Cell-to-cell contact by locally differentiated surfaces in conjugation of Blepharisma

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium. When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Isolation and partial characterization of gamone 1 of Euplotes octocarinatus

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10⁻¹⁵M.     

Propagation of meiosis and other nuclear changes in multicellular complexes of Blepharisma

Meiosis and other nuclear changes in conjugation of Blepharisma japonicum regularly occur when cells of complementary mating types I and II unite (heterotypic union). But no nuclear changes occur if unions are induced between cells of the same mating type (homotypic union). Similarly, chains of homotypically united cells, induced by treating type II cells of a doublet strain (mutant with two attachment points) with gamone of mating type I, do not undergo nuclear changes. However, if a type I cell unites at one end of such a chain, the nuclear changes of conjugation occur not only in the doublet to which the type I cell unites but also in other doublets in the same chain. We examined the mode of propagation of nuclear activation by surgically separating all cells in the chain and observing the subsequent occurrence of nuclear changes in these isolated cells. The nuclear activation began at the site of heterotypic union and propagated from cell-to-cell without skipping. In chains of a given length, the propagation slowed down as it proceeded in the chain. If compared at the corresponding site of the chain, the propagation was slower in longer chains. We conclude that meiosis and other nuclear changes in conjugation are initiated by a substance originating at the site of heterotypic union and transferable to other cells through the united regions of the cells.     

Requirement of protein synthesis in the initiation of meiosis and other nuclear changes in conjugation of Blepharisma.

In conjugation of Blepharismajaponicum, cell contact between complementary mating types induces meiosis and other nuclear changes. How long the cells must be in contact in order to be induced to undergo these nuclear changes (activated) can be ascertained by surgically separating the united cells at different times after the onset of cell union and then examining the occurrence of the nuclear changes. Applying this technique to cycloheximide-treated cells, we investigated the role of protein synthesis in the activation. Cycloheximide was used at the concentration which was found to inhibit most incorporation of amino acid into protein in this ciliate. Newly formed conjugant pairs were incubated with and without cycloheximide, washed free of the inhibitor and surgically separated. Although untreated controls were activated in 1.8 h after cell-cell contact, no activation was observed in cycloheximide-treated cells after 5 h of contact. Removal of cycloheximide from the paired cells resulted in an activation delayed by the interval of exposure time to the inhibitor. If the pairs were first incubated in normal medium and then exposed to cycloheximide, operated, activated cells appeared and increased very slowly (activation rate, about $\text{1}\text{10}$ of the control). Protein synthesis is therefore required for the initiation of meiosis and other nuclear changes. We propose that heterotypic cell union induces and maintains the synthesis of a protein, whose accumulation to a certain threshold is required for activation.     

Isolation of the mating-inducing factor of the ciliate Euplotes

Numerous strains of different mating types of the marine ciliate Euplotes raikovi have been found to be autonomous excreters into the surrounding medium of specific mating-inducing factors (gamones) (Luporini, P et al., J exp zool 226 (1983) 1 [9]). The gamone from the mating type represented by strain 13 has been isolated and identified as a glycoprotein with a molecular weight (MW) of about 12 kD and a pI of 4. It has been termed euplomone r 13. At a concentration of 3 × 10⁻¹² M, euplomone r 13 specifically induces cells of a complementary mating type to unite in conjugation within 2 h.     

Inhibition of conjugation in Tetrahymena pyriformis by cerulenin. Possible requirement for de novo lipid synthesis

Conjugation in Tetrahymena pyriformis is induced by the mixing of two starved complementary mating types. Addition of the antibiotic cerulenin, a specific inhibitor of de novo lipid synthesis, upon mixing of the mating types inhibited the conjugation process. The inhibition of conjugation was found to be reversible upon washing the cells.Cerulenin inhibited [¹⁴C]acetate incorporation into the lipid fraction of the cells, while it did not affect the incorporation of [³H]leucine into proteins. Analysis of the fatty acid composition of the whole cells revealed that during conjugation the ratio of saturated to unsaturated fatty acids is markedly changed. While the ratio of saturated:unsaturated fatty acids is 0.30 in unconjugated cells, it reached a value of 0.45 in conjugated cells.     

Alternative gene expression in type I and type II cells may enable further nuclear changes during conjugation of Blepharisma japonicum

In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.     

Behavioural changes induced by the conjugation-inducing pheromones,gamone 1 and 2, in the ciliate Blepharisma japonicum

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.     

Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica.

Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Inhibition of conjugation in Tetrahymena pyriformis by cerulenin. Possible requirement for de novo lipid synthesis.

