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. In other experiments (not shown) the plasmodial curves the axis corresponding to one plasmodium per plate. The time after the exponential phase are seen to reach a maximum and corresponding to the intersection is designated tI and referred then decline. The decline may result from the fusion of small to as the "time when plasmodium production commences." plasmodia with one another as well as the loss, during the Whether or not the first plasmodium on a plate actually arises wash-off procedure, of larger, more fragile plasmodia. at this time is not known, and would in fact be difficult to de- Because the plasmodial curve (Fig. 2) rises so steeply, we termine. Nevertheless, the time for the appearance of the first believe that the plasmodia arise principally or exclusively from plasmodium must be close to t1. Thus, the two strains CH357 Downloaded by guest on October 1, 2021 1122 Cell Biology: Youngman et al. Proc. Natl. Acad. Sci. USA 74 (1977)

Nuclepore filters \A Amoeba (sporse)+bacteria Amoeba (dense) bacteria

FIG 4. Schematic representation of cells and filters in an in- duction experiment. The upper and lower cultures have the same initial number of bacteria.

10 filters that were, in turn, placed on the surface of an agar plate. The filters, bearing the cells on their upper surfaces, were re- moved and replaced with a second set of culture-bearing filters E / that carried amoebae at a predifferentiation growth stage. The z formation of plasmodia on the second set of filters was moni- 102 tored by periodically removing a filter, washing it, and plating dilutions of the suspension on assay plates. Plasmodium pro- duction on these filters was indistinguishable from that on A control filters that had been transferred to either fresh agar or bacteria only rather than to the spots vacated by the first set of cultures. Thus, the agar beneath a filter-borne culture of dif- 31: 391 47, not contain demonstrable in- 0 ferentiating CL amoebae did 30 40 50 60 70 ducing activity by this test. Time (hours) Transfilter Induction. The filter replacement experiment FIG. 3. Kinetics of plasmodium production with varying initial described above might not be adequate to detect an inducing food supply. The number of bacteria per puddle were as follows: substance that was either unstable, or required in the high (0, *) 5 X 107; (A, A) 4 X 108; (0, 0) 1.6 X 109; X refers to the average concentrations that would be found only in the immediate of amoebic points, which were within 10% of one another. proximity of cells releasing it. Thus, we turned to experiments in which the sparse amoebal culture was placed directly on top and CL may be said to differ by 9 hr in the time at which of a denser culture. Several additional refinements were also plasmodium formation begins (Fig. 2). ultimately incorporated. Millipore filters (thickness, 125 ,Am) Dependence of ti on Initial Numbers of Amoebae and were replaced by the thinner (10 ,m) Nuclepore filters with Bacteria. We observed in a series of kinetics experiments that 0.2 ,um pores. Paired filters were used, which permit the upper the less the initial inoculum of amoebae on a differentiation filter to be removed without carrying with it any cells from the plate, the larger was the value of ti. The number of amoebae culture growing on the agar surface. Formalin-killed bacteria at the time t1, on the other hand, was approximately constant. were used to eliminate the possibility that any apparent in- One possible explanation of this phenomenon is that amoebae ductive effects would have to do with either altered bacterial differentiate when the food supply is exhausted. As a test of this growth or substances released by the bacteria. The experimental hypothesis, we started a series of cultures of the Colonia strain arrangement after transfer of the filter-borne cultures to the with a constant number of amoebae but a varying number of inducing cultures is shown in the accompanying sketch (Fig. the bacteria that are used by the amoebae as food. Formalin- 4). killed bacteria were used, to avoid the complications that would The results of such an experiment are presented in Fig. 5. The have arisen if live bacteria and varying amounts of soluble inducing cultures, which were inoculated with 104 amoebae bacterial nutrients had been used. Measurements on the pro- per puddle, exhibited the same growth and differentiation duction of plasmodia by these cultures are shown in Fig. 3. As curves seen earlier (Fig. 2). The filter cultures were begun with predicted by this hypothesis, tI was smallest for the culture fed about 50 amoebae per filter and with the same amount of food with the smallest number of bacteria. as the inducing cultures. The filter-borne amoebae also grew The results on the variation of tI with initial numbers of exponentially although at a lower growth rate than the amoebae amoebae and bacteria have two important implications for the directly on agar. In control filter cultures, which were trans- design of experiments aimed at the detection of induction ef- ferred to vacant agar, tI was in excess of 100 hr. In the experi- fects. First, it is possible to prepare amoebic cultures that would mental cultures, which were transferred to inducing cultures not ordinarily differentiate for a long period of time by starting at 36 hr, plasmodium production began at about 60 hr. We them with small inocula of amoebae. Such cultures can serve hypothesize that the accelerated plasmodium production in the as "probes" for the detection of postulated inducing substances. experimental filter cultures is due to a substance that diffuses Second, one must determine whether any observed stimulation through the pores of the Nuclepore filters from the inducing of plasmodium production is in fact due to induction, or merely culture below. to a limitation on the number of bacteria. Very few cells, if any, leave the area of a "puddle." Never- Initial Attempts to Detect Inducer. Our first attempt to theless, we cannot, on the basis of the observations above, ab- detect an inducer involved experiments in which the thickness solutely rule out the possibility that the plasmodia recovered of the agar plates was varied. When plates were made as thin from the upper filter were in fact wandering, committed as 2 mm, Colonia amoebae growing on them differentiated plasmodia from the inducing culture. Two experiments de- faster than on thicker plates. It was unclear whether tj was signed to explore this possibility utilized a mutant (mth apt1) affected, but the number of plasmodia about 10 hr after t1 was of Colonia that is deficient in the ability to form plasmodia (4). certainly greater on the thinner plates. We next carried out The experimental design was like that of the experiment whose experiments in which cultures that would be expected to release results are shown in Fig. 5, but with the substitution of the the postulated inducer were grown on the surface of Millipore mutant for either the upper or lower culture. When the upper Downloaded by guest on October 1, 2021 Cell Biology: Youngman et al. Proc. Natl. Acad. Sci. USA 74 (1977) 1123 107

