Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1120-1124, March 1977 Cell Biology An extracellular inducer of asexual plasmodium formation in Physarum polycephalum (differentiation/cell commitment/cell interaction) PHILIP J. YOUNGMAN, PAUL N. ADLER*, THOMAS M. SHINNICK, AND CHARLES E. HOLT Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139 Communicated by Joseph G. Gall, January 10, 1977 ABSTRACT Asexual conversion of amoebae to plasmodia live bacteria, the amoebae were grown on lawns of live Esch- was studied in the Colonia isolate of the myxomycete, Physarum erichia coli on liver infusion agar. For experiments with for- polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the malin-killed bacteria (7), the amoebae were subcultured serially appearance of cells committed to the plasmodial state. The onset on formalin-killed E. coli covered buffer-streptomycin plates of plasmodium production appears to be related to amoebic to ensure elimination of live bacteria. nutrition since cultures supplied with fewer bacteria display Kinetic Experiments. At time zero, replicate amoebic cul- earlier differentiation. For a period of time after differentiation tures on dPRM agar plates (15 X 100 mm) were prepared from is initiated, conversion of amoebae to plasmodia is rapid and a single suspension of plasmodia-free amoebae. The inoculum proceeds as an exponential function of time. A filter-transmis- sible substance, apparently released by differentiating cells, is to each plate consisted of 0.05-0.1 ml of suspension placed in implicated in the control of this rapid conversion. the middle of the plate and spread to a diameter of 21-24 mm before drying. The disc containing the bacteria and amoebae The myxomycete Physarum polycephalum displays two is referred to as a "puddle" and the dPRM plate bearing it as strikingly different vegetative forms: microscopic, uninucleate, a "differentiation plate." The set of cultures was incubated at colorless amoebae and macroscopic, multinucleate, yellow 26°. At appropriate times, the cells on individual differentiation plasmodia (1, 2). Amoebae of the Colonia isolate, which carry plates were harvested and assayed for numbers of amoebae and the allele mth at the mating type locus, readily undergo an plasmodia by the following procedure. The surface of the plate asexual conversion to the plasmodial state. Because the change was flooded with sterile water and rubbed with a glass rod. The occurs without genetic alteration (3) and results in major, stable resulting suspension was diluted serially and samples of the phenotypic alterations, the material provides a model system dilutions were spread onto liver infusion agar or dPRM7 agar for studies on the control of cell differentiation. Mutants af- plates (assay plates). The assay plates were incubated at a fecting the differentiation can be isolated (refs. 4 and 5; L. temperature (30°) that is severely inhibitory to the amoebic- Davidow and C. E. Holt, manuscript in preparation; P. N. plasmodial transition in Colonia amoebae (6) but is favorable Adler, manuscript in preparation), and the present work pro- for plasmodial development and growth. The numbers of vides a beginning for physiological and biochemical studies on amoebic and plasmodial plaques on the assay plates (Fig. 1) the process of commitment to the plasmodial state. were counted. The numbers from the assay plates derived from We report here the development of a technique which per- a single suspension varied linearly with the dilution; thus, the mits a quantitative analysis of the time course and extent of formation of the plaques is not dependent on interactions differentiation in a Colonia culture. With this technique, we among cells on the assay plates. Amoebic plaques were counted have demonstrated that a differentiating culture of Colonia cells after 5-8 days of incubation. Plasmodial plaques were counted can induce early differentiation in a neighboring culture sep- between 2 and 3 days of incubation with the use of a dissection arated by filters which prevent direct cell contact between the microscope. The assay of the number of plasmodia in a cell two populations. The results favor the conclusion that differ- suspension containing a much larger number of amoebae ini- entiating cells elaborate a diffusible inducer of differentia- tially presented a difficult problem. When such a suspension tion. was plated in a concentrated form such that the assay plate received an inoculum of more than 105 amoebae, the plasmodia MATERIALS AND METHODS that arose were unhealthy and their numbers were not repro- Media. Dilute plasmodial rich medium (dPRM) agar and ducible. When the suspension was diluted enough so that the liver infusion agar were made as described previously (4, 6). assay plates prepared from it gave rise to normal plasmodial Agar containing dPRM adjusted to pH7 (dPRM7) rather than