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.     

Immunological Characterization of Gamete Autolysins in Chlamydomonas reinhardtii

Polyclonal antibodies were raised in rabbits against the C. reinhardtii cell wall lytic enzyme, autolysin, which dissolves the cell wall of gametes prior to cell fusion. The purified immunoglobulins react with both the native and the deglycosylated forms of this gametic autolysin and specifically inhibit enzyme activity. In addition, the immunoglobulins selectively detect the gametic autolysin in immunoblots of crude extracts and do not cross-react with the autolysin of the vegetative cells. The antibodies have been used to study the time of synthesis of the enzyme during gametogenesis and to compare gametic autolysins of different strains of Chlamydomonas.     

Wall loss and origin of soluble carbohydrates during mating of Chlamydomonas reinhardi

In the mating reaction between gametes of the green alga Chlamydomonas reinhardi, a lytic factor which solubilizes the cell wall is released. It has been shown that carbohydrates accumulate in the supernatant of mating gametes. We present here data which support the notion that the released carbohydrates arise from solubilized gametic cell walls. The evidence is based, in part, on the comparison of the carbohydrates and amino acids in the acid hydrolysates of cell-free supernatants to the reported composition of isolated cell walls. In both cases the three predominant sugars are mannose, arabinose and galactose, and also, in both cases, large amounts of the amino acid hydroxyproline are present. In addition, it is shown that if gametic cell walls are removed prior to mating interactions by treatment with a preparation of lytic factor, much less carbohydrate is subsequently released into the supematant, when such ‘nake’ gametes are mated.     

Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes

A quantitative assay for cell wall release in Chlamydomonas has been used to study the timing of release of cell wall degrading enzyme (lysin) during adhesion. Lysin activity, which shows a broad pH range and requires divalent cations, is released as a pulse within 1–2 min after mixing of mt^? and mt⁺ gametes. Thereafter, there is no further lysin release. Gametes of both mating types release the activity during aggregation with isolated gametic flagella of the opposite mating type, although mt⁺ gametes appear to release more lysin activity than mt^? gametes. Electrophoretic analysis of cell wall proteins before and after lysin degradation indicate that the major wall proteins are unchanged after wall breakdown.     

Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall

Chlamydomonas lytic enzyme of the cell wall, which is released during agglutination of gametes of opposite mating types, has been characterized as a metalloprotease. The purified enzyme contains zinc. Removal of zinc with EDTA results in an inactive, metal-free apoenzyme, and Co2+ restores the activity most effectively. Among various protease inhibitors of microbial origin, pepstatin A, chymostatin, antipain, leupeptin, and E-64 do not inactivate the enzyme, whereas phosphoramidon causes a complete loss of lytic activity. Cysteine, histidine, aspartic acid, and glutamic acid also inhibit the activity. The lytic enzyme splits casein and RNase A into several polypeptides of lower molecular masses. To determine which polypeptides of the cell wall are sensitive to the lytic enzyme, we first separated the intact cell walls into sodium perchlorate-soluble and -insoluble components, treated them with enzyme, and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. We conclude that only 2 of 16 polypeptides are digested by exposure to the enzyme and that the sensitive polypeptides belong to the salt-insoluble component of the cell wall. The mechanism of cell wall digestion with the lytic enzyme is discussed.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

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.     

Two Novel Endopeptidases Released into the Medium during Mating of Gametes of Chlamydomonas reinhardtii

During the mating reaction between mt⁺ and mt^- gametes of Chlamydomonasreinhardtii, two novel endopeptidases, each of which was ableto digest the B chain of insulin, were released into the culturemedium, together with a gamete lytic enzyme (GLE) which is responsiblefor digestion of the gametic cell walls. Both endopeptidasesand GLE were copurified from the mating medium by column chromatographyon DEAE-cellulose and concanavalin A. Gel filtration separatedthe peptidases, which were unable to digest gametic cell walls,into two fractions, endopeptidase-1 and endopeptidase-2. Theseenzymes were also separated from GLE, which was unable to digestthe B chain of insulin. Endopeptidase-1, with a molecular massof about 200 kDa, cleaved the B chain of insulin at the Ala¹⁴-Leu¹⁵peptide bond, and this activity was inhibited by EDTA. Endopeptidase-2,with a molecular mass of about 110 kDa, digested the peptideat the Leu¹⁵-Tyr¹⁶ peptide bond and was sensitive to iodoacetateand chymostatin. When the cell walls of gametes of either mating-typewere digested prior to mating with exogenously added GLE, thetwo endopeptidases were released into the medium, a result thatsuggests that they are stored, like GLE, outside the plasmalemma. (Received March 25, 1994; Accepted June 13, 1994)     

