Development 105, 753-759 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

753

Evidence that elevated intracellular cyclic AMP triggers spore maturation in

Dictyostelium

ROBERT R. KAY

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Summary

Spore maturation occurs during normal development inDictyostelium when environmental influences induce amigrating slug to transform into a fruiting body. As theamoeboid prespore cells turn into refractile spores thereis a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis ofpreformed components of the spore coat. Evidence ispresented here that this process is triggered by anelevated intracellular cAMP concentration.

First, a number of rapidly developing (rde) mutants,whose cAMP metabolism had been investigated pre-viously, are shown to be able to form spores in sub-merged monolayers, whereas wild-type strains are not.The phenotypes of these mutants are best explained by aderepression of the signal transduction pathway utilizingintracellular cAMP.

Second and more direct, it is shown that the permeantcAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore

differentiation in wild-type amoebae incubated in sub-merged monolayers. These analogues also stimulateaccumulation of UDP-galactose epimerase in slug cellstransferred to shaken suspension.

The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique thatcan be exploited in various ways. For instance, sporedifferentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neithercell contact nor additional soluble inducers are necessaryin these conditions. In contrast NC4 cells may require anadditional inducer. Spore differentiation is inhibited bythe stalk- specific inducer DIF-1 suggesting that DIF-1inhibits a target downstream of intracellular cAMP inthe signal transduction pathway inducing spore differen-tiation.

Key words: Dictyostelium, spore maturation, intracellularcAMP, 8-bromo-cyclic AMP.

Introduction

The migrating slug of Dictyostelium discoideum is anarrested stage of development which can persist fordays before it is triggered by suitable environmentalconditions to transform into a mature fruiting body. Asthis transformation proceeds, there is a radical reorgan-ization of the slug, accompanied by the overt differen-tiation of stalk and spore cells from their amoeboidprecursors. The prestalk cells sequentially vacuolateand lay down cellulose as they move into the growingtip of the stalk. The prespore cells are carried aloft bythe stalk and transform synchronously into refractile-walled spores in a process that involves a burst ofaccumulation of enzymes such as UDP-galactose epi-merase (Newell & Sussman, 1970) and, at a later stage,the exocytosis of preformed components of the sporecoat (Hohl & Hamamoto, 1969; Maeda & Takeuchi,1969).

The transformation of a slug into a fruiting body(culmination) can be triggered by overhead light, adrop in humidity or the loss of ammonia from the

aggregate (Newell et al. 1969; Schindler & Sussman,1977). In addition the slugs appear to accumulate a lowMr metabolite, possibly a weak acid, that is necessaryfor culmination (Sussman et al. 1978). Presumably theseinfluences must be transduced into the individual cellswithin the slug and integrated to trigger changes in bothgene expression and cellular morphogenesis.

The initial differentiation of prespore cells appears tobe induced by extracellular cAMP (Kay et al. 1978; Kay,1979) and, since there is evidence that cAMP signallingpersists to later stages of development (Bonner, 1949;Nestle & Sussman, 1972; Schaap & Wang, 1984), somemodulation of cAMP signalling may be involved intriggering spore maturation. cAMP signals are trans-duced into at least three intracellular second messen-gers: cAMP itself, cGMP and inositol phosphates(Gerisch, 1987; Europe-Finner & Newell, 1987). How-ever, the individual roles of the second messengers inproducing changes in gene expression, cell movementand morphogenesis have not yet been clearly dis-tinguished.

The hypothesis will be advanced here that spore

754 R. R. Kay

maturation is triggered by elevated levels of intracellu-lar cAMP. This idea was first suggested by the pheno-types of certain mutants facilitated in spore maturationand then powerfully supported by the use of permeantcAMP analogues to induce spore formation in wild-typestrains. The techniques for spore induction then al-lowed further questions to be asked about the possiblerole of signals, such as cell-cell contact, in wild-typespore differentiation.

