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 was studied in the Colonia isolate of the myxomycete, Physarum polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the appearance of cells committed to the plasmodial state. The onset of plasmodium production appears to be related to amoebic nutrition since cultures supplied with fewer bacteria display earlier differentiation. For a period of time after differentiation is initiated, conversion of amoebae to plasmodia is rapid and proceeds as an exponential function of time. A filter-transmissible substance, apparently released by differentiating cells, is implicated in the control of this rapid conversion. The myxomycete Physarum polycephalum displays two strikingly different vegetative forms: microscopic, uninucleate, colorless amoebae and macroscopic, multinucleate, yellow plasmodia (1, 2). Amoebae of the Colonia isolate, which carry the allele mth at the mating type locus, readily undergo an asexual conversion to the plasmodial state. Because the change occurs without genetic alteration (3) and results in major, stable phenotypic alterations, the material provides a model system for studies on the control of cell differentiation. Mutants affecting the differentiation can be isolated (refs. 4 and 5; L. Davidow and C. E. Holt, manuscript in preparation; P. N. Adler, manuscript in preparation), and the present work provides a beginning for physiological and biochemical studies on the process of commitment to the plasmodial state. We report here the development of a technique which permits a quantitative analysis of the time course and extent of differentiation in a Colonia culture. With this technique, we have demonstrated that a differentiating culture of Colonia cells can induce early differentiation in a neighboring culture separated by filters which prevent direct cell contact between the two populations. The results favor the conclusion that differentiating cells elaborate a diffusible inducer of differentiation.
MATERIALS AND METHODS Media. Dilute plasmodial rich medium (dPRM) agar and liver infusion agar were made as described previously (4, 6). Agar containing dPRM adjusted to pH7 (dPRM7) rather than pH 4.6 was also used. Buffer-streptomycin agar was made by adding 0.25 g streptomycin sulfate (Sigma Chemical Co.) and 10 ml of 1 M citrate buffer (pH 5) to 1 liter of 1.5% agar. Final concentrations were 250 ,ug/ml and 0.01 M, respectively. Preparation of Amoebae. Plasmodia-free amoebae for use in starting kinetics experiments were prepared by growing the amoebae on agar plates at 300 and harvesting the amoebae prior to the onset of plasmodium formation. For experiments with Abbreviation: dPRM, dilute plasmodial rich medium. * Present address: Center for Pathobiology, University of California, Irvine, Calif. 92717.
live bacteria, the amoebae were grown on lawns of live Escherichia coli on liver infusion agar. For experiments with formalin-killed bacteria (7), the amoebae were subcultured serially on formalin-killed E. coli covered buffer-streptomycin plates to ensure elimination of live bacteria. Kinetic Experiments. At time zero, replicate amoebic cultures on dPRM agar plates (15 X 100 mm) were prepared from a single suspension of plasmodia-free amoebae. The inoculum to each plate consisted of 0.05-0.1 ml of suspension placed in the middle of the plate and spread to a diameter of 21-24 mm before drying. The disc containing the bacteria and amoebae is referred to as a "puddle" and the dPRM plate bearing it as a "differentiation plate." The set of cultures was incubated at 26°. At appropriate times, the cells on individual differentiation plates were harvested and assayed for numbers of amoebae and plasmodia by the following procedure. The surface of the plate was flooded with sterile water and rubbed with a glass rod. The resulting suspension was diluted serially and samples of the dilutions were spread onto liver infusion agar or dPRM7 agar plates (assay plates). The assay plates were incubated at a temperature (30°) that is severely inhibitory to the amoebicplasmodial transition in Colonia amoebae (6) but is favorable for plasmodial development and growth. The numbers of amoebic and plasmodial plaques on the assay plates (Fig. 1) were counted. The numbers from the assay plates derived from a single suspension varied linearly with the dilution; thus, the formation of the plaques is not dependent on interactions among cells on the assay plates. Amoebic plaques were counted after 5-8 days of incubation. Plasmodial plaques were counted between 2 and 3 days of incubation with the use of a dissection microscope. The assay of the number of plasmodia in a cell suspension containing a much larger number of amoebae initially presented a difficult problem. When such a suspension was plated in a concentrated form such that the assay plate received an inoculum of more than 105 amoebae, the plasmodia that arose were unhealthy and their numbers were not reproducible. When the suspension was diluted enough so that the assay plates prepared from it gave rise to normal plasmodial plaques, the number of assay plates required for good statistics was in excess of what is practical on a routine basis. We found ultimately that assay plates receiving concentrated suspension can be used reliably, as long as the plasmodia on them are counted between 2 and 3 days of incubation. The plasmodia on such plates are spherical, rather than fan-shaped, and often die after further incubation. The identity of the structures as plasmodia was established by comparison of their number with the number of normal plasmodia on assay plates receiving diluted suspension. Filter Experiments. Millipore filters (25 mm circles, 0.45 ,um pore diameter, Millipore Filter Corp.) and Nuclepore filters (25 mm circles, 0.2 or 0.05 ,um pore diameter, Nuclepore Corp.) 1120
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Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 1. Amoebic and plasmodial plaques. Three amoebic and one 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. were sterilized by autoclaving (15 pounds/square inch, 20 min) and rinsed in sterile water before use. Cells were removed from the upper filter for assay by placing the filter in a large test tube containing 4 ml of water and shaking the tube on a vortex
mixer.
