The sequence of regulatory events in the sporulation control network of Physarum polycephalum analysed by time-resolved somatic complementation of mutants

The developmental decision for sporulation of Physarum polycephalum plasmodia is under sensory control by environmental factors like visible light or heat shock and endogenous signals like glucose starvation. Several hours after perceiving an inductive stimulus, plasmodia become committed to sporulation; thereby, they lose their unlimited replicative potential and execute a developmental program that involves differentiation into various cell types required to form a mature fruiting body. Plasmodia are multinuclear single cells which spontaneously fuse upon physical contact. Fusion of mutant plasmodia and cytoplasmic mixing allows complementation studies to be performed at the functional level. Mutant cells altered in their ability to sporulate in response to phytochrome activation by far-red light were cured by fusion with wild-type or other mutant plasmodia. Phytochrome activation in one plasmodium and subsequent fusion with a non-induced plasmodium revealed that complementation of the two mutations depended on (i) which of two genetically distinct plasmodial cells was stimulated; and (ii) on the delay time elapsed between stimulation and cytoplasmic mixing. Such experiments allow us to determine the kinetics and the causal sequence of the regulatory events tagged by mutation.     

Detecting functional interactions in a gene and signaling network by time-resolved somatic complementation analysis

Somatic complementation by fusion of two mutant cells and mixing of their cytoplasms occurs when the genetic defect of one fusion partner is cured by the functional gene product provided by the other. We have found that complementation of mutational defects in the network mediating stimulus-induced commitment and sporulation of Physarum polycephalum may reflect time-dependent changes in the signaling state of its molecular building blocks. Network perturbation by fusion of mutant plasmodial cells in different states of activation, and the time-resolved analysis of somatic complementation effects can be used to systematically probe network structure and dynamics. Time-resolved somatic complementation quantitatively detects regulatory interactions between the functional modules of a network, independent of their biochemical composition or subcellular localization, and without being limited to direct physical interactions.     

Isolation of Physarum polycephalum plasmodial mutants altered in sporulation by chemical mutagenesis of flagellates

Plasmodia are giant, multinucleate single cells which develop from mononucleate amoebae during the developmental cycle of Physarum polycephalum. In visible light, starving plasmodia lose their unlimited replicative potential and terminally differentiate into fruiting bodies (sporulation). Aiming at genetic dissection of the circuits controlling commitment and differentiation, we worked out a standardized procedure for the generation and screening of plasmodial mutants altered in sporulation by mutagenesis with ethylnitrosourea. To obtain a homogeneous population of cells of those strains which cannot grow axenically, we describe a protocol for preparing a suspension of flagellates to be used as starting material for mutagenesis. Flagellates can transform into plasmodia via the amoebal stage. Pilot phenotypic screening yielded plasmodial mutants altered in the photocontrol of sporulation or with disturbed developmental program. The existence of mutants with a disturbed developmental program indicates that the sequence and synchrony of morphogenetic steps of fruiting body formation can be uncoupled through mutation. Complementation testing by plasmodial fusion identified three complementation groups of non-sporulating mutants. The work described provides an experimental basis for performing mass screens for Physarum mutants altered in sporulation.     

THE DESTRUCTION OF XYLEM BY A PLASMODIAL PARASITE IN SEEDLINGS OF PISUM SATIVUM

The seedlings of Pisum sativum var. ‘Alaska’ grown either in complete darkness or in partial red light are often afflicted by a plasmodial parasite, which either partially or completely destroys the tracheids. The vegetative stage of the organism consists of small cells connected with each other by hyaline filaments. These cells are seen inside the tracheids, where they presumably hydrolyze cellulose to separate the lignified helical wall thickenings from the cell wall. The spirals are then incorporated into a plasmodium. The plasmodia are found both inside and outside the plant tissue. The younger portions of the seedlings often contain large plasmodia with engulfed helical wall thickenings, which are gradually dissolved, presumably by means of a peetinolytic enzyme, at the time of sporulation. The spores (4–5μ) are borne in small numbers, ca. 10–16 per well-defined oval or elongate sporangium. The pitted vessels are not destroyed by the organism. Pea seedlings can be badly infected without showing externally visible pathology. Unless the xylem is destroyed and the plant tissues are filled with plasmodia, recovery from the infection is possible. Pisum sativum, a homozygous variety ‘L’ grown aseptically in light on an agar medium, also contained all the stages in the development of this organism.     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.     

