Mating type and the differentiated state in Physarum polycephalum.

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

Mitochondrial inheritance patterns in Didymium iridis are not influenced by stage of mating competency

To test whether the timing of transition to mating competency affected mitochondrial transmission patterns in D. iridis. Reciprocal crosses were made by combining mating compatible strains that differed in their competency to mate. The results were compared to crosses where both mating strains were competent at the time of combining and crosses where somatic fusion of plasmodia was allowed. The results show that the mating competency of the parental strains at the time of confronting a compatible mate does not affect mitochondrial transmission patterns, mating efficiency or the likelihood of biparental inheritance. However the timing of plasmodial formation is delayed when precompetent and competent strains are mated compared to when both strains are competent at the time of mixing. We also observed that somatic fusion of plasmodia did not appreciably increase the incidence of biparental inheritance compared to crosses where individual plasmodia were isolated. These results provide additional evidence of the variable nature of mitochondrial inheritance in D. iridis within crosses and between mating trials.     

A Gene, ALCA, Affecting the Life Cycle Form Expressed in PHYSARUM POLYCEPHALUM

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the sexual mechanism less efficiently than wild-type amoebae. The various effects of an alc mutation seem to derive from mutation of a single gene, since reversion for one effect is always accompanied by reversion for the other effects. Moreover, a mutation, aptA1, that blocks direct plasmodium formation by alcA amoebae, also increases their growth rate to near normal. The manner of plasmodium formation in alcA strains differs significantly from that in another class of mutants, the gad mutants. Unlike gad amoebae, alcA amoebae need not reach a critical density in order to differentiate directly into plasmodia and do not respond to the extracellular inducer of differentiation. In addition, alcA differentiation is not prevented by a mutation, npfA1, that blocks direct differentiation by most gad amoebae.     

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.     

Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.     

Aminopeptidases of Physarum polycephalum. Activity, isoenzyme pattern, and synthesis during differentiation.

In extracts from both growing and differentiating (spherulating) plasmodia of the true slime mold Physarum polycephalum, high aminopeptidase activities were found. The specificity of the aminopeptidases changed during differentiation with a higher relative activity towards hydrophobic NH2-terminal amino acids. This change in specificity was found to be the result of a shift in the isoenzyme spectrum during differentiation as was tested by isoelectric focusing in sucrose gradients. Three different classes of isoenzymes were found: one band which was present in both growing and differentiating cultures; two bands which were found only in growing cultures; and four bands which were detectable only in differentiating plasmodia. If cycloheximide was applied during the induction of differentiation, only one band, the one present in both types of plasmodia, was found in the isoelectric focusing. Density labeling experiments using deuterated amino acids revealed that the bands which are present in differentiated plasmodia only are synthesized de novo during this differentiation.     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

The effect of proteases on plasmodium formation in Didymium iridis.

An improved assay for quantitatively measuring the number of plasmodia formed with time is presented. Using this assay we have investigated the effects of three proteases, subtilisin PBN', subtilisin carlsberg and alpha-chymotrypsin. We have shown that 1) plasmodium formation is sensitive to protease treatment only during the first 2 h after mixing amoebae of compatible mating type but not after, 2) amoebae are protease sensitive when treated 1 h prior to mixing, 3) the two clones used have different sensitivities to protease treatment and 4) these effects are due to enzymatic activity and have little effect on viability. The meaning of these results in relation to recent evidence for a diffusible inducer of plasmodium formation is discussed.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