RESULTS Kinetics of Plasmodium Formation. The kinetics of production of plasmodia in cultures of growing amoebae was studied by the use of a biological assay. The assay permits measurement of amoebic and plasmodial numbers over a wide range. We note that the assay for plasmodia detects cells committed to the plasmodial state rather than cells necessarily having the appearance of plasmodia at the time that assay plates are prepared. Fig. 2 displays the results of kinetics experiments for two strains, Colonia or CL (mth) and a mutant (CH357) of CL. During an initial growth phase, the number of amoebae increase exponentially with time and no plasmodia are produced. (In these experiments, the level of sensitivity was such that four plasmodia per plate would have been detected.) Plasmodium production then begins, and the number of plasmodia increases exponentially with time until there are about 105 plasmodia per plate. In other experiments (not shown) the plasmodial curves after the exponential phase are seen to reach a maximum and then decline. The decline may result from the fusion of small plasmodia with one another as well as the loss, during the wash-off procedure, of larger, more fragile plasmodia. Because the plasmodial curve (Fig. 2) rises so steeply, we believe that the plasmodia arise principally or exclusively from
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 extrapolated 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, A) strain CH357.
amoebae rather than from division of preexisting plasmodia. The doubling time of the plasmodial curve is 1.1 (CH357) or 1.6 (CL) hr, and that of the amoebic curve, 8 hr. Under optimal conditions, the doubling time for plasmodial cultures is 9-12 hr (8). The hypothesis that the plasmodia arise mainly by conversion of amoebae predicts that when a sufficient number of plasmodia have formed, a decrease in the number of amoebae should be observed. A slight decrease of about the right magnitude is seen for CL. The decrease is much more apparent in the data for strain CH357, which is a spontaneous variant (P. N. Adler, unpublished) of Colonia that carries a mutation (rap) that is unlinked to mt (L. Davidow, unpublished). In this strain, early amoebic growth is indistinguishable from that of the wild type (Fig. 2 upper curves), but plasmodium production begins sooner. The number of plasmodia becomes significant relative to the number of amoebae at a relatively early time and the predicted reduction in the number of amoebae is seen (Fig. 2). Measurements of the extent and timing of differentiation, when performed as described above, are highly reproducible. Differences between strains, such as those displayed in Fig. 2, are consistently obtained. A parameter that is useful in discussing these differences may be defined as follows. Data are plotted as shown in Fig. 2 and the exponential portion of the plasmodium curve is extrapolated downward until it intersects the axis corresponding to one plasmodium per plate. The time corresponding to the intersection is designated tI and referred to as the "time when plasmodium production commences." Whether or not the first plasmodium on a plate actually arises at this time is not known, and would in fact be difficult to determine. Nevertheless, the time for the appearance of the first plasmodium must be close to t1. Thus, the two strains CH357
Cell Biology: Youngman et al.
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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 induction experiment. The upper and lower cultures have the same initial number of bacteria. 10
/
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70 60 50 Time (hours) FIG. 3. Kinetics of plasmodium production with varying initial food supply. The number of bacteria per puddle were as follows: (0, *) 5 X 107; (A, A) 4 X 108; (0, 0) 1.6 X 109; X refers to the average of amoebic points, which were within 10% of one another.
and CL may be said to differ by 9 hr in the time at which plasmodium formation begins (Fig. 2). Dependence of ti on Initial Numbers of Amoebae and Bacteria. We observed in a series of kinetics experiments that the less the initial inoculum of amoebae on a differentiation plate, the larger was the value of ti. The number of amoebae at the time t1, on the other hand, was approximately constant. One possible explanation of this phenomenon is that amoebae differentiate when the food supply is exhausted. As a test of this hypothesis, we started a series of cultures of the Colonia strain with a constant number of amoebae but a varying number of the bacteria that are used by the amoebae as food. Formalinkilled bacteria were used, to avoid the complications that would have arisen if live bacteria and varying amounts of soluble bacterial nutrients had been used. Measurements on the production of plasmodia by these cultures are shown in Fig. 3. As predicted by this hypothesis, tI was smallest for the culture fed with the smallest number of bacteria. The results on the variation of tI with initial numbers of amoebae and bacteria have two important implications for the design of experiments aimed at the detection of induction effects. First, it is possible to prepare amoebic cultures that would not ordinarily differentiate for a long period of time by starting them with small inocula of amoebae. Such cultures can serve as "probes" for the detection of postulated inducing substances. Second, one must determine whether any observed stimulation of plasmodium production is in fact due to induction, or merely to a limitation on the number of bacteria. Initial Attempts to Detect Inducer. Our first attempt to detect an inducer involved experiments in which the thickness of the agar plates was varied. When plates were made as thin as 2 mm, Colonia amoebae growing on them differentiated faster than on thicker plates. It was unclear whether tj was affected, but the number of plasmodia about 10 hr after t1 was certainly greater on the thinner plates. We next carried out experiments in which cultures that would be expected to release the postulated inducer were grown on the surface of Millipore
filters that were, in turn, placed on the surface of an agar plate. The filters, bearing the cells on their upper surfaces, were removed and replaced with a second set of culture-bearing filters that carried amoebae at a predifferentiation growth stage. The formation of plasmodia on the second set of filters was monitored by periodically removing a filter, washing it, and plating dilutions of the suspension on assay plates. Plasmodium production on these filters was indistinguishable from that on 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 differentiating CL amoebae did not contain demonstrable inducing activity by this test. Transfilter Induction. The filter replacement experiment described above might not be adequate to detect an inducing substance that was either unstable, or required in the high concentrations that would be found only in the immediate proximity of cells releasing it. Thus, we turned to experiments in which the sparse amoebal culture was placed directly on top of a denser culture. Several additional refinements were also ultimately incorporated. Millipore filters (thickness, 125 ,Am) were replaced by the thinner (10 ,m) Nuclepore filters with 0.2 ,um pores. Paired filters were used, which permit the upper filter to be removed without carrying with it any cells from the culture growing on the agar surface. Formalin-killed bacteria were used to eliminate the possibility that any apparent inductive effects would have to do with either altered bacterial growth or substances released by the bacteria. The experimental arrangement after transfer of the filter-borne cultures to the inducing cultures is shown in the accompanying sketch (Fig. 4). The results of such an experiment are presented in Fig. 5. The inducing cultures, which were inoculated with 104 amoebae per puddle, exhibited the same growth and differentiation curves seen earlier (Fig. 2). The filter cultures were begun with about 50 amoebae per filter and with the same amount of food as the inducing cultures. The filter-borne amoebae also grew exponentially although at a lower growth rate than the amoebae directly on agar. In control filter cultures, which were transferred to vacant agar, tI was in excess of 100 hr. In the experimental cultures, which were transferred to inducing cultures at 36 hr, plasmodium production began at about 60 hr. We hypothesize that the accelerated plasmodium production in the experimental filter cultures is due to a substance that diffuses through the pores of the Nuclepore filters from the inducing culture below. Very few cells, if any, leave the area of a "puddle." Nevertheless, we cannot, on the basis of the observations above, absolutely rule out the possibility that the plasmodia recovered from the upper filter were in fact wandering, committed plasmodia from the inducing culture. Two experiments designed to explore this possibility utilized a mutant (mth apt 1) of Colonia that is deficient in the ability to form plasmodia (4). The experimental design was like that of the experiment whose results are shown in Fig. 5, but with the substitution of the mutant for either the upper or lower culture. When the upper
Cell Biology: Youngman et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
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F IG 5. Transfilter induction of plasmodium formation: formalin-killed bacteria. Sparse (upper) cultures of CL amoebae on 0.2gqm Nuclepore filters were transferred at the time shown by the arrow to dense (lower) cultures of CL or to vacant agar: (0, 0) lower culture; (3, *) upper culture, transferred to lower culture; (a, w) upper culture, transferred to vacant agar.
culture was mutant, no plasmodia whatsoever were recovered from the upper filter, even though the amoebae in the upper culture grew normally and the amoebae in the lower culture differentiated normally. When the lower culture was mutant, amoebae in the upper culture were induced. In fact, induction occurred even earlier with the mutant underneath than with the wild type underneath, suggesting that the mutant produces active levels of the putative inducer earlier (P. J. Youngman, B. Smith, T. M. Shinnick, and C. E. Holt, manuscript in preparation). The growth rate of the filter-borne amoebae grown on formalin-killed bacteria was less than normal. This lowered growth rate is not the sole cause of the premature plasmodium production by the amoebae, because control filter cultures transferred to vacant agar showed the same lowered growth rate and no early plasmodium production. It could be postulated, nevertheless, that only slowly growing amoebae can be induced. That this is not the case is shown by the results of an induction experiment conducted with live rather than formalin-killed bacteria. In this case, the amoebic growth rate on the filters was not reduced, and induction still occurred (Fig. 6). It is to be noted that the lawn of live bacteria is established in a short time relative to the duration of the experiment, and that the lawn for the upper culture is grown on one plate (before the filters are transferred) and the lawn for the lower culture on another. As controls to determine whether the apparent induction involved bacterial growth or bacterial metabolic products, filters were transferred to amoeba-free bacteria or to cultures of Dictyostelium discoideum amoebae. (D. discoideum amoebae, strain JM41, were grown in puddles under exactly the same conditions as the P. polycephalum amoebae and grew somewhat faster but to nearly the same extent as P. polycephalum amoebae. The former do not, of course, form plasmodia.) Plasmodium production occurred with the same kinetics in the cultures transferred to vacant agar, bacteria only, and D. discoideum.
20
80 100 120 60 Time (hours) FIG. 6. Transfilter induction of plasmodium formation: live bacteria. See legend to Fig. 5. 40
In other filter experiments similar to those described above, the ability of the putative inducer to pass through different types of filters was studied. Induction was observed with two, but not three, stacked Millipore filters (pore size 0.45 Am, thickness 125 ,gm). Induction was observed when the two 0.2 tim Nuclepore filters used routinely were replaced by two Nuclepore filters having a pore size of 0.05 ,um. In addition, reduced induction was found with a filter pair consisting of a 55 mm square of dialysis membrane and one 0.2 ,tm Nucleopore filter.
DISCUSSION Dense amoebic cultures induce the differentiation of sparse amoebic cultures when the two are separated by a pair of Nuclepore filters with a pore size of 0.05 tim. The effect occurs both with live and with formalin-killed food-bacteria. The effect is not caused by passage of cells from one culture to the other. The effect is not seen when the dense culture is replaced by bacteria only or by amoebae of a cellular slime mold. We conclude that the effect is mediated by a diffusible substance elaborated by the P. polycephalum cells. The results are compatible with the hypothesis that the diffusible substance is an inducer released by the dense, differentiating culture. Two observations raise the possibility that the diffusible substance has a short range of action. First, only when plates are as thin as 2 mm is any effect seen on the kinetics of differentiation. Second, induction does not occur through three Millipore filters. However, other explanations of these observations are possible, and further work will be needed to establish the range over which the inducer acts. The steepness and exponential nature of the plasmodial curve for the Colonia strain introduce the possibility that the differentiation process in this strain may be cooperative. It is plausible that a population of amoebae derives an advantage from a concerted transition to the plasmodial state. The social activity of organisms such as D. discoideum (9,10), myxobacterium (11), and yeast (12) provide ample precedent. This cooperativity could be achieved by the autocatalytic production, by differentiating cells, of a diffusible signal which induces neighboring amoebae to join the differentiating population. Such a hy-
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pothesis is supported by our discovery that differentiating cells do in fact possess the capacity to induce a neighboring culture to initiate differentiation. Our additional finding that food depletion instigates early differentiation indicates that starvation may function as .a stimulus to initiate differentiation, perhaps by promoting the manufacture or release of an inducing substance. An alternative hypothesis not absolutely excluded by our findings is one which.would attribute the induction effect to the destruction, by differentiating cells, of a diffusible inhibitor of differentiation which is made constitutively by cells in a sparse population. We tend to discount the hypothesis for two reasons. First, extensive dilution and replating at low density does not stimulate cells to differentiation. Second, reducing the thickness of differentiation plates promotes rather than hinders differentiation. Clearly, the work raises many questions about the cell state in which the putative inducer is produced, the time course of the production of the inducer, the responsiveness of cells to the inducer, its stability, its rate of diffusion and its chemical nature. Success in isolating, from a culture of Colonia cells, an active preparation of inducer would be a major step toward answering these questions.
Proc. Natl. Acad. Sci. USA 74 (1977) The authors are grateful to Barbara Smith for help in the conduct of experiments and to Jeanne Margolskee for the culture of D. discoideum. This work was supported by National Science Foundation Grant BMS75-15604 to C.E.H; P.J.Y and T.M.S are supported by Training Grant NIH-5-TO1-GM00515 to the Department of Biology; P.N.A was supported by National Institutes of Health Training Grant 5-T01BM11710 to the Department of Biology. 1. Gray, W. D. & Alexopoulos, C. J. (1965) Biology of the Myxomycetes (Ronald Press, New York). 2. Olive, L. S. (1975) The Mycetozoans (Academic Press, New York). 3. Cooke, D. & Dee, J. (1974) Genet. Res. Camb. 23,307-318. 4. Wheals, A. E. (1973) Genet. Res. Camb. 21, 79-86. 5. Adler, P., Davidow, L. & Holt, C. (1975) Science 190,65-67. 6. Adler, P. N. & Holt, C. E. (1974) Genetics 78, 1051-1062. 7. Haugli, F. B. (1971) Ph.D. Dissertation, Univ. of Wisconsin. 8. Sachsenmaier, W. & Rusch, H. P. (1964) Exp. Cell Res. 36, 124-133. 9. MacHac, M. A. & Bonner, J. T. (1975) J. Bacteriol. 124, 16241625. 10. O'Day, D. H. & Lewis, K. E. (1975) Nature 254,431-432. 11. Wireman, J. W. & Dworkin, M. (1975) Science 189,516-522. 12. Hicks, J. & Herskowitz, I. (1976) Nature 260, 246-248.
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 was studied in the Colonia isolate of the myxomycete, Physarum polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the appearance of cells committed to the plasmodial state. The onset of plasmodium production appears to be related to amoebic nutrition since cultures supplied with fewer bacteria display earlier differentiation. For a period of time after differentiation is initiated, conversion of amoebae to plasmodia is rapid and proceeds as an exponential function of time. A filter-transmissible substance, apparently released by differentiating cells, is implicated in the control of this rapid conversion. The myxomycete Physarum polycephalum displays two strikingly different vegetative forms: microscopic, uninucleate, colorless amoebae and macroscopic, multinucleate, yellow plasmodia (1, 2). Amoebae of the Colonia isolate, which carry the allele mth at the mating type locus, readily undergo an asexual conversion to the plasmodial state. Because the change occurs without genetic alteration (3) and results in major, stable phenotypic alterations, the material provides a model system for studies on the control of cell differentiation. Mutants affecting the differentiation can be isolated (refs. 4 and 5; L. Davidow and C. E. Holt, manuscript in preparation; P. N. Adler, manuscript in preparation), and the present work provides a beginning for physiological and biochemical studies on the process of commitment to the plasmodial state. We report here the development of a technique which permits a quantitative analysis of the time course and extent of differentiation in a Colonia culture. With this technique, we have demonstrated that a differentiating culture of Colonia cells can induce early differentiation in a neighboring culture separated by filters which prevent direct cell contact between the two populations. The results favor the conclusion that differentiating cells elaborate a diffusible inducer of differentiation.
MATERIALS AND METHODS Media. Dilute plasmodial rich medium (dPRM) agar and liver infusion agar were made as described previously (4, 6). Agar containing dPRM adjusted to pH7 (dPRM7) rather than pH 4.6 was also used. Buffer-streptomycin agar was made by adding 0.25 g streptomycin sulfate (Sigma Chemical Co.) and 10 ml of 1 M citrate buffer (pH 5) to 1 liter of 1.5% agar. Final concentrations were 250 ,ug/ml and 0.01 M, respectively. Preparation of Amoebae. Plasmodia-free amoebae for use in starting kinetics experiments were prepared by growing the amoebae on agar plates at 300 and harvesting the amoebae prior to the onset of plasmodium formation. For experiments with Abbreviation: dPRM, dilute plasmodial rich medium. * Present address: Center for Pathobiology, University of California, Irvine, Calif. 92717.
live bacteria, the amoebae were grown on lawns of live Escherichia coli on liver infusion agar. For experiments with formalin-killed bacteria (7), the amoebae were subcultured serially on formalin-killed E. coli covered buffer-streptomycin plates to ensure elimination of live bacteria. Kinetic Experiments. At time zero, replicate amoebic cultures on dPRM agar plates (15 X 100 mm) were prepared from a single suspension of plasmodia-free amoebae. The inoculum to each plate consisted of 0.05-0.1 ml of suspension placed in the middle of the plate and spread to a diameter of 21-24 mm before drying. The disc containing the bacteria and amoebae is referred to as a "puddle" and the dPRM plate bearing it as a "differentiation plate." The set of cultures was incubated at 26°. At appropriate times, the cells on individual differentiation plates were harvested and assayed for numbers of amoebae and plasmodia by the following procedure. The surface of the plate was flooded with sterile water and rubbed with a glass rod. The resulting suspension was diluted serially and samples of the dilutions were spread onto liver infusion agar or dPRM7 agar plates (assay plates). The assay plates were incubated at a temperature (30°) that is severely inhibitory to the amoebicplasmodial transition in Colonia amoebae (6) but is favorable for plasmodial development and growth. The numbers of amoebic and plasmodial plaques on the assay plates (Fig. 1) were counted. The numbers from the assay plates derived from a single suspension varied linearly with the dilution; thus, the formation of the plaques is not dependent on interactions among cells on the assay plates. Amoebic plaques were counted after 5-8 days of incubation. Plasmodial plaques were counted between 2 and 3 days of incubation with the use of a dissection microscope. The assay of the number of plasmodia in a cell suspension containing a much larger number of amoebae initially presented a difficult problem. When such a suspension was plated in a concentrated form such that the assay plate received an inoculum of more than 105 amoebae, the plasmodia that arose were unhealthy and their numbers were not reproducible. When the suspension was diluted enough so that the assay plates prepared from it gave rise to normal plasmodial plaques, the number of assay plates required for good statistics was in excess of what is practical on a routine basis. We found ultimately that assay plates receiving concentrated suspension can be used reliably, as long as the plasmodia on them are counted between 2 and 3 days of incubation. The plasmodia on such plates are spherical, rather than fan-shaped, and often die after further incubation. The identity of the structures as plasmodia was established by comparison of their number with the number of normal plasmodia on assay plates receiving diluted suspension. Filter Experiments. Millipore filters (25 mm circles, 0.45 ,um pore diameter, Millipore Filter Corp.) and Nuclepore filters (25 mm circles, 0.2 or 0.05 ,um pore diameter, Nuclepore Corp.) 1120
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FIG. 1. Amoebic and plasmodial plaques. Three amoebic and one 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. were sterilized by autoclaving (15 pounds/square inch, 20 min) and rinsed in sterile water before use. Cells were removed from the upper filter for assay by placing the filter in a large test tube containing 4 ml of water and shaking the tube on a vortex
mixer.
RESULTS Kinetics of Plasmodium Formation. The kinetics of production of plasmodia in cultures of growing amoebae was studied by the use of a biological assay. The assay permits measurement of amoebic and plasmodial numbers over a wide range. We note that the assay for plasmodia detects cells committed to the plasmodial state rather than cells necessarily having the appearance of plasmodia at the time that assay plates are prepared. Fig. 2 displays the results of kinetics experiments for two strains, Colonia or CL (mth) and a mutant (CH357) of CL. During an initial growth phase, the number of amoebae increase exponentially with time and no plasmodia are produced. (In these experiments, the level of sensitivity was such that four plasmodia per plate would have been detected.) Plasmodium production then begins, and the number of plasmodia increases exponentially with time until there are about 105 plasmodia per plate. In other experiments (not shown) the plasmodial curves after the exponential phase are seen to reach a maximum and then decline. The decline may result from the fusion of small plasmodia with one another as well as the loss, during the wash-off procedure, of larger, more fragile plasmodia. Because the plasmodial curve (Fig. 2) rises so steeply, we believe that the plasmodia arise principally or exclusively from
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 extrapolated 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, A) strain CH357.
amoebae rather than from division of preexisting plasmodia. The doubling time of the plasmodial curve is 1.1 (CH357) or 1.6 (CL) hr, and that of the amoebic curve, 8 hr. Under optimal conditions, the doubling time for plasmodial cultures is 9-12 hr (8). The hypothesis that the plasmodia arise mainly by conversion of amoebae predicts that when a sufficient number of plasmodia have formed, a decrease in the number of amoebae should be observed. A slight decrease of about the right magnitude is seen for CL. The decrease is much more apparent in the data for strain CH357, which is a spontaneous variant (P. N. Adler, unpublished) of Colonia that carries a mutation (rap) that is unlinked to mt (L. Davidow, unpublished). In this strain, early amoebic growth is indistinguishable from that of the wild type (Fig. 2 upper curves), but plasmodium production begins sooner. The number of plasmodia becomes significant relative to the number of amoebae at a relatively early time and the predicted reduction in the number of amoebae is seen (Fig. 2). Measurements of the extent and timing of differentiation, when performed as described above, are highly reproducible. Differences between strains, such as those displayed in Fig. 2, are consistently obtained. A parameter that is useful in discussing these differences may be defined as follows. Data are plotted as shown in Fig. 2 and the exponential portion of the plasmodium curve is extrapolated downward until it intersects the axis corresponding to one plasmodium per plate. The time corresponding to the intersection is designated tI and referred to as the "time when plasmodium production commences." Whether or not the first plasmodium on a plate actually arises at this time is not known, and would in fact be difficult to determine. Nevertheless, the time for the appearance of the first plasmodium must be close to t1. Thus, the two strains CH357
Cell Biology: Youngman et al.
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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 induction experiment. The upper and lower cultures have the same initial number of bacteria. 10
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70 60 50 Time (hours) FIG. 3. Kinetics of plasmodium production with varying initial food supply. The number of bacteria per puddle were as follows: (0, *) 5 X 107; (A, A) 4 X 108; (0, 0) 1.6 X 109; X refers to the average of amoebic points, which were within 10% of one another.
and CL may be said to differ by 9 hr in the time at which plasmodium formation begins (Fig. 2). Dependence of ti on Initial Numbers of Amoebae and Bacteria. We observed in a series of kinetics experiments that the less the initial inoculum of amoebae on a differentiation plate, the larger was the value of ti. The number of amoebae at the time t1, on the other hand, was approximately constant. One possible explanation of this phenomenon is that amoebae differentiate when the food supply is exhausted. As a test of this hypothesis, we started a series of cultures of the Colonia strain with a constant number of amoebae but a varying number of the bacteria that are used by the amoebae as food. Formalinkilled bacteria were used, to avoid the complications that would have arisen if live bacteria and varying amounts of soluble bacterial nutrients had been used. Measurements on the production of plasmodia by these cultures are shown in Fig. 3. As predicted by this hypothesis, tI was smallest for the culture fed with the smallest number of bacteria. The results on the variation of tI with initial numbers of amoebae and bacteria have two important implications for the design of experiments aimed at the detection of induction effects. First, it is possible to prepare amoebic cultures that would not ordinarily differentiate for a long period of time by starting them with small inocula of amoebae. Such cultures can serve as "probes" for the detection of postulated inducing substances. Second, one must determine whether any observed stimulation of plasmodium production is in fact due to induction, or merely to a limitation on the number of bacteria. Initial Attempts to Detect Inducer. Our first attempt to detect an inducer involved experiments in which the thickness of the agar plates was varied. When plates were made as thin as 2 mm, Colonia amoebae growing on them differentiated faster than on thicker plates. It was unclear whether tj was affected, but the number of plasmodia about 10 hr after t1 was certainly greater on the thinner plates. We next carried out experiments in which cultures that would be expected to release the postulated inducer were grown on the surface of Millipore
filters that were, in turn, placed on the surface of an agar plate. The filters, bearing the cells on their upper surfaces, were removed and replaced with a second set of culture-bearing filters that carried amoebae at a predifferentiation growth stage. The formation of plasmodia on the second set of filters was monitored by periodically removing a filter, washing it, and plating dilutions of the suspension on assay plates. Plasmodium production on these filters was indistinguishable from that on 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 differentiating CL amoebae did not contain demonstrable inducing activity by this test. Transfilter Induction. The filter replacement experiment described above might not be adequate to detect an inducing substance that was either unstable, or required in the high concentrations that would be found only in the immediate proximity of cells releasing it. Thus, we turned to experiments in which the sparse amoebal culture was placed directly on top of a denser culture. Several additional refinements were also ultimately incorporated. Millipore filters (thickness, 125 ,Am) were replaced by the thinner (10 ,m) Nuclepore filters with 0.2 ,um pores. Paired filters were used, which permit the upper filter to be removed without carrying with it any cells from the culture growing on the agar surface. Formalin-killed bacteria were used to eliminate the possibility that any apparent inductive effects would have to do with either altered bacterial growth or substances released by the bacteria. The experimental arrangement after transfer of the filter-borne cultures to the inducing cultures is shown in the accompanying sketch (Fig. 4). The results of such an experiment are presented in Fig. 5. The inducing cultures, which were inoculated with 104 amoebae per puddle, exhibited the same growth and differentiation curves seen earlier (Fig. 2). The filter cultures were begun with about 50 amoebae per filter and with the same amount of food as the inducing cultures. The filter-borne amoebae also grew exponentially although at a lower growth rate than the amoebae directly on agar. In control filter cultures, which were transferred to vacant agar, tI was in excess of 100 hr. In the experimental cultures, which were transferred to inducing cultures at 36 hr, plasmodium production began at about 60 hr. We hypothesize that the accelerated plasmodium production in the experimental filter cultures is due to a substance that diffuses through the pores of the Nuclepore filters from the inducing culture below. Very few cells, if any, leave the area of a "puddle." Nevertheless, we cannot, on the basis of the observations above, absolutely rule out the possibility that the plasmodia recovered from the upper filter were in fact wandering, committed plasmodia from the inducing culture. Two experiments designed to explore this possibility utilized a mutant (mth apt 1) of Colonia that is deficient in the ability to form plasmodia (4). The experimental design was like that of the experiment whose results are shown in Fig. 5, but with the substitution of the mutant for either the upper or lower culture. When the upper
Cell Biology: Youngman et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
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F IG 5. Transfilter induction of plasmodium formation: formalin-killed bacteria. Sparse (upper) cultures of CL amoebae on 0.2gqm Nuclepore filters were transferred at the time shown by the arrow to dense (lower) cultures of CL or to vacant agar: (0, 0) lower culture; (3, *) upper culture, transferred to lower culture; (a, w) upper culture, transferred to vacant agar.
culture was mutant, no plasmodia whatsoever were recovered from the upper filter, even though the amoebae in the upper culture grew normally and the amoebae in the lower culture differentiated normally. When the lower culture was mutant, amoebae in the upper culture were induced. In fact, induction occurred even earlier with the mutant underneath than with the wild type underneath, suggesting that the mutant produces active levels of the putative inducer earlier (P. J. Youngman, B. Smith, T. M. Shinnick, and C. E. Holt, manuscript in preparation). The growth rate of the filter-borne amoebae grown on formalin-killed bacteria was less than normal. This lowered growth rate is not the sole cause of the premature plasmodium production by the amoebae, because control filter cultures transferred to vacant agar showed the same lowered growth rate and no early plasmodium production. It could be postulated, nevertheless, that only slowly growing amoebae can be induced. That this is not the case is shown by the results of an induction experiment conducted with live rather than formalin-killed bacteria. In this case, the amoebic growth rate on the filters was not reduced, and induction still occurred (Fig. 6). It is to be noted that the lawn of live bacteria is established in a short time relative to the duration of the experiment, and that the lawn for the upper culture is grown on one plate (before the filters are transferred) and the lawn for the lower culture on another. As controls to determine whether the apparent induction involved bacterial growth or bacterial metabolic products, filters were transferred to amoeba-free bacteria or to cultures of Dictyostelium discoideum amoebae. (D. discoideum amoebae, strain JM41, were grown in puddles under exactly the same conditions as the P. polycephalum amoebae and grew somewhat faster but to nearly the same extent as P. polycephalum amoebae. The former do not, of course, form plasmodia.) Plasmodium production occurred with the same kinetics in the cultures transferred to vacant agar, bacteria only, and D. discoideum.