The Structure and Origin of the Plasmodium of Rhopalura ophiocomae(Phylum Orthonectida)

Abstract Adult orthonectids develop from germinal cells within a cytoplasmic matrix called a plasmodium. This is generally assumed to be formed by the parasite. In the case of Rhopalura ophiocomae, which lives in the brittle star Amphipholis squamata, the plasmodia occupying the perivisceral coelom are closely associated with the walls of the genital bursae or the gut, and they are covered by peritoneum. They have been reported to contain scattered small nuclei distinct from those within germinal cells, embryos, and adults, but the results of the present study indicate that such nuclei probably do not exist. Furthermore, electron micrographs show that some plasmodia are in continuity with the cytoplasm of contractile cells that lie beneath the peritoneum of a genital bursa or the gut of the host. The matrix of a plasmodium of R. ophiocomaeappears, therefore, to consist of cytoplasm of a contractile cell. It is proposed that after a contractile cell has been entered by an infective cell of the parasite, it hypertrophies, bulging progressively farther into the perivisceral coelom and lifting up the peritoneum, which remains in intimate contact with it.     

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.     

Viability of Physarum polycephalum spores and ploidy of plasmodial nuclei.

Amoebae of Physarum polycephalum carrying the mth mating-type allele may differentiate into plasmodia in the absence of mating. Such plasmodia are haploid and, upon sporulation, produce mainly inviable spores. We have asked whether the viable spores arise from meiotic or mitotic divisions. Using a microfluorometric measurement of the deoxyribonucleic acid content of individual nuclei, we found the fraction of viable spores to be correlated with the proportion of rare, diploid nuclei containing in the generally haploid plasmodium. When homozygous diploid plasmodia were created by heat shocking, spore viability increased dramatically. We suggest that viable spores are produced via meiosis in mth plasmodia, that the mth allele has no effect on sporulation per se, and that the normal source of viable haploid spores is a small fraction of diploid nuclei ubiquitous in haploid plasmodia.     

Complementation of Amoebal-Plasmodial Transition Mutants in PHYSARUM POLYCEPHALUM

Amoebae of the Myxomycete Physarum polycephalum differentiate to yield plasmodia in two ways: in crossing, haploid amoebae of appropriate genotypes fuse to form diploid plasmodia; in selfing, plasmodia form without amoebal fusion or increase in ploidy. Amoebae carrying the mating-type allele matAh (formerly mt_h) self efficiently, but occasionally give rise to mutants that self at very low frequencies. Such "amoebal-plasmodial transition" mutants were mixed in pairs to test their ability to complement one another in the formation of plasmodia by crossing. The pattern of crossing permitted 33 mutants to be assigned to four complementation groups (aptA^-, npfA^-, npfB^- and npfC^-). Similar tests had previously proved only partially successful, as crossing had occurred only rarely in mixtures of compatible strains. The efficiency of complementation was greatly increased in the current work by mixing strains that carried different alleles of a newly-discovered mating-compatibility locus, matB; this locus had no effect on the specificity of complementation. A possible interpretation of the complementation behavior of the mutants is suggested.     

A morphogen for the sporulation of Physarum polycephalum detected by cell fusion experiments

The light stimulus, which under conditions of starvation induces the development of sporangia in the slime mold Physarum polycephalum, can be transferred from the light-exposed part to the unexposed part of a plasmodium by means of plasma circulation. A small quantity of protoplasm from a sporulating donor plasmodium, which had passed through the premorphogenetic phase, was transferred by a short period fusion with a briefly starved, light-induction-incompetent acceptor plasmodium. This led to sporulation and even to a reduction of the premorphogenetic phase from 9 down to 3 h in the acceptor plasmodium. After fusion with a sporulating plasmodium, a highly starved plasmodium from a non-sporogenic culture line or a growing plasmodium from a normal line prevents further morphogenesis of sporangia in the sporulating partner.     

Theory of time-resolved somatic complementation and its use to explore the sporulation control network in Physarum polycephalum

Mutants of Physarum polycephalum can be complemented by fusion of plasmodial cells followed by cytoplasmic mixing. Complementation between strains carrying different mutational defects in the sporulation control network may depend on the signaling state of the network components. We have previously suggested that time-resolved somatic complementation (TRSC) analysis with such mutants may be used to probe network architecture and dynamics. By computer simulation it is now shown how and under which conditions the regulatory hierarchy of genes can be determined experimentally. A kinetic model of the sporulation control network is developed, which is then used to demonstrate how the mechanisms of TRSC can be understood and simulated at the kinetic level. On the basis of theoretical considerations, experimental parameters that determine whether functional complementation of two mutations will occur are identified. It is also shown how gene dosage-effect relationships can be employed for network analysis. The theoretical framework provided may be used to systematically analyze network structure and dynamics through time-resolved somatic complementation studies. The conclusions drawn are of general relevance in that they do not depend on the validity of the model from which they were derived.     

