Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium...

Differentiation (1987) 34: 79-87 Differential ion 0 Springer-Verlag 1987

Original articles

Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium discoideum Margaret Clarke *, Samuel C. Kayman and Kristina Riley Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA

Abstract. The synthesis of the lectin, discoidin I, by vegetative cells of Dictyostelium discoideum (strain NC4) was monitored using immunoblot analysis and indirect immunofluorescence. Suspension cultures were used, so that the D. discoideurn cell density and the concentration of bacteria could be controlled. Discoidin-I production was found to be a function of the relative densities of D. discoideum cells and food bacteria. Synthesis was initiated in exponentially growing D. discoideum cells approximately three generations before depletion of the food supply. In the growth medium of cells producing discoidin I, a soluble activity was detected that caused low-density cells to begin discoidin-I synthesis. This activity was not dialyzable and was destroyed by heat. A similar activity was produced by AX3 cells during axenic growth. Density-dependent induction of other 'early developmental' proteins was also detected in wild-type cells. These findings suggest that the expression of several 'early developmental' genes is regulated by a mechanism that measures cell density relative to food supply, not by starvation per se.

Introduction

The eukaryotic microorganism, Dictyostelium discoideum, exists as free-living amoebae as long as a food supply is available. The natural food is bacteria, and D. discoideum cells are commonly propagated in the laboratory in con- junction with bacteria on nutrient agar plates. When starved, the amoebae initiate a developmental program that results in cell aggregation and the formation of multicellular fruiting bodies. The growth and development of D. discoideum have been reviewed by Loomis [22, 231.

Among the proteins whose synthesis is greatly increased in starving cells is the lectin, discoidin I. The synthesis of this family of related proteins peaks within a few hours after the cells have been shifted to starvation conditions, and then decreases during later development [25, 32, 341. The function of discoidin I has been a source of interest and controversy. Early studies suggested a role in cell-cell adhesion (reviewed in [2]). Recent work has shown that discoidin I has some sequence homology to fibronectin and is involved in cell-substratum adhesion as well as in cell streaming during aggregation [9, 351.

* To whom offprint requests should be sent

Earlier studies detected trace amounts of discoidin-I protein [34] and mRNA [32] in vegetative cells; however, the significance of these observations was not clear, because these studies used cells growing in association with bacteria on nutrient agar plates, a condition subject to regional vari- ation with respect to the time at which the food supply is depleted (for further discussion, see [32]).

In the present study, we monitored the production of discoidin I in a continuously mixing suspension culture of wild-type D. discoideum cells and food bacteria. These conditions permit an accurate determination of the growth state of D. discoideum cells and the level of the food supply. We found that exponentially growing cells do synthesize discoidin I, although not constitutively. The production of discoidin I is regulated by the density of D. discoideum cells relative to that of the food bacteria.

Methods

Strains and culture conditions. Dictyostelium discoideum, strain NC4, was grown in association with Klebsiella aerogenes. The bacteria were spread on SM agar plates [22], incubated at 22" C for 2-5 days, and rinsed from the plates with 17 mM potassium phosphate buffer (pH 6.4). The final volume was adjusted to an optical density at 660 nm of 8 for the standard (1 x ) bacterial suspension; this corre- sponded to approximately 35 ml buffer per 100-mm plate. Dictyostelium discoideum cells were mixed with freshly prepared bacterial suspension as spores or amoebae at a density of lo3 to lo4 cells/ml and incubated at 21" C on a rotary shaker (180 rpm) until they had reached the desired density. The generation time was 2.8 h under these conditions; the cell number was determined by counting in a hemacytometer. Unless otherwise indicated, all experiments were carried out with NC4 cells. For an experiment involv- ing axenic cells, the axenic mutant AX3 was grown on HL5 medium as previously described [7].