Membrane differentiations at sites specialized for cell fusion

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.     

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.     

Developmental changes in ciliary composition during gametogenesis in Chlamydomonas

Chlamydomonas reinhardtii transitions from mitotically dividing vegetative cells to sexually competent gametes of two distinct mating types following nutrient deprivation. Gametes of opposite mating type interact via their cilia, initiating an intraciliary signaling cascade and ultimately fuse forming diploid zygotes. The process of gametogenesis is genetically encode, and a previous study revealed numerous significant changes in mRNA abundance during this life-cycle transition. Here we describe a proteomic analysis of cilia derived from vegetative and gametic cells of both mating types in an effort to assess the global changes that occur within the organelle during this process. We identify numerous membrane- and/or matrix-associated proteins in gametic cilia that were not detected in cilia from vegetative cells. This includes the pro-protein from which the GATI-amide gametic chemotactic modulator derives, as well as receptors, a dynamin-related protein, ammonium transporters, two proteins potentially involved in the intraciliary signaling cascade-driven increase in cAMP, and multiple proteins with a variety of interaction domains. These changes in ciliary composition likely directly affect the functional properties of this organelle as the cell transitions between life-cycle stages.     

Factors affecting the mating competence in the unicellular green algaChlamydomonas eugametos (Volvocales)

Routinely prepared gametes (by flooding 3 week-old agar cultures) showed about 80% mating competence if the opposite sexual partners were mixed together. The mating competence exhibited a strict dependence on the composition of the solution in which the cells were suspended before mixing; it decreased progressively with increasing concentration of nitrates. In contrast, no inhibiting effect was found if urea was used as the source of nitrogen. Other ions present in nutrient media did not show any effect. Mating activity varied according to the spectral composition of light, being higher with a blue light than with a red one. Blue light caused accumulation ofvis-à-vis pairs, which were blocked to form zygotes. Freshly released daughter cells in vegetatively grown synchronous cultures had a dual nature—vegetative and sexual one. In these daughter cells, similar rules were found for governing of mating competence to those valid for standard gametes obtained from flooded agar cultures. High mating competence was found in daughter cells released the during dark period in distilled water, nitrate-free media, in the presence of Mg²⁺ or Ca²⁺ ions, or in media containing urea. The conditions during which daughter cells are released and the conditions under which they mate can be considered crucial for expression of gametic nature as a mating competence.     

Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas

New methods have been developed for the purification and characterization of the cell wall degrading enzyme, lysin, which is released into the medium during the mating reaction of the biflagellated alga Chlamydomonas reinhardtii. A quantitative spectrophotometric assay that detects the number of cells losing walls was used to devise a procedure for the 60-fold purification of lysin from the medium of mating gametes with a 30% yield of activity. Molecular sieve and ion exchange chromatography in combination with SDS-PAGE showed that lysin was a single polypeptide with an Mr of 60,000. High-performance liquid chromatography and sucrose density gradient centrifugation of lysin activity were used to obtain an estimate of 66,000 D for the nondenatured molecular weight of lysin, indicating that lysin behaves as a monomer.     

Activation of the cell wall degrading protease, lysin, during sexual signalling in Chlamydomonas: the enzyme is stored as an inactive, higher relative molecular mass precursor in the periplasm

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.     

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).     

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

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.     

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.     

Mating-induced shedding of cell walls, removal of walls from vegetative cells, and osmotic stress induce presumed cell wall genes in Chlamydomonas

The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3',5'-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.     

Nutritional control of sexuality in Chlamydomonas reinhardi

1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated.