Inhibition of conjugation in Tetrahymena pyriformis by concanavalin A : Binding of concanavalin A to material secreted during starvation and to washed cells

A group of glycoproteins which form an insoluble complex with concanavalin A (ConA) are secreted during starvation of the mating types strain, as well as by the non-mating types of Tetrahymena pyriformis. These glycoproteins were isolated by Sepharose-ConA, characterized, and their relevance to the conjugation process studied. The isolated ConA binding proteins (CBM) contain about 26% of their total weight, a phenol sulfuric acid-positive material, presumably carbohydrates, and exhibit about 5–8 major bands by gel electrophoresis analysis. The possibility that ConA inhibits the conjugation process by precipitating with this material was tested. Addition of isolated CBM restored conjugation previously inhibited by ConA. However, incubation of isolated CBM with either of the mating types or with both did not have any effect on the kinetics of the conjugation process. Antibodies prepared against CBM-secreted by both mating types did not prevent conjugation when added to the conjugation medium. The data suggest that CBM does not directly participate in the conjugation process. Inhibition of conjugation by ConA is probably due to its interaction with specific membrane-bound glycoproteins.     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

DURATIONS OF GAMETE MOTILITY AND CONJUGATION ABILITY OF ULVA COMPRESSA (ULVOPHYCEAE)1

The present study was designed to develop a technique for crossing and to gain insight into how sexual reproduction contributes to the maintenance of local populations of Ulva compressa L. To examine the durations of gamete motility and conjugation ability, freshly released gametes were incubated for various periods of time prior to mixing both mating types. The conjugation ability of the gametes gradually declined after being released from the thalli when the gametes were incubated without mixing with the opposite mating type. The ability to conjugate decreased by half after 6 h, although most of the gametes remained motile. The gametes released 4 h later had the same level of conjugation ability when mixed immediately after releasing. When the mature thalli were wrapped in a moist paper towel to prevent gametes from being released, the gametes were preservable for 7 h without a significant decrease in their conjugation ability. Conjugation occurred soon after mixing gametes of both mating types and reached a plateau after 30 s. However, conjugation rates did not exceed a rate of ～70%, even though freshly released gametes were used. Interestingly, a portion of the gametes newly conjugated 30 min after mixing both mating types, and conjugation rates reached a second plateau at ～90%. Gametes with delayed conjugation are provided some period of time that allows them to be transported away and increases their chances of mating with more distant populations, thus contributing to the maintenance of genetic variation.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

The wound-healing responses of Antithamnion nipponicum and Griffithsia pacifica (Ceramiales, Rhodophyta) monitored by lectins

The binding of FITC labeled lectins to repair cells of Antithamnion nipponicum Yamada et Inagaki and Griffithsia pacifica Kylin, and their physiological effects on somatic cell fusion have been studied. Results indicate that repair cells strongly bind the lectins ConA and LCA, whereas other lectins did not bind to the cell, The binding of these lectins to the dead cell wall shows ConA and LCA specific substances are secreted from the tip of the repair cells. When fluorescently labeled ConA or LCA was added at various time intervals after wounding, it firstly bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair ceils. Intense labeling at these sites continued throughout the wound-healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion, When added to plants prior to wounding and with continued monitoring, these same lectins were found to act as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. These results suggest that a signal glycoprotein with α-D-mannosyl residues is involved in the wound-healing process of Antithamnion nipponicum. Lectins conjugated with visible tags can be used as a very fast and useful tool to monitor these signal substances.     

Cell Pairing during Mating in Tetrahymena: I. Does Phagocytosis or a Cell Surface Receptor Participate in Con A Block?1

The lectin, Concanavalin A (Con A), inhibits cell pairing during mating in Tetrahymena and binds to the surface of pairing cells via receptors concentrated around the conjugation junction. Concanavalin A is also ingested in large amounts into food vacuoles. To dispel the possibility that Con A inhibits pairing via uptake into food vacuoles or through induction of food vacuole formation and to strengthen the idea that pairing is blocked through binding of Con A to cell surface receptors, we have conducted three types of experiments: 1) attempts to inhibit pairing by feeding with nutrients and with tantalum, a non-nutritive reagent; 2) a temporal analysis of the presence of food vacuoles in mating cells fed with tantalum; and 3) analysis of the restoration of pairing following the addition of α-methyl mannoside to cells previously treated with inhibitory concentrations of Con A. The results of these studies support the idea that Con A inhibits pairing by binding to receptors located on the cell surface and not by induction of or uptake into food vacuoles. We also present evidence that cells grown in an enriched proteose peptone medium are able to pair and undergo morphogenesis more readily than cells grown in 2% proteose peptone.     

Inhibition of ConA erythroagglutination by alkali-treated lipopolysaccharide

Bacterial lipopolysaccharide treated by hydrolysis in basic solution (hLPS) and then added to suspensions of human erythrocytes markedly inhibited erythroagglutination by concanavalin A (ConA), soybean agglutinin (SBA), and Phaseolus vulgaris phytohemagglutinin (PHA). Likewise, red cells presensitized by exposure to hLPS and then suspended in hLPS-free buffer were not agglutinated by ConA. However, when hLPS was not added to buffer both SBA and PHA strongly agglutinated erythrocytes presensitized by hLPS. Also, the binding of ³H-labeled ConA to red cells was unaffected by presensitization of the cells with hLPS. It appears, therefore, that membrane-associated hLPS localizes in the ConA receptor regions of erythrocyte membranes and inhibits ConA erythroagglutination by disruption of receptor responses to bound ConA or alteration of the active subunit structure of bound ConA.     

Effect of Adenine on Paramecium multimicronucleatum*

SYNOPSIS. The addition of adenine to the culture medium of Paramecium multimicronucleatum caused an inhibition of conjugation and fission and also an increase in the number of food vacuoles. The inhibition of conjugation was observed at the stage of adhesion of cell membranes. With adenine, the duration of fission was prolonged and L-shaped cells were formed. Adenine apparently repressed the excretion of food vacuoles and thus caused an accumulation of food vacuoles within the cells. All these effects were observed at similar concentrations of adenine higher than 2 mM. The addition of uridine to the adenine-containing medium reversed the inhibition of fission; conjugation and vacuole accumulation remained unaffected.     

Morphological and Mutational Analysis of Mating in Ustilago hordei

Martinez-Espinoza, A. D., Gerhardt, S. A., and Sherwood, J. E. 1993. Morphological and mutational analysis of mating in Ustilago hordei. Experimental Mycology 17, 200-214. Ustilago hordei is a basidiomycete that causes covered smut on barley. Mating in U. hordei, which is controlled by a single locus with two alleles, results in the conversion of haploid, nonpathogenic yeast-like sporidia to dikaryotic, pathogenic mycelia. When sporidia of the opposite mating type were mixed and placed on water agar, both cell types produced conjugation tubes within 2 h at 21°C. Growth of conjugation tubes was directed toward the tip of tubes arising from cells of the opposite mating type. These tubes fused and the dikaryotic mycelium emerged from the conjugation bridge. Sporidia separated by a dialysis membrane were still capable of inducing conjugation tube formation by cells of the opposite mating type, indicating the involvement of diffusible small-molecular-weight mating factors (pheromones). Numerous nutritional and environmental variables were examined in order to optimize conjugation tube induction. Twenty-six mutants that fail to form dikaryotic mycelium have been isolated and characterized. These mutants were arranged into classes based on their ability to form conjugation tubes, the ability to induce conjugation tube formation by opposite mating-type cells, and cell morphology. These mutants provide an indication of the genetic complexity involved in this critical phase of the U. hordei life cycle.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method. Combined transmission and scanning electron microscopic study of ConA binding sites in mouse macrophages

Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-point-dried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the three-dimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0 degree C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0 degree C were further incubated at 37 degrees C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cell-bound ligands.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method

Summary Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-pointdried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the threedimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0° C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0° C were further incubated at 37° C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cellbound ligands.This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, and the Japan Private School Promotion Foundation     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Inhibition of Formation of Escherichia coli Mating Pairs by f1 and MS2 Bacteriophages as Determined with a Coulter Counter

The effect of male-specific filamentous deoxyribonucleic acid (f1) and isometric ribonucleic acid (MS2) bacteriophages on the formation of mating pairs in Escherichia coli conjugation was examined directly in the Coulter counter. When a sufficient multiplicity of infection (MOI) was used, the f1 phage immediately and completely inhibited the formation of mating pairs. On the other hand, the MS2 phage at a relatively high MOI also inhibited the formation of mating pairs significantly although not completey. The inhibitory effect of MS2 phage was dependent on the time of addition and the MOI used. At relatively low MOI (<20), the MS2 phage showed some inhibitory effect when added to a male culture prior to mixing with females, whereas no effect was observed when phages were added after mating pair formation had already commenced. At a high MOI (>400) MS2 phage disrupted the mating pairs already formed. Some preformed mating pairs were resistant to the high MOI of MS2 phages, however, and the "sensitive" (to high MOI) mating pairs seem to mature into "resistant" mating pairs as a function of time. We conclude that the tip of an F pilus is the specific attachment site for mating. The following process of mating pair formation has been formulated by deduction. (i) The sides of F pili weakly contact female cells, (ii) then the tips of F pili attach to the specific receptor sites to form initial mating pairs, and (iii) those pairs mature into mating pairs that are resistant to the high MOI of MS2 phages. The high MOI of MS2 prevents the first step, whereas f1 phages affect the second step-the binding between the tips of F pili and the receptor sites.     

Life Cycle of Paramecium bursaria Syngen 1 in Nature

ABSTRACT. Studies were completed on the natural population density of Paramecium bursaria syngen 1 and on the life cycle stages to which the individuals belonged. Green paramecia were collected from two streams once every 20 days for over one year: 413 individuals on 26 collection dates in Mikumarikyo stream and 83 individuals on 23 collection dates in Momijidani-gawa stream. Individuals in nature did not maintain at a steady density but fluctuated greatly depending on the month. It seems that conjugation occurred from April to June in the Mikumarikyo stream and from May to June in the Momijidani-gawa stream. The appearance of individuals with mating ability might be related closely to increasing population so that sexual reproduction probably occurred near the peak of the population density. The 413 individuals from Mikumarikyo stream were examined to determine their position within the life cycle; 309 (74%) were immature, 55 (13%) were adolescent, and 49 (12%) were mature. No senile individuals were observed. The fraction of individuals with mating ability was generally less than 30% at any collection. Four mating types were observed occurring with about equal frequencies in mature individuals. The results show the frequencies of the recessive genes for mating types (a and b) are higher than for dominant genes (A and B). Of 83 individuals from Momijidani-gawa stream, 44 (52%) were immature, 21 (25%) were adolescent, and 18 (21%) were mature. Again, no senile individuals were observed. Because only two mating types were found, II and III (genotypes aaB- and aabb), it seems possible that the dominant gene A was rare or absent in the Momijidani-gawa population.     

The Relation of Concanavalin A Receptor Distribution to the Conjugation Process in Tetrahymena thermophila

By using fluorescent isothiocyanate-conjugated concanavalin A (FITC-Con A), cell surface events were examined under a light microscope during the early period of the conjugation process in Tetrahymena thermophila. Until the two complementary mating types (D-III and IV) were mixed, Con A-binding activities were hardly detected on the cell surface of ciliates. After mixing, however, the FITC-Con A (25 μg/ml) bound especially to the anterior cell surface at the early stage of conjugation, followed by characteristic changes of the Con A-binding pattern and, subsequently, by formation of a bright fluorescent ring around the area of contact between conjugants. Such alterations of FITC-Con A-binding pattern were found to be interrupted or eliminated by cycloheximide (2 μg/ml). These findings are related to the onset and subsequent conjugation in T. thermophila.     

The endocytosis of concanavalin A by Dictyostelium discoideum cells

Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α- mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α- mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [¹²⁵I]ConA, uncompetable by methyl-α- mannopyranoside in our and other binding studies, may be this intracellular ConA.     

利用铁葡聚糖分离纤毛虫接合对的方法

介绍一种制备微小铁葡聚糖颗粒以及利用微小铁葡聚糖颗粒分离纤毛虫接合对的方法,通过本方法可以获得接合率达95%以上,且发育基本同步的实验材料。     

Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.     

Concanavalin A mediated attachment and ingestion of red blood cells by macrophages.

The interaction of red blood cells and macrophages mediated by Concanavalin A (ConA) was studied using mouse peritoneal macrophages and fresh, homologous red cells. Erythrocytes exposed to ConA at 0.5 μg/ml, a condition that leads to a saturation of 3% of the ConA sites, were bound by macrophages at 22 °C. The ConA inhibitor, α-methylmannoside, prevented this attachment of red cells and largely reversed it when added to preformed macrophage-red cell rosettes up to 90 min. However, red cell attachment was essentially irreversible by 3 h. Electron microscopy showed a progressive increase in the degree of contiguity between red cells and macrophages with time, some macrophage projections distorting and partially encircling red cells at 3 h. Macrophages pretreated with high concentrations of ConA (25 μg/ml) also bound red cells. However, phagocytosis of adherent red cells did not occur at either 22 or 37 °C, even when both red cells and macrophages were pretreated with ConA. In contrast, phagocytosis of attached red cells was observed when preformed rosettes were exposed to ConA at a concentration of 5 μg/ml, and it was complete with ConA at a concentration of 25 μg/ml. These studies demonstrate that ConA in low concentration on red cells is detected by macrophages which form a progressively tighter bond with the red cell surface. However, it appears that phagocytosis can occur only under conditions in which a high density of ConA is established on the surface of the red cell.     

Middle aged wasps mate through most of the year, without regard to body size, ovarian development and nestmateship: a laboratory study of the primitively eusocial wasp Ropalidia marginata

We studied the mating behaviour of the primi-tively eusocial wasp Ropalidia marginata and the factors that may influence sperm transfer. By introducing a male and a female R. marginata into ventilated transparent plastic boxes, we were able to observe mating behaviour, and it involved mounting and short or long conjugation of the wasps. Dissection of female wasps after the observation indicated that long conjugation is a good behavioural predictor of sperm transfer. This finding makes it possible to obtain mated females without dissecting them every time. We tested the effect of age, season, relatedness, body size and female’s ovarian status on mating. Under laboratory conditions, mating success declined rapidly below and above the ages 5–20 days. Within this age range mating success was significantly low in December compared to other months tested. There was no nestmate discrimination, and there was no influence of male and female body size or of the ovarian state of the female on the probability of sperm transfer.