plaques, the number of assay plates required for good statistics pH 4.6 was also used. Buffer-streptomycin agar was made by was in excess of what is practical on a routine basis. We found adding 0.25 g streptomycin sulfate (Sigma Chemical Co.) and ultimately that assay plates receiving concentrated suspension 10 ml of 1 M citrate buffer (pH 5) to 1 liter of 1.5% agar. Final can be used reliably, as long as the plasmodia on them are concentrations were 250,ug/ml and 0.01 M, respectively. counted between 2 and 3 days of incubation. The plasmodia Preparation of Amoebae. Plasmodia-free amoebae for use on such plates are spherical, rather than fan-shaped, and often in starting kinetics experiments were prepared by growing the die after further incubation. The identity of the structures as amoebae on agar plates at 300 and harvesting the amoebae prior plasmodia was established by comparison of their number with to the onset of formation. For experiments with the number of normal plasmodia on assay plates receiving di- plasmodium luted suspension. Abbreviation: dPRM, dilute plasmodial rich medium. Filter Experiments. Millipore filters (25 mm circles, 0.45 ,um * Present address: Center for Pathobiology, University of California, pore diameter, Millipore Filter Corp.) and Nuclepore filters Irvine, Calif. 92717. (25 mm circles, 0.2 or 0.05 ,um pore diameter, Nuclepore Corp.) 1120 Downloaded by guest on October 1, 2021 Cell Biology: Youngman et al. Proc. Natl. Acad. Sci. USA 74 (1977) 1121 0 / 0" F As * 31 0 0 20 40 60 80 Time (hours) FIG 2. Kinetics of plasmodium production in strains CL and CH357. Amoebic cultures on live bacteria were started at time zero and amoebae and plasmodia assayed at the times shown. Results are expressed as number of cells per differentiation plate. The extrapo- lated times, t1, are shown just below the axis for one plasmodium per plate. In this and subsequent figures, amoebae are represented by open symbols and plasmodia by closed symbols: (0, 0) strain CL; (A, FIG. 1. Amoebic and plasmodial plaques. Three amoebic and one A) strain CH357. plasmodial plaque are shown on a dPRM7 agar assay plate. The amoebic plaques are about 1.5 mm in diameter. Plasmodial plaques appear to be free of amoebae. amoebae rather than from division of preexisting plasmodia. The doubling time of the plasmodial curve is 1.1 (CH357) or were sterilized by autoclaving (15 pounds/square inch, 20 min) 1.6 (CL) hr, and that of the amoebic curve, 8 hr. Under optimal and rinsed in sterile water before use. Cells were removed from conditions, the doubling time for plasmodial cultures is 9-12 the upper filter for assay by placing the filter in a large test tube hr (8). containing 4 ml of water and shaking the tube on a vortex The hypothesis that the plasmodia arise mainly by conversion mixer. of amoebae predicts that when a sufficient number of plas- modia have formed, a decrease in the number of amoebae should be observed. A slight decrease of about the right mag- RESULTS nitude is seen for CL. The decrease is much more apparent in Kinetics of Plasmodium Formation. The kinetics of pro- the data for strain CH357, which is a spontaneous variant (P. duction of plasmodia in cultures of growing amoebae was N. Adler, unpublished) of Colonia that carries a mutation (rap) studied by the use of a biological assay. The assay permits that is unlinked to mt (L. Davidow, unpublished). In this strain, measurement of amoebic and plasmodial numbers over a wide early amoebic growth is indistinguishable from that of the wild range. We note that the assay for plasmodia detects cells com- type (Fig. 2 upper curves), but plasmodium production begins mitted to the plasmodial state rather than cells necessarily sooner. The number of plasmodia becomes significant relative having the appearance of plasmodia at the time that assay plates to the number of amoebae at a relatively early time and the are prepared. predicted reduction in the number of amoebae is seen (Fig. Fig. 2 displays the results of kinetics experiments for two 2). strains, Colonia or CL (mth) and a mutant (CH357) of CL. Measurements of the extent and timing of differentiation, During an initial growth phase, the number of amoebae in- when performed as described above, are highly reproducible. crease exponentially with time and no plasmodia are produced. Differences between strains, such as those displayed in Fig. 2, (In these experiments, the level of sensitivity was such that four are consistently obtained. A parameter that is useful in dis- plasmodia per plate would have been detected.) Plasmodium cussing these differences may be defined as follows. Data are production then begins, and the number of plasmodia increases plotted as shown in Fig. 2 and the exponential portion of the exponentially with time until there are about 105 plasmodia per plasmodium curve is extrapolated downward until it intersects plate.