Synaptonemal complexes and meiosis in myxomycetes

Synaptonemal complexes (SC) have been observed in spores 18–24 hr past cleavage in natural fruitings of Physarum cinereum, P. bogoriense, Hemitrichia stipitata, Tubifera ferruginosa, and Arcyria incarnata. Laboratory fruitings of Arcyria cinerea, Stemonitis herbatica, and a homothallic isolate of Physarum pusillum also have SC's present in spores during the same postcleavage period. The presence of these paired chromosomes of meiotic prophase in spores of species collected in nature and in a diversity of taxa suggests that the usual position of meiosis in Myxomycetes is inside the postcleavage spore. Criteria are proposed for evaluating the validity of the SC as an indicator of meiosis.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

Adenylate cyclases in Physarum polycephalum: Inhibition of a nuclear enzyme by polyamines

Two distinct adenylate cyclase enzymes have been found in Physarum polycephalum. One is derived from isolated nuclei and is potently inhibited by an equimolar combination of the three polyamines, putrescine, spermidine, and spermine. The second enzyme is particulate, derived from the cytoplasmic compartment, and is insensitive to inhibition by the polyamines. These observations support a potential role for the polyamines in the control of adenosine 3′,5′-monophosphate synthesis in P. polycephalum nuclei.     

Advanced initiation of the first synchronous mitosis following coalescence of starved, UV-irradiated microplasmodia of Physarum polycephalum

The microplasmodia of the slime mold, Physarum polycephalum, coalesce readily upon contact. The nuclei of the resulting macroplasmodia divide in synchrony approx. 6–8 h after coalescence. If prior to coalescence the microplasmodia are maintained on non-nutrient salts solution, followed by continued starvation of the resulting macroplasmodia, the nuclei also will eventually divide, although at a much later time. This mitosis occurs earlier if the starved microplasmodia are irradiated with UV light prior to coalescence. The most pronounced advancement of mitosis was found in plasmodia which were obtained by coalescence of irradiated, starved microplasmodia with non-irradiated ones.     

Fine Structure of Five Species of Myxomycetes with Clustered Spores*

SYNOPSIS. Electronmicroscopic studies were made of 5 species of Myxomycetes with clustered spores to determine the exact mechanism holding the spores together. In Dianema corticatum the neighboring spore walls were closely appressed and appeared to be fused at numerous points. In Trichia synsporum there was a substance between the outer spore walls of adjacent spores which seemed to cement them together. In Badhamia nitens the spore walls were completely fused along their closely appressed surfaces. The adjacent spores of Badhamia versicolor appeared to be free, or if any fusion point was present it was only at the tips of the ornamentation. In Physarum bogoriense such tips were in contact with those of neighboring spores and were occasionally fused at these points.     

A mechanism of propagule release from unilocular reproductive structures in brown algae

Summary The structure of unilocular sporangia inP. littoralis was investigated along with several other species of brown algae in order to study the mechanism by which propagules are released from unilocular reproductive structures. Unilocular sporangia inP. littoralis are composed of a spherical cell wall of two distinct layers and contain a number of zoospores. The mass of spores is surrounded and permeated by mucilaginous carbohydrates. It is suggested that the production of these carbohydrates generates the necessary pressure to weaken the sporangial wall. In addition, ultrastructural observations indicate that further weakening seems to occur due to digestion of the inner wall layer. Walls of sporangia were mechanically broken just prior to normal spore release in order to investigate whether internal pressure exists, and if it can effect spore discharge. Results show that an internal pressure does exist prior to normal spore discharge and that this pressure is not generated by turgor pressure of the spores themselves or by a semi-permeable wall osmoticum system. The discharge of spores seems to occur when the carbohydrate around the spores swells. The adsorption of water when plants are immersed by the incoming tide thus seems a likely mechanism of spore discharge. The similarities of unilocular reproductive structures and spore release in several brown algal species suggests common mechanisms of propagule discharge for members of thePhaeophyta.     

Isolation and Characterization of a Galactosamine Wall from Spores and Spherules of Physarum polycephalum

The myxomycete, Physarum polycephalum, can be induced under laboratory conditions to form two different hard-walled forms, spores and spherules. Characterization of both types of walls revealed only a single sugar, galactosamine. It was identified after acid hydrolysis of the isolated walls by chromatography in three solvent systems, by its positive reaction with ammoniacal silver nitrate, ninhydrin, Galactostat, and the Elson-Morgan test, and by ninhydrin degradation to lyxose. Galactosamine was present as a polymer with solubility characteristics the same as the β1-4–linked glucosamine polymer (chitosan). The walls were also found to contain about 2% protein. Spherule walls revealed a single glycoprotein on gel electrophoresis. Spore walls contained a similar protein component. The phosphate content of isolated spherule walls was 9.8%, and that of spore walls was 1.4%. Spore walls also contained about 15% melanin which was shown to be similar to fungal melanin. A novel method was used to measure the rate of mature spherule formation based on the loss of extractability of P. polycephalum natural pigment. The presence of a rare galactosamine polymer in P. polycephalum spore and spherule walls as the only carbohydrate suggests that the myxomycetes are not closely related to the fungi or the protozoa.     