°104 Xg,

2 0 6 01 I 103

E 2 0 102 10

10-~ ~ /J*~~~~~ 4 0 o 20 40 60 80 100 120 0 Time (hours) 0 20 40 60 80 100 120 Time (hours) FIG. 6. Transfilter induction of plasmodium formation: live F IG 5. Transfilter induction of plasmodium formation: forma- bacteria. See legend to Fig. 5. lin-killed bacteria. Sparse (upper) cultures of CL amoebae on 0.2gqm Nuclepore filters were transferred at the time shown by the arrow to In other filter experiments similar to those described above, dense (lower) cultures of CL or to vacant agar: (0, 0) lower culture; the ability of the putative inducer to pass through different (3, *) upper culture, transferred to lower culture; (a, w) upper cul- ture, transferred to vacant agar. types of filters was studied. Induction was observed with two, but not three, stacked Millipore filters (pore size 0.45 Am, 125 Induction was observed when the two culture was mutant, no plasmodia whatsoever were recovered thickness ,gm). 0.2 from the upper filter, even though the amoebae in the upper tim Nuclepore filters used routinely were replaced by two culture grew normally and the amoebae in the lower culture Nuclepore filters having a pore size of 0.05 ,um. In addition, reduced induction was found with a filter differentiated normally. When the lower culture was pair consisting of a mutant, 55 mm square of membrane and one 0.2 amoebae in the upper culture were induced. In fact, induction dialysis ,tm Nucleo- occurred even earlier with the mutant underneath than with pore filter. the wild type underneath, suggesting that the mutant produces active levels of the putative inducer earlier (P. J. Youngman, DISCUSSION B. Smith, T. M. Shinnick, and C. E. Holt, manuscript in prep- Dense amoebic cultures induce the differentiation of sparse aration). amoebic cultures when the two are separated by a pair of Nu- The growth rate of the filter-borne amoebae grown on for- clepore filters with a pore size of 0.05 tim. The effect occurs malin-killed bacteria was less than normal. This lowered growth both with live and with formalin-killed food-bacteria. The ef- rate is not the sole cause of the premature plasmodium pro- fect is not caused by passage of cells from one culture to the duction by the amoebae, because control filter cultures trans- other. The effect is not seen when the dense culture is replaced ferred to vacant agar showed the same lowered growth rate and by bacteria only or by amoebae of a cellular slime mold. We no early plasmodium production. It could be postulated, nev- conclude that the effect is mediated by a diffusible substance ertheless, that only slowly growing amoebae can be induced. elaborated by the P. polycephalum cells. The results are com- That this is not the case is shown by the results of an induction patible with the hypothesis that the diffusible substance is an experiment conducted with live rather than formalin-killed inducer released by the dense, differentiating culture. bacteria. In this case, the amoebic growth rate on the filters was Two observations raise the possibility that the diffusible not reduced, and induction still occurred (Fig. 6). It is to be substance has a short range of action. First, only when plates noted that the lawn of live bacteria is established in a short time are as thin as 2 mm is any effect seen on the kinetics of differ- relative to the duration of the experiment, and that the lawn entiation. Second, induction does not occur through three for the upper culture is grown on one plate (before the filters Millipore filters. However, other explanations of these obser- are transferred) and the lawn for the lower culture on another. vations are possible, and further work will be needed to establish As controls to determine whether the apparent induction in- the range over which the inducer acts. volved bacterial growth or bacterial metabolic products, filters The steepness and exponential nature of the plasmodial curve were transferred to amoeba-free bacteria or to cultures of for the Colonia strain introduce the possibility that the differ- Dictyostelium discoideum amoebae. (D. discoideum amoebae, entiation process in this strain may be cooperative. It is plausible strain JM41, were grown in puddles under exactly the same that a population of amoebae derives an advantage from a conditions as the P. polycephalum amoebae and grew some- concerted transition to the plasmodial state. The social activity what faster but to nearly the same extent as P. polycephalum of organisms such as D. discoideum (9,10), myxobacterium (11), amoebae. The former do not, of course, form plasmodia.) and yeast (12) provide ample precedent. This cooperativity Plasmodium production occurred with the same kinetics in the could be achieved by the autocatalytic production, by differ- cultures transferred to vacant agar, bacteria only, and D. dis- entiating cells, of a diffusible signal which induces neighboring coideum. amoebae to join the differentiating population. Such a hy- Downloaded by guest on October 1, 2021 1124 Cell Biology: Youngman et al. Proc. Natl. Acad. Sci. USA 74 (1977) pothesis is supported by our discovery that differentiating cells The authors are grateful to Barbara Smith for help in the conduct do in fact possess the capacity to induce a neighboring culture of experiments and to Jeanne Margolskee for the culture of D. discoi- to initiate differentiation. Our additional finding that food deum. This work was supported by National Science Foundation Grant depletion instigates early differentiation indicates that star- BMS75-15604 to C.E.H; P.J.Y and T.M.S are supported by Training vation may function as .a stimulus to initiate differentiation, Grant NIH-5-TO1-GM00515 to the Department of Biology; P.N.A was perhaps by promoting the manufacture or release of an in- supported by National Institutes of Health Training Grant 5-T01- ducing substance. BM11710 to the Department of Biology. An alternative hypothesis not absolutely excluded by our findings is one which.would attribute the induction effect to 1. Gray, W. D. & Alexopoulos, C. J. (1965) Biology of the Myx- the destruction, by differentiating cells, of a diffusible inhibitor omycetes (Ronald Press, New York). of differentiation which is made constitutively by cells in a 2. Olive, L. S. (1975) The Mycetozoans (Academic Press, New sparse population. We tend to discount the hypothesis for two York). reasons. First, extensive dilution and replating at low density 3. Cooke, D. & Dee, J. (1974) Genet. Res. Camb. 23,307-318. does not stimulate cells to differentiation. Second, reducing the 4. Wheals, A. E. (1973) Genet. Res. Camb. 21, 79-86. thickness of differentiation plates promotes rather than hinders 5. Adler, P., Davidow, L. & Holt, C. (1975) Science 190,65-67. differentiation. 6. Adler, P. N. & Holt, C. E. (1974) Genetics 78, 1051-1062. Clearly, the work raises many questions about the cell state 7. Haugli, F. B. (1971) Ph.D. Dissertation, Univ. of Wisconsin. in which the putative inducer is produced, the time course of 8. Sachsenmaier, W. & Rusch, H. P. (1964) Exp. Cell Res. 36, 124-133. the production of the inducer, the responsiveness of cells to the 9. MacHac, M. A. & Bonner, J. T. (1975) J. Bacteriol. 124, 1624- inducer, its stability, its rate of diffusion and its chemical nature. 1625. Success in isolating, from a culture of Colonia cells, an active 10. O'Day, D. H. & Lewis, K. E. (1975) Nature 254,431-432. preparation of inducer would be a major step toward answering 11. Wireman, J. W. & Dworkin, M. (1975) Science 189,516-522. these questions. 12. Hicks, J. & Herskowitz, I. (1976) Nature 260, 246-248. Downloaded by guest on October 1, 2021