987. J. Cell Biol. 105:2279-2293) that cAMP plays a key role in this process: gametic flagellar adhesion elicits a sharp increase in intracellular cAMP, and presentation of dibutyryl-cAMP to unmated gametes elicits all known mating responses. The present study evaluates the role of Ca2+ in this system. We document that the mating-induced increase in cAMP, and hence the mating responses themselves, are blocked by a variety of drugs known to interfere with Ca(2+)-sensitive processes. These data suggest that Ca(2+)-mediated events may couple adhesion to the generation of cAMP. Such events, however, appear to be localized to the flagellar membrane; we find no evidence for the mating-related increase in cytosolic free Ca2+ that has been postulated by others. Indeed, by monitoring the length of the Ca(2+)-sensitive centrin-containing nucleus-basal body connector, we show that cytosolic free Ca2+ levels, if anything, decrease in response to cAMP signaling. We confirm a previous report that Ca2+ levels increase in the mating medium, but document that this represents a response to augmented cAMP levels and not a prelude. Finally, we show that IP3 levels remain constant throughout the mating reaction. These results are discussed in terms of the various signal transduction systems that have now been identified in Chlamydomonas.     

DEEP-ETCH ANALYSIS OF ADHESION COMPLEXES BETWEEN GAMETIC FLAGELLAR MEMBRANES OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The ultrastructure of adhesion complexes between gametic flagellar membranes of Chlamydomonas reinhardtii Dangeard was analyzed using the quick-freeze deep-etch technique. The sexual agglutinin fibrils interact by forming hybrid fibers that frequently branch, forming extensively cross-bridged meshworks. This pattern of interaction mimics a prominent mode of cell wall formation in Chlamydomonas, supporting the notion that the agglutinins evolved from cell wall proteins and that sexual adhesion and cell wall assembly are homologous events.     

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.     

The relationship of flagellar length to sexual signalling in Chlamydomonas

The flagella of Chlamydomonas reinhardi are required for the initiation of mating between opposite mating type gametes. It has been suggested that flagellar length is a crucial factor in a cell's ability to transmit and receive the sexual signals necessary for fusion. Mating type + (mt⁺) cells of gam-5, a mutant which is characterized by variable length, paralyzed flagella, were mated with wild-type, mt⁻ cells. Activation of the mating structures of the gam-5 gametes, and therefore successful signalling, was demonstrated for cells with flagella as short as 1.5 μm (less than 1/6 normal length). Because this mutant displays aberrant axonemal structures, and because various mutants with other defects in axonemal structure are also able to mate, it seems likely that the flagellar membrane may provide the main conduit for gametic sexual signals.     

Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction

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.     

Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion

To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.     

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.