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.

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 aggutinins. 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 reductionJalkylation--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 first cell-cell recognition mechanisms developed by eukaryotes were probably used in fertilization. It follows that the modern sexual protozoa, although clearly "highly evolved" in their own right, may offer clues as to what kinds of cell surface molecules were originally used to mediate gametic fusion with the appropriate partner. In the unicellular alga Chlamydomonas reinhardi, the key sexual recognition molecule has been shown to be a large hydroxyproline-rich fibrous glycoprotein (1, 2). Since similar proteins appear to function in the organization of the extracellular matrix in higher plants (3), and since the collagens, also rich in hydroxylated amino acids, are widely implicated in organizing the tissues of the metazoa (4), an understanding of the Chlamydomonas agglutinin structure may yield evolutionary insights into the origins of cell-cell interactions in eukaryotes.This paper first presents a detailed analysis of the purified agglutinin protein using the quick-freeze, deep-etch techniques developed by Heuser (5,6). We compare the structure 924 niques developed by Heuser (5, 6). We compare the structure of the mating-type plus (rot +) and mating-type minus (rot-) proteins, and describe alterations in their structure induced by proteolytic digestion and disulfide reduction/alkylation. In the course of this study we h...

Abstract. 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 FITCstreptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/mi) 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 GPI into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Ch/amydomonas 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.

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A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 12~I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas o~-and fl-tubulins; we also demonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane. KEY WORDS lz~I-protein Aantigen-antibody complexes autoradiography tubulin membranesThe characterization of cellular components using immunological approaches has been hampered by the fact that many polypeptides of interest (e.g., tubulin, actin) are poor antigens, eliciting weak and/or nonprecipitating antisera that are often difficult to analyze. Moreover, many structures of interest (e.g., cell membranes) carry a complex array of components, some more antigenic than others. Antisera raised against intact membranes therefore contain a complex array of antibody species at varying titers, and such antisera have heretofore been of limited experimental value.We report here a simple, rapid, and highly sensitive method, which utilizes 125I-labeled protein A from Staphylococcus aureus for analyzing antisera against cellular components. We then show how the procedure can be applied to basic biological questions, presenting experiments which demonstrate interspecific relationships between a-and/3-tubulin polypeptides and experiments which reveal both the major and minor polypeptides of a cell surface membrane. MATERIALS AND METHODSProtein A (Pharmacia Inc., Piscataway, N. J.) was labeled with ~25I (New England Nuclear, Boston, Mass.) by the chloramine-T method of Greenwood et al. (5) except that the chloramine-T was added in four aliquots at 15-s intervals. The labeled product (sp act = 1.5 x 10 ~ cpm/tzg protein) was separated on a G-50 Sephadex column pre-run with 20 mg of bovine serum albumin. Greater than 60% of the radioactivity was covalently bound to protein A under these conditions, and recovery from the column was essentially complete.Sea urchin sperm tail axonemes and antitubulin antisera (raised in rabbits against vinblastine sulfate crystals of sea urchin [Strongylocentrotus] egg cytoplasmic tubulin [4]) were generous gifts of Dr. K. Fujiwara (Harvard Medical School, Boston, Mass.). Flagella were isolated and purified from Chlamydomonas reinhardi gametes by the pH-shock procedure of Witman et al. (12). Antiserum against C. reinhardi mt § gametic flagella (a-G +) was raised in a rabbit by standard procedures; details will be described elsewhere (U. W. Goodenough and D. Jurivich, J. Cell Biol., in press).Samples for gel electrophoresis were solubilized in lysis buffer [3% wt/vol sodium dodecyl sulfate (SDS), 5% vol/vol 2-mercaptoethanol, 10% glycerol, 0.01% wt/vol bromphenol blue, 6.25 mM Tris-HCl, pH 6.8) and boiled for 2 min before loading. Electrophoresis was carried out in slab gels (1 mm • 1 cm • 18 cm) as described in the figure legends, using the discontinuous buffer system of Lae...