Materials and methods

8-(4-chlorophenylthio)-cyclic AMP was from Boehringer,cAMP (cAMP), 2'-deoxy-cyclic-AMP and 8-bromo-cAMP(Br-cAMP) were from Sigma. Br-cAMP was also synthesizedby bromination of cAMP with bromine water (Muneyama etal. 1971). The precipitate was purified by first dissolving inwater by bringing to pH~8 with KOH and then reprecipitat-ing with HC1. The product remained pale brown after 3 cyclesof purification but did not contain detectable impurities onTLC (Muneyama et al. 1971, system B) and behaved identi-cally with the commercial material in the experiments to bedescribed. DIF-1 was synthesized as described (Masento et al.1988), and synthetic discadenine was a kind gift from Dr Y.Tanaka (Abe et al. 1976).

The sporogenous mutant HM15 derives ultimately fromstrain V12M2 and was selected from its immediate parent,HM2, by virtue of its ability to form detergent-resistant sporeswhen incubated in a monolayer with cAMP (Town etal. 1976;Kay et al. 1978; Kay, 1987). The rapidly developing mutantsHTY 217, HTY 506, HTY 507 and HTY 509 were a kind giftfrom Drs K. Abe and K. Yanagisawa (Abe & Yanagisawa,1983) and Frl7 (Sonneborn et al. 1963) from Dr C. D. Town.Cells were grown on Klebsiella aerogenes and prepared fordevelopment as previously described (Kay, 1987). Slugs wereobtained by allowing cells to develop on 1-8% L28 agar(Oxoid) containing 20 mM-KCl, 20mM-NaCl, 1 mM-CaCl2 andafter 18 h harvested, partially disaggregated by syringingthrough a 19-gauge needle and resuspended at a nominal celldensity of 2x10^ cells ml"1. Suspensions were shaken inconical flasks at 180 revs min"1. The medium for develop-ment, in suspension or in monolayers in Sterilin tissue culturedishes, was 10mM-2-(N-morpholino)-ethanesulphonic acid,20 mM-KCl, 20mM-NaCl, lmM-MgCl2, lmM-CaCl2 pH6-2containing 15/igmP1 tetracycline, 200/igml"1 streptomycinsulphate and cyclic nucleotides as indicated ('spore medium',Kay, 1982, 1987). Cell differentiation was monitored byphase-contrast microscopy.

UDP-galactose-4-epimerase (EC 5.1.3.2) was assayed by acoupled spectrophotometric assay (Telser & Sussman, 1971)at 35°C and protein by a dye-binding assay (Bradford, 1976).

Results

Mutants in spore maturationThe initial clue linking intracellular cAMP to sporematuration came from the phenotypes of two sets ofindependently isolated mutants in which spore matu-ration is facilitated or 'derepressed'. The sporogenousmutants were isolated because they are able to formspores in submerged monolayers with cAMP, whereastheir parents arrest as amoeboid prespore cells andnever (<1 in 105) form spores in the same conditions

Table 1.

% cell type

Strain

V12M2 (parent)HM15 (sporogenous)NC4 (parent)Frl7 (rdeA)HTY507 (rdeA)HTY509 (rdeA)HTY506 (rdeC)HTY217 (rdeC)

Stalk

56-757-0

05-61-01-600

Spore

034-5

066-840-342-943-065-1

Rapidly developing (rde) mutants are sporogenous (differentiateinto spores in submerged monolayers in the presence of cAMP).The ultimate parent of the rde mutants is NC4 and this strain isincluded for comparison, as is an authentic sporogenous mutant,HM15, and its ultimate parent V12M2. Monolayers of cells of eachgenotype were submerged at lO'cm"2 in a simple salts mediumsupplemented with 5 IHM-CAMP (see Materials and methods). After48 h stalk and spore cell differentiation was scored microscopicallyand the results given as a percentage of total cells. Results aremeans of 2-6 separate plates