The intranuclear microtubule organizing centre in the plasmodium of the myxomycete Physarum polycephalum

Physarum possesses two different microtubule cytoskeletons. In amoebae, cytoplasmic and mitotic microtubules are nucleated by a typical centrosome. In contrast, it has been reported that plasmodia have an intranuclear spindle organizing centre (SPOC) devoid of centrioles. We present genetic evidence suggesting that the SPOC located in the centrosome is very similar to the intranuclear plasmodial SPOC. The immunostaining properties of a new monoclonal antibody against Physarum centrosome has been used to compare these different MTOCs. Moreover, a dense plasmodial microtubule network was present in interphase plasmodia and absent in plasmodia undergoing mitosis. MTOCs responsible for the nucleation of the cytoplasmic microtubule network and intranuclear SPOCs were located in two different compartments of the plasmodium.     

Comparative Study of Ultrastructure of Interlamellar and Intralamellar Types of Henneguya exilis Kudo from Channel Catfish*†

SYNOPSIS. The inter- and intralamellar types of Henneguya exilis Kudo (Myxosporida) infections from channel catfish are similar in spore structure and sporogenesis, but differ in the structure of their plasmodium wall and surface coat and in their relationship with the host cells. The 2 clinical types differ also in the sites of development and growth patterns of plasmodia within a gill filament. Interlamellar plasmodia are limited by 2 outer unit membranes which give rise to both single-and double-membraned pinocytic canals. Intralamellar plasmodia are limited by a single outer unit membrane which gives rise to single-membraned pinocytic canals. Interlamellar plasmodia are covered by a fine granular coat of highly variable thicknesses; in some regions there is direct contact between the parasite and cells of the host. There is some evidence that host cell cytoplasm as well as interstitial material are taken in by interlamellar plasmodia. In contrast, intralamellar plasmodia are covered by a fine granular coat of almost uniform thickness, which prevents direct contact between the parasite and cells of the host; probably only interstitial material is taken by these plasmodia.     

Ultrastructure of the plasmodial slime moldPerichaena vermicularis

Summary Mature plasmodia ofPerichaena vermicularis require a light period to induce sporulation. In this paper the ultrastructure and acid phosphatase localization of the mature plasmodium ofPerichaena vermicularis are investigated. Acid phosphatase is localized in vacuoles containing remnants of bacteria and cell organelles. Morphological and histochemical evidence support the interpretation that these vacuoles constitute two types of lysosomes called respectively heterophagic and autophagic vacuoles.Coated vesicles which apparently originate from smooth endoplasmic reticulum are dispersed throughout the plasmodium and frequently associated with lysosomes. Several dumbbellshaped mitochondria are observed in the plasmodium at the onset of fruiting but not during later stages of plasmodiocarp development. Cytoplasmic microtubules are identified inPerichaena vermicularis. Some of these are closely associated with microfilaments.This work was supported by National Science Foundation grants (GB-5884 and GB-8537) to Dr.Ian K.Ross, NSF grant (GB 12371) to Dr.James Cronshaw, and an NSF Traineeship (GZ 445 and 796) to I.Charvat.This constitutes a portion of a thesis presented to the Regents of the University of California by the first author in partial fulfillment of the requirements for the Ph. D. degree.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Summary Mutant (APT) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.     

HAPLOID SPORE FORMATION FOLLOWING ARRESTED CELL FUSION IN CHLAMYDOMOXAS (CHLOROPJIYTA) 1

Sexual fusion of haploid Chlamydomonas gametes produces a diploid zygote which undergoes sporulation (maturation). We have used a combination of genetic and cellular approaches to evaluate the role(s) of gametic cell and nuclear fusion in the progression of sporulation. A fusion-arrested strain, zym-26–3. was obtained following ultraviolet irradiation of vegetative haploid cells of the homothallic species Chlamydomonas monoica Strehlow. Using the DNA-specific fluorochrome, DAPI, we determined that diploidy was rarely achieved although nuclear migration to the base of the cytoplasmic bridge connecting the gametes and attempted transit through the tubule could be easily documented. Unusual cytoplasmic‘buds’which developed adjacent to the cytoplasmic bridge in sporulating haploids were usually found to contain a migrant nucleus. Using transmission electron microscopy, we determined that ultrastructural changes typically associated with sporulation of a diploid zygote (e.g. spore wall formation; plastid dedifferentiation and associated lipid accumulation; nuclear migration and heterochromatization) could occur following arrested cell fusion despite the absence of nuclear fusion. Genetic analysis of the zym-26–3 strain revealed two unlinked mutations: cf-1 responsible for the failure to complete cell fusion; and ger-8, a mutant allele not affecting cell fusion, but interfering with late stages of spore maturation and germination.‘Cytoplasmic budding’was observed in strains carrying each of these mutations singly and may be a common secondary consequence of disturbances in the relative timing of interrelated processes required for spare wall assembly.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

Cell-cell interactions in developmental lysis of Myxococcus xanthus

The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.