Conjugation in Tetrahymena pyriformis is induced by the mixing of two starved complementary mating types. Addition of the antibiotic cerulenin, a specific inhibitor of de novo lipid synthesis, upon mixing of the mating types inhibited the conjugation process. The inhibition of conjugation was found to be reversible upon washing the cells. Cerulenin inhibited [14C]acetate incorporation into the lipid fraction of the cells, while it did not affect the incorporation of [3H]leucine into proteins. Analysis of the fatty acid composition of the whole cells revealed that during conjugation the ratio of saturated to unsaturated fatty acids is markedly changed. While the ratio of saturated:unsaturated fatty acids is 0.30 in unconjugated cells, it reached a value of 0.45 in conjugated cells.     

Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor

A diffusible substance, α factor, is produced constitutively by haploid yeast cells of α mating type and this factor specifically inhibits the division of a mating type cells. Experiments are presented which demonstrate that α factor arrests a cells as unbudded, mononucleate cells prior to the initiation of DNA synthesis in the cell cycle. Studies with temperature-sensitive mutants defective in one of thirteen different cell cycle functions suggest that although arrested a cells continue to enlarge they do not perform functions required for the next cell cycle. The arrest is reversible and a partially synchronized round of DNA replication is observed upon removal of α factor from arrested cells. We propose that this factor is one element of a regulatory system that functions to assure the synchronization of a and α haploid cell cycles prior to conjugation.     

The mechanism of regulation of fibroblastic cell replication. III. A central role for nutrient limitation: a conditioning effect due to serum imprinting of the cell response

Haploid Saccharomyces cerevisiae cells of mating type a, but not α, produce and secrete a diffusible substance, designated a factor. The a factor transiently arrests cells of mating type α, but not a, at a very early stage of the cell cycle, prior to budding and to the initiation of DNA synthesis. While the cells are arrested at this stage, few, if any, of the functions required for the ensuing cell cycle are carried out. This stage of the cell cycle coincides with the stage at which α factor, produced by cells of mating type a, specifically arrests cells of mating type a [2]. It seems probable that the reciprocally acting a and α factors together provide the mechanism by which haploid cells are synchronized to the appropriate stage of the cell cycle as a prelude to conjugation.     

Mating Types in Aspidisca sp. (Ciliophora, Hypotrichida): a Cluster of Cryptic Species

A mating-type analysis was performed on 78 stocks of the marine hypotrich ciliate, Aspidisca sp., from a sufficient number of diverse geographic locations, some widely separated. Evidence is provided for the existence of a binary mating system in this "morphospecies." The collected stocks have been challenged by the most rigorous criterion, namely breeding affinity in the laboratory, and have yielded at least four reproductively, not necessarily geographically, isolated groups that are in fact "biological species," here referred to as "syngens." Different syngens contain different pairs of mating types. Syngens are morphologically indistinguishable; hence Aspidisca sp. can be considered a conservative taxon comprising a number of "cryptic" or "sibling species." Information is also presented about the mating behavior and the pattern of nuclear events at conjugation in Aspidisca sp. Search for soluble pheromones of the mating types gave only negative results. Hence, direct contact with potential partners is postulated to play a critical role in preparing individuals to mate. Mating reaction and mating which actually involves cross-fertilization (conjugation, sensu stricto) are completely inhibited by 10 μg/ml cycloheximide, suggesting the necessity of protein synthesis for recognition and union in conjugation of potential partners.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

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.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.     

Mouse teratocarcinoma and embryonic development. Two-dimensional protein patterns in nerve differentiation

The transition in mouse teratocarcinomas of pluripotential stem cells to histogenetically determined ones of the neurogenic cell lineage was analysed at the total cellular protein level by two-dimensional gel electrophoresis. The change in morphology and function was found to be reflected by extensive shifts in the protein synthesis patterns. From a total of about 1000 resolved polypeptides that are synthesized in embryoid bodies of a multidifferentiating teratocarcinoma, about 12% (117 proteins) are not found or only present at a very much reduced level in two teratocarcinoma-derived neuroblastomas. On the other hand, the change in phenotype is accompanied by the de novo or greatly enhanced synthesis of another 69 proteins (about 7%) in the neurogenic tumors. In a screening for differentiation-specific proteins this set was compared with the protein synthesized in neural tissue of maturing brain and muscle tissue of developing limbs at three different postimplantation stages covering the onset of organogenesis. This comparison disclosed that a great portion of the differentiation-related proteins (26 proteins) are synthesized in both brain and muscle and are probably required by a differentiated cell in general like cytostructural proteins. Seventeen polypeptides, however, are synthesized in a cell type specific manner. In particular, 4 proteins that are synthesized in both neurogenic tumors and in brain but not in muscle tissue were tentatively called nerve-specific proteins (NSP) and are the most promising in further analyses of differentiation-specific proteins.