Antibodies. The antiserum specific for discoidin I was a gift from Dr. Samuel Barondes. The antiserum was received in a lyophilized form and was rehydrated in water to the original strength. It was then diluted (1 : 20) in TBS (20 m M Tris-HC1 and 150 mM NaCl, pH 7.4) and preabsorbed with K. aerogenes cells. Bacteria from 2 ml bacterial suspension prepared as described above were collected by centrifugation ( 5 min at 3000 g), fixed in 10 ml methanol at - 15" C for 5min, and washed three times in TBS. The washed

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bacteria were suspended in 2 ml TBS and mixed with 100 p1 1 :20 antibody against discoidin I. After 1 h at room temperature, the bacteria were removed by centrifugation. The secondary antibody [fluorescein-isothiocyanate (F1TC)- conjugated goat anti-rabbit IgG; Cooper Biomedical; diluted 1:100] was preabsorbed with bacteria in the same manner. In addition, the secondary antibody was preabsorbed with glutaraldehyde-fixed D. discoideum cells, prepared as follows: cells were washed with 17 mM phosphate buffer (Na,HPO, and KH,PO,, pH 6.4), and lo8 cells were suspended in 1 ml phosphate buffer, mixed with 1 ml 4% glutaraldehyde, and incubated for 5 min at room temperature. The cells were washed three times in phosphate buffer and suspended in 0.1 M glycine ethyl ester for 1 h at room temperature. They were then washed three times in TBS and mixed with antibody at the ratio of 5 x lo7 cells per milliliter of diluted antibody. After 1 h at room temperature, the cells were removed by centrifugation.

Indirect immunofuorescence. Dictyostelium discoideum cells were harvested by centrifugation at 200 g for 3 min. For each sample, 2 x lo6 cells were collected. The cell pellet was washed by vortexing in 10 ml17 mM phosphate buffer, pH 6.4, and was then centrifuged again. Three washes were carried out. The final cell pellet, which was essentially free of bacteria, was resuspended in 0.5 ml of the same buffer, and a drop of the cell suspension was placed on a glass coverslip in a moist chamber. After 5 min to allow the cells to settle and attach, the cells were covered with a small square of agarose prepared as described elsewhere [36], and the excess liquid was removed by blotting. The coverslip was immersed in a solution of 1 % (v/v) formaldehyde prepared by dilution of a 37% stock into absolute methanol; fixation was carried out at - 15" C for 5 min. The fixed cells were washed by three 5-min rinses in TBS at room temperature; during these rinses, the agarose layer was lifted off and discarded. Each cell sample was covered with 50 pl antibody against discoidin I (diluted 1 : 200 in TBS), incubated at 37" C for 30 min, and washed as described above. Each sample was then covered with 50 pl of a 1 : 400 dilution of FITC-conjugated anti-rabbit IgG and incubated in the dark for 30 min at 37" C. After a final series of washes conducted in the dark, each sample was rinsed in distilled water and mounted as described elsewhere [36]. The samples were observed and photographed using a Zeiss standard microscope equipped with epifluorescence illumination and phase-contrast optics. For each experiment, all fluorescence photographs were taken and printed using identical expo- sure settings.

Electrophoresis and immunoblot procedures. Polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS) was carried out using gels containing 12% acrylamide and the discontinuous buffer system described by Laemmli [ 191. Gels were stained with Coomassie Brilliant Blue, or used unstained for immunoblot experiments. Immunoblot analysis was carried out as previously described [31] using a solution of 5% nonfat dry milk in TBS for the blocking and dilution of antibodies. The primary antibody was the antibody against discoidin I, which was preabsorbed as described above and diluted (1 : 50) in TBS. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Cooper Biomedical; not further purified) at a dilution of 1 : 500.

Protein determination. Protein was measured using a modification [17] of the Lowry assay [24].

Enzyme assays. NC4 cells were grown on a 1 x bacterial suspension and harvested by centrifugation at densities of 5 x lo5 and 5 x lo6 cells/ml. The cells were washed free of bacteria by differential centrifugation (four cycles at 200 g) using 17 mM potassium phosphate buffer (pH 6.5); the final pellets were suspended in membrane buffer [50mM Tris-HCl, pH 7.5, 20 mM MgSO,, 10 m M p-tosyl-L-argi- nine methyl ester (TAME), and 10 mM benzamidine], counted, and then pelleted again. For the assay of membrane-bound CAMP phosphodiesterase, cells were resuspended in membrane buffer at a density of 10' cells/ml, and membranes were prepared and assayed as previously described [3]. For the assay of lysosomal enzymes, cells were suspended at a density of 10' cells/ml in dH,O containing 10 m M TAME and 20 m M benzamidine, and then lysed by freezelthaw and sonication. Aliquots were assayed as described below. The starving cells had been grown on a 1 x bacterial suspension and harvested at a density of 5 x lo5 cells/ml. They were washed free of bacteria as described above, except that the fourth wash was with starvation buffer (3 mM potassium phosphate buffer, 2.5 m M KCI, 0.65 mM MgCl,, and 100 pg/ml dihydrostreptomycin sulfate). The final cell pellet was resuspended in starvation buffer at a density of 1 x lo7 cells/ml; the cell suspension was shaken at 180 rpm for 8 h at 21" C, washed once with 17 mM potassium phosphate buffer, and then processed as described above.