Ultrastructural localization of β-galactan in the nuclei of the myxomycete Physarum polycephalum

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated β-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei ofP. polycephalum by electron microscopy using a specific “sandwich” technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactin was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasma and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum

An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein.Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker.A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.     

Meiotic cytokinetic apparatus in the formation of the linear spore tetrads of Conocephalum japonicum (Bryophyta)

Most bryophytes produce tetrahedral spore tetrads. However, linear spore tetrads have been reported to occur in Conocephalum japonicum (Thunb.) Grolle. In this study, the distribution of microtubules (MTs) during meiosis in C. japonicum was examined to determine the division pattern resulting in a linear tetrad. Spore mother cells in the pre-meiotic stage were cylindrical with randomly distributed cytoplasmic MTs. In the prophase-metaphase transition, spindle MTs replaced cytoplasmic MTs and a barrel-shaped spindle with two flattened poles developed. Cortical MT arrays were not detectable throughout meiosis. Although a phragmoplast appeared between sister nuclei in telophase-I, it disappeared without expanding to the parental cell wall. Metaphase-II spindles oriented parallel to the long axis of the cell and in tandem to each other resulted in a linear arrangement of telophase nuclei. Radial arrays of MTs developed from the nuclear surfaces and three phragmoplasts appeared among the four nuclei to produce four spores. Two phragmoplasts separating the paired sister nuclei appeared prior to the appearance of a phragmoplast between non-sister nuclei. The MT cycle is basically the same as that reported in meiosis of C. conicum, which produces non-linear tetrads. A morphometric study indicated that the difference in the division pattern between C. conicum and C. japonicum is due to a difference in the shape of spore mother cells. The cylindrical shape of sporocytes of C. japonicum restricts the orientation of spindles and phragmoplasts so that the four resultant spores are arranged linearly. Received: 22 April 1998 / Accepted: 15 May 1998     

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.     

Cell-Cycle-Specific Activity of Cyclic Nucleotide Phosphodiesterase In Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. the activities of plasmodial homogenate and of selected subcellular fractions were measured. the results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. the whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 × 10^-4 m cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. the K_m values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Production and degradation of β-Poly-L-malate in cultures of Physarum polycephalum

β-Poly-L-malate (PMA) is synthesized by plasmodia of Physarum polycephalum during growth and secreted into the culture medium. There it is degraded to L-malate after growth has ceased. Its concentration is highest in cell nuclei, where it probably performs a plasmodium-specific function.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

Assay of adenylate cyclase by use of polyacrylamide-boronate gel columns

A method is described for covalent attachment of up to 1.1 mmol of m-aminobenzeneboronic acid/g dry weight of polyacrylamide beads. Small (4 ml) columns of the derivatized beads have been used to separate quantitatively cyclic AMP from ATP in a single-step procedure. Columns of the polyacrylamide-boronate gel have been used as the basis of a convenient new assay of the soluble adenylate cyclase prepared from nuclei of Physarum polycephalum.     

Subunit structure of chromosomes in mitotic nuclei of Physarum polycephalum

We have investigated the subunit structure of mitotic chromosomes of the acellular slime mould Physarum polycephalum, using the nuclease susceptibility of isolated mitotic nuclei as a probe. A characteristic pattern of DNA digestion products is obtained, containing approximately integral multiples of a basic 140 base pair DNA segment that resembles very closely the pattern in G₂ phase nuclei of Physarum and of calf lymphocyte nuclei. These results demonstrate that during the process of chromosome condensation there is no alteration at the primary level of chromatin structure that is responsible for the characteristic DNA digestion pattern.     

Annular structures in isolated nuclei of Physarum polycephalum

Whole-mount preparations of isolated interphase nuclei of Physarum polycephalum show annular structures. The outer and inner diameters are about 1 080 and 580 Å respectively. Ultrastructural features of the annuli are described; an association of the rings with fibers of various diameters, probably of chromatin nature, is also reported.     

ADP-ribosylation of the nuclear matrix in Physarum polycephalum

Less than 10% of the total ADP-ribosylation in isolated nuclei of Physarum polycephalum are bound to the nuclear matrix. In S-phase the matrix-associated ADP-ribosylation is almost twice as high as compared with the G2-period of the cell cycle. Inhibitors of DNA- and RNA-synthesis and the mutagen N-methyl-N′-nitro-N-nitrosoguanidine increase the percentage of matrix-associated ADP-ribosylation.     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.     

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm³) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.