ABSTRACT
Sensory control of sporulation in Physarum polycephalum plasmodia is mediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants defective in the pathway were isolated and three phenotypes obtained: blue-blind, general-blind and light-independent sporulating. When plasmodia of the blue-blind mutant Blu1 were exposed to a pulse of blue light and subsequently fused to non-induced wild-type plasmodia, the resulting heterokaryons sporulated, indicating a functional blue-light photoreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between induction by light and time of fusion, suggesting that Nos1 can be bypassed by the time-dependent formation of a downstream signal-transduction intermediate. Phenotype expression in constitutively sporulating (Cos) mutants depended on starvation. The Cos2 product was titrated by fusing mutant plasmodia of different sizes to wild-type plasmodia of constant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somatic complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway without prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.
Introduction
Fundamental cellular functions, such as metabolic control, regulation of the cell cycle, differentiation and motility, rely on information-processing mechanisms mediated by networks of interacting proteins and other compounds. Our current view of the function of these networks is based largely on genetic and biochemical studies. Analysis of genetic interactions between genes involved in signal processing generates (epi)static models of signalling pathways, in most cases leaving the assignment of gene products to a well-defined regulatory function at the system level, a difficult task, to be solved. In this respect, it is important that biochemical studies on isolated components should be correlated with the in vivo function by experimental evidence rather than be predicted on the basis of sequence homology to related proteins. The background to our approach is to explore the potential of time-resolved somatic complementation analysis to study the architecture and function of signal-processing regulatory networks in vivo. Experimentally, the approach is based on the fusion of cells carrying mutations in a signalling pathway to wild-type cells or to other mutants. The conceptual difference from genetic complementation analysis is that the signalling pathway is triggered in one of the complementing partners at a defined time before the cells are fused. From the concentration-and time-dependence of the complementation efficiency observed in the resulting heterokaryons, mutations could be functionally characterized in detail even before the isolation of a gene is envisaged. As an experimental system, we use multinuclear plasmodia of the true slime mould Physarum polycephalum, which spontaneously fuse as they make physical contact with each other.
During its life cycle, P. polycephalum goes through several well-defined states of cellular differentiation (Burland et al. 1993; see references therein). The life cycle starts when haploid amoebae hatch from mature spores. The amoebae feed on bacteria and multiply by mitosis. Cells of two amoebal clones of different mating type can fuse to give a diploid zygote. Upon continuous growth and upon multiple nuclear divisions, the zygote develops into a macroscopic, diploid and multinuclear plasmodium. The plasmodium behaves like a single giant cell since all of its nuclei are perfectly synchronized with respect to the cell cycle and differentiation (Rusch et al. 1966). The plasmodium continues to grow as long as nutrition is available. Starving plasmodia can encapsulate by forming macrocysts, so-called sclerotia or, in an alternative developmental pathway, they can sporulate. During sporulation, the whole plasmodial mass differentiates into fruiting bodies, each of which finally contains hundreds of haploid spores. The generation and analysis of mutants in P. polycephalum is facilitated by a temperature-sensitive mutation that allows haploid amoebae to develop directly into a multinuclear haploid plasmodium at the permissive temperature (apogamic development; Wheals, 1970). This allows screening for both dominant and recessive non-lethal mutations without the need for back-crossing.
In P. polycephalum, sporulation can be triggered by ultraviolet or visible light (Schreckenbach, 1984; see references therein). This photomorphogenesis occurs upon activation of a phytochrome-type photoreceptor that is responsive to far-red light or, alternatively, by an additional blue-light photoreceptor (Starostzik and Marwan, 1995a,b). The two photoreceptors are linked to a common signalling pathway that is also responsive to heat shock and to the degree of starvation of the plasmodium (Starostzik and Marwan, 1995a). The plasmodium is irreversibly committed to sporulation if a starvation (competence) signal and one of the photoreceptor (or heat shock) signals are present at the same time. Approaching the commitment point (the so-called point of no return) involves the formation of a titratable morphogenetic signal. The morphogenetic signal is defined and can be detected in a plasmodial fusion assay by its ability to drive plasmodia to sporulation that by themselves are neither competent for the induction of sporulation nor have been exposed to light. A minimal model of the signal-transduction pathway explaining the sensory control of sporulation (Starostzik and Marwan, 1995a) serves as a basic framework for the characterization of signalling mutants in the context of this study (Fig. 1A).