The buffer used for the assay of the lysosomal enzymes, a-mannosidase-I and p-galactosidase-2, was citric-acid phosphate (CP) prepared as described in the Documenta Geigy Scientific Tables. a-Mannosidase activity was measured at pH 3.5 [12, 131. ,!I-Galactosidase activity was measured at both pH 2.5 and 5.0; the activity of p-galactosidase-2 was calculated from the difference of these two values, as described elsewhere [lo]. The reaction mixtures contained 50 p1 cell lysate (diluted in dH,O), 100 p1 CP buffer, and 150 p1 substrate added to start the reaction. The substrates were p-nitrophenyl-a-u-mannopyranoside (final concentration, 10 mM) or p-nitrophenyl-p-D-galacto- pyranoside (final concentration, 20 mM; Sigma, St. Louis, MO). The reaction mixtures were incubated at 35" C for 20 or 60 min, and the reaction was stopped by the addition of 300 pl 1.0 M Na,CO,. The samples were centrifuged for 3 min at 13000 g to remove insoluble material, and the absorbance of the supernatant fluid at 410 nm was determined. Hydrolysis was measured from the release of p-ni- tro-phenol (molar extinction coefficient, 1.62 x lo4). Values were the average from duplicate samples. Each enzyme was measured in at least two different cell preparations; the results in these cases were always similar.

Results

Discoidin-I production by wild-type D. discoideum cells during vegetative growth. We used antiserum specific for discoidin I in indirect-immunofluorescence assays as a sensitive means to detect the induction of discoidin-I synthesis. In the course of these studies, we observed that, under some conditions, wild-type (NC4) cells produced discoidin I during vegetative growth. To confirm that the antigen being detected was actually discoidin I, we purified discoidin I as

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I O O L ! I I I I , , , , , A B

s 1 2 3 1 2 3

C D

1 2 3 1 2 3 Fig. 1A-D. Immunoblot detection of discoidin I in vegetative NC4 cells. NC4 cells were harvested from bacterial suspension culture at densities of 3 x lo5 and 3 x lo6 cells/ml and washed free of bacteria. The total protein from 8 x lo5 cells at each density was applied to adjacent wells of an SDS gel next to an aliquot of purified discoidin I. A The three samples stained for protein; B identical samples transferred to nitrocellulose and stained with an antibody against discoidin I as described in Methods. A, B : lane 1, cells at 3 x lo5 cells/ml; lane 2, cells at 3 x 106 cells/ml; lane 3, discoidin I (1.0 pg in A; 0.2 pg in B); lane S, molecular-weight standards (phosphorylase b, 94000; bovine serum albumin, 67000; ovalbu- min, 43000; carbonic anhydrase, 30000; soybean trypsin inhibitor, 20100; a-lactalbumin, 14400). C, D A similar experiment in which NC4 cells were grown on three concentrations of bacteria; cells were harvested at a density of 3 x lo6 cells/ml and washed free of bacteria. Equal aliquots of cells from the 1/2 x (lane I ) , 1 x (lane 2), and 2 x (lane 3) bacterial suspensions are shown. C protein stain; D immunoblot. The arrow marks the migration position of discoidin I

previously described [S] and tested the specificity of the antiserum by immunoblot analysis. The results (shown in Fig. 1 A and B) confirmed that the antiserum recognized the purified protein and detected only a single co-migrating species in crude cell lysates. The antigen was not detectable in low-density cultures, but was detectable in cultures of

W Q

2 4 6 8 TIME (h)

4 ' 6 ' ' ' ' ' ' ' ' ' Fig. 2. Growth rate of NC4 cells on different concentrations of bacteria. NC4 cells from an exponentially growing culture were inoculated at a density of 1 x lo3 cells/ml into three different concentrations of bacteria, i.e., l j2 x (.), 1 x (o), and 2 x (A). (The 1 x concentration of bacteria corresponds to an optical density at 660 nm of 8, and represents our standard growth conditions.) Cell counts were performed at intervals beginning the following morning; the first count was designated time zero. The UYYOWS

mark times when samples were taken for immunofluorescence (see Fig. 3)

moderate density. The effect of cell density was further examined as described below.

Discoidin-I production as a function of the relative levels of D. discoideum cells and,food bacteria. The immunoblot results described above indicated that the density of the D. discoideum cells affected discoidin-I production. We tested whether cell density was the only significant variable, or whether the level of the food supply was also important. In order to vary the ratio of D. discoideum cells to food bacteria in a controlled manner, we prepared suspensions of K. aerogenes by rinsing bacteria from nutrient agar plates (see Methods). Our standard (1 x ) bacterial suspension had an optical density at 660nm of 8; bacterial suspensions that were half as concentrated (1/2 x ) and twice as concentrated (2 x ) were also prepared. D. discoideum cells were inoculated at a density of 1 x lo3 cells/ml into these three concentrations of bacteria and were allowed to grow overnight.

Samples from these three cultures were taken for immunoblot analysis when the D. discoideum cells had reached a density of 3 x lo6 cells/ml. Figure 1 C and D show that the concentration of food bacteria did affect discoidin-I levels; more discoidin I was produced at lower concentrations of bacteria. Since all three cultures were in log-phase growth at the time that they were sampled, there could not have been any shortage of food. This was examined more critically in a second experiment.

NC4 cells were inoculated into the three concentrations of bacteria described above; the cell number was determined at intervals by direct counting. The rate of cell growth in the three concentrations of bacteria was identical, at least up to a cell density of 6 x lo6 cells/ml (Fig. 2). In other experiments (not shown), we determined that expo- nential growth continued to a density of 1-2 x lo7 cells/ml for cells growing on a 1 x bacterial suspension. Thus, cells at a density of 3 x lo6 cells/ml were growing exponentially in the presence of an ample food supply.

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Fig. 3A-L

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Fig. 4A-D. Regulation of discoidin-I synthesis by a soluble cell product. NC4 cells were grown on a standard bacterial suspension culture. When the cells had reached a density of 5 x lo6 cellsjml, the cells and bacteria were removed by centrifugation, and the conditioned buffer was used to prepare fresh bacteria at the 1 x concentration. NC4 cells were grown overnight on this conditioned medium and on a regular bacterial suspension. Both cultures were tested for discoidin-I production while still at low density: A, B control culture, 2 x lo5 cells/ml; C, D conditioned culture, 1 x lo5 cells/ml. In each case, the same field of cells is shown by indirect immunofluorescence of discoidin I and by phase-contrast microscopy. Bur, 20 pm

Cells from the three cultures monitored in Fig. 2 were tested for discoidin-I production by indirect immunofluorescence. This technique requires 500-fold less antibody than immunoblotting, and it offers the additional advan- tage of providing information regarding the behavior of individual cells. Samples were taken at two time points (marked by arrows in Fig. 2) corresponding to cell densities of 7 x lo5 and 3 x lo6 cells/ml. The results are shown in Fig. 3. The upper and lower rows of immunofluorescence images illustrate discoidin-I production as a function of D. discoideum cell density: more cells were producing discoidin I in the higher-density cultures. The immunofluorescence images across each row illustrate the effect of bacterial concentration; at a constant D. discoideum cell density, more cells were producing discoidin I in the presence of lower concentrations of food bacteria. Both the immunofluorescence and immunoblot techniques indicated that a population of D. discoideum cells can measure its own density relative to that of its food supply, and turn on discoidin- I synthesis while still in log-phase growth.

Production of a soluble substance that regulates discoidin-I synthesis. The simplest model to explain the data presented above is a two-component system. The D. discoideum cells could monitor their own density by elaborating a substance that accumulates in the medium in proportion to their cell density. The production of this substance or its activity could be modulated by the concentration of the food bacteria. If such a mechanism operates, the growth medium from cells at a high enough density to be producing discoidin I should be ‘conditioned’ with the inducing substance. As shown in Fig. 4, this proved to be the case. The medium from cells that were producing discoidin I was freed of cells and bacteria by centrifugation (10 min at 12000 g ) and then used to prepare a new suspension of freshly harvested bacte-

ria. Cells inoculated into this medium produced discoidin I even at very low cell densities (Fig. 4). Other experiments showed that several hours were required for the induction of discoidin-I synthesis by conditioned medium. Medium ‘conditioned’ by low-density cells that were not producing discoidin I did not induce discoidin-I synthesis (not shown).

