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.

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.

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.

During fertilization in the biflagellated alga, Chlamydomonas reinhardtii, gametes of opposite mating types adhere to each other via agglutinin molecules located on their flagellar surfaces, generating a sexual signal that induces several cellular responses including cell wall release. This cell contact-generated signal is mediated by cAMP and release of the wall, which is devoid of cellulose and contains several hydroxyproline-rich glycoproteins, is due to the activation of a metalloprotease, lysin. Although we originally assumed that lysin would be stored intracellularly in a compartment structurally separate from its substrate, recently we showed that lysin is stored in the periplasm as an inactive, higher relative molecular mass precursor, prolysin (Buchanan, M.J., S. H. Imam, W. A. Eskue, and W. J. Snell. 1989. J. Cell Biol. 108:199-207). Here we show that conversion of prolysin to lysin is due to a cellular, nonperiplasmic enzyme that has the properties of a serine protease. Release of this serine protease into the periplasm is induced by incubation of gametes in dibutyryl cAMP. This may be one of the few examples of regulated secretion of a protease in a eucaryotic microorganism and a novel example of regulated secretion in a plant system.

During the mating reaction in Chlamydomonas reinhardtii mating type plus and mating type minus gametes adhere to each other via adhesion molecules on their flagellar surfaces. This adhesive interaction induces a sexual signal leading to release of a cell wall degrading enzyme, lysin, that causes wall release and degradation. In this article, we describe the preparation of a polyclonal antibody against the 60,000-Mr lysin polypeptide excised from SDS-PAGE gels. After absorption of the IgG with cell walls to remove antibodies against a carbohydrate epitope common to several Chlamydomonas glycoproteins, the immune IgG reacted with the 60,000-Mr polypeptide, and a 47,000-Mr species that we show here was immunologically cross-reactive with the 60,000-Mr molecule. By use of several fractionation methods including ion exchange and molecular sieve chromatography, sucrose gradient centrifugation, and affinity chromatography, we showed that the 60,000-Mr antigen copurified with lysin activity, thereby demonstrating that the antibody was indeed directed against the enzyme. Immunoblot experiments on suspensions of nonmating and mating gametes showed that the 60,000-Mr antigen was missing in the nonmating gametes. Instead, they contained a 62,000-Mr antigen that was not present in suspensions of mating gametes that had undergone sexual signalling. Furthermore, nonmating gametes whose walls were removed with exogenously added lysin did not contain either form of the antigen. We also found that the 62,000-Mr form of the antigen, which could be released from gametes by freeze-thawing, did not have wall degrading activity. These results indicate that lysin in gametes is stored in the periplasm as a higher relative molecular mass, inactive precursor and also that sexual signalling induces conversion of this molecule to a lower relative molecular mass, active enzyme. This may be a novel example of processing of an extracellular protease induced by cell contact.

The Chlamydomonas cell wall is a multilayered, extracellular matrix containing 20-25 proteins and glycoproteins, many of which are highly enriched in hydroxyproline. 80-90% of the wall protein is located in a crystalline portion of the wall that is soluble in sarkosyl-urea solutions as well as in chaotropic salts. Although the wall has no cellulose it contains a noncrystalline, highly insoluble framework portion that is responsible for the integrity and overall shape of the wall. In the present report we show that the framework of the wall is composed of two components that are acted upon by lysin, a wall degrading enzyme released by mating gametes. One, which makes up the major portion of the framework, is insoluble upon boiling in SDS-PAGE sample buffer. Lysin treatment of this portion leads to its physical degradation and the concomitant appearance of several SDS-dithiothreitol-soluble polypeptides ranging in relative molecular mass from greater than 400,000 to less than 60,000. The second component is the flagellar collar. This hollow cylinder composed of striated fibers aligned in parallel array serves as the tunnel in the wall through which the flagella protrude. Our evidence indicates that the primary collar polypeptide is a 225,000-Mr molecule that itself has at least two functional domains. One domain, contained in a 185,000-Mr fragment, permits the self-association of the molecules to form the main body of the collar. The second part of the molecule anchors the collar to the wall framework via sarkosyl-urea-insensitive, SDS-dithiothreitol-sensitive linkages.

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.

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

A flagellar adhesion-induced signal sent during the mating reaction of the biflagellate alga, Chlamydomonas reinhardtii, initiates release of cell-wall-degrading enzymes, activation of mating structures, and cell fusion. The nature of this signal is unknown, but it may be mediated by an adhesion-induced change (activation) of flagellar tips. The studies reported here show that lidocaine, a local anesthetic that is reported to interfere with the movement of divalent cations across cell membranes, reversibly blocks cell wall loss and gametic fusion without blocking adhesion or flagellar tip activation. In these experiments lidocaine inhibited both the initial rates and the extent of wall loss and zygote formation. Studies with gametes of a paralyzed flagellar mutant, pf 17, revealed that lidocaine also blocked flagellar surface motility (visualized as movement of polystyrene beads) at concentrations of the inhibitor which also prevented gametic fusion. The concentration of lidocaine required to block cell fusion was dependent on the concentration of calcium or magnesium in the medium. In the absence of added calcium, 0.5 mM lidocaine inhibited fusion by 70%. In 0.5 mM calcium, 0.5 mM lidocaine had no effect on fusion and 2 mM lidocaine was required for 90% inhibition. The results suggest that divalent cations may play a critical role in sexual signalling in Chlamydomonas.

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.

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.