20
80 100 120 60 Time (hours) FIG. 6. Transfilter induction of plasmodium formation: live bacteria. See legend to Fig. 5. 40
In other filter experiments similar to those described above, the ability of the putative inducer to pass through different types of filters was studied. Induction was observed with two, but not three, stacked Millipore filters (pore size 0.45 Am, thickness 125 ,gm). Induction was observed when the two 0.2 tim Nuclepore filters used routinely were replaced by two Nuclepore filters having a pore size of 0.05 ,um. In addition, reduced induction was found with a filter pair consisting of a 55 mm square of dialysis membrane and one 0.2 ,tm Nucleopore filter.
DISCUSSION Dense amoebic cultures induce the differentiation of sparse amoebic cultures when the two are separated by a pair of Nuclepore filters with a pore size of 0.05 tim. The effect occurs both with live and with formalin-killed food-bacteria. The effect is not caused by passage of cells from one culture to the other. The effect is not seen when the dense culture is replaced by bacteria only or by amoebae of a cellular slime mold. We conclude that the effect is mediated by a diffusible substance elaborated by the P. polycephalum cells. The results are compatible with the hypothesis that the diffusible substance is an inducer released by the dense, differentiating culture. Two observations raise the possibility that the diffusible substance has a short range of action. First, only when plates are as thin as 2 mm is any effect seen on the kinetics of differentiation. Second, induction does not occur through three Millipore filters. However, other explanations of these observations are possible, and further work will be needed to establish the range over which the inducer acts. The steepness and exponential nature of the plasmodial curve for the Colonia strain introduce the possibility that the differentiation process in this strain may be cooperative. It is plausible that a population of amoebae derives an advantage from a concerted transition to the plasmodial state. The social activity of organisms such as D. discoideum (9,10), myxobacterium (11), and yeast (12) provide ample precedent. This cooperativity could be achieved by the autocatalytic production, by differentiating cells, of a diffusible signal which induces neighboring amoebae to join the differentiating population. Such a hy-
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Cell Biology: Youngman et al.
pothesis is supported by our discovery that differentiating cells do in fact possess the capacity to induce a neighboring culture to initiate differentiation. Our additional finding that food depletion instigates early differentiation indicates that starvation may function as .a stimulus to initiate differentiation, perhaps by promoting the manufacture or release of an inducing substance. An alternative hypothesis not absolutely excluded by our findings is one which.would attribute the induction effect to the destruction, by differentiating cells, of a diffusible inhibitor of differentiation which is made constitutively by cells in a sparse population. We tend to discount the hypothesis for two reasons. First, extensive dilution and replating at low density does not stimulate cells to differentiation. Second, reducing the thickness of differentiation plates promotes rather than hinders differentiation. Clearly, the work raises many questions about the cell state in which the putative inducer is produced, the time course of the production of the inducer, the responsiveness of cells to the inducer, its stability, its rate of diffusion and its chemical nature. Success in isolating, from a culture of Colonia cells, an active preparation of inducer would be a major step toward answering these questions.
Proc. Natl. Acad. Sci. USA 74 (1977) The authors are grateful to Barbara Smith for help in the conduct of experiments and to Jeanne Margolskee for the culture of D. discoideum. This work was supported by National Science Foundation Grant BMS75-15604 to C.E.H; P.J.Y and T.M.S are supported by Training Grant NIH-5-TO1-GM00515 to the Department of Biology; P.N.A was supported by National Institutes of Health Training Grant 5-T01BM11710 to the Department of Biology. 1. Gray, W. D. & Alexopoulos, C. J. (1965) Biology of the Myxomycetes (Ronald Press, New York). 2. Olive, L. S. (1975) The Mycetozoans (Academic Press, New York). 3. Cooke, D. & Dee, J. (1974) Genet. Res. Camb. 23,307-318. 4. Wheals, A. E. (1973) Genet. Res. Camb. 21, 79-86. 5. Adler, P., Davidow, L. & Holt, C. (1975) Science 190,65-67. 6. Adler, P. N. & Holt, C. E. (1974) Genetics 78, 1051-1062. 7. Haugli, F. B. (1971) Ph.D. Dissertation, Univ. of Wisconsin. 8. Sachsenmaier, W. & Rusch, H. P. (1964) Exp. Cell Res. 36, 124-133. 9. MacHac, M. A. & Bonner, J. T. (1975) J. Bacteriol. 124, 16241625. 10. O'Day, D. H. & Lewis, K. E. (1975) Nature 254,431-432. 11. Wireman, J. W. & Dworkin, M. (1975) Science 189,516-522. 12. Hicks, J. & Herskowitz, I. (1976) Nature 260, 246-248.
