Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins.

The unicellular alga Chlamydomonas reinhardtii is surrounded by a cell wall composed entirely of hydroxyproline-rich glycoproteins (HRGPs). When the walls of vegetative cells are removed with the enzyme gamete autolysin (g-lysin), they regenerate a matrix within 3-4 hr. In vitro translation of mRNAs isolated from g-lysin-treated cells showed significant increases and decreases in abundance of several mRNAs encoding proline-rich polypeptides. Because the population of up-regulated mRNAs is likely to include species encoding cell wall components, expression of genes for two outer wall HRGPs (GP1 and GP2) was analyzed during wall regeneration by using cDNAs isolated from a C. reinhardtii lambda gt11 library. Transcripts encoding GP1 and GP2 were elevated severalfold within the first hour or regeneration, suggesting that upregulation of HRGP mRNAs is a primary response to cell wall removal by g-lysin. Cell wall regeneration in Chlamydomonas provides an accessible system to study HRGP gene expression during matrix development.

Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales.

The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373-2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae.

Nucleated assembly of Chlamydomonas and Volvox cell walls.

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.

Characterization of the purified Chlamydomonas minus agglutinin.

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.

Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Characterization of Chlamydomonas sexual agglutinins.

Molecules (agglutinins) mediating mating-type-specific adhesion of Chlamydomonas reinhardtii plus and minus gametes have been purified and shown to be homologous by several criteria. Both are large, extrinsic glycoproteins, rich in hydroxyproline and serine, which to a variety of inactivating agents. Direct visualization of agglutinins by the rapid-freeze deep-etch technique of Heuser reveals that both are extremely asymmetric, fibrous molecules with similar but distinct topographies. Each has a globular head, a rigid domain, a relatively flexible region and a terminal hook. In situ images show that the molecules associate with the flagellar surface via the latter (hook) domain, projecting the globular head distally. A functional role for the head is implied from inactivation studies (thermolysin digestion and reduction/alkylation) showing a selective modification of this domain. A library of monoclonal antibodies, raised against the plus agglutinin, has been used to probe the structural organization of this molecule by immunotopographic mapping. Two Two distinct classes of monoclonal antibodies are shown to react with carbohydrate epitopes localized to the two termini (head and hook) of the plus agglutinin while a third recognizes a polypeptide determinant that repeats along the rod-like portion of the molecule. The latter class is gamete (but not mating-type)-specific while the other two classes display distinct patterns of cross reactivity. Finally, the Chlamydomonas agglutinins show a striking compositional and structural resemblance to the major cell wall glycoproteins of this organism. Wall glycoproteins, which are also rich in hydroxyproline and serine and capable of self-assembly, may therefore share a common evolutionary heritage with the agglutinins.

Chlamydomonas agglutinin conjugated to agarose beads as an in vitro probe of adhesion.

Flagellar sexual agglutinins are responsible for the primary recognition and adhesion events of mating in Chlamydomonas reinhardi which culminate in zygotic union of plus and minus gametes. Recent studies in this laboratory have shown the plus agglutinin to be an extremely large (greater than 10(6) D) and asymmetric glycoprotein containing a high proportion of hydroxyproline and serine residues [14, 27, 28]. This paper reports an improved method for in vitro investigations of the adhesive nature of this molecule. Purified agglutinin is covalently attached to an insoluble (Affi-gel 15 agarose bead) support and shown to retain potent agglutination activity when presented to living minus gametes, which rapidly and extensively adhere to the coated bead surface by their flagella. The specificity of the response is documented by the lack of interaction of plus gametes with the immobilized plus agglutinin (IA+). Using this simple yet sensitive bioassay, we have subjected IA+ beads to various enzymatic, chemical and physical treatments and assessed the effects on agglutinin activity. These studies reveal that Chlamydomonas plus agglutinin is sensitive to thermolysin or trypsin digestion, alkaline borohydride reduction, periodate oxidation, thiol reduction and heating at 65 degrees C, but unaffected by treatment with chymotrypsin, endo- or exoglycosidases, or incubation with isolated minus agglutinin. The implications of these results for agglutinin structure and possible functional interactions in initial recognition/adhesion events are discussed.

Chlamydomonas agglutinin is a hydroxyproline-rich glycoprotein.

The mt(+) sexual agglutinin from Chlamydomonas reinhardi is shown to contain approximately 12% hydroxyproline, and two inhibitors of hydroxyproline formation, alpha,alpha'-dipyridyl and 3,4-dehydroproline, are shown to block the production of agglutinin activity in an in vivo bioassay system. These results indicate that the agglutinin glycoprotein may be related to a class of hydroxyproline-rich glycoproteins found in the extracellular matrix of higher plants, several of which have been shown to have lectin activity.

Identification and visualization of the sexual agglutinin from the mating-type plus flagellar membrane of Chlamydomonas.

Sexual agglutinins located on the flagellar membranes of Chlamydomonas gametes mediate a mating-type-specific adhesion reaction that brings complementary gametes together for zygotic cell fusion. We identify the mating-type plus agglutinin, using a combination of biochemical and genetic analysis, as a glycopolypeptide with an apparent molecular weight of greater than 10(6) by SDS-polyacrylamide gel electrophoresis. Its core polypeptide migrates as a approximately 480-kd species, and it is estimated to be present in approximately 30 copies per gametic flagellum. The agglutinin is present in the wild type, in a mutant that agglutinates but cannot fuse, and in a complementing diploid, whereas it is absent from four nonagglutinating mutants and from a noncomplementing diploid. Electron microscopy shows the purified agglutinin to be a highly asymmetric molecule, 220 X 4 nm. To our knowledge, this is the first reported purification and visualization of a membrane-associated cell-cell recognition protein.