After induction by light of a competent plasmodium, there is a premorphogenetic phase of approximately 10 h. Visible morphogenesis then starts when the plasmodial strands cleave into nodular structures that culminate and finally form the fruiting bodies (Fig. 1B). Distinct morphogenetic intermediate stages are accompanied by a cascade-like differential expression of mRNAs (Putzer et al. 1984; Werenskiold et al. 1988). During the premorphogenetic phase, early transcripts can be detected by differential display reverse transcription polymerase chain reaction (DD-RT-PCR). These transcripts are associated with the point of no return, which is passed approximately 6 h after induction (R. Kroneder and W. Marwan, unpublished results).
Here, we report the isolation of mutants with defects in the signalling pathway that controls the commitment of the plasmodium to sporulation and the functional characterization of the mutants by time-resolved somatic complementation analysis.
Materials and methods
Growth of amoebal and plasmodial cultures
Amoebae of Physarum polycephalum were grown on live Escherichia coli using liver infusion agar (LIA; Cooke and Dee, 1975) or diluted semidefined medium (DSDM) agar (Dee et al. 1989). Plasmodia were grown axenically on nutrient agar plates or as microplasmodia in shaken cultures in nutrient broth (Daniel and Baldwin, 1964). Plasmodia competent for sporulation were obtained on starvation agar plates (Daniel and Rusch, 1962). All cultures were grown in complete darkness. The growth temperature was 24 °C for amoebae and plasmodia. Amoebae carrying the gadAh mutation were grown at 30 °C to delay apogamic development of plasmodia (Wheals, 1970).
Strains used
The strains used for this study are listed in Table 1. The amoebal strain CS310 was derived from a sporulated LU352 × LU897 plasmodium to be used for mutagenesis. Amoebae of CS310 (matA2, fusA1, gadAh, whiA+) can form a highly light-sensitive haploid plasmodium by apogamic development.
Mutagenesis and screening for sporulation mutants
Amoebal cultures of strain CS310 growing exponentially on LIA plates were exposed to far ultraviolet light (254 nm, UVIS, Desaga, Heidelberg, Germany) for 60 s. This particular ultraviolet exposure produced approximately one sporulation-defective mutant out of 100 clones screened. After mutagenesis, growth was continued for 3 days on the same plate at 30 °C to allow the cells to proliferate several times following the mutagenic event. Cells were then resuspended in 3 ml of 15 % glycerol, diluted and plated on LIA. After 4 days of growth at 30 °C, single colonies were picked in duplicate and inoculated on 24-well plates containing DSDM–agar (deep-well tissue culture plate, no. 3047, Falcon, USA) and preinoculated with live E. coli to obtain two parallel sets of multiwell plates. One set was incubated at 30 °C to generate amoebal stocks. The other set was incubated at 23 °C to allow apogamic development of plasmodia, which occurred within 6–12 days. These plasmodia contain many, but genetically identical, haploid nuclei. Individual small plasmodia together with the suspending agar plug were transferred to a nutrient agar plate (9 cm in diameter) that had been overlayed with four quarters of sterile filter paper, and growth was continued until the plasmodium completely covered the agar surface. Residual bacteria were eliminated by plasmodial phagocytosis. Plasmodia together with the suspending filter paper were then transferred to small starvation agar plates (4.5 cm in diameter). After 6 days of starvation in complete darkness, the cultures were scored for spontaneously sporulated plasmodia and for blue-blind, far-red-blind or general-blind phenotypes.
To identify colour-blind and general-blind mutants, light conditions were chosen to activate selectively either the far-red-absorbing phytochrome type or the blue-light photoreceptor. Far red light (⩾700 nm; 13 W m−2) was produced by Concentra Weißlicht lamps (Osram, Munich, Germany) passed through an Orange 478 combined with a Blau 627 acrylic filter (Röhm, Darmstadt, Germany). For selective activation of the blue-light photoreceptor, plasmodia were exposed to white light (24 W m−2) produced by Fluora (Lichtfarbe 77) fluorescent lamps (Osram, Munich, Germany). The emission spectrum of these fluorescent lamps consists of two bands centred in the blue (around 440 nm) and the red (around 650 nm) regions of the spectrum. When applied to plasmodia, the blue emission band excites both the blue-light photoreceptor and the blue-absorbing band of the phytochrome, thereby converting the far-red-light-absorbing form of phytochrome (Pfr) to the red-light-absorbing form (Pr), which induces sporulation. Unwanted photoaccumulation of the Pr intermediate is partially suppressed by the red light emission band that reconverts Pr to Pfr. Since, according to the action spectrum for the induction of sporulation (Starostzik and Marwan, 1995b), the red-light-absorbing band of the Pr intermediate greatly dominates the blue-light-absorbing band of the Pfr intermediate, a photoequilibrium with a low photostationary concentration of Pr is expected. Blue-blind mutants obtained using this approach supported the validity of the rationale. For the sake of simplicity, light emitted from Fluora lamps is referred to as blue light in this work.
