Biomedical subjects
U W Goodenough
Publications and source records attributed to U W Goodenough.
Cilia, flagella and the basal apparatus.
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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.
Molecular characterization of a zygote wall protein: an extensin-like molecule in Chlamydomonas reinhardtii.
The green alga Chlamydomonas reinhardtii elaborates two biochemically and morphologically distinct cell walls during its life cycle: one surrounds the vegetative and gametic cell and the other encompasses the zygote. Hydroxyproline-rich glycoproteins (HRGPs) constitute a major component of both walls. We describe the isolation and characterization of a zygote-specific gene encoding a wall HRGP. The derived amino acid sequence of this algal HRGP is similar to those of higher plant extensins, rich in proline and serine residues and possessing repeating amino acid motifs, notably X(Pro)3 and (Ser-Pro)n. Antiserum against this zygote wall protein detected common epitopes in several other zygote polypeptides, at least one of which is also encoded by a zygote-specific gene. We conclude that there is one set of HRGP wall genes expressed only in zygotes and another set that is specific to vegetative and gametic cells.
High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits.
A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.
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.
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.
Transcription of novel genes, including a gene linked to the mating-type locus, induced by Chlamydomonas fertilization.
Six cDNA clones have been identified that are complementary to transcripts present in young zygotes of Chlamydomonas reinhardtii but absent from vegetative and gametic cells. Five early transcripts are synthesized within 5 to 10 min of fertilization; the sixth, late, transcript is not synthesized until 90 min following fertilization. Synthesis of both classes requires cell fusion between gametes. Cycloheximide fails to inhibit early mRNA synthesis, indicating that transcription factors must preexist in the gametes and be activated by cytoplasmic confluence. By contrast, cycloheximide blocks synthesis of the late transcript, suggesting that an early protein product(s) is required for expression of the late gene. Restriction fragment length polymorphism analysis of inter- and intraspecific genetic crosses demonstrates that one of the early genes is very tightly linked to the mating-type locus.
Experimental analysis of the adhesion reaction between isolated Chlamydomonas flagella.
Protocols are presented for producing reproducible in vitro agglutination between Chlamydomonas flagella, based on previous studies by Kohle et al. The results indicate that the adhesive interactions occur between glutaraldehyde cross-linked flagella and can be cross-linked by glutaraldehyde. They are sensitive to divalent cations and, to a lesser extent, to monovalent ions, an inhibition that is fully reversed when the ions are removed. The ambiguities inherent in in vivo studies, particularly in a system where the adhesion molecules have been shown to be in a dynamic state of turnover and inactivation, are eliminated, and numerous studies involving enzyme digestion, antibodies, lectins and the like should now be possible with this adhesion system.
Crystals of the Chlamydomonas reinhardtii cell wall: polymerization, depolymerization, and purification of glycoprotein monomers.
Two of the three major outer layers of the Chlamydomonas reinhardtii cell wall (W6 and W4) can be solubilized from living cells with sodium perchlorate or other chaotropes and will repolymerize in vitro to form milligram amounts of wall crystals. Conditions for optimal crystalization are presented, and conditions that fail to induce polymerization are exploited to maintain monomers in aqueous solution for ion-exchange chromatography. The four major glycoproteins of the complex (GP1, 1.5, 2, and 3) have in this way been purified to apparent homogeneity and have been characterized morphologically by transmission electron microscopy using the quick-freeze, deep-etch technique and by amino acid composition. Three of the four are hydroxyproline-rich species that copolymerize to form the W6 layer. The fourth (GP1.5) is a glycine-rich species that binds to the interior of the in vitro crystal; it is apparently equivalent to the granules within the W4 layer in situ.
Outer and inner dynein arms of cilia and flagella.
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Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella.
The substructure of the components of the axoneme interior--the inner dynein arms, the radial spokes, and the central pair/projection complex--was analyzed for Chlamydomonas. Tetrahymena, Strongelocentrotus, and Mnemiopsis using the quick-freeze, deep-etch technique. The inner arms are shown to resemble the outer arms in overall molecular organization, but they are disposed differently on the microtubule and have two distinct morphologies--dyads with two heads and triads with three. The dyads associate with spokes S3 and S2; the triads associate with S1. The spokes form a three-start right-handed helix with a 288-nm rise; the central pair makes a shallow left-handed twist. The spoke heads are shown to be made up of four major subunits; two bind to the spoke shaft and two bind to a pair of central-sheath projections.
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.
The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.
Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.
The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique.
Using the quick-freeze, deep-etch technique, we have analyzed the structure of the intact cell wall of Chlamydomonas reinhardi, and have visualized its component glycoproteins after mechanical shearing and after depolymerization induced by perchlorate or by the wall-disrupting agent, autolysin. The intact wall has previously been shown in a thin-section study (Roberts, K., M. Gurney-Smith, and G. J. Hills, 1972, J. Ultrastruct. Res. 40:599-613) to consist of a discrete central triplet bisecting a meshwork of fibrils. The deep-etch technique provides additional information about the architecture of each of these layers under several different experimental conditions, and demonstrates that each layer is constructed from a distinct set of components. The innermost layer of the central triplet proves to be a fibrous network which is stable to perchlorate but destabilized by autolysin, disassembling into fibrillar units we designate as "fishbones." The medial layer of the triplet is a loose assemblage of large granules. The outer layer is a thin, crystalline assembly that is relatively unaffected by autolysin. It depolymerizes into two glycoprotein species, one fibrous and one globular. The wall glycoproteins prove to be structurally similar to two fibrous proteins that associate with the flagellar membrane, namely, the sexual agglutinins and the protomers of a structure we designate a "hammock." They are also homologous to some of the fibrous components found in the extracellular matrices of multicellular plants and animals. The quick-freeze, deep-etch technique is demonstrated to be a highly informative way to dissect the structure of a fibrous matrix and visualize its component macromolecules.
Genetic analysis of mating locus linked mutations in Chlamydomonas reinhardii.
The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt+) or the minus (mt-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt+ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt+/mt- diploids.
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.
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.