Topography of Chlamydomonas: fine structure and polypeptide components of the gametic flagellar membrane surface and the cell wall.

Surface polypeptide components of the flagellar membrane of Chlamydomonas reinhardi Dang. gametes are identified by their accessibility to in-vivo vectoral labeling by glucose oxidase-coupled lactoperoxidase-dependent (125)I iodination. Vectoral labeling is accomplished without observable adverse effects on cell viability or gametic function. Flagella isolated from labeled wild-type cells carry about 3% of the total incorporated label, which is found by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be distributed among 16 identifiable polypeptide bands. The most prominent surface-labeled species migrates in the Mr (relative molecular weight) 350 k region of the gel; each of the remaining iodinated polypeptides, which range in Mr from 25 k to 500 k, carries only a small proportion of incorporated label. To determine which polypeptides are unique to the flagellum and which are contaminants from the cell wall, wild-type profiles were compared with those of mutant strains and of mechanically isolated cell walls. Identification of contaminants was also facilitated by two-dimensional peptide mapping. We conclude that only 11 of the labeled bands are contributed by flagellar polypeptides; the remaining five bands are shown to be contaminants from the cell wall, and additional cell-wall polypeptides are found to co-migrate with flagellar species. A polypeptide designated as a possible membrane tubulin in preliminary studies is shown here to be different from tubulin in its peptide map. The 11 polypeptides assigned as specific flagellar surface components are candidate participants in such biological events as sexual adhesion, flagellar surface motility, and sensory signalling.

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization.

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.

Membrane-membrane and membrane-ligand interactions in Chlamydomonas mating.

Our investigations of the mating reaction of Chlamydomonas revealed a surprisingly intricate series of interrelated events. Adhering sites are moved to the flagellar tips in a fashion highly reminiscent of the capping of surface ligands over the centriolar regions of lymphocytes (28). Tipping is prevented by the gam-1 mutation and by agents that interact with tubulin; the molecular mechanism(s) for the inhibition effects are currently being sought. Tip locking appears to be accompanied by the accumulation of a dense material beneath the tip membrane, a postulated alteration of axonemal structure, and an immobilization of component(s) involved in surface motility. Two mating signals are then transduced to the locked-in cells who respond by shedding cell walls, activating mating structures, and fusing together. Signal transmission and/or reception is sensitive to such agents as trypsin, chymotrypsin, and cold temperature. Once zygotic cell fusion has occurred, tip unlocking and a reversal of the tip activation response appear to occur in parallel. Since all of these events can occur within 30 sec, the mating reaction serves as an experimental paradigm for studying rapid cellular responses to specific membrane-membrane interactions.

Localization of cellular antigens in sodium dodecyl sulfate-polyacrylamide gels.

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins; we also domonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane.

Induced alteration in uptake properties of Tetrahymena and its association with the development of mating competency.

The rate of uridine uptake in Tetrahymena declines by an order of magnitude by two hours after shiftdown to a non-nutrient buffer. This alteration in uptake properties cannot be accounted for by an increase in the intracellular pool of uridine, an increase in apparent Km for uptake or a decline in the rate in which uridine is processed intracellularly. It is argued that the decrease in uridine uptake is due to a reduction in numbers of functional transport molecules exposed at the cell surface and is a reflection of a developmentally related cell surface transformation. In addition, the putative decline in functional transport molecules cannot be entirely explained by metabolic turnover of these molecules in the absence of replacement, nor does it require the synthesis of new protein. We discuss the possibility that a shift in equilibrium between accessible and inaccessible transporters is operating. Finally, a close correlation between conditions which elicit the transport alteration and those which allow the development of mating competency suggests that the two phenomena may be coordinately regulated.

A cytological study of micronuclear elongation during conjugation in Tetrahymena.

Micronuclear elongation is the first major event in a series of nuclear changes occurring during the sexual stage of the life cycle of Tetrahymena. Beginning at about one hour after cells of complementary mating types have conjugated, the micronucleus leaves its recess in the macronucleus and swells slightly. This is accompanied by a reorganization of its chromatin from a reticular to a solid body. In the next stage the micronucleus assumes an egg shape, a development concomitant with the appearance of microtubules. While the chromatin "spins out" from the dense body, and microtubules increase in number, the nucleus assumes a spindle shape. During the elongation, which increases the length of the nucleus some fifty fold, microtubules are prominent in clusters just internal to the nuclear membrane, and parallel to the longitudinal axis of the nucleus. When elongation is completed the nucleus is curved around the macronucleus. Internally, partially condensed strands of chromatin are located off-center, towards the macronuclear side, and the density of the microtubules is diminished. At all the stages, DNA is located throughout the nucleus; neither discrete chromosomes nor synaptonemal complexes are seen. Occasionally cytoplasmic membrane systems are seen fused to the nuclear envelope which retains the typical appearance of a double membrane with pores.