(Town et al. 1976; Kay et al. 1978). These mutants aretherefore considered to be facilitated in the maturationof prespore cells into spores. The rapidly developing(rde) mutants were isolated because they form sporesprematurely in normal development (Sonneborn et al.1963; Kessin, 1977; Abe & Yanagisawa, 1983) and wereof interest because their lesion had been linked to analtered intracellular cAMP metabolism (see below) andbecause of their phenotypic resemblance to some of thesporogenous mutants. For instance, in conditions suit-able for normal development, both the sporogenousmutant HM15 and the rdeC mutants arrest as moundsand produce spores several hours earlier than theirrespective parents. Similarly HM18 and the rdeA mu-tants arrest as early culminates and spore differen-tiation is again premature. These similarities suggestedthat some of the rde and sporogenous mutants might beallelic. Unfortunately a direct genetic test of this idea isdifficult, because the two groups of mutants wereisolated in the V12 and NC4 backgrounds, which areincompatible in parasexual crosses (Robson & Wil-liams, 1979). However, it has already been shown thatthe rdeA mutant Frl7 is sporogenous (Town et al. 1976)and Table 1 shows that all rde mutants of both availablecomplementation groups (rdeB is lost) are sporogen-ous, that is they make spores in monolayers whenincubated with cAMP. Kessin (1977) suggested that therde phenotype might be due to an overproduction ofintracellular cAMP, which in turn acted as an inducer ofdevelopmental gene expression. Altered cAMP metab-olism in the rde mutants was subsequently confirmed bydirect measurement (Coukell & Chan, 1980; Abe &Yanagisawa, 1983). Rde A mutants have elevatedintracellular cAMP levels as expected, but surprisinglyrdeC mutants have very low levels. However, thisparadoxical property of rdeC mutants can be explainedwithin the original hypothesis, since in both yeast andmammalian cells cAMP levels are controlled by nega-tive feedback acting through the cAMP-dependent

Induction of spore maturation by cAMP in Dictyostelium 755

protein kinase (Nikawa et al. 1987; Gettys et al. 1987).Thus a constitutively active protein kinase would feedback to inhibit adenyl cyclase and produce low cAMPlevels, as seen in the rdeC mutants, while producing thedownstream effects of elevated cAMP levels.

The results with the spore maturation mutantssuggest that elevated intracellular levels of cAMPinduce spore maturation, but a more direct test of thisidea was required.

Spore induction in wild-type strains by permeantcAMP analoguesIn mammalian cells, many of the effects of hormonesthat use intracellular cAMP as a second messenger canbe mimicked using high extracellular concentrations ofcertain cAMP analogues. These analogues can bypassthe relevant surface receptor by penetrating the plasmamembrane and activating cAMP-dependent proteinkinase directly. The most potent analogues, such asthose with an 8-substitution of the adenine ring, areeffective because they are both more resistant tohydrolysis by cAMP-phosphodiesterase and better ableto activate the cAMP-dependent protein kinase thancAMP itself (Simon et al. 1973; Miller et al. 1975). Themost promising analogue for Dictyostelium cells seemedto be 8-bromo-cAMP (Br-cAMP) which has about a7-fold increased Km for the phosphodiesterase and a3-fold decreased KA for the protein kinase compared tocAMP (Van Haastert et al. 1983; de Wit et al. 1982).Concentrations of 0-1-1 mM-Br-cAMP are usually

necessary with mammalian cells but higher concen-trations were also explored with Dictyostelium cells,because of their relative impermeability.

It is apparent from Fig. 1 that high concentrations ofBr-cAMP can induce greater than 70% spore forma-tion amongst amoebae of strain V12M2 incubated fromthe start of development with the inducer in submergedmonolayers. In these experiments, spore formationstarted after about 16 h. Spores could also be induced instrain NC4 though less efficiently (see later). Theinduced spores stain with a spore-specific antibody(Takeuchi, 1963) and retain full viability after detergenttreatment, which kills all amoebae (0-3 % cemulsol for2h; not shown). Spore formation can be detected at5mM-Br-cAMP and is half-maximal at llmM-Br-cAMP. Of a number of other analogues tested over arange of concentrations only 8-chlorophenylthio-cAMPwas active. It was roughly as potent as Br-cAMP butunfortunately it could not be used above 8 ITIM due toprecipitation in the incubation medium. The followingwere inactive at up to 40 ITIM: CAMP, dibutyryl-cAMP,8-bromo-cGMP, dibutyryl-cGMP, 2-deoxy cAMP.

The experiments described so far show clearly thatBr-cAMP can induce starving cells to differentiate intospores, but do not indicate when Br-cAMP (rather thancAMP) acts to do this. Three observations suggest thatit is the maturation of prespore cells into spores that canbe specifically promoted by Br-cAMP but not bycAMP. First, cAMP is able to induce starving cells todifferentiate as far as prespores but not spores in similarmonolayer incubation conditions (Kay et al. 1978; Kay,

0 10 20 308-Br-cAMP concentration (ITIM)

Fig. 1. Induction of spore differentiationby Br-cAMP in submerged monolayers ofcells of strain V12M2. Left, dose-responsecurve with cells at a density of SxlC^cm"2.Right, phase-contrast micrographs of cellsat lff'cm"2 incubated without Br-cAMP(top) or with 20mM-Br-cAMP (bottom).Amoebae were incubated for 48 h in tissueculture plates containing spore mediumplus 100 ng ml"1 BSA and the appropriateconcentrations of Br-cAMP. Results from 2dose-response experiments are pooled.

756 R. R. Kay

10 r

00

00a.QD

30 60 90Time (min)

120

Fig. 2. Induction of UDP-galactose epimerase, a markerfor culmination, by Br-cAMP. Migrating slugs at t18 werepartially disaggregated in spore medium and the suspensionshaken at 180revmin~L with the additions indicated (cyclicnucleotides were 15 ITIM). Duplicate l'5ml portions wereassayed for enzyme activity and proteins as described inMaterials and methods. The experiment is representative of4.

1982). Second, Br-cAMP induces prespore cells, takenfrom migrating slugs, to differentiate into spores with adelay of only 3-4 h compared to the 16 h delay withvegetative cells. Again, spores do not form with cAMP(not shown). Finally a biochemical marker for sporematuration, UDP-galactose epimerase (Newell & Suss-man, 1970), is rapidly induced when Br-cAMP is addedto slug cells in shaken suspension (Fig. 2). In theseconditions cAMP does not induce the enzyme, thoughit does stabilize existing levels.

Mode of action of Br-cAMPSeveral arguments indicate that Br-cAMP cannot beinducing spore maturation solely by occupation of theknown surface cAMP receptor: (1) receptor saturatingconcentrations of Br-cAMP (2mM, about 20 times thereceptor Ko for Br-cAMP; Van Haastert & Kein, 1983)do not induce spore maturation (Fig. 1); (2) highconcentrations of agonists (cAMP, 2'-deoxy-cAMP)with a much greater affinity for the surface receptorthan Br-cAMP are without effect; (3) spore inductionby Br-cAMP is not inhibited by equimolar cAMP,though this should displace nearly all the Br-cAMPfrom the surface receptor (the K& for cAMP is about450-fold lower than that for Br-cAMP; van Haastert &Kein, 1983; result not shown). It therefore seems mostlikely that Br-cAMP works by penetration of the cellmembrane and activation of the intracellular responsemachinery in Dictyostelium, as in mammalian cells.