Properties of the inducer of discoidin-I synthesis. We examined some of the properties of the discoidin-I inducer in conditioned medium using the methods illustrated in Fig. 4 to detect activity. The activity passed through a 0.2-pm Nalgene filter, but was retained by dialysis tubing (Spectra- por l , molecular-weight cut-off, 6000-8000). It was destroyed by heating at 70” C for 15 min, although it survived heating at 50” C for 15 min. It was adsorbed by activated charcoal. These properties are appropriate for a macromol- ecule such as a protein or glycoprotein.

We measured the amount of protein present in conditioned medium and found it to be extremely low, i.e., approximately 8 pg/ml. We tested whether the addition of a comparable concentration of purified discoidin I to a low- density cell culture could mimic the effect of conditioned medium; it had no detectable effect. Attempts to purify the inducing substance from conditioned medium have thus far resulted in loss of activity, possibly because the concentration of the substance is so low.

Induction of discoidin-I synthesis in axenic cells. Although wild-type D. discoideum cells can grow only by the phagocy- tosis of bacteria, mutants have been isolated that are capa- ble of growth on liquid (‘axenic’) medium (for a review of the properties of such mutants, see [6]). Axenic mutants have been shown to produce discoidin I (and certain other proteins whose levels are elevated in starving cells) during growth on axenic medium, but not during growth on bacte-

Fig. 3A-L. Discoidin-I production as a function of the relative densities of NC4 cells and food bacteria. Discoidin-I production was monitored by indirect immunofluorescence of cells growing on three different concentrations of bacteria as described in the legend to Fig. 2 : 1/2 x (A, D, G, J), 1 x (B, E, H, K), and 2 x (C, F, I, L). Each culture was sampled at two different cell densities, i.e., 7 x lo5 cells/ml (A-F) and 3 x lo6 cells/ml (G-L). In each case, the same field of cells is shown by indirect immunofluorescence (above) and phase-contrast microscopy (below). Bur, 20 pm

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Fig. 5A-L

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Table 1. Effect of cell density and starvation on enzyme activity

Growth state of NC4 cells" Specific activityb

a-mannosidase-I P-galactosidase-2 Membrane-bound CAMP phosphodiesterase

Growing cells

5 x 1o6/ml

Starving cells

5 x 0.096 0.36 0.91 0.98

3.0 0.9

20.0 2.9 33.0

a Cells growing on a 1 x bacterial suspension were harvested at the indicated density. Starving cells were starved in suspension for 8 h at a density of 1 x lo7 cells/ml

For a-mannosidase-I and B-galactosidase-2, the data represent nanomoles per minute per milligram of cell protein ; for membrane-bound CAMP phosphodiesterase, the results are given as nanomoles per minute per microgram of membrane protein

ria [4, 3 1, 331. We considered the possibility that the axenic induction of discoidin-I synthesis is due to the same regulatory mechanism found in wild-type cells. If so, the density- dependent induction of discoidin-I synthesis would also take place in axenically growing cells, but at a lower cell density than is seen with wild-type cells, because of the absence of bacteria. We tested this prediction using cells of the axenic mutant AX3, growing on the standard axenic growth medium, HL5.

As detected by indirect immunofluorescence, axenically growing AX3 cells produced little or no discoidin I at very low cell density, but began to synthesize discoidin I as their density increased (Fig. 5A-C). The density required to induce discoidin-I synthesis in axenically growing cells was approximately one order of magnitude lower than that required for bacterially growing cells. This is consistent with the earlier observation that more discoidin can be purified from high-density than low-density axenic cells [33].

If the axenically growing cells were responding to the same regulatory mechanism as wild-type cells, a further prediction was that HL5 medium taken from AX3 cells that were producing discoidin I should be able to induce low- density wild-type cells to produce discoidin I. This proved to be the case, while HL5 alone had no effect (Fig. 5G, H). As determined from its effect on low-density wild-type cells, the inducing activity present in AX3-conditioned HL5 medium was retained by dialysis tubing and was destroyed by heating at 70" C (Fig. 51). It therefore seems likely that the inducing substances produced by axenically growing and bacterially growing cells are identical. This would confirm that D. discoideum cells themselves (and not the bacteria) are the source of the inducing activity.

