Selection for conditional gametogenesis in Chlamydomonas reinhardi.

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella . By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone . (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actinfilled fertilization tubule during activation . Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse . The imp-1 mutation is linked to the mating-type plus (mt + ) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome . (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse . The imp-11 mutation converted a wild-type minus cell into a pseudoplus strain ; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.Biological membranes do not ordinarily fuse with one another : the eucaryotic cell is filled with membrane systems and organelles that maintain their integrity . The important exceptions to this rule include endocytosis, the rough endoplasmic reticulum (RER) --* Golgi --* exocytosis transit, the fertilization of gametes, and the formation of such syncytia as myofibrils . In each case, the relevant membranes fuse in a highly specific fashion, and only in response to well defined physiological signals (e.g. receptor clustering, Ca" fluxes, or-possibly-the presence of a clathrin coat) .Fusion between gametic cells of Chlamydomonas reinhardi (reviewed in references 8 and 9) is particularly well suited to experimental analysis. (a) The discrete sector of the plasma membrane that is specialized for this event can be readily identified in either thin section or freeze-fracture replicas (2,7,13,23,24) . (b) Fusion specificity resides in this membrane sector : mating-type plus (mt+) gametes will fuse only with minus (mt) partners of the same species. (c) These membranes acquire the capacity to fuse in response to a defined physiological signal, namely, the adhesion of flagellar membranes (1,12,18,22,25). (d) Chlamydomonas can be readily manipulated genetically (4,20), offering the possibility of isolating nonfusing mutants. 378Previous studies from this laboratory (13, 24) defined several stages in the acquisition of fusion capacity . The present report exploits the ability of fixation in the presence of tannic acid and en bloc staining with uranyl acetate to reveal new features of th...

Section: The Imp-11 Mutationmentioning

confidence: 99%

Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

Goodenough¹,

Detmers²,

Hwang³

1982

“…Two of the nonagglutinating impotent mutants (imp-2 and imp-8) (6,19,20), crossed into apf-18 background, were used in several experiments. The conditional nonsignaling mutant gam-1 (16) was assayed at both the permissive temperature (25°C) and the restrictive temperature (35*C) .…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Experimental dissection of flagellar surface motility in chlamydomonas

Hoffman¹,

Goodenough²

1980

Experiments have explored the possible relationships between the flagellar surface motility of Chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol . 15 :983-989), and the capacity of gametic flagella to participate in the mating reaction . While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those on gametic flagella exhibit no such preference . This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, /. Cell Biol. 79 :680-693) . Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-ß-tubulin antiserum are all able to inhibit the binding of beads to the flagellar surface. Trypsin digestion and an antiserum directed against whole Chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile . These results suggest that at least two flagellar activities participate in surface motility : (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e .g . an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signalto-fuse from the flagellar tips to the cell body . Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella .Movement of marker particles on the surface of biological membranes has been widely used to study membrane fluidity and cell migration (1,3,4,10,13,23,24,26) . Recently, discussion has centered on possible connections between membranes and underlying cytoskeletal structures (i .e., axonemes, microtubules, microfilaments, and actomyosin complexes) (11,12,31) . It therefore becomes of interest to learn whether surface particle movements are driven by known components of the cytoskeleton, and whether these movements can serve to transmit biological information to the cell interior.We have addressed these questions by analyzing surface motility associated with the flagellar membrane of the unicellular eukaryote Chlamydomonas reinhardi. As described and discussed in detail by Bloodgood and co-workers (7,8), the flagella will bind polystyrene microspheres, small fragments of debris, or bacterial cells suspended in methyl cellulose, and these will be rapidly transported up and down the length of the flagellum . Each bead moves linearly along the flagellum, and if several beads are bound to the same flagellum they can pass ea...

“…A particularly effective method for studying gametogenesis appears to be the analysis of mutants defective in the various gamete-specific properties required for zygote formation. One such mutant, gam-1, carries a temperature-sensitive defect: it can agglutinate but not fuse at the restrictive temperature (35~ while behaving normally at the permissive temperature (25~ (5). When gam-1 cells that have undergone gametogenesis at 35~ are shifted down to 25~ zygotes are first detected after -45 min, although at least lt/2 h is needed before a large number of zygotes are seen.…”

mentioning

confidence: 99%

Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.

Forest¹,

Goodenough²,

Goodenough³

1978

Self Cite

The temperature-sensitive gametogenesis-defective mutant, gain-1 is sex-limited, expressed only in mating type minus (rot-), and can sexually agglutinate but not fuse at the restrictive temperature (35~ with gametes of wild type (wt) mt +.Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gain-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35~ each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gain-1 flagella. The two mutant phenotypes displayed by gain-1 have been denoted gam-l-I and gam-l-II. An agglutination reaction involving gam-I-I cells does not result in activation of the wt mt § mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-l-II cells, but because the gam-1 rot-mating structure still fails to activate, cell fusion is precluded. We conclude that the gain-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.KEY WORDS mutant temperature sensitivity flagellar membrane sexual agglutination signaling mating structureThe sexual cycle of the single-celled eukaryoteChlamydomonas reinhardi represents an easily controlled developmental process. Gametogenesis is initiated when the haploid cells are starved for nitrogen (11). Both mating types then develop the ability to agglutinate by their flagellar tips (6, 11), a reaction that occurs immediately when gametes of opposite mating types are mixed. During gamete formation, mating-type-specific mating structures also develop in association with the plasma membrane (2,6,9,14,17). These mating structures remain unactivated unless stimulated by some sort of sexual agglutination. The activated structure of mating-type plus (mt +) is a long, 74 J. CELL BIOLOGY ~ The Rockefeller University Press