Involvement of other signalsThe technique just described for inducing wild-typecells to differentiate into spores allows a number offurther questions to be asked about the factors control-ling spore differentiation. For instance, spore differen-

102 103 104

Cell density (cells cm"2)

Fig. 3. Cell-density dependence of spore differentiation instrain V12M2. Cells were plated at the stated densities intissue culture dishes containing spore medium plus 15 ITIM-Br-cAMP, 100 jig ml"1 BSA and 10/igmP1 of the sporegermination inhibitor discadenine. Cell differentiation wasscored microscopically at t^. At low cell densities most cellsbecome spores, whereas at high density DIF accumulatesand stalk cells differentiate in consequence. Spores:• • ; stalk cells: A • .

tiation might require, in addition to Br-cAMP, someform of interaction between the cells in the monolayer.The interaction might require either cell-cell contact orthe accumulation of a diffusible inducer but in eithercase it would be attenuated at low compared to high celldensity. Fig. 3 shows that spore differentiation in strainV12M2 is in fact very efficient at low densities, wherethe cells are all single. This result is similar to thatobtained previously with various sporogenous mutants(Kay, 1982) and seems to preclude any essential role inspore induction for cell-cell contact or diffusibleinducers in these conditions. The reduced efficiency ofspore formation at high cell density is probably due toaccumulated DIF diverting the amoebae to stalk forma-tion (see Fig. 4). Spore formation by cells of strain NC4is always less efficient than with V12M2 cells, beingrarely greater than 30 % at high cell density and fallingto zero at 103 cells cm"2 (not shown). One contributingfactor is that the NC4 spores tend to hatch out to giveamoebae soon after they form. Hatching can bereduced by including 10^M-discadenine (a spore germi-nation inhibitor, Abe et al. 1976) in the medium, buteven in this case NC4 cells do not form spores at lowdensity, suggesting that an additional factor is necessary(see Grabel & Loomis, 1978; Mehdy & Firtel, 1985;Berks & Kay, 1988). The putative factor has not beencharacterized but preliminary experiments indicate thatit is not methionine or ammonia, which do not improvethe efficiency of spore formation by low-density NC4cells at 5mM and 20 mM, respectively (not shown;Gibson & Hames, 1988; Gross et al. 1983).

DIF-1 (l-[3,5-dichloro-2,6-dihydroxy-4-methoxy-phenyl]hexan-l-one; Morris et al. 1987) is an endogen-ous stalk-specific inducer which has been shown toinhibit prespore and spore differentiation, diverting the

Induction of spore maturation by cAMP in Dictyostelium 757

3? 100

5 10DIF concentration (nM)

15

Fig. 4. DIF-1 diverts cells of strain V12M2 from spore tostalk cell differentiation. Vegetative cells were incubated intissue culture dishes at a density of SxKh'cm" in sporemedium supplemented with 100 /.ig ml"1 BSA, 20mM-Br-cAMP and DIF-1 as indicated and spores scoredmicroscopically after 40 h. The results of 2 experiments,each done with duplicate plates, are combined. • •spore cells; • • stalk cells. DIF-1 also suppressedspore formation when slug-stage cells were incubated underthe same induction conditions (not shown).

cells to differentiate instead into stalk cells (Kay &Jermyn, 1983). It has been suggested that the inhibitionof spore differentiation by DIF-1 is a consequence of aninhibition of cAMP binding to its surface receptor(Wang et al. 1986). Such an inhibition would bebypassed by Br-cAMP if it acts intracellularly. How-ever, since spore formation induced by Br-cAMP is stillsensitive to inhibition by DIF-1 (Fig. 4), it appears thatDIF-1 must also have a target further down the signaltransduction pathway than intracellular cAMP, at leastat the time of spore maturation.

Discussion

The hypothesis underlying the experiments describedhere is that spore maturation can be triggered by anespecial elevation in intracellular cAMP levels. Thishypothesis was first suggested by the altered cAMPmetabolism in mutants where spore maturation occursmore readily than in the wild type and is stronglysupported by the induction of spore maturation in wild-type strains by permeant cAMP analogues. There areseveral further consequences of this hypothesis.