Density-dependent induction of other 'developmentally regulated' proteins. In preliminary experiments, we measured

the levels of certain other proteins whose levels or activities increase early in the developmental phase of the D. discoideum life cycle. For these experiments, we used exponentially growing wild-type cells at densities of 5 x lo5 and 5 x 10' cells/ml, and also cells that had been washed free of bacteria and starved in suspension for 8 h. We assayed two enzymes whose activity is known to be elevated in both starving cells and axenically growing cells (also the case for discoidin I), i.e., a-mannosidase-1 [l , 211 and p-galactosidase-2 [lo]. The specific activity of both of these enzymes was greater in the higher-density cultures; the values ob- tained were intermediate between those of the lower-density cells and 8-h starved cells. The latter values were consistent with previously reported values for vegetative cells and starving cells [lo, 211. We also assayed membrane-bound cyclic AMP phosphodiesterase, whose activity is elevated in starving cells but not in axenically growing cells [26]: its activity did not increase in the higher-density cultures but actually diminished somewhat. This, too, is consistent with the behavior of this enzyme in axenically growing cells, in which its activity is lower than in bacterially growing cells [3]. The data from these experiments are summarized in Table 1.

Discussion

Many earlier studies have shown that specific proteins or mRNAs are preferentially synthesized at certain stages of the D. discoideum life cycle [5, 18, 20, 22, 301. In wild-type cells, there appears to be a clear demarcation between genes and proteins expressed during growth (or during growth and development) and those induced by starvation, which has been considered to be the trigger for initiating the developmental phase of the life cycle. Among the earliest group

Fig. 5A-L. Regulation of discoidin-I synthesis in axcnically growing cells. AX3 cells growing on HL5 medium were monitored for discoidin-I production as a function of cell density. Cells were washed twice in either 17 mM phosphate buffer or filtered HL5, and then processed for immunofluorescence. Cell densities at the time of harvest were 4 x lo4 (A, D), 1 x lo5 (B, E) and 1 x lo6 cells/ml (C, F). AX3 cells were also tested for production of a soluble substance that induced discoidin-] synthesis in wild-type cells. HL5 growth medium from an AX3 culture at a density of 5 x lo6 cells/ml was freed of cells by centrifugation, filter sterilized, and dialyzed overnight against 17 mM potassium phosphate buffer. A portion of the conditioned medium was heated at 70" C for 15 min. Bacterial suspensions were prepared using fresh HL5 that had also been filtered and dialyzed (C, J), conditioned HL5 (H, K), or conditioned HL5 that had been heated (I, L). These bacterial suspensions were inoculated with NC4 cells, which were grown overnight and harvested at a density of 3 x l o5 cells/ml. Discoidin-1 production was monitored by indirect immunofluorescencc. Bar, 20 pm

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of genes expressed in starving cells is discoidin I (reviewed in [18]).

In the present study, we showed that discoidin-I synthesis is actually initiated in exponentially growing cells approximately three generations before depletion of the food supply. Furthermore, studies in progress indicate that at least two other proteins whose levels increase greatly in starving cells are also affected (cr-mannosidase-1 and P-galactosidase-2), suggesting that this is a general regulatory mechanism. Thus, several genes previously considered to be ‘early developmental’ may actually be induced not by starvation per se, but by a mechanism that measures cell density relative to food supply. Such genes would be expressed by exponentially growing cells at a density that was a function of food level. Under our standard suspension culture conditions, discoidin-I synthesis is induced at a density of about 1 x lo6 cells/ml in cells growing on bacteria, and at a density of about 1 x lo5 cells/ml in cells growing on axenic medium.

The simplest regulatory system consistent with our data would consist of two components, one elaborated by the D. discoideum cells and the other by the food bacteria. An interaction between these two substances could provide a means of measuring the relative concentrations of D. discoideum cells and bacteria. The present data indicate that the D. discoideum component is a soluble substance. Compara- ble experiments have failed to detect a bacterial product (soluble or bacteria associated) that can neutralize the inducing activity present in conditioned medium (unpublished observations). It therefore seems likely that food bacteria inhibit the production of the inducing substance or the ability of D. discoideum cells to respond to it, rather than inacti- vating it directly. The actual regulatory pathway and the chemical nature of the soluble component produced by D. discoideum cells remain to be defined.