HETEROTHALLISM AND HOMOTHALLISM IN TWO MYXOMYCETES

Collins , O'Neil Ray . (Queens Coll., New York City.) Heterothallism and homothallism in two Myxomycetes. Amer. Jour. Bot. 48(8): 674–683. Illus. 1961.—Single-spore studies of 2 Myxomycetes, Didymium iridis and Fuligo cinerea, revealed that the former is heterothallic and the latter is homothallic. In D. iridis, 256 single-spore isolations were made from sporangia which developed in mass-spore cultures. Of these, 101 germinated and 22 yielded plasmodia that later fructified in most cases. The remaining 79 single-spore cultures produced clones of myxamoebae and swarm cells only. When 18 of the 79 clones were mated in all possible combinations, plasmodia developed in a pattern which showed that the clones were either (+) or (–) with regard to mating type. Fructifications were readily obtained from these plasmodia. Fifty-three single spores of the F₁ generation were isolated. Of the 44 that germinated, 9 yielded plasmodia in monospore cultures, and 35 produced clones of myxamoebae and swarm cells only. Twenty-five of the F₁ clones were back-crossed with their parents. Results of the back crosses show that each F₁ clone is capable of yielding plasmodia with either the (+) or the (–) parent, never with both. When 14 of the F₁ clones were mated among themselves, a (+) and (–) mating type system was again revealed. Most of the 22 original single-spore cultures which produced plasmodia, later formed sporangia. From these sporangia, 88 spores were isolated. Seventy-two of these germinated and yielded large populations of swarm cells and myxamoebae, but none produced plasmodia. Twenty of the 72 clones were then mated among themselves. Some matings resulted in plasmodial formation, but the pattern was difficult to interpret. However, when these 20 clones were mated with known (+) and (–) clones, the results appear to be in keeping with a (+) and (–) mating type system. In F. cinerea, 219 single spores were isolated from aethalia derived from mass-spore cultures. Of these, 144 germinated and the same number yielded plasmodia. Fructifications were easily obtained from such plasmodia. Thirty-five second-generation single spores were isolated, of which 15 germinated and 15 yielded plasmodia. These results indicate that F. cinerea is homothallic.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

The ability to develop an activity that transfers histones onto sperm chromatin is acquired with meiotic competence during oocyte growth.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, transferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to SrCl2. One-third of treated oocytes produced at least one Ca2+ oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the Ca2+ chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a Ca2+-dependent process.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

Mutations Increasing Asexual Plasmodium Formation in PHYSARUM POLYCEPHALUM

Rare plasmodia formed in clones of heterothallic amoebae were analyzed in a search for mutations affecting plasmodium formation. The results show that the proportion of mutants varies with both temperature (18°, 26° or 30°) and mating-type allele (mt1, mt2, mt3, mt4). At one extreme, only one of 33 plasmoida formed by mt2 amoebae at 18° is mutant. At the other extreme, three of three plasmodia formed by mt1 amoebae at 30° are mutant. The mutant plasmodia fall into two groups, the GAD (greater asexual differentiation) mutants and the ALC (amoebaless life cycle) mutants. The spores of GAD mutants give rise to amoebae that differentiate into plasmodia asexually at much higher frequencies than normal heterothallic amoebae. Seven of eight gad mutations analyzed genetically are linked to mt and one (gad-12) is not. The gad-12 mutation is expressed in strains with different alleles of mt. The frequency of asexual plasmodium formation is heat sensitive in some (e.g., mt3 gad-11 ), heat-insensitive in two (mt2 gad-8 and mt2 gad-9) and cold-sensitive in one (mt1 gad-12) of twelve GAD mutants analyzed phenotypically. The spores of ALC mutants give rise to plasmodia directly, thereby circumventing the amoebal phase of the life cycle. Spores from five of the seven ALC mutants give rise to occasional amoebae, as well as plasmodia. The amoebae from one of the mutants carry a mutation (alc-1) that is unlinked to mt and is responsible for the ALC phenotype in this mutant. Like gad-12, alc-1 is expressed with different mt alleles. Preliminary observations with amoebae from the other four ALC mutants suggest that two are similar to the one containing alc-1; one gives rise to revertant amoebae, and one gives rise to amoebae carrying an alc mutation and a suppressor of the mutation.     

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.     

A Comparative Study of Mitosis in Amoebae and Plasmodia of the True Slime Mold Didymium nigripes

SYNOPSIS. Studies comparing mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes reveal that at the time of differentiation pronounced changes occur in the mitotic process. Not only does the amount of time required for division of the 2 stages differ, but plasmodial mitosis is characterized by persistence of the nuclear membrane and the apparent lack of centrioles. The origin of multinucleate plasmodia from uninucleate cells which have already undergone cytoplasmic differentiation is described. Division time in a population of amoebae becomes more uniform after those cells which are destined to form plasmodia have differentiated.
The observations and data presented indicated that differences in mitotic behavior also occur between amoebae of 3 stocks with differences in plasmodial structure and behavior. Comparison of mitosis in the plasmodia of these 3 stocks revealed no significant differences.