First, elevated intracellular cAMP levels may triggerspore maturation during normal development as well asduring monolayer incubation. In support of this, severalstudies, including one where the aggregates were indi-vidually staged, show that cAMP levels increase 2- to3-fold as spores mature during culmination (Brenner,1978; Abe & Yanagisawa, 1983; Merkle et al. 1984). It ispossible that in the single exception, where only a smallrise in cAMP levels was detected, the strain A3 used didnot develop with sufficient synchrony to produce astrong increase in cAMP levels (Brenner, 1978). Therise in cAMP levels during culmination could bebrought about by a modulation of the basic cAMPsignalling system by some other signal. For instance, a

drop in ammonia levels can trigger culmination(Schindler & Sussman, 1977) and would be expected toproduce an elevation in intracellular cAMP levels(Williams et al. 1984).

Second, the hypothesis suggests a number of lesionsthat might account for the sporogenous and rde pheno-types. Since all the mutants tested are geneticallyrecessive, they could represent the knock-out of differ-ent inhibitory elements in the cAMP signal transductionpathway. Targets might include a G( protein affectingadenyl cyclase, the regulatory subunit of cAMP-depen-dent protein kinase and intracellular cAMP phosphodi-esterase.

Finally, intracellular cAMP may stimulate differen-tiation at other stages of development apart fromduring culmination (Sampson etal. 1978; Kessin, 1977).This idea is attractive even though it has been shownthat the expression of certain aggregative and postag-gregative genes can be induced without the normaloscillatory increases in intracellular cAMP (Wurster &Bumann, 1981; Oyama & Blumerg, 1986). Even inthese cases, adenyl cyclase is sufficiently active toproduce intracellular concentrations of cAMP in the fiurange, which should be adequate to stimulate fully thecAMP-dependent protein kinase or other cAMP-bind-ing proteins (Sampson, 1977; de Gunzberg & Veron,1982; Tsang & Tasaka, 1986). A role for intracellularcAMP at earlier stages of development is furthersuggested by the acceleration of early gene expressionin the rde mutants (Sonneborn etal. 1963; Kessin, 1977;Abe & Yanagisawa, 1983).

Spore differentiation by monolayers of wild-type cellshas not been described before (a preliminary reportappeared in Kay et al. 1988) and this technical advanceallows a number of further questions to be asked aboutthe control of spore differentiation. One question iswhether cell-cell contact is necessary for cell differen-tiation, as has been suggested by indirect experiments(Mehdy etal. 1983; Chisholm etal. 1984). Cell contact isclearly not necessary in strain V12M2 since isolatedcells at great dilution form spores efficiently in the Br-cAMP medium (see also Kay & Trevan, 1981; Kay,1982, for similar results with sporogenous mutants).Strain NC4 differs from V12M2 in that both spore andstalk cell differentiation (Berks & Kay, 1988) are veryinefficient at low cell density. The reason for this is notknown but it could be due to a stringent requirement fora soluble factor early in development whereas inV12M2 cells this requirement is more relaxed (Grabel& Loomis, 1978; Mehdy & Firtel, 1985). A secondquestion is where is the target for the inhibition of sporecell differentiation by DIF-1 (Kay & Jermyn, 1983). Inprinciple, this target could be at any point in the signaltransduction pathway leading from extracellular cAMPto overt spore differentiation and the cAMP receptorhas been suggested as a potential target (Wang et al.1986). However, the present results suggest an ad-ditional target below intracellular cAMP in the path-way. Finally it has been suggested that cell-cycle phaseat the time of starvation may determine whether anindividual cell differentiates toward a stalk or a spore

758 R. R. Kay

cell (Gomer & Firtel, 1987). In the experiments de-scribed here amoebae can be switched from about 90 %spore to 90 % stalk cell differentiation merely by addingDIF-1 to the starvation medium. This, and many otherresults showing cell-type regulation during develop-ment (e.g. Raper, 1940), indicate that cell-type differ-entiation is regulated by interactions between the cellsand is not predetermined by intrinsic differences be-tween them.

I should like to thank Jeff Williams, Mary Berks, DavidTraynor, Ines Carrin, Robert Insall, Jenny Brookman andMark Bretscher for comments on the manuscript.

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{Accepted 13 January' 1989)