There have been several studies of soluble factors that are produced by starving cells and affect gene expression, although there is no previous report of such a factor being produced by exponentially growing cells. Two groups have examined soluble factors produced very early in development. Grabel and Loomis [I51 found that the accumulation of an early developmental enzyme, N-acetylglucosaminidase, required extremely high cell density (greater than 3 x lo7 cells/ml) as well as starvation: the requirement for high cell density could be by-passed if low-density starving cells ( 5 x lo6 cells/ml) were exposed to medium conditioned by high-density starving cells. The effector molecule was small enough to pass through a dialysis membrane and was heat stable, both of which properties differ from those described here. In the study of Grabel and Loomis, the ‘low ’-density cells, which failed to accumulate enzyme, were at a higher density than those from which we could isolate conditioned medium. Thus, there are fundamental differences between their observations and ours, suggesting that different regulatory systems are involved. Interestingly, Grabel and Loomis [15] noted that axenically growing cells produced conditioning activity, although the heat stability and dialyzability of this activity were not reported. It is therefore possible that the axenically produced activity was identical to that described in the present study.

Mehdy and co-workers [28, 291 have also detected a factor secreted by cells during early development. Their conditioned medium factor, or CMF, appears to be required for preaggregation gene expression, which in turn

renders cells competent for the induction of prestalk and prespore gene expression [14]. Although the properties of CMF have not been well defined, preliminary studies have suggested that it is a small, dialyzable molecule [28], possibly identical to the developmentally regulated, dialyzable ‘D factor’ described by Hanna and Cox [16]. The dialyzability of these factors differs from the behavior of the inducing activity produced by growing cells, but the information available about the properties of CMF is insufficient to exclude the possibility of a relationship [18a].

The regulatory system described in the present study may account for the elevated levels of several ‘early developmental ’ proteins present in axenically growing cells [l, 41. Among the limited number of proteins that we have examined so far, there is a correlation between density- dependent induction in vegetative wild-type cells and elevated levels in axenically growing cells. Discoidin I, cl-mannosidase-I , and /$galactosidase-2 exhibit elevated levels under both conditions, whereas the level of membrane- bound cyclic AMP phosphodiesterase is not elevated under either of these conditions, but only in starving cells. Because the enzyme levels reached in moderate-density exponentially growing cells are intermediate between those of low- density cells and starved cells, the density effect can most clearly be seen in enzymes that manifest a sharp increase. Such enzymes were chosen for our initial analysis, although others must also be examined to confirm this apparent correlation.

The situation in axenic cells is complicated by the effects of the axenic mutations, which are only poorly understood. Some aspects of this additional complexity have been pointed out by Oyama and Blumberg [30] in their studies of developmental gene expression in axenically vs. bacterially grown cells, as well as by our laboratory [6] in preliminary studies of the effects of the axenic linkage groups I1 and I11 on the levels of axenically produced discoidin I. Further work is needed to understand how the axenic mutations interact with the regulatory system described here.

Indirect immunofluorescence provides a sensitive and informative technique for monitoring the production of discoidin I. It demonstrated that the response of a clonal cell population to a uniform set of environmental conditions is extremely heterogeneous. Our data suggest that the low levels of discoidin-I mRNA found in populations of bacterially grown vegetative cells [32] do not represent a uniform low level of transcription, but rather a high level in a few cells. This variability is difficult to attribute to differences in microenvironment, because in the present study, samples were taken from a continuously mixing culture. It is possible that the same cells that are precocious in turning on discoidin I (and presumably other proteins subject to the same regulatory mechanism) will later be the initiators of aggregation centers. The ability to initiate centers is affected by position in the cell cycle [27], which might also be a factor in the induction of discoidin-I synthesis. This possibility remains to be tested.

Acknowledgements. We are grateful to Dr. Samuel Barondes for generously supplying the discoidin-I antiserum used in this study, and we thank Deborah Lans and Richard Birchman for expert technical assistance. Deborah’s experiments led to our recognition of the phenomenon characterized here. This work was supported by grants from the National Institutes of Health to M.C. (GM11301 and GM29723).

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1931265-275

Received February 1987 / Accepted May 4, 1987

Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium...

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Transcript of Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium...