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.     

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.     

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.     

Graviresponse of Physarum — Investigations in actual weightlessness

The graviresponse of Physarum plasmodia was investigated in actual weightlessness during a Space Shuttle Mission, where the contractile activity of plasmodial strands was observed. The simultaneous and subsequent inductions of the opposing light and gravity responses and their mutual influences on one another provided insights into the acceleration-stimulus reaction chain in this non-sensory cell.     

A Bryophyte Flora of Berkshire and Oxfordshire

Abstract

In temperate North American forests, collections were made of myxomycetes (plasmodial slime molds) for which bryophytes served as the primary substrate for fruiting. Most associations among the 52 myxomycete species and 55 bryophyte species, represented in these collections, appeared to be coincidental but two species of myxomycetes (Barbeyella minutissima and Lepidoderma tigrinum) were evidently bryophilous, preferring leafy hepatics on rotten coniferous logs. Bryophytes obviously provide exposed surfaces convenient for myxomycete sporulation. Whether bryophytes also sustain the feeding phases of the myxomycete life-cycle (swarm cells, myxamoebae, and plasmodia) is not yet known.     

Pigmentation and sporulation in selected Myxomycetes

Chemical, chromatographic and spectrometric methods are used to characterize plasmodial pigments and determine relationships between pigmentation and sporulation in selected Myxomycetes. In Physarum gyrosum (white) a single pigment is identified and characterized as a flavone. Physarum polycephalum (yellow) and Didymium iridis (brown) contain four and six components, respectively, in their plasmodial pigments which test negatively for flavones but show the presence of some type of phenolic compound. No detectable component is identified in the white plasmodium of Didymium squamulosum which proved to be independent of light for fruiting. The absorption spectra of all species that were light sensitive for fruiting showed common peaks in the 300–400-mμ region, among others. Pigment changes associated with light absorption are reported for some white, yellow and brown plasmodial types. In Physarum gyrosum a yellow pigment forms in light which did not show the characteristic flavones present in the white plasmodial stage. Changes in absorption spectra are reported for Physarum polycephalum, Didymium iridis and Didymium squamulosum as the plasmodial pigments change prior to fruiting. Results show a close relationship between the physiology of plasmodial pigmentation and sporulation in the Myxomycete species studied.     

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.     

Gene expression kinetics in individual plasmodial cells reveal alternative programs of differential regulation during commitment and differentiation

During its life cycle, the amoebozoon Physarum polycephalum forms multinucleate plasmodial cells that can grow to macroscopic size while maintaining a naturally synchronous population of nuclei. Sporulation‐competent plasmodia were stimulated through photoactivation of the phytochrome photoreceptor and the expression of sporulation marker genes was analyzed quantitatively by repeatedly taking samples of the same plasmodial cell at successive time points after the stimulus pulse. Principal component analysis of the gene expression data revealed that plasmodial cells take different trajectories leading to cell fate decision and differentiation and suggested that averaging over individual cells is inappropriate. Queries for genes with pairwise correlated expression kinetics revealed qualitatively different patterns of co‐regulation, indicating that alternative programs of differential regulation are operational in individual plasmodial cells. At the single cell level, the response to stimulation of a non‐sporulating mutant was qualitatively different as compared to the wild type with respect to the differentially regulated genes and their patterns of co‐regulation. The observation of individual differences during commitment and differentiation supports the concept of a Waddington‐type quasipotential landscape for the regulatory control of cell differentiation. Comparison of wild type and sporulation mutant data further supports the idea that mutations may impact the topology of this landscape.     

Calcineurin Subunit B Is Required for Normal Vegetative Growth inNeurospora crassa

A series ofNeurospora crassamutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulatedccg-1gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted genecnb-1was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca²⁺and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth inN. crassaand suggest that thecnb-1mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca²⁺may play an important role in regulating fungal morphology.     

Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.     

THE CHANGING PATTERN OF BIREFRINGENCE IN PLASMODIA OF THE SLIME MOLD, PHYSARUM POLYCEPHALUM

Plasmodia of the acellular slime mold, Physarum polycephalum, reveal a complex and changing pattern of birefringence when examined with a sensitive polarizing microscope. Positively birefringent fibrils are found throughout the ectoplasmic region of the plasmodium. In the larger strands they may be oriented parallel to the strand axis, or arranged circularly or spirally along the periphery of endoplasmic channels. Some fibrils exist for only a few minutes, others for a longer period. Some, particularly the circular fibrils, undergo changes in birefringence as they undergo cyclic deformations. In the ramifying strand region and the advancing margin there is a tendency for fibrils of various sizes to become organized into mutually orthogonal arrays. In some plasmodia the channel wall material immediately adjacent to the endoplasm has been found to be birefringent. The sign of endoplasmic birefringence is negative, and its magnitude is apparently constant over the streaming cycle. The pattern of plasmodial birefringence and its changes during the shuttle streaming cycle of Physarum are considered in the light of several models designed to explain either cytoplasmic streaming alone or the entire gamut of plasmodial motions. The results of this and other recent physical studies suggest that both streaming and the various other motions of the plasmodium may very likely be explained in terms of coordinated contractions taking place in the fibrils which are rendered visible in polarized light.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

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.     

Developmental Coordination of Gamete Differentiation with Programmed Cell Death in Sporulating Yeast

The gametogenesis program of the budding yeast Saccharomyces cerevisiae, also known as sporulation, employs unusual internal meiotic divisions, after which all four meiotic products differentiate within the parental cell. We showed previously that sporulation is typically accompanied by the destruction of discarded immature meiotic products through their exposure to proteases released from the mother cell vacuole, which undergoes an apparent programmed rupture. Here we demonstrate that vacuolar rupture contributes to de facto programmed cell death (PCD) of the meiotic mother cell itself. Meiotic mother cell PCD is accompanied by an accumulation of depolarized mitochondria, organelle swelling, altered plasma membrane characteristics, and cytoplasmic clearance. To ensure that the gametes survive the destructive consequences of developing within a cell that is executing PCD, we hypothesized that PCD is restrained from occurring until spores have attained a threshold degree of differentiation. Consistent with this hypothesis, gene deletions that perturb all but the most terminal postmeiotic spore developmental stages are associated with altered PCD. In these mutants, meiotic mother cells exhibit a delay in vacuolar rupture and then appear to undergo an alternative form of PCD associated with catastrophic consequences for the underdeveloped spores. Our findings reveal yeast sporulation as a context of bona fide PCD that is developmentally coordinated with gamete differentiation.     

Role of cell cohesion in Myxococcus xanthus fruiting body formation.

Dsp mutants of Myxococcus xanthus have a complex phenotype with abnormal cell cohesion, social motility, and development. All three defects are the result of a single mutation in the dsp locus, a region of DNA about 14 kilobases long. Cohesion appears to play a central role in social motility, since nonsocial mutants exhibit weak agglutination or, in the case of Dsp cells, no agglutination (L. J. Shimkets, J. Bacteriol. 166:837-841, 1986). However, Dsp cells can be agglutinated by cohesive strains of M. xanthus. This provided the opportunity to examine the role of cohesion during development by comparing the developmental phenotype of Dsp cells with that of Dsp cells mixed with cohesive strains. Dsp mutants were unable to complete any of the developmental behaviors: aggregation, fruiting body formation, developmental autolysis, and sporulation. Contact with cohesive strains seemed to restore some developmental characteristics to the Dsp cells. When allowed to develop with wild-type cells, Dsp cells accumulated in fruiting bodies and underwent developmental autolysis, but did not form a significant portion of the spore population. Igl mutants, which may be similar to the previously described frizzy mutants, are cohesive strains that are unable to form fruiting bodies. Mixing Igl cells with Dsp cells under developmental conditions resulted in fruiting body formation, although the Dsp cells were unable to form significant levels of myxospores. In spite of their inability to sporulate under developmental conditions, Dsp mutants did not appear to be defective in the sporulation process. In fact, they formed normal levels of myxospores in response to the chemical inducer glycerol.     

Isolation of cell cycle mutants of Physarum polycephalum

Summary Asynchronous amoebal cultures of temperature-sensitive mutants of Physarum polycephalum were examined cytologically, and two cell cycle mutants were identified. Genetic analysis indicated that each mutant carried a single mutation that was expressed in both amoebal and plasmodial phases. Thus it is possible to isolate cell cycle mutations expressed in plasmodia by initial isolation and analysis of amoebal mutants, a quicker procedure than the alternative of isolating plasmodial mutants directly. The two mutants were studied further by measuring nuclear DNA contents and synthesis of macromolecules. Both mutants gave results consistent with a block in nuclear division.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

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.