Preparation of plasmodia for somatic complementation
To obtain large numbers of plasmodia of uniform size, sensitivity and degree of starvation, strains were grown as microplasmodia in shaken liquid cultures. After 4 days of growth, the plasmodial mass was harvested by centrifugation (200 g, 5 min, 20 °C) and washed twice with an equal volume of sporulation medium (Daniel and Rusch, 1962). The plasmodial mass was then put onto five layers of filter paper, which absorbed excess liquid, and finally applied to starvation agar plates (4.5 cm in diameter) as a ring of 2 cm diameter using a 20 ml syringe (Braun Melsungen, Melsungen, Germany). All manipulations were carried out under sterile conditions and under a green safelight, as described by Starostzik and Marwan (1994). During the first day of starvation, microplasmodia spontaneously fused to develop a veined macroplasmodium that migrated to cover the entire agar surface evenly. Unless indicated otherwise, plasmodia were starved for 6 days in the dark to gain sporulation competence. For somatic complementation experiments, pieces of defined size were cut off a plasmodium including the supporting agar slice. Spontaneous fusion of the plasmodia was achieved by placing two agar slices side by side in a Petri dish. Statistical evaluation of the data was performed as described by Koller (1956). Error bars given in the figures correspond to a confidence interval of 95 %.
Results
Isolation of mutants impaired in the photosensory control of the developmental switch to sporulation
When several competent wild-type plasmodia are irradiated with a non-saturating light pulse, only some of the plasmodia sporulate. The percentage of sporulated plasmodia depends on the intensity of the light pulse (Starostzik and Marwan, 1995b). Wild-type plasmodia behave uniformly with respect to sporulation. If the induction is sufficient, the entire plasmodial mass differentiates into sporangia. If induction is insufficient, sporulation does not occur, which means that not even a single sporangium is formed. It is concluded that sporulation is under the sensory control of a developmental switch that is active throughout the entire plasmodium.
Haploid amoebae of the light-sensitive strain CS310 were mutagenized by ultraviolet light, and plasmodia were grown by apogamic development from individual amoebal clones. Plasmodia were screened for defects in the photosensory control of sporulation. In a total of 950 clones screened, three classes of phenotype were obtained: colour-blind, general-blind and light-independent sporulating strains.
In light-independent sporulating mutants, some of the plasmodia prepared from the same culture did not sporulate if the phenotype was not severe. However, sporulation in these strains was always an all-or-none process within a given plasmodium (see below). Qualitatively, the same phenomenon was observed in mutants that were not completely blind, i.e. showed reduced sensitivity to light. It is concluded that mutations exist that do not influence the developmental switch per se or related downstream events, but instead affect the sensory control of the probability that the switch will be set to the sporulation pathway.
Identification of early signal-transduction mutants by time-resolved somatic complementation analysis
Somatic complementation was performed by allowing two plasmodia of different strains to fuse spontaneously and analysing the probability that the resulting heterokaryon would sporulate. Time-resolved complementation studies were performed by exposing one of the two plasmodia to a short pulse of light and fusing the plasmodium to a second, non-irradiated plasmodium. Optionally, the time elapsing between the inductive light pulse and the fusion event could be varied (Fig. 2). This experimental approach was used to characterize colour-blind or general-blind mutants.
The blue-blind mutant Blu1 shows reduced sensitivity to blue light but normal responsiveness to induction of sporulation by far-red light (results not shown). Upon induction by blue light, only a small percentage of Blu1 plasmodia sporulated. The slight effectiveness of blue light may be due to the blue-absorbing band of the P. polycephalum phytochrome rather than to the residual activity of the blue-light photoreceptor. For somatic complementation, plasmodia of Blu1, irradiated with a pulse of blue light, were then fused to a non-irradiated wild-type plasmodium. Sporulation of the fusion product was measured on the seventh day of starvation. When the irradiated mutant was fused to a wild-type plasmodium, the mutation was cured and sporulation occurred with the same efficiency as in the irradiated wild-type control (Fig. 3). It is concluded that this mutant is equipped with a functional blue-light photoreceptor and that the mutation causing the blue-blind phenotype must therefore be due to a downstream component of the blue-light-specific signal-transduction pathway.
The mutant Nos1 is general-blind. There was no sporulation in response to far-red or blue light. When the mutant was irradiated with either far-red or blue light and immediately after irradiation fused to a non-irradiated wild-type plasmodium, there was no sporulation (Fig. 4). However, when the wild-type was irradiated and immediately fused to a non-irradiated mutant plasmodium, there was a small sporulation response. This indicates that the Nos1 part of the heterokaryon actively suppressed the ability of the wild-type part to sporulate in response to irradiation. When the delay after pulse induction of the wild-type plasmodium until fusion was lengthened (see Fig. 2), the probability of the fusion product sporulating increased to a value of up to 70 % (Fig. 5). It is concluded that the ability of the Nos1 part of the heterokaryon to inhibit sporulation can be overcome by time-dependent signal-transduction processes occurring in the wild-type plasmodium before fusion and cytoplasmic mixing take place. Hence, the results argue strongly against the suggestion that Nos1 confers photoreceptor deficiency, but rather suggest a dominant block in signal-transduction processes downstream from the photoreceptors.
Mutations causing light-independent sporulation: occurrence, titration and bypassing by somatic complementation
In the mutagenesis screen, some constitutively (i.e. light-independent) sporulating mutants were obtained. These mutants sporulate as they become competent during progressive starvation without requiring the activation of photoreceptors (Fig. 6A). Wild-type plasmodia exposed to a blue light pulse and then starved in complete darkness show a similar time-dependence of sporulation (Starostzik and Marwan, 1994). In the context of the minimal model of the sensory control of sporulation (Fig. 1), the time course of sporulation of pre-irradiated wild-type plasmodia reflects the acquisition of sporulation competence by starvation (since the photoreceptor signal is already present in pre-irradiated plasmodia). As expected, sporulation of plasmodia of the constitutively sporulating mutants starved for 3 days could be suppressed by refeeding them on the third day of starvation (Fig. 6B), indicating that expression of the constitutive phenotype indeed depends on the metabolic state. Refeeding before the third day of starvation suppressed sporulation completely in all three strains tested (not shown). The starvation-dependence of phenotype expression suggests that the mutation affects a component upstream from the point of integration of the photoreceptor and starvation signals. If so, unstarved mutant plasmodia should be as responsive to the morphogenetic signal as wild-type plasmodia because the morphogenetic signal functions downstream from the point of integration of the photoreceptor and starvation signals (see Fig. 1; Starostzik and Marwan, 1995a). Consequently, a light-induced wild-type plasmodium should be able to induce sporulation in a heterokaryon formed with a non-starved, and therefore incompetent, mutant plasmodium. In addition, the sporulation probability should increase with the time elapsed between irradiation and fusion. As shown in Fig. 7, the efficiency of the irradiated wild-type plasmodium in causing sporulation of incompetent Cos3 plasmodia increased with a time course similar to that of the formation of the morphogenetic signal (compare Starostzik and Marwan, 1995a).
To estimate the effectiveness of the mutation conferring the Cos2 phentopype, wild-type plasmodia of equal size were allowed to fuse with Cos2 plasmodia of various sizes. Although consisting of differently sized plasmodia, the heterokaryons still retained perfect synchrony and homogeneity with respect to sporulation, i.e. the developmental decision to sporulate always included the entire plasmodium. However, the probability that constitutive sporulation would occur depended strongly on the percentage of Cos2 nuclei within the heterokaryon (Fig. 8). This indicates that a small change in the concentration of Cos2 determines whether a cell will sporulate. Somatic complementation by plasmodial fusion makes titration of the effectiveness of mutations an easy task.
Discussion
We have isolated plasmodial mutants of Physarum polycephalum with defects in the sensory control of sporulation. Mutagenesis was carried out at the haploid amoebal stage. Haploid, multinuclear plasmodia were then regenerated by apogamic development of cloned single amoebal cells and screened for phenotypes of interest. Three classes of mutants were obtained in the initial screen: blue-blind, general-blind and light-independent (constitutively) sporulating plasmodia.
Genetic complementation analysis is usually carried out by crossing mutants with other mutants or with the wild type and analysis of the phenotype of the heterozygous progeny. In contrast to genetic complementation, somatic complementation can be performed by plasmodial fusion, which generates so-called heterokaryons. Because of the two genetically different nuclear populations of a heterokaryon, the gene products specified by the different alleles of a gene mix, as in a heterozygous diploid cell. However, using somatic complementation, one can introduce two additional parameters compared with genetic complementation: concentration-and time-dependence.
By complementing plasmodia of a constitutively sporulating mutant with wild-type plasmodia of different size, we have shown that the probability of the phenotype occurring in the complemented plasmodium depends on the ratio of mutant to wild-type plasmodial mass. The slope of the titration curve and the ratio of protoplasmic mass required to obtain 50 % complementation efficiency allows a quantitative estimate of the effectiveness of a mutation and the concentration-dependence of the related phenotype. This is an advantage compared with genetic complementation analysis, in which we can discriminate dominant, co-dominant and recessive mutations. The concentration-dependence of the mutation in causing the phenotype may yield important information, especially for understanding the function of gene products that play a role in cellular regulation.
The second parameter, time, is even more important for the functional analysis of components of regulatory pathways. We have performed time-resolved mutant complementation experiments and, taking Blu1 and Nos1 as examples, now discuss how they can be used to analyse cellular signalling.
When a blue-blind mutant plasmodium (Blu1) was irradiated with blue light and, after the inductive light pulse, fused with a non-irradiated wild-type plasmodium, sporulation of the heterokaryon occurred. This demonstrates that the mutant is equipped with a functional blue-light photoreceptor that had been activated. Hence, the blue-blind phenotype must be caused by mutation of a downstream component of the blue-light-specific signalling pathway and not by a photoreceptor mutation. This experimental approach allows the easy identification of mutants in the early branches of the signalling pathway by discriminating them from photoreceptor-or chromophore-biosynthesis mutants.
When a general-blind plasmodium (Nos1) was irradiated and fused with a non-irradiated wild-type plasmodium, there was no sporulation. When the wild-type plasmodium was irradiated and subsequently fused with a non-irradiated mutant plasmodium, the mutant part suppressed sporulation of the heterokaryon. This experiment characterizes the mutation as dominant. However, the dominant effect of the mutation was lost as a function of time elapsing between induction by light of the wild-type part and fusion. This time-dependent loss of dominant sporulation repression can be explained by assuming that the mutation has been bypassed by an intermediate of the wild-type signalling pathway (Fig. 9). If the fusion event occurs too early after light induction of the wild type, the dominant mutation of the mutant part prevents the formation of the downstream signalling intermediates. As a result, the heterokaryon will not sporulate, exhibiting the blind phenotype of the mutant. However, if plasmodial fusion occurs at a later time, a signalling intermediate in the wild type has been formed that is downstream from the signalling intermediate whose activation is (dominantly) blocked in the mutant (and the heterokaryon). This bypasses the mutation because the rest of the signalling pathway can be successfully followed. This concept (after minimizing the current experimental limitations discussed below) may prove to be powerful in characterizing the regulatory role of mutated intermediates of signal-processing pathways. It should be possible to order the temporal activation of the signal-transduction intermediates affected in a set of mutants, revealing the functional architecture of the pathway. In combination with titration experiments, this should allow conclusions to be drawn regarding the kinetic properties of signal-transduction intermediates defined by mutation even before the genes have been identified and isolated. Using time-resolved somatic complementation, those mutants that display a clearly defined regulatory function at the system level can be identified within a large collection of mutants. This approach may be useful if, in addition to the biochemistry, the biophysics of regulatory processes is of interest.
Regardless of the possible potential of time-resolved somatic complementation analysis, some experimental limitations must be overcome. In the experiments described here, the temporal resolution was limited by the time required for complete mixing of the cytoplasm of the two complementing partners. This time has to be minimized and it has to be determined experimentally. One possibility is to fuse a white and a yellow plasmodium and to determine the time required for the yellow pigment to distribute evenly in the heterokaryon. An alternative, more elegant but not yet realized, approach is to use mutant transgenic strains expressing two different forms of the green (or blue) fluorescent protein.
A second limitation lies in the design of the genetic screen for sporulation mutants. In the pilot screen reported here, we have mutagenized haploid amoebae that are capable of apogamic development of a haploid plasmodium by growth and multiple nuclear division. Using these haploid plasmodia, recessive mutations can easily be identified since no back-crossing is necessary to observe the phenotype. However, this procedure yields only those mutations that are not lethal and those that do not block the apogamic transition of the amoebal to the plasmodial stage. Therefore, future screens will include dominant mutations in a diploid background.
ACKNOWLEDGEMENTS
We thank Mrs Nicole Müller and Mrs Sabine Sibler for excellent technical assistance, Drs Juliet Bailey, Timothy Burland and Jennifer Dee for sending amoebal strains and Professor Dieter Oesterhelt for generous support in his laboratory. This work was supported by the Deutsche Forschungsgemeinschaft (